by ALENCIA V. WOODARD-GRICE SUSAN L. BELLIS, CHAIR DANIEL BULLARD STUART FRANK DENNIS KUCIK JOANNE MURPHY-ULLRICH DOUGLAS WEIGENT A DISSERTATION

Transcription

1 HYPOSIALYLATION REGULATES α4β1 INTEGRIN BINDING TO VCAM-1 by ALENCIA V. WOODARD-GRICE SUSAN L. BELLIS, CHAIR DANIEL BULLARD STUART FRANK DENNIS KUCIK JOANNE MURPHY-ULLRICH DOUGLAS WEIGENT A DISSERTATION Submitted to the graduate faculty of The University of Alabama at Birmingham, in partial fulfillment of the requirements for the degree of Doctor of Philosophy BIRMINGHAM, ALABAMA 2008

2 ii HYPOSIALYLATION REGULATES α4β1 INTEGRIN BINDING TO VCAM-1 ALENCIA V. WOODARD-GRICE CELLULAR & MOLECULAR PHYSIOLOGY ABSTRACT During monocyte activation and differentiation along the macrophage lineage, the activity of α4β1 integrins is upregulated, which promotes extravasation from the vasculature and migration through inflamed tissues. In this dissertation research, we investigated the mechanisms regulating α4β1 activation, utilizing U937 promonocytic cells as a model system for studying the monocyte/macrophage transition. When treated with PMA, these cells acquire functions characteristic of mature phagocytes. We demonstrate that PMA induces a rapid, yet transient, activation of α4β1 receptors, which is followed by a more pronounced and sustained increase in α4β1 binding activity. We hypothesize that this second increase in binding allows sustained recruitment of monocytes, which are known to traffic to sites of inflammation much later than neutrophils. Our results suggest that this latter adhesive phase occurs as a consequence of a structural change in α4β1 receptors, namely, the expression of a β1 integrin subunit that lacks α2,6 linked sialic acids. During in vitro differentiation of both U937 cells and primary CD14 + human monocytes, the ST6Gal-I sialyltransferase is markedly downregulated, which leads to hyposialylation of the β1 subunit. The expression of hyposialylated integrins is temporally correlated with increased cell adhesion to VCAM- 1. In addition, pharmacological intervention, as well as ectopic expression of activated MEK, in U937 cells reveal that ST6Gal-I downregulation, integrin hyposialylation, and VCAM-1 binding are all directed by a PKC/ras/ERK signaling cascade, a pathway

3 iii known to be in involved in monocyte differentiation. Interestingly, the activity of BACE1, a protease not previously identified in monocytes, appears to be responsible for decreased ST6Gal-I levels. We find that in U937 cells and primary monocytes, differentiation along the macrophage lineage dramatically increases the expression of BACE1 (via a PKC/ras/ERK signaling cascade), which in turn mediates ST6Gal-I cleavage and shedding from the cell. Importantly, preventing ST6Gal-I downregulation, through both BACE1 inhibition and constitutive overexpression of ST6Gal-I, eliminates α4β1 dependent VCAM-1 binding. In addition, ST6Gal-I overexpression also prevents a PMA-induced conformational change of the β1 integrin into a more activated state. Taken together, these results describe a novel mechanism for regulation of α4β1 integrins, and further suggest a previously unanticipated role for BACE1 in immune cell function.

4 iv DEDICATION I dedicate this work to my parents, Emanuel and Sandra Woodard. My mother, the best science project designer ever, instilled in me, at a very young age, a love of education and a drive for perfection. Thank you for never accepting any cheap A s. My father taught me the true meaning of hard work and responsibility and the healing power of a good laugh. Thank you for all you have done to ensure that all my dreams could come true.

5 v ACKNOWLEDGEMENTS When I first entered graduate school, a former student (and now one of my closest friends) Dr. Chastity Bradford told me, Learning what not to do is still learning. Truer words have not been spoken of scientific research. This simple, yet powerful, adage gave me the strength to accept the pitfalls and failures as vital experiences. I thank Dr. Susan Bellis, my mentor, for patiently guiding me through my quest to become an independent scientist. I am a better writer and a more meticulous researcher because of your constant training. I am grateful to the members of my committee for the time they invested in my professional development and for the invaluable suggestions for my project. I must thank my husband, Joseph, the love of my life, for his constant support and encouragement. Thank you for listening intently to my countless presentations, even if you did not find integrin glycosylation to be a thrilling topic. Without you, I could have never balanced the pressures and requirements of graduate school with the joys and responsibilities of motherhood and marriage. I also thank my son, Joseph Emanuel, for his astounding ability to make even the worst day brighter and worthwhile with one smile, hug, funny face or big kiss. You are my driving force. Everything I have done and sacrificed has been to make all of your dreams come true. I thank my big sister, Adrienne, for being my personal cheerleader. From teaching me to read at the age of 2 to paving the way to Vanderbilt and ultimately UAB, you have always been my hero. I strive constantly to make you proud.

6 vi To my innumerable family, friends and prayer warriors, thank you for every supportive word you have uttered. Your prayers made all of this possible. Thank you for the frequent babysitting, the invitations to dinner, cleaning my house and everything else you did to help. I appreciate these kind acts more than you will ever know. Throughout my graduate career, numerous faculty, staff and students here at UAB have offered a great deal of assistance. However, two students in particular have made my graduate experience much better. I thank both Clintoria Richards-Williams and Kristin Hennessy for their constant words of encouragement, laughs, hugs and prayers. Most of all, I thank my Lord and Savior Jesus Christ for without you none of this would be possible. Thank you for being all encompassing, for being in the in between and taking care of even the small things. For God is not a man that He should lie, nor a son of man, that He should repent. Has He said, and will He not do? Or has He spoken, and will He not make it good? (Numbers 23:19)

7 vii TABLE OF CONTENTS Page ABSTRACT... ii DEDICATION... iv ACKNOWLEDGMENTS...v LIST OF TABLES... ix LIST OF FIGURES...x LIST OF ABBREVIATIONS... xii INTRODUCTION...1 Immune Cells...1 Lymphocytes...2 Granulocytic Cells...3 Mast Cells...4 Dendritic Cells...4 Follicular Dendritic Cells...5 Mononuclear Phagocytes...5 Innate Immunity...6 Inflammation...6 Leukocyte Extravasation...6 Integrins: Structure, Function and Regulation...13 Function of the α4β1 Integrin...19 N-Linked Glycosylation...20 ST6Gal-I Sialyltransferase...24 Regulation of ST6Gal-I Expression...25 ST6Gal-I in Immuunology...29 Regulation of β1 Integrins by Differential Sialylation...31 Role of Variant Sialylation in Regulating Monocyte/Macrophage Function...34 Rationale for Study...36

8 viii TABLE OF CONTENTS (Continued) Page PROTEOLYTIC SHEDDING OF ST6GAL-I BY BACE1 REGULATES THE GLYCOSYLATION AND FUNCTION OF α4β1 INTEGRINS...37 A POTENTIAL ROLE FOR BACE1 IN IMMUNOLOGY...72 CONCLUSIONS...86 FUTURE DIRECTIONS...96 GENERAL REFERENCES...100

9 ix LIST OF TABLES Table Page 1 Integrin receptors...14

10 x Figure LIST OF FIGURES INTRODUCTION Page 1 The leukocyte adhesion casaced Integrin structure and conformation Integrins expressed by immune cells Steps of N-glycosylation Glycosyltransferases have similar secondary topology...23 PROTEOLYTIC SHEDDING OF ST6GAL-I BY BACE1 REGULATES THE GLYCOSYLATION AND FUNCTION OF α4β1 INTEGRINS 1 α4β1 dependent VCAM-1 binding temporally correlates to hyposialylated β1 integrin expression The α4 subunit is not a substrate for ST6Gal-I VCAM-1 binding is regulated by a PCK/ras/ERK cascade Forced ST6Gal-I expression blocks PMA-induced β1 integrin hyposialylation and VCAM-1 binding Forced ST6Gal-I expression blocks PMA-induced conformational changes of the β1 integrin BACE1 expression is upregulated by a PKC/ras/ERK signaling cascade Upregulation of BACE1 expression temporally correlates with ST6Gal-I downregulation and secretion BACE1 inhibition prevents PMA-dependent ST6Gal-I downregulation and secretion, as well as VCAM-1 binding Differentiation of CD14 + monocytes induces increased VCAM-1 binding...61

11 xi Figure LIST OF FIGURES (continued) CONCLUSIONS Page 1 Working model...95

12 xii LIST OF ABBREVIATIONS Aβ AD APP APR BACE1 camek dbsa ECM ER ERK ESAM EV FN FRET GalNAc GlcNAc Glc-T GnT-V HSC amyloid β Alzeheimer s Disease amyloid precursor protein acute phase response β site APP-cleaving enzyme1 constitutively active MEK expressing U937 cells denatured bovine serum albumin extracellular matrix endoplasmic reticulum extracellular signal regulated kinase endothelial cell-selective adhesion molecule empty vector U937 cells fibronectin fluorescence resonance energy transfer N-acetylgalactosamine N-acetylglucosamine glucosyltransferase ß1,6-N-Acetylglucosaminyltransferase V hematopoietic stem cells

13 xiii HRP ICAM-1 ICAM-2 I-domain I-EGF IFN-γ IL-1 IL-1R2 JAK horseradish peroxidase intercellular adhesion molecule-1 intercellular adhesion molecule-2 inserted domain integrin epidermal growth factor-like interferon- γ interleukin-1 interleukin-1 type II receptor Janus kinase JAK-2 Janus kinase 2 JAM LAD LEC LDL machr MAdCAM-1 MIDAS NF-κB NK NSAIDs OPN OST Par junctional adhesion molecule leukocyte adhesion deficiency Long-Evans Cinnamon low density lipoprotein muscarinic acetylcholine receptor mucosal addressin cell adhesion molecule-1 metal-ion dependent adhesion site nuclear factor-κ B natural killer nonsteroidal anti-inflammatory drugs osteopontin oligosaccharyltransferase parental U937 cells

14 xiv PECAM-1 PMA PKC PPARγ PSGL-1 PSI SNA ST6 ST3Gal ST6GalNAc platelet-endothelial cell adhesion molecule-1 phorbol myristate acetate protein kinase C peroxisome proliferators-activated receptor-γ P-selectin glycoprotein ligand-1 plexin/semaphorin/integrin Sambucus Nigra Agglutinin U937 cells constitutively expressing ST6Gal-I β galactoside α2,3-sialyltransferase (α-n-acetyl-neuraminyl-2,3-β-galactosyl-1,3)-nacetylgalactosaminide α-2,6-sialyltransferase ST6Gal ST6Gal-I ST6Gal-II ST8Sia STAT STAT1 TCR T C T H T reg VE-cadherin VCAM-1 β galactoside α2,6-sialyltransferase β galactoside α2,6-sialyltransferase-i β galactoside α2,6-sialyltransferase-ii α-n-acetyl-neuraminide α-2,8-sialyltransferase signal transducer and activator of transcription signal transducer and activator of transcription-1 T cell receptors T cytotoxic T helper T regulatory vascular-endothelial cadherin vascular cell adhesion molecule-1

15 xv VLA very late activating

16 1 INTRODUCTION Multicellular organisms possess an immune system as a defense against pathogens. This highly adaptable system employs a host of cells and molecules to achieve a suitable scheme of recognition and response. The immune system has two distinct collaborative components; innate and adaptive immunity. Innate immunity is highly effective as the body s first line of defense, utilizing molecules and cells that circulate prior to the onset of infection. Most infections are prevented or eliminated within hours of initial exposure by the innate immune system. On the other hand, adaptive immunity, which initiates in response to infection, is specialized to recognize, eliminate and remember pathogens. Dependent on innate immunity, adaptive immunity commences a few days following the initial infection and provides a second, broad line of defense to eradicate pathogens that either escape the innate responses or persevere in spite of them. Immune Cells Various white blood cells, or leukocytes, circulate within the blood and lymph and populate the lymphoid organs. These cells, like all blood cells, evolve from hematopoietic stem cells (HSCs) during a process termed hematopoiesis. Hematopoesis begins in the embryonic yolk sac, the fetal liver and the spleen during the early months of gestation. However, after the seventh month of gestation, the majority of HSC

17 2 differentiation occurs in the bone marrow. Self-renewing multipotent HSCs can either proliferate or differentiate along one of two pathways, into a lymphoid or myeloid progenitor cell. Lymphoid progenitor cells develop into T, B and natural killer (NK) cells. Myeloid progenitor cells give rise to progenitors of red blood cells (erythrocytes), many of the white blood cells, and megakaryocytes, which generate platelets. Lymphocytes Lymphocytes comprise approximately 30% of the body s white blood cells and account for 99% of the cells in the lymph. They are classified into 3 major populations: T, B and NK cells, based on their function and membranous components. B and T lymphocytes each express individual sets of antigen receptors and are considered the major cells of adaptive immunity. However, the body s small population of large granular NK cells are a part of innate immunity and do not express the cell surface markers that distinguish B and T cells. Mature B cells uniquely synthesize and express membranous antibodies. When a naive B cell first encounters and binds the antigen that correspond with its antibody, it rapidly divides and its progeny differentiate into plasma cells and memory B cells. While memory B cells, which have a longer lifespan than naive cells, express the same membranous antibody as their parent B cell, plasma cells produce a secreted form of the same antibody. During maturation, T cells express T cell receptors (TCRs), which are unique membranous molecules that only recognize antibody that is bound to cell membrane proteins. Upon antigen recognition, the T cell proliferates and differentiates into effector

18 3 and memory T cells, which are classified as T helper (T H ), T cytotoxic (T C ) and T regulatory (T reg ) cells. T H cells, which display CD4 glycoproteins, differentiate into effector cells that facilitate the activation of other immune cells, such as B cells, T C cells and macrophages. T C cells, which express CD8 glycoproteins, proliferate and differentiate into memory cells that monitor and eliminate any cells that display foreign antigen, such as virus infected cells or tumor cells. Unlike T H and T C cells, T reg cells, which express both CD4 and CD25, negatively regulate the immune system. Natural killer cells recognize and exhibit cytotoxic activity against an extensive range of tumor cells and some viruses, without expressing antigen specific receptors. Instead, NK cells employ two different mechanisms to recognize potential target cells. NK cells can utilize NK-cell receptors to recognize abnormalities. In addition, NK cells can utilize CD16 to attach to antitumor or antiviral antibodies on target cells from a previously mounted CD16 mediated antibody response against antigens on the surface of these cells. NKT cells display characteristics of both NK cells and T cells. They express TCRs as well as CD16 and other NK receptors. NKT cells also possess the ability to kill target cells, as well as secrete cytokines. Granulocytic Cells Granulocytes are classified by their morphology and cytoplasmic staining into 3 subpopulations: basophils, eosinophils and neutrophils. Basophils have lobed nuclei and profoundly granulated cytoplasm which stains with methylene blue, a basic dye. These cells, which release pharmacologically active molecules, play a major role in certain allergic responses. Eosinophils have bilobed nuclei and granulated cytoplasm that stains

19 4 with eosin red, an acidic dye. Like neutrophils, eosinophils exhibit phagocytic activity that is thought to be involved in defense against parasitic organisms. Neutrophils, which are also called polymorphonuclear (PMN) leukocytes, constitute approximately 60% of circulating white bloods cells and contain multilobed nuclei and granulated cytoplasm which stains with both acidic and basic dyes. These cells, which are generally the first cell types to adhere to inflamed endothelium, are specialized for phagocytosis. Mast Cells Mast cells, which are very similar to basophils, contain granules rich in histamine and heparin. However, mast cells only differentiate after they migrate from the bloodstream into tissue. Although best known for their role in the development of allergies and anaphalaxis, mast cells also play an important protective role, being directly involved in wound healing and pathogen defense (160). Dendritic Cells The four major categories of dendritic cells (Langerhans, interstitial, monocytederived, and plasmacytoid-derived) arise from HSCs through different mechanisms in different locations. These cells, which occur in many forms, function as antigenpresenting cells. Once immature dendritic cells capture antigen, they migrate to the lymph nodes and present the antigen to T cells (169).

20 5 Follicular Dendritic Cells Follicular dendritic cells, which do not originate in bone marrow, are entirely different from the dendritic cells previously discussed. These cells are found in lymph follicles and do not function as antigen-presenting cells. However, these cells express high levels of membrane receptors for antibodies, which allows them to interact with and play an important role in the maturation and differentiation of B cells (133). Mononuclear Phagocytes Circulating monocytes and tissue macrophages constitute the mononuclear phagocytic system. Granulocyte-monocyte progenitor cells differentiate into promonocytes, which leave the bone marrow and differentiate into mature monocytes in the blood. Monocytes enlarge 5 to 10 fold while circulating and then migrate into specific tissues, where they are termed macrophages. Some macrophages become fixed in individual tissues, while others remain motile. During an immune response, multiple types of stimuli are known to activate macrophages. Phagocytosis or receptor contacts are often initial activating stimuli, while T H cell secreted cytokines can enhance macrophage activity. Macrophages employ several antipathogen strategies. In addition to phagocytosis of pathogens, macrophages serve as antigen-presenting cells to T H cells, secrete various cytokines and produce complement proteins that promote inflammation.

21 6 Innate Immunity The innate immune system provides immediate defense against infection, usually within minutes or hours. However, it does not supply long term or protective immunity. The major functions of innate immunity include cytokine mediated immune cell recruitment to inflammatory and infectious sites, complement cascade activation, foreign substance identification and removal, and activation of the adaptive immune system via antigen-presentation. Inflammation Inflammation, which is one of the immune system s first responses to infection, is stimulated by chemicals released by injured cells. This complex cascade of events serves to create a physical barrier against the spread of infection and to promote healing of damaged tissue. Within a few hours, leukocytes begin to extravasate into the infected tissues, phagocytose pathogens, and release cytokines for the recruitment and activation of effector cells. Leukocyte Extravasation Leukocyte extravasation, which was originally described by nineteenth century pathologists, is an extremely regulated process involving various cell adhesion molecules whose expressions are upregulated by cytokines and other inflammatory mediators as the inflammatory response progresses. This process takes place mainly in post-capillary venules, where haemodynamic shear forces are reduced (126). Selectin-mediated rolling, chemoattractant induced activation, and integrin-mediated arrest and adhesion

22 7 contributed to combinatorial specificity in the original model of the leukocyte adhesion cascade (24, 189). Transendothelial migration was not initially included in the leukocyte adhesion cascade as the mechanisms underlying this process have only recently been elucidated (91). In fact, recent insights gained over the past decade have led to the expansion of the original three-step model of the leukocyte adhesion cascade, which now include slow rolling, adhesion strengthening, intravascular crawling, transcellular and paracellular migration, and basement membrane migration (91, 109, 120, 152) (Figure 1). Figure 1 The leukocyte adhesion cascade. Cell Adhesion Molecules In general, there are four families of leukocyte-endothelial adhesion molecules; selectins, selectin ligands, integrins and Ig superfamily receptors. The selectin family consists of three-carbohydrate recognizing transmembrane molecules, which all interact with P-selectin glycoprotein ligand 1 (PSGL-1). While L-selectin and E-selectin

23 8 expression occurs only on leukocytes and activated endothelium respectively, P-selectins are present on platelets and activated endothelium (145, 203). Selectin ligands are richly glycosylated via O-linked side chains (146, 208) and N-linked carbohydrates (191). The Ig superfamily receptors, intercellular adhesion molecule-1 (ICAM-1), intercellular adhesion molecule-2 (ICAM-2) and vascular cell adhesion molecule-1 (VCAM-1), represent the largest family of endothelial adhesion molecules (189). ICAM-1 ligands include leukocyte specific β2 integrins, while VCAM-1 associates with α4β1 and α4β7 integrins (15). β2 integrins are expressed by all leukocytes. In addition, monocytes, eosinophils and lymphocytes also express β1, and α4 integrins. β7 integrins are also expressed by eosinophils and lymphocytes (90). Leukocyte capture Leukocytes have a tendency to circulate in a position close to the endothelial surface rather than in the central blood stream due to a phenomenon termed margination. Margination depends on red blood cell aggregation (67, 154) and interaction with white blood cells in small venules (174). Selectin mediated capture initiates the interaction between endothelial cells and leukocytes (127), a process which is believed to be L- selectin dependent, allowing leukocytes that lack E- and P-selectin to reach inflammatory sites (125) (Figure 1). In vitro, α4 integrins have been shown to initiate lymphocyte capture under shear and in the absence of selectins (14).

24 9 Selectin-mediated rolling Selectins associate and disassociate with their ligands at an exceptionally high rate, allowing leukocytes to adhere to activated endothelium under blood flow conditions. The most important rolling molecules, E- and P-selectin, are expressed on endothelial cells (Figure 1). However, inhibition of E-selectins appears to have limited effects on the inflammatory process, which can be explained by the redundancy of E- and P-selectin function with respect to rolling (99). Interestingly, selectin binding can initiate signals in both the selectin and ligand expressing cells. However, the specific signaling pathways are only recently beginning to emerge (1, 119, 185, 228). Integrin-mediated rolling It has been demonstrated that integrins also contribute to leukocyte rolling. In vivo, eosinophils use α4 integrins to roll in cytokine-treated venules (190). Lymphocytic cell lines use α4β1 and α4β7 integrins to reversibly interact with VCAM-1 (4, 14, 99) and Mucosal Addressin Cell Adhesion Molecule-1 (MAdCAM-1) (10, 14) respectively (Figure 1). α4β1-dependent rolling is typically observed with monocytes and monocytic cell lines, T cells and T cell lines (14, 25, 87, 186). α4 integrin-dependent rolling occurs at high rolling velocities indicating that it has a much lower efficiency than selectindependent rolling (125). Leukocyte rolling is also supported by β2 integrins. In vitro, E-selectin engagement with neutrophils induces an αlβ2 conformation with intermediate affinity, which allows αlβ2 to transiently bind to ICAM-1 (29, 168) (Figure 1). αlβ2 integrins expressed by K562 leukaemia cells support rolling on ICAM-1 (183). Recent studies

25 10 indicate that, under shear stress, αlβ2 can alter its conformation and increase ligand binding (8). Furthermore, in vivo slow rolling has been demonstrated to involve αlβ2 or αmβ2 integrin engagement, in addition to E-selectin (53, 100, 114). Leukocyte activation and arrest Rolling leukocytes are in close contact with the endothelium for extended periods of time, which allows them to be activated by mediators secreted from or bound to activated endothelium. This activation initiates leukocyte arrest, or firm adhesion, via integrin binding to endothelial ICAM-1 and VCAM-1. β1 and β2 integrins are the most pertinent to leukocyte arrest. Although additional mechanisms exist, neutrophil firm adhesion is largely β2 integrin-dependent. Neutrophils use both αmβ2 and αlβ2 for adhesion (125), while monocytes, lymphocytes and eosinophils can bind to VCAM-1 using their α4β1 integrins (55) (Figure 1). In addition, integrin ligand affinity is considered a key step in chemokine induce leukocytic arrest (25, 66, 109). For example, monocytic increases in α4β1 affinity for VCAM-1 involves conformational changes (25, 31). Transendothelial migration The final step in leukocyte emigration is transmigration through venular walls into inflamed tissues. This process can actually occur with negligible disruption to the structure of the vessel wall. Platelet-Endothelial Cell Adhesion Molecule-1 (PECAM-1) is required for leukocyte transmigration both in vitro and in vivo (207) (Figure 1). β2 integrins have also been shown to be required for transmigration in several in vitro

26 11 systems (214). However, in cases of myocardial damage, significant neutrophil emigration continues when β2 is neutralized or absent (19). Crawling. Neutrophils and monocytes crawl inside blood vessels prior to crossing the postcapillary venule walls via ICAM-1 and αmβ2 (158, 173) (Figure 1). In fact, transmigration is delayed when crawling is hindered (158). When leukocytic αmβ2 associates with endothelial ICAM-1, leukocyte membrane extensions begin to protrude into the endothelial cell and junctions. In addition, adherent leukocytes can stimulate the formation of endothelial cell projections containing ICAM-1, VCAM-1, as well as cytoplasmic and cytoskeletal components. These projections, which are referred to as docking structures or transmigratory cups, may initiate transendothelial cell migration via a paracellular or transcellular pathway. Paracellular migration. Most studies have determined that leukocytic transendothelial migration occurs primarily along the paracellular pathway at endothelial junctions (132) (Figure 1). The field has focused on endothelial junctional molecules, such as CD99, ICAM-1&2, platelet/endothelial-cell adhesion molecule 1 (PECAM-1), junctional adhesion molecules (JAM-A,B&C) and endothelial cell-selective adhesion molecule (ESAM) (152). Transmigrating leukocytes must traverse endothelial junctions which contain multiple proteins involved in selective permeability and cell-cell adhesion, such as vascular endothelial-cadherin (VE-cadherin) (117). VE-cadherin establishes a complex with several cytosolic molecules. This complex, which provides stability by linking to the actin cytoskeleton, is thought to regulate leukocyte transmigration (98,

27 12 136). Specifically, neutrophils and monocytes can induce displacement of VE-cadherin during transmigration (181, 192), suggesting that paracellular leukocyte transmigration is regulated by compounded signaling mechanisms. Transcellular migration. The paracellular paradigm for leukocyte transmigration has been recently challenged by several studies that provide evidence for a transcellular pathway whereby leukocytes migrate through endothelial cells (22). Although only a marginal amount of migrating leukocytes utilize the transcellular pathway, the response can be very rapid (33). Following the extensions of leukocytic protusions during crawling, ICAM-1 association initiates signaling cascades in endothelial cells that result in the formation of channels through which leukocytes can migrate (126) (Figure 1). Transcellular neutrophil migration is associated with areas of endothelial-cell thinning in vivo (59). Moreover, the same molecules that regulate paracellular migration may be involved in transcellular migration (56). Extravasation Following transmigration, leukocytes migrate through the extracellular matrix (ECM) to reach target sites of inflammation. Many of the lymphocytic β1 integrins bind ECM proteins. Although neutrophils do not express the full assortment of β1 integrins (113), they may be able to use β2 integrins to bind ECM proteins (17, 215) (Figure 1).

28 13 Integins: Structure, Function and Regulation Integrins, a family of heterodimeric glycoproteins, are composed of two noncovalently linked type I transmembrane glycoproteins, referred to as α and β subunits. In vertebrates, there are currently 18α and 8β species known (86), forming at least 24 different receptors (134). One of the most ubiquitously expressed subunits, the β1 (CD29) integrin, is known to pair with at least 12 different α subunits (89). The pairing of these subunits determines the ligand specificity of each integrin (Table 1). For example, α4β1 binds VCAM-1 and FN, while α5β1 binds primarily FN. Integrin α and β subunits have large extracellular domains. Excluding the β4 integrin subunit, integrin cytoplasmic domains are relatively short (46). The N-terminal regions of the receptor form a globular, extracellular ligand-binding domain. This globular region stands on 2 legs which connect to the transmembrane and cytoplasmic domains (134) (Figure 2a). Initial X-ray crystallography of the extracellular domain of the αvβ3 integrin revealed that the legs of the heterodimer were severely bent with the head domain close to the leg portion near the membrane (222, 223). Subsequently, several studies have demonstrated that this bent conformation represents the low affinity state of the integrin. Priming and ligand binding cause a global conformational change in which the integrin opens in a switchblade-like motion (107, 197, 199, 221) (Figure 2b&c). The N-terminal region of the α subunit contains 7 segments of approximately 60 amino acids that fold into a 7-bladed β propeller domain (188, 221, 222). Roughly half of the known α subunits contain a functional domain of approximately 200 amino acids, referred to as the inserted (I) domain or the von Willebrand factor A domain. The I- domain lies between β sheets 2 and 3 of the β propeller (134) (Figure 2a). In integrins

29 14 Family αβ pair Receptor I Ligand RGD Distribution domain recognition VLA α1β1 VLA-1 + C,L? ECs, monocytes macrophages, activated T/B cells α2β1 VLA-2 + C,L? ECs, EPs, activated T cells α3β1 VLA-3 - C,L, FN? ECs, EPs α4β1 VLA-4 - FN, VCAM-1, - Leukocytes ICAM-1 α5β1 VLA-5 - FN, invasin + ECs, EPs, lymphocytes, monocytes, macrophages α6β1 VLA-6 - L + ECs, EPs, T lymphocytes, mast cells α7β1 - α8β1 - FN, VN, TN α9β1 - FN, VN, TN EPs, muscle cells α10β1 + α11β1 + αvβ1 - + Leukocyte αlβ2 LFA-1 + ICAM-1, -2, -3 Leukocytes αmβ2 MAC-1 + FB, ICAM-1 - Monocytes, macrophages, granulocytes, Langerhans cells, neutrophils αxβ2 P150, Monocytes, macrophages, granulocytes, Langerhans cells, neutrophils αdβ2 + ICAM-3 - Macrophage, lymphocytes α4β7 - MAdCAM-1 Lymphocytes αεβ7 + E-cadherin Lymphocytes Cytoadhesin αvβ3 - VN, FB, FN, + Ubiquitous OPN, VWF, TN αiibβ3 - FB, FN, VWF, + Platelets, ECs VN - Others αvβ5 - VN, FN + Ubiquitous αvβ6 - FN, TN? Lung-EPs αvβ8 - VN + α6β4 - L + EPs Table 1 Integrin receptors. VLA=very late activating, C=collagen, L=laminin, FN=fibronectin, VN=vitronectin, VCAM-1=vascular cell adhesion molecule-1, TN=tenascin, ICAM=intercellular adhesion molecule, FB=fibrinogen, MAdCAM-1=mucosal addressin cell adhesion molecule-1, OPN=osteopontin, EC=endothelial cell, EP=epithelial cells

30 15 in which it is present, I-domain sites are speculated to be responsible for direct ligand binding (49) (Figure 2c). These I-domains contain a metal ion-dependent adhesion site (MIDAS), which coordinates a divalent cation and facilitates integrin binding to negatively charged residues within ligands (182, 198). Manganese and magnesium are divalent cations that are known to activate integrins, while calcium is thought to be inhibitory. The β integrin subunits have a domain similar to that of the αi domain, termed an Ilike domain, which also contains a metal ion-binding motif similar to the α subunit MIDAS (198). In integrin heterodimers that do not contain I-domains on the α subunit, the βi-like domain is thought to bind ligand directly (Figure 2b) (198). In αi-domaincontaining integrins, the β I-like domain is thought to allosterically regulate ligand A I-domain α β-propeller domain Thigh Calf-1 Genu PSI Calf-2 Cytoplasmic domain TM β β I domain Hybrid I-EGF β-tail Cytoplasmic domain Extrinsic ligand B β- propeller β I domain C I domain Intrinsic ligand β I domain Thigh Genu Hybrid PSI Calf-1 I-EGF1-4 Calf-2 β-tail TM α β α β Bent conformation Open conformation Low affinity High affinity Figure 2 Integrin structure and conformation. TM α β Bent conformation Low affinity α β Open conformation High affinity

31 16 binding (Figure 2c). I-domain containing integrins include the β2 family and collagen binding β1 integrins (α1β1, α2β1, etc.). Integrins such as α4β1 and α5β1 do not have αi domains. The leg of the α subunit contains 3 β sandwich domains; the upper leg containing the thigh domain and the lower leg consisting of the calf-1 and calf-2 domains. The α subunit genu, which is the pivotal domain for the switchblade opening in the α subunit, is composed of a small Ca 2+ -binding loop between the thigh and calf-1 domains. The hybrid domain, which composes the upper portion of the upper β leg, is connected to the βi domain. The hybrid domain, in turn, is connected to the plexin/semaphorin/integrin (PSI) domain. Four integrin epidermal growth factor-like (I-EGF) domains and a β tail domain form the remainder of the β subunit leg (134). The bend, or knee, in the β leg is located between I-EGF domains 1 and 2(198) (Figure 2b-c). Weak interfaces between the headpiece and lower legs, the lower α and β legs, and the transmembrane and cytoplasmic domains stabilize the bent conformation. Integrins were named for their ability to integrate extracellular and intracellular environments. They serve as receptors for mediating cellular adhesion, both to extracellular matrix and/or other cells or pathogens. They also interact with the cell s actin-cytoskeleton and other intracellular components and activate many intracellular signaling pathways. Integrins are exceptional in that their activity can be regulated by inside-out signaling or priming. In these processes, cytoplasmic and transmembrane association, with other proteins and receptors (76, 115), as well as cytoplasmic phosphorylation can alter an integrin s avidity for extracellular ligands (38, 81, 97). In addition, integrins also facilitate outside-in signaling in which ligand binding results in

32 17 signal transduction signals into the cytoplasm that regulate cellular growth, survival, shape and migration. Integrin clustering, which is defined as lateral redistribution on the cell surface, is also thought to strengthen integrin adhesiveness and play a major role in outside-in signaling (23, 108). Figure 3 Integrins expressed by immune cells. Integrins perform critical roles in several immune related functions; including leukocyte trafficking and migration, signaling, cell polarization and phagocytosis. At least 11 integrin heterodimers, belonging to the β1, β2 and β7 subfamilies, are expressed by immune cells (Figure 3). β1 integrins are ubiquitously expressed, however, β2 and β7 integrins are exclusively expressed on leukocytes. The dynamic signaling transduced by integrins is vital to their involvement in immune function. In fact, leukocyte migration

33 18 and normal immune responses may be repressed by integrin inhibitors or mutations. The importance of integrins in leukocyte trafficking is verified by leukocyte adhesion deficiency (LAD), an autosomal recessive disease characterized by repeated bacterial infections and impaired wound healing. Talin-1 is the most widely studied actin-binding protein implicated in initiating the upregulation of integrin affinity. The head of talin-1 wedges between the integrin cytoplasmic tails to induce or stabilize the high affinity conformation of the extracellular domains (196, 218). The α4 integrin subunit cytoplasmic tail binds paxillin, a 68 kda signaling adaptor molecule, only when serine-988 is dephosphorylated. This occurs when α4β1 integrin is in a high affinity conformation (70, 88, 129) (Figure 2b). α4- paxillin interaction in outside-in signaling is pertinent to leukocyte migration. Mice with a mutation in the α4 chain that blocks paxillin binding exhibit impaired mononuclear leukocyte recruitment to inflammation (70, 88, 129). Although integrins in the bent conformation are able to bind ligands, the ligandbinding site is not in an optimal orientation for binding. Additionally, in the resting state, the α and β subunit cytoplasmic tails are relatively close together. However, inside-out activation, including protein kinase C (PKC), talin binding to the β cytoplasmic tail, and G protein-coupled receptor stimulation, leads to spatial separation of the cytoplasmic tails (134). Moreover, the extracellular addition of both Mn 2+ and soluble ICAM-1, which causes integrin extension, also induces α and β cytoplasmic tail separation (107). Cytoplasmic and transmembrane domain separation results in separation of the lower legs, which destabilizes the headpiece-lower legs interface, and resultantly integrin extension (134). In addition, fluorescence resonance transfer (FRET) studies have

34 19 demonstrated that integrin conformational change can also be initiated by outside-in signaling (107). Several studies have shown that ligand binding stabilizes the extended conformation (134). Function of the α4β1 Integrin The β1 integrin subfamily, also known as the VLA (very late activating) subfamily, consists of twelve members (89). These integrins display a wide tissue distribution and primarily interact with extracellular matrix proteins, such as collagen, laminin and FN. VLA-4 (CD49c/α4β1) is expressed on monocytes, basophils, T- and B- lymphocytes, and eosinophils but is not thought to be present on circulating neutrophils. Although α4β1 binds FN and the bacterial protein invasin (144), the main ligand for α4β1 is VCAM-1, which is a member of the immunoglobulin superfamily and is found on activated endothelial cells. VCAM-1 plays a role in the localization of T- and B-cells and regulates endothelial adhesion of monocytes, lymphocytes, basophils and eosinophils (54, 124). Leukocytes constitutively express α4β1 integrins in a low ligand-binding state in order to circulate in the blood stream with only low numbers extravasating through the endothelial wall. However, upon presentation of an inflammatory stimulus, the local release of inflammatory mediators stimulates endothelial cells to overexpress cell adhesion molecules, including VCAM-1. Inflammation also causes activation and differentiation of monocytes along the macrophage lineage, which promotes a drastic increase in cell adhesiveness to facilitate transmigration through the endothelium and tethering at sites of inflammation. This increased adhesiveness is due largely to the

35 20 activation of integrins. However, integrin activity is not limited to an individual stage of leukocyte migration, as α4 integrins appear to participate in all aspects of the adhesion cascade (Figure 1). During inflammation, the α4β1 integrins expressed by monocytes associate with VCAM-1 on the surface of endothelial cells. This event is known to be crucial for monocyte transmigration through the vascular wall. Following transmigration, monocytic α4β1 and α5β1 heterodimers then facilitate cell migration through the stroma by interacting with FN in the extracellular matrix (Figure 1). N-linked Glycosylation Several post-translational modifications have been well documented as crucial mediators of protein folding, trafficking, stability and function. Glycosylation is one such modification in which saccharides are added to proteins and lipids. Glycolipids provide energy, membrane stability, cell attachment and markers for cellular recognition. Glycoproteins have many diverse functions, including structural, lubrication, transport, enzymatic and immunologic. Two types of glycosylation exist: O-linked glycosylation to the hydroxyl oxygen of serine and threonine side chains and N-linked glycosylation to the amide nitrogen of asparagine side chains (209). The initial step in N-glycosylation involves the synthesis of a dolichol oligosaccharide precursor. The structure of the precursor, which is conserved among eukaryotes, contains 2 N-acetylglucosamine, 9 mannose and 3 glucose residues (3, 82, 85, 112, 130, 163, 164, 166, 167, 187). Oligosaccharyltransferase (OST), a multisubunit ER membrane protein, transfers the oligosaccharide precursor from the lipid carrier to an

36 21 asparagine residue on a growing polypeptide chain. The minimal N-glycosylation consensus sequence surrounding the acceptor asparagine is Asn-X-Ser/Thr, where X could be any amino acid except Pro. Asn-X-Cys is also a target for N-glycosylation in rare instances (61, 103, 104, 147, 184) (Figure 4a). Following the covalent attachment of the oligosaccharide prescursor to asparagine, glucosidases I and II, which are present in the ER lumen, sequentially remove the 3 glucose residues, a processs which is involved in protein folding mechanisms. If the glycoprotein is improperly folded, it is reglucosylated by a glucosyltransferase (Glc- T) and retained in the ER for proper folding by calnexin. Subsequently, glycosidases within the ER and Golgi remove glucoses and specific mannoses from the oligosaccharide precursor to form either high-mannose or complex oligosaccharides. Although high-mannose oligosaccharides are simply 2 N-acetylglucosamines with many mannose residues, complex oligosaccharides can contain several other types of saccharides, including N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), galactose, fucose and sialic acid (209) (Figure 4a). As the protein is trafficked through the medial and trans-golgi, glycosyltransferases sequentially modify the trimannosyl core. Figure 4b depicts the addition of an N-acetyllactosamine unit by the enzyme, ß1,6-N- Acetylglucosaminyltransferase V (GnT-V), a process which can be repeated. Following the addition of these units, the terminal galactose may remain unmodified or be capped by a sugar such as sialic acid. The transfer of a monosaccharide unit from an activated sugar phosphate to an acceptor molecule on the oligosaccharide is mediated by several glycosyltransferases, which are classified into families based on the types of sugars they

37 22 Figure 4 Steps of N-glycosylation. P=precursor, OST=oligosaccharyltransferase, GNt-V= ß1,6-N- Acetylglucosaminyltransferase V transfer (Figure 4b) (156).. While other organisms utilize an extensive range of nucleotide donors, mammals only use 9: UDP-GlcNAc, UDP-GalNAc, UDP-glucose, UDP-galactose, UDP-xylose, UDP-glucuronic acid, GDP-mannose, GDP-fucose, and CMP-sialic acid (209). These donors and their respective glycosyltransferases add to the vast diversification of N-glycans. Although glycosyltransferases have virtually no sequence homology, they all exhibit similar secondary structures. These enzymes are type II transmembrane glycoproteins consisting of a short NH 2 -terminal cytosolic domain, a hydrophobic transmembrane domain, a stem region and a COOH-terminal catalytic domain in the

38 23 Golgi lumen. The Golgi retention signal is specified by the hydrophobic transmembrane domain as well as adjacent cytoplasmic and luminal sequences (39) (Figure 5). Figure 5 Glycosyltransferases have similar secondary topology. Similar to many Golgi enzymes, glycosyltransferases are often proteolytically cleaved and secreted by cells. This cleavage usually occurs in the stem region releasing a catalytically active enzyme. Interestingly, certain inflammatory stimuli can upregulate the production of soluble glycosyltransferases. However, since these soluble enzymes lack adequate donor sugar nucleotides, which are primarily intracellular, they cannot catalyze transfer reactions. For this reason, the physiological significance of soluble glycosyltransferases has not been elucidated.

39 24 ST6Gal-I Sialyltransferase Sialyltransferases, which transfer sialic acids to nascent oligosaccharides, are a family of approximately 20 different glycosyltransferases which are grouped and distinguished by the acceptor structure on which they act and by the type of sugar linkage they form. The four general sialyltransferase categories are nomenclated: β galactoside α2,3 sialyltransferase (ST3Gal), β galactoside α2,6 sialyltransferase (ST6Gal), (α-nacetyl-neuraminyl-2,3-β-galactosyl-1,3)-n-acetylgalactosaminide α2,6 sialyltransferase (ST6GalNAc) and α-n-acetyl-neuraminide α2,8 sialyltransferase (ST8Sia). Sialyltransferases exhibit very little sequence homology except for three consensus sequences called the sialylmotifs: L, S and VS. The L-sialylmotif contributes to the binding of the sugar donor, while the S-sialylmotif contributes to the binding of both the acceptor and donor subtrates. The VS-sialylmotif may participate in the catalytic activity of the sialyltransferase (72). While ST6GalNAc enzymes are restricted to O-linked glycans and glycolipids, sialic acid may be added to N-glycans in mammals either via an α2,3 or an α2,6 linkage to galactose, an α2,6 linkage to N-acetylgalactosamine, or an α2,8 linkage to another sialic acid, forming polysialic acid(40). The α2,3 linkage, which contributes to the formation of Sialyl Lewis structures, is the most common sialic acid linkage found on most vertebrate cell types and tissues. However, α2,6 and α2,8 linkages are less ubiquitous, being found in various cell types. Although several sialyltransferases can add α2,3 and α2,8 linked sialic acids, there are only two known to add α2,6 linked sialic acids: ST6Gal-I and ST6Gal-II. ST6Gal-I catalyzes the sialic acid transfer to a terminal galactose presented as a free disaccharide or as a terminal N-acetyllactosamine unit of an

40 25 O- or N-linked oligosaccharide (72). Unlike ST6Gal-I, ST6Gal-II is largely localized in the brain, favors free oligosaccharides and exhibits weak glycoprotein and no glycolipid activity (200). The human ST6Gal-I gene has been mapped to chromosome 3. Promoters and alternative splicing result in 3 distinct mrna isoforms which contain varying exons. One transcript is ubiquitously expressed, while another is expressed in B-lymphocytes. A third form represents the major liver transcript but has also been detected in colonic tissues(39). Although ST6Gal-I is rather widely expressed, its expression is dramatically regulated during various cellular processes. This regulation occurs transcriptionally, translationally and post-translationally. Regulation of ST6Gal-I Expression In mammals, ST6Gal-I tissue distribution is widespread. However, these levels of protein expression vary, with liver, lactating mammary gland, intestinal epithelia, and activated B cells having the highest expression (7, 42). These differences in tissue expression are the result of differential usage of several promoters that control the ST6Gal-I gene (148, 194, 216, 217, 220). Although two promoters, P1 and P3, are active in liver, P1 is restricted to the liver and serves as the major supplier to the hepatic ST6Gal-I mrna pool and is functioning when ST6Gal-I becomes up-regulated during the acute phase response (APR) (43, 84). P3 seems to be utilized in a nonspecific tissue manner (194). Promoter regulatory regions P2a, P2b, P2c and P3 mediate ST6Gal-I expression in B cells, while P4 mediates ST6Gal-I expression in lactating mammary glands (42, 220).

41 26 Several post-translational modifications affecting ST6Gal-I activity have been described. The rat liver ST6Gal-I can form catalytically inactive disulfide-bonded dimers in the endoplasmic reticulum (ER) (137). Moreover, the rat liver ST6Gal-I is expressed as two distinct isoforms with a single amino acid difference in the catalytic domain. The isoform that possesses a tyrosine at amino acid 123 is more catalytically active, but is more rapidly cleaved and secreted than the isoform that possesses a cysteine at the same position, which also has an enhanced ability to oligomerize (27, 60, 138). Both isoforms are phosphorylated on luminal serine and threonine residues, the tyrosine isoform to a greater extent (139). Interestingly, N-glycosylation of ST6Gal-I is necessary for activity, especially the soluble form (57). In addition, the luminal stem of ST6Gal-I, which plays a role in Golgi retention, is sensitive to proteases (111). For over 2 decades, it has been known that plasma ST6Gal-I levels are dramatically enhanced during the acute phase response. However, the physiological relevance of soluble ST6Gal-I is unclear. Although the soluble protein is catalytically active, it could scarcely facilitate sialylation since there is a lack of sugar nucleotide donor in the plasma. It has recently been shown that ST6Gal-I is a cleavage substrate of Alzheimer s β-secretase (BACE1) (110). This trans-golgi aspartic protease is responsible for the cleavage of amyloid precursor protein (APP) to produce amyloid β peptide (Aβ), a crucial process for the pathogenesis of Alzheimer s disease. Although BACE1 expression is enriched in the brain, Vassar et al. demonstrated that low levels of BACE1 mrna are expressed in most peripheral tissues, including a pooled leukocyte population (210). However, no protein levels were evaluated in this study. Still, not much is known about the function of BACE1 outside the brain.

42 27 Interestingly, BACE1 null mice have increased rates of death in the first few weeks after birth, and surviving mice are smaller in size (50). It was speculated in this study that loss of BACE1 expression resulted in some type of unidentified impairment in the immune response. This concept is in line with our hypothesis that ST6Gal-I downregulation, via BACE1 cleavage, is necessary for optimal trafficking of monocytes/macrophages during inflammation. BACE1 Regulation The BACE1 promoter includes several putative transcription factor binding sites, including signal transducer and activator of transcription-1 (STAT1), Sp1, YY1, nuclear factor-κ B (NF-κB), and peroxisome proliferators-activated receptor-γ (PPARγ) and four GATA sites. While nothing is known about the regulation of BACE1 in leukocytes, there is a wealth of literature from neuronal and glial cells regarding transcription factor regulation of BACE1 expression. For example, studies suggest that STAT1, Sp1, YY1 serve as activators of BACE1 transcription (32, 118). However, NF-kB signaling appears to be more complex. Data suggest that NF-κB displays a suppressor role in BACE1 transcription in neurons, but acts as a stimulator in activated astrocytes (18, 118). Embryonic fibroblasts isolated from mice deficient in PPAR- γ exhibited an upregulation in BACE1 transcription and promoter activity. This finding suggests that PPAR- γ could also be a repressor of BACE1. PPAR- γ gene transcription is extremely reduced by inflammatory cytokines, a process that can be prevented by incubation with ibuprofen (171). In addition, neuroblastoma cells treated with nonsteroidal anti-inflammatory drugs (NSAIDs) displayed decreased BACE1 mrna levels, protein expression and enzymatic

43 28 activity (170). In addition, cholesterol is also known to upregulate BACE1 expression in neurons (65). BACE1 is also regulated by cholinergic receptor signaling. BACE1 protein expression increases two-fold when M1 muscarinic acetylcholine receptor (machr) is selectively stimulated in neuroblastoma cells (233). In addition, these authors have demonstrated that direct PKC stimulation with phorbol esters produces similar BACE1 upregulation which can be blocked by MEK/ERK inhibition in these cells. However, M2 machr activation, or activation of its PKA-mediated downstream signaling cascade, leads to a reduction of BACE1 expression (233). Interestingly, injections of interferon-γ (IFN-γ) into mouse brains increased BACE1 expression in astrocytes (32). Cho et al. demonstrated in this study that IFN-γ activates Janus kinase 2 (JAK2) and ERK 1/2, which leads to the phosphorylation of STAT1. Phosphorylated STAT1 binds to putative STAT1 binding sites in the BACE1 promoter region leading to increased BACE1 transcription (32). Transcriptional and intracellular signaling regulations of BACE1 expression are complemented by post-transcriptional modulations of BACE1 activity. In vitro, altered BACE1 protein degradation and translational control have been found to control BACE1 protein levels. Ceramide, the lipid second messenger, has been found to increase the half life of BACE1 (161). The BACE1 5 untranslated region (5 UTR) is 446 nucleotides in length, contains 3 upstream open reading frames (UORF), and has a GC-content of 77% (47, 116, 165). These characteristic are often found in mrnas with tight translational control. In vitro translational experiments revealed that the presence of the 5 UTR decreased BACE1 translation by 90%.

44 29 ST6Gal-I in Immunology It has been well documented that cytokine regulated alterations in the sialylation of various plasma proteins accompany severe inflammatory conditions (6, 11, 26, 94, 140, 141). Changes in the glycosylation states of these glycoproteins serve as predictive indexes in several diseases which have an inflammatory component, such as rheumatoid arthritis (9, 74, 142), diabetes mellitus (80), and cancer (41, 73, 141). In fact, ST6Gal-I upregulation and secretion are considered fundamental components of the systemic inflammatory response, particularly the hepatic APR (95, 102). As a result, several studies have proposed a direct inflammatory role for ST6Gal-I. The ST6Gal-I null mice created by Marth s group exhibit a normal phenotype except for a B cell related immunodeficiency (78). Specifically, ST6Gal-I deficiency inhibited the activation and proliferation of B cells, which resulted in a lack of B cell antibody production in response to T cell-independent and T cell-dependent antigens (78). This study demonstrated the importance of ST6Gal-I and the production of α2,6- sialylated oligosaccharides in promoting B lymphocyte activation and immune function. In a recent study, Marino et al reported that ST6Gal-I deficient mice exhibit decreased thymic cellularity, suggesting that ST6Gal-I also regulates T cell development (143). However, as previously described, ST6Gal-I is intricately regulated, which suggests an even more complex physiological contribution of ST6Gal-I than suggested by the ST6Gal-I null mice results reported by Marth and Marino. An explanation was previously offered by Varki s group in which the authors propose that since glycan diversity is regulated by exogenous stressors, such as viral and bacterial pathogens,

45 30 certain mutant animals, such as the ST6Gal-I null mice, commonly thrive free of any symptoms because of the lack of these exogenous pressures in their pathogen-free environments (62). Lau s group shed some light on this idea by partially inactivating Siat1, the mouse ST6Gal-I gene, by disrupting the liver specific P1 transcriptional promoter, allowing the B cell response to remain intact (7). This study examined the consequence of P1 disruption on the hepatic APR and on the response to bacterial challenge. The authors reported that these promoter specific ST6Gal-I-deficient mice are unable to elevate hepatic Siat1 mrna as part of the turpentine induced inflammatory response. Furthermore, as compared to their WT counterparts, these mice had significantly greater numbers of leukocytes in the peritoneum and infected organs upon challenge with a bacterial pathogen (7). In a later study, Lau s group utilized the thioglycollate model of peritonitis to demonstrate that both ST6Gal-I P1 deficient and null mice displayed enhanced neutrophil acute inflammation. In addition, ST6Gal-I deficient mice exhibited a significantly greater pool of myeloid cells within the bone marrow than in the marrow of wild-type mice. This study demonstrates a model in which an increased reservoir of available inflammatory cells and an increased granulopoietic capacity to replenish this pool exist in ST6Gal-I deficient animals. Moreover, data collected from ST6Gal-I deficient mice, in this study, demonstrate a role for ST6Gal-I in regulating inflammation, circulating neutrophil homeostasis, and myeloid differentiation (153). The immunosuppression of ST6Gal-I transgenic knockout mice indicates a fundamental role for α2,6-sialylated structures in immune response regulation. One such oligosaccharide is CDw75, a cell surface ST6Gal-I dependent carbohydrate epitope

46 31 expressed by most B cells, whose expression corresponds with B cell maturation (39). Additionally, CD22, also known as Siglec-2, is a sialic acid binding lectin expressed on the surface of B lymphocytes that is known to negatively regulate the B cell receptor(45). Interestingly, on resting B cells, α2,6-sialylation of N-linked chains of CD22 abrogates its lectin binding activity (20, 180). However, in ST6Gal-I deficient mice, the immunosuppressed response suggests that, in the absence of its primary ligand, CD22 may be wielding a stronger negative regulation of B cell signaling (78). More recently, Ghosh et al. have demonstrated a direct involvement of CD22 and α2,6-linked sialic acid in the homing of recirculating B cells to the bone marrow (64). ST6Gal-I may also govern the release of myeloid cells from the bone marrow into the peripheral blood. During the late stage of myeloid maturation, there is a dramatic increase in cell surface α2,6-sialylation and ST6Gal-I mrna. These changes are associated with decreased myeloid cell binding to fibronectin and cultured bone marrow stroma (122). In response to inflammatory stimuli, there is a marked increase in the expression of acute-phase serum proteins, as well as hepatic and plasma ST6Gal-I. Although sugar nucleotide donor abundance is limited in the plasma, inflammation induces an increase in α2,6-sialylation of certain serum glycoproteins (227). The biological relevance of soluble ST6Gal-I remains unclear. However, recent studies suggest a possible mechanism by which proteolytically cleaved ST6Gal-I sialylates serum glycoproteins inside the Golgi lumen prior to secretion (193).

47 32 Regulation of β1 Integrins by Differential Glycosylation Integrins are regulated by multiple mechanisms such as signal transductionmediated conformational changes, phosphorylation, and proteolytic cleavage. The role of variant glycosylation in regulating integrin function is less well-accepted, although, in fact, there is a substantial amount of evidence supporting this mechanism. For more than 15 years, it has been documented that, as various cell types undergo phenotypic changes, the molecular weight of the β1 integrin becomes altered, as represented by a mobility shift upon western blotting for β1 (13). Enzymatic cleavage of N-linked glycans eliminates this difference in mobility, suggesting that the shifts are due to variant N- glycosylation. Glycosylation-dependent mobility shifts have been observed for β1 integrins expressed by monocytes, epithelial cells, fibroblasts, T cells, cytotrophoblasts and keratinocytes, supporting the concept that variant glycosylation is a wide-spread and fundamental cellular event (13). However, our knowledge of the functional significance of altered glycosylation has been limited by the fact that the specific changes in sugar composition had not previously been well-defined. Our laboratory has determined that the β1 integrin subunit (but not β3 or β5) is a substrate for the ST6Gal-I sialyltransferase. We have shown in both epithelial cells and monocytes that activation of ras-dependent signaling cascades leads to: 1) an altered ST6Gal-I expression, 2) a corresponding change in integrin sialylation, and 3) altered activity of integrin receptors. We have also shown that enzymatic manipulation of sialic acids on purified integrin receptors alters ligand binding in a cell-free assay system, providing definitive proof that sialylation directly regulates integrin function (176, 177, 179). Sialic acid, a negatively charged sugar, could affect positioning of the ligand

48 33 within the ligand-binding domain, or could influence the coordination of divalent cations, a requisite event in integrin activation. Alternatively, sialylation may modulate integrin conformation. Recent evidence from Springer s group supports the hypothesis that differential glycosylation regulates integrin conformation. In these studies, an artificial N-glycosylation site was engineered into the I-like domain of the β1 subunit, a region of the molecule that plays a key role in ligand binding. It was found that the addition of an N-glycan at this site caused the integrin to assume a more extended (activated) conformation, which was, in turn, correlated with increased integrin activity (135). More recently, our laboratory has determined that the native β1 molecule carries three N-linked glycans within the I-like domain and 7 other gylcans distributed throughout the extracellular domain (177). This finding provided a significant advance in our understanding of integrin structure, given that the sites of N-glycosylation had never previously been mapped. The addition of N-glycans is important for proper protein folding. Zheng et al. have demonstrated that N-glycosylation of both the α and β subunits of α5β1 is required for these subunits to heterodimerize (231). In addition, N-glycosylation on the α5 subunit β-propeller domain is necessary for heterodimerization, maturation and α5β1 mediated biological functions (92). However, sialylation occurs in the trans-golgi, after the α and β subunits have already been folded and heterodimerized. Therefore, we speculate that differential sialylation does not affect integrin function by regulating subunit folding and/or association, but by regulating the conformation of the heterodimerized receptor.

49 34 Role of Variant Sialylation in Regulating Monocyte/Macrophage Function The U937, THP-1, and HL-60 cell lines have been widely used as model systems for studying monocytic differentiation of myeloid/promonocytic cells. Treatment with phorbol myristate acetate (PMA) causes these cells to acquire functions characteristic of mature phagocytes, including increased respiratory burst activity, phagocytosis and adhesiveness to several integrin ligands. Our laboratory has found that PMA-stimulated differentiation of U937 and THP-1 cells induces ST6Gal-I downregulation, which leads to the expression of hyposialylated β1 integrin isoforms (179). The expression of these isoforms is temporally correlated with markedly increased cell adhesion to FN. Consistent with these results, we reported that the enzymatic de-sialylation of purified α5β1 integrins increases their binding to FN (178). In other studies, it was shown that the enzymatic removal of sialic acids from the surface of HL-60 myeloid cells stimulates the binding of integrins to FN (159). Finally, Villavicencio-Lorini et al. showed that when HL60 myeloid cells were biochemically engineered to express an unnatural variant of sialic acid, the α5β1 and α4β1 integrins expressed by these cells showed increased binding activity (212). Collectively these studies provide compelling evidence that sialylation regulates the function of α5β1. In the present dissertation, we now demonstrate that this pathway regulates α4β1 function, as well. It is thought that PKC signaling through ERK is essential for macrophage differentiation (75, 83, 105, 150, 201), and activation of this pathway is known to regulate integrin function. For example, heterologous expression of constitutive active ras or MEK in myeloid/promonocytic cells strongly stimulates cell adhesion to FN (75, 79, 83, 105, 150, 176, 230). However, the molecular mechanisms directing increased

50 35 integrin activity have not been elucidated. Our lab has found that ST6Gal-I downregulation during PMA-induced U937 differentiation is directed by a PKC/ras/ERK signaling cascade. PMA-induced cell adhesion to FN, ST6Gal-I downregulation, and integrin hyposialylation are all blocked by pharmacologic inhibitors of PKC, ras or MEK, whereas expression of constitutively active MEK stimulates these same events (177). Thus, regulation of integrin sialylation is mediated by the same signaling mechanism known to be involved in the increased cell adhesiveness associated with monocyte/macrophage differentiation. However, the mechanisms by which differential sialylation regulates integrin function remain to be elucidated. Sialic acid may alter integrin ligand binding, conformation, or both. It has been well documented that ligand charge is an important factor in ligand binding. In fact, several integrin binding motifs contain amino acids which are negatively charged, such as RGD (Table 1). When present, the negatively charged sialic acid may interfere with the integrin s ability to bind ligand by causing charge repulsion. In addition to being negatively charged, sialic acid is a relatively large residue. For this reason, sialic acid may cause steric hindrance in the association of ligands in the ligand binding domain, as well as interfere with the global conformational changes required for integrin activity. Our work has focused primarily on the role of sialylation in regulating integrin binding to traditional ligands. However, we suspect that differential sialylation will also be a major factor in lectin biology. In fact, several mammalian lectins recognize α2,6 linked sialic acids as a ligand. Actually, α2,6 sialic acid is the only binding ligand for Siglec-2, which is also known as CD22. In contrast, α2 6 sialylation negatively

51 36 regulates the binding of galectins. Although not much lectin/sialic acid research has been done in monocytes, this interaction is a crucial factor in T and B-cell signaling. In particular, galectin 1 and 3 induce T cell apoptosis (5, 128). Rationale for Study We have previously shown that the activation of a PKC/ras/ERK signaling cascade in differentiating promonocytic cells induces downregulation of ST6Gal-I (βgalactoside α2-6 sialyltransferase), a trans-golgi enzyme that adds α2-6 sialic acids to glycoproteins. Furthermore, we have identified β1 integrins as a substrate for ST6Gal-I and have shown that, in response to the activation of PKC/ras/ERK, β1 integrins become hyposialylated (176, 178, 179). This expression is associated with enhanced monocyte binding to fibronectin (FN), due to the activation of α5β1 integrins (178, 179). We therefore hypothesized that ST6Gal-I downregulation regulates macrophage adhesion by altering integrin sialylation. As a result, the overall goal of this dissertation was to determine whether α4β1 integrins are also regulated by sialylation and whether hyposialylation of these integrins plays a key role in regulating monocyte adhesion to (VCAM-1). The first objective was to determine whether sialylation directly effects α4β1 integrin function. After which we sought to elucidate the mechanisms regulating differentiation induced downregulation.

52 37 PROTEOLYTIC SHEDDING OF ST6GAL-I BY BACE1 REGULATES THE GLYCOSYLATION AND FUNCTION OF α4β1 INTEGRINS ALENCIA V. WOODARD-GRICE, ALEXIS C. McBRAYER, JOHN K. WAKEFIELD, YA ZHUO, and SUSAN L. BELLIS Accepted (pending minor revision) to Journal of Biological Chemistry Format adapted for dissertation

53 38 Abstract Differentiation of monocytes into macrophages is accompanied by increased cell adhesiveness, due, in part, to the activation of α4β1 integrins. Herein we report that the sustained α4β1 activation associated with macrophage differentiation results from expression of β1 integrin subunits that lack α2-6-linked sialic acids, a carbohydrate modification added by the ST6Gal-I sialyltransferase. During differentiation of U937 monocytic cells and primary human CD14 + monocytes, ST6Gal-I is downregulated, leading to β1 hyposialylation and enhanced α4β1-dependent VCAM-1 binding. Importantly, ST6Gal-I downregulation results from cleavage by the BACE1 secretase, which we show is dramatically upregulated during macrophage differentiation. BACE1 upregulation, ST6Gal-I shedding, β1 hyposialylation and α4β1-dependent VCAM-1 binding are all temporally correlated, and share the same signaling mechanism (PKC/ras/ERK). Preventing ST6Gal-I downregulation (and therefore integrin hyposialylation), through BACE1 inhibition or ST6Gal-I constitutive overexpression, eliminates VCAM-1 binding. Similarly, preventing integrin hyposialylation inhibits a differentiation-induced increase in the expression of an activation-dependent conformational epitope on the β1 subunit. Collectively these results describe a novel mechanism for α4β1 regulation, and further suggest an unanticipated role for BACE1 in macrophage function. Introduction Upon exposure to inflammatory stimuli, circulating monocytes become activated and begin differentiating along the macrophage lineage. As part of this process, the cells

54 39 become significantly more adhesive, which facilitates extravasation through the endothelium and migration through subendothelial tissues. Increased monocyte adhesiveness is due, in part, to the activation of the integrin family of cell adhesion receptors. During inflammation, α4β1 integrins expressed by monocytes associate with vascular cell adhesion molecule-1 (VCAM-1) on the surface of endothelial cells, an event crucial for monocyte transmigration (28, 36, 51, 58). In vitro modeling of monocyte activation and differentiation can be accomplished by treating the U937 and THP-1 promonocytic cell lines with phorbol myristate acetate (PMA), which causes cells to acquire functions characteristic of mature phagocytes, including increased α4β1- dependent adhesion to VCAM-1. However, the mechanisms underlying enhanced α4β1 activity remain unclear. Integrins are regulated by multiple mechanisms including signal transductionmediated conformational changes ( inside-out signaling ), phosphorylation, proteolytic cleavage and differential glycosylation (2, 16, 18, 21, 32, 53). Previously, our lab reported that the β1 integrin subunit from PMA-treated U937 and THP-1 cells lacked α2 6-linked sialic acid glycans, due to the PMA-induced downregulation of the β galactoside α2 6-sialyltransferase, ST6Gal-I (50). The expression of hyposialylated β1 integrins was associated with markedly increased cell adhesion to fibronectin (FN). We further showed that the enzymatic de-sialylation of purified FN-binding integrins (α5β1) significantly increased binding to FN (50), and that this effect could be reversed by re-addition of sialic acids by recombinant ST6Gal-I (49). Consistent with our studies, Pretzlaff et al. reported that de-sialylation of cell surface glycoproteins expressed by HL- 60 myeloid cells stimulated the binding of integrins to FN (40). Finally, Villavicencio-

55 40 Lorini showed that the incorporation of unnatural sialic acid variants into cell surface proteins caused HL-60 cells to acquire enhanced adhesiveness to both FN and VCAM-1 (57). Taken together, these results suggest that sialic acids directly regulate the function of β1-containing integrin heterodimers. The mechanisms controlling differential α2 6 sialylation are poorly understood, however most studies have focused on transcriptional regulation of ST6Gal-I (9). Interestingly, ST6Gal-I has recently been identified as a cleavage substrate for the β site APP-cleaving enzyme1 (BACE1) secretase (27), which is responsible for the production of amyloid β peptide in Alzheimer s disease. BACE1 expression is enriched in the brain as compared with most other tissues, although low levels of BACE1 mrna have been observed in a pooled population of leukocytes (30, 56). In the present study, we tested whether BACE1 activity in monocytes might be responsible for decreased ST6Gal-I levels, leading to the synthesis of hyposialylated α4β1 integrins with greater binding activity toward VCAM-1. We now show that in both U937 cells and primary CD14 + human monocytes, differentiation along the macrophage lineage dramatically upregulates the expression of BACE1, which in turn mediates ST6Gal-I cleavage. Importantly, preventing ST6Gal-I downregulation, through both BACE1 inhibition and constitutive overexpression of ST6Gal-I, eliminates α4β1-dependent VCAM-1 binding, indicating that α4β1 hyposialylation is required for optimal activity.

56 41 Experimental Procedures Cell Culture A U937 subclone, selected for sensitivity toward granulocyte/macrophage colonystimulating factor, was obtained from Dr. Elizabeth Eklund (Northwestern University). The cells were maintained in Dulbeccos modified Eagle s medium containing 10% fetal bovine serum, 1% amphotericine B (Cellgro) and 1% penicillin-streptomycin (Cellgro). U937 cells that constitutively express ST6Gal-I ( ST6 ) were generated by infecting cells with a lentivirus expressing a V5-tagged ST6Gal-I construct (this plasmid was a generous gift from Dr. Karen Colley, University of Illinois at Chicago). Cells were also transduced with an empty vector lentivirus ( EV ) as a control. Stably-transduced cells were obtained by puromycin selection. Expression of the ST6Gal-I construct was verified by immunoblotting for the V5 tag (not shown). Of note, the EV and ST6 cell lines represent pooled populations of cells stably transduced with the lentiviral vectors. U937 cells expressing constitutively active MEK were generated as previously described (49). Primary CD14 + monocytes were purchased from Clonetics. These cells were differentiated by incubation for two weeks in Dulbeccos modified Eagle s medium containing 30% human serum, 1% amphotericine B and 1% penicillin-streptomycin. Enzyme Inhibitor Studies Cells were incubated for 20 min at 37ºC with one of the following enzyme inhibitors: 10µM R (Calbiochem), 30µM manumycin A (Sigma), 50µM PD98059 (Calbiochem), or 30nM wortmannin (Sigma). PMA was then added to a final

57 42 concentration of 100ng/ml, and cells were incubated in non-attaching dishes in the presence of both PMA and the selected inhibitor overnight at 37ºC. Cell Attachment Assays Cells were treated with or without 100ng/ml PMA in non-attaching dishes, and also with pharmacologic inhibitors in some trials, and then seeded onto tissue culture dishes that had been precoated with either 10µg/ml VCAM-1 (R&D Systems), or with 2% denatured bovine serum albumin (dbsa) as a control for nonspecific binding. Cells were allowed to adhere for 1h, and adhesion was quantified as described previously using a crystal violet staining method (50). For some experiments, cells were pre-incubated for 1h with a function-blocking antibody, and function-blocking antibodies were also present throughout the adhesion assays. The following antibodies were used: 20μg/ml anti-β1 (Millipore), 40μg/ml anti-α4 (Biodesign), 40μg/ml anti-vcam-1 (BD Pharmingen), or two isotype control antibodies; IgG1 (Chemicon), and IgG2B (Chemicon), both used at 40μg/ml. Western Blotting Cells were treated with or without 100ng/ml PMA for a range of timepoints. Cells were then lysed in 50mM Tris buffer (ph 7.4) containing 1% Triton X-100, 4mM sodium fluoride, 200µM sodium orthovanadate, and a protease inhibitor cocktail (Roche). Protein concentrations in the lysates were determined using a modified Bradford assay (Sigma). Lysates were resolved by reducing SDS-PAGE and β1 integrins were Western blotted using a monoclonal antibody from BD Transduction Laboratories. Western blot

58 43 analysis of ST6Gal-I was accomplished using a monoclonal antibody developed by the University of Alabama at Birmingham s Hybridoma Core Facility. BACE1 was immunoblotted with an antibody from Abcam, and phosphoerk was detected with a polyclonal antibody from Cell Signaling. Immunoprecipitation and SNA Blotting Cell lysates were incubated overnight at 4ºC with antibodies specific for either the α4 (Chemicon) or β1 (Chemicon) integrin subunits. Protein A/G-coupled agarose beads (Santa Cruz) were then added, and samples were incubated for an additional 2 hours at 4ºC with rotation. Antibody/glycoprotein complexes were collected by brief centrifugation and washed with lysis buffer. Glycoproteins were resolved by SDS-PAGE and transferred to PVDF membrane. The membrane was incubated with Sambucus Nigra Agglutinin (SNA) lectin conjugated to horseradish peroxidase (HRP) (EY Labs), and α2 6 sialylated proteins were visualized by enhanced chemiluminescence. As well, immunoprecipitated samples were blotted for the α4 and β1 integrin subunits to insure that equal amounts of integrins were precipitated (anti-α4 blotting antibody was from Santa Cruz; anti-β1 from Transduction Labs). SNA Precipitation Cell lysates were incubated for 2 hours with SNA-conjugated agarose beads (EY labs). The α2 6 sialylated proteins were precipitated by centrifugation and washed extensively with lysis buffer. The sialylated/lectin conjugates were then resolved by SDS-PAGE and immunoblotted for β1 as described previously.

59 44 Flow Cytometry Cells were resuspended in PBS containing 1% (wt/vol) BSA (Sigma-Aldrich). Cells were pre-incubated with non-labeled human IgG (Sigma-Aldrich) to block Fc receptors. For surface staining of β1 integrins, cells were incubated for 1h at 4 C with anti-integrin β1 clone 12G10 (Chemicon), FITC-conjugated anti-β1 clone CBL481F (Chemicon), or with isotype control antibodies, all at the recommended dilutions. After washing, 12G10 labeled cells-were incubated with the Alexa Fluor 488 conjugate to the F(ab ) 2 fragment of rabbit anti-mouse (Invitrogen). Stained cells were washed and were analyzed with FACSCalibur (Becton-Dickinson) by the UAB Arthritis and Musculoskeletal Center Analytic and Preparative Core Facility (APCF). BACE1 Inhibition Cells were incubated for 24 hours with a BACE1 inhibitor (β-secretase Inhibitor IV; Calbiochem) at a concentration of 10μM. Cells were then incubated overnight with or without 100ng/ml PMA in the presence of BACE1 inhibitor. BACE1 Activity Assays Cells were treated with 100ng/ml PMA for appropriate time points and subsequently lysed. BACE1 activity was detected with the β Secretase (BACE1) Activity Detection Kit (Fluorescent) (Sigma Aldrich) according to the manufacturer s protocol. Briefly, cell lysates were incubated with a fluorescent tagged BACE1 substrate. The fluorescent signal is enhanced after the substrate is cleaved by the BACE1 present in the cell lysate. Fluorescence was measured using a standard fluorescent plate reader.

60 45 Statistical Analysis Unless otherwise indicated, at least three independent experiments, each executed in triplicate, were performed for all protocols. Results were evaluated by independent student s t-tests, and P-values of 0.05 or less were considered significant. Results Expression of hyposialylated β1 integrins is temporally correlated with cell adhesion to VCAM-1. We previously determined that the onset of hyposialylated β1 integrin expression occurs approximately 7-9 hours following the initiation of PMA treatment, and by hours, most of the wild-type integrin has been replaced by the hyposialylated glycoform (50). To examine whether hyposialylated β1 integrin expression was temporally correlated with enhanced integrin activity, we established a time course for PMAdependent U937 cell adhesion to VCAM-1. Briefly, U937 cells were treated with PMA for varying time points, and then seeded onto VCAM-1-coated tissue culture wells and monitored for adhesion using a standard colorimetric assay. Consistent with other reports (6, 7, 13, 17), PMA stimulated rapid cell binding to VCAM-1 (Fig.1A), however adhesiveness returned to baseline levels by 3 hours. Cell adhesiveness toward VCAM-1 then began to increase again at 7-9 hours following the initiation of PMA treatment, and continued to rise throughout the 15 hours assayed. We speculate that the rapid, transient phase of α4β1 activation is due to traditional forms of inside-out signaling, given that it occurs too rapidly to be explained by the expression of differentially-sialylated β1 subunits. However, the delayed, sustained phase of VCAM-1 binding, occurring between

61 Figure 1 α4β1 dependent VCAM-1 binding temporally correlates to hyposialylated β1 integrin expression. (A)U937 cells were treated with PMA for varying time intervals and then allowed to attach to VCAM-1 coated tissue culture wells for 1 hour. Data represent the means and SEMs from three independent experiments, each performed in triplicate. (B) To establish a direct role for α4β1 integrins in PMA-dependent VCAM- 1 binding, PMA-treated cells were pre-incubated for 1 hour with function blocking antibodies. Cells were then seeded onto VCAM-1 in the presence of function blocking antibodies for adhesion assays. IgG1 is the nonspecific isotype control for anti-β1 and anti-vcam-1. IgG2 is the nonspecific isotype control for anti-α4. Control and PMA treated cells were allowed to adhere to denatured BSA as a negative control. Data represent the means and SEMs from three independent experiments, each performed in triplicate. * denotes significant difference from PMA-treated cells (P<0.05). 46

62 hours, correlates well with the expression of hyposialylated β1, and is also consistent with the prolonged kinetics associated with macrophage differentiation (12, 15, 39). To verify that the delayed, sustained phase of VCAM-1 binding was mediated by α4β1 integrins, cells were treated overnight with PMA and then subjected to adhesion assays in the presence of function-blocking antibodies. These experiments revealed that U937 cell binding to VCAM-1 was blocked by antibodies specific for the α4 and β1 integrin subunits, as well as an antibody against VCAM-1 (Fig.1B). In addition, we observed that PMA did not significantly increase binding to denatured BSA, further suggesting that enhanced cell adhesiveness is due to integrin activation, rather than nonspecific mechanisms. The α4 subunit is not α2-6 sialylated. There is little known regarding the spectrum of ST6Gal-I substrates. While we have shown that β1 is modified by α2-6 sialylation (46, 47, 49, 50), it is interesting that neither the β3 nor β5 integrin subunits appear to carry this moiety (47). To determine whether the α4 subunit is differentially sialylated during macrophage differentiation, α4 integrin subunits (and also β1) were immunoprecipitated from control and PMA-treated U937 cell lysates. The immunoprecipitates were resolved by SDS-PAGE, transferred to PVDF membrane, and the membranes were subsequently incubated with HRP-conjugated SNA, a lectin that specifically recognizes α2 6 linked sialic acids. SNA-reactive bands were visualized by enhanced chemiluminescence. As shown in Fig.2A, β1 integrins from control, but not PMA-treated, U937 cells exhibited SNA reactivity, consistent with prior

63 48 results showing that PMA induces expression of β1 integrins that lack α2 6 sialic acids (50). Figure 2 The α4 subunit is not a substrate for ST6Gal-I. (A) α4 and β1 integrin subunits were immunoprecipitated from control and PMA treated U937 cell lysates, and then blotted with SNA-HRP to reveal α2 6 sialylated proteins. (B) Immunoprecipitated samples were also blotted for either α4 or β1 to confirm that equal amounts of protein were immunoprecipitated. However, no SNA-reactive bands were observed for α4 immunoprecipitates, suggesting that this integrin species is not a substrate for ST6Gal-I. To confirm that the immunoprecipitation protocol was effective, immunoprecipitated samples were also blotted for either α4 or β1 (Fig.2B). As shown, equivalent amounts of both the α4 and β1 subunits were immunoprecipitated from control and PMA-treated cell lysates. Also important, the mature β1 integrin isoform (the functional receptor) from PMA-treated cells had a smaller apparent molecular mass, due to the loss of sialic acid (of note, the

64 49 precursor β1 isoform is not α2 6 sialylated, since it does not traffic through the trans- Golgi). In contrast to the β1 integrin subunit, the α4 integrin did not exhibit any shift in electrophoretic mobility following PMA treatment, consistent with the observation that this integrin subunit does not appear to be differentially sialylated. PMA-dependent VCAM-1 binding is directed by a PKC/ras/ERK signaling cascade. In prior studies we determined that PMA-dependent expression of hyposialylated integrins is directed by a PKC/ras/ERK signaling cascade (49), which is the same signaling pathway associated with macrophage differentiation (19, 20, 25, 37, 54). To test the hypothesis that this pathway mediates cell adhesion to VCAM-1, cells were pretreated with pharmacologic inhibitors, stimulated with PMA (plus inhibitor) overnight, and then subjected to standard binding assays. These experiments (Fig.3A) showed that PMA-dependent VCAM-1 binding was blocked by R (a PKC inhibitor), manumycin A (a compound that blocks ras activity by preventing farnesylation), and PD98059 (a MAPK kinase (MEK) inhibitor). In contrast, binding was not inhibited by the PI3K inhibitor, wortmannin. These results are consistent with our prior studies showing that PMA-dependent ST6Gal-I downregulation and β1 hyposialylation are blocked by R , manumycin A, and PD98058, but not by wortmannin (49). To verify that sialylation-dependent integrin function is regulated by an ERKdependent signaling cascade, we generated U937 cells that stably express constitutively active MEK (camek). These cells have downregulated ST6Gal-I and hyposialylated β1

65 50 integrins, as previously reported (49). Exploiting this cell line, we now show that expression of constitutively active MEK stimulates cell adhesion to VCAM-1 (Fig.3B). Figure 3 VCAM-1 binding is regulated by a PKC/ras/ERK cascade. (A) To determine the signaling mechanisms underlying PMA-dependent VCAM-1 binding, U937 cells were treated for 20 minutes with one of the following pharmacological inhibitors prior to PMA treatment: R (R031), manumycin A (mana), PD98059 (PD98), or wortmannin (wort). Cells were then allowed to attach to VCAM-1 coated tissue culture wells for 1 hour. Data represent the means and SEMs from three independent experiments performed in triplicate. * denotes significant difference from control cells (P<0.05). (B) To determine if ERK was a sufficient activator of VCAM-1 binding, adhesion assays were performed with parental U937 cells and U937 cells expressing a constitutively active MEK construct (camek). Data represent the means and SEMs from four independent experiments performed in triplicate. *P<0.05

66 51 Taken together, these results indicate that changes in α4β1 sialylation and function are directed by a shared signaling mechanism, namely a PKC/ras/ERK pathway. Forced expression of ST6Gal-I blocks PMA-induced β1 integrin hyposialylation and VCAM-1 binding. In order to more directly examine the effects of differential sialylation on integrin activity, we created U937 cells that stably express an ST6Gal-I construct (ST6) that cannot be downregulated by PMA, and then monitored integrin structure and function. We also evaluated integrin activity in cells transduced with an empty vector construct as a control (EV). Parental (Par), EV or ST6 cells were incubated overnight in the presence or absence of PMA, and then lysed and monitored for expression of hyposialylated integrins. As shown in β1 immunoblots (Fig.4A), the mature β1 integrins from PMAtreated Par and EV cells have a smaller apparent molecular mass, reflecting expression of the hyposialylated glycoform. In contrast, β1 integrins expressed by PMA-treated ST6 cells do not exhibit an electrophoretic mobility shift, consistent with the fact that constitutive expression of ST6Gal-I prevents PMA-induced integrin hyposialylation. To more definitively evaluate integrin sialylation levels, α2 6 sialylated proteins were precipitated with agarose-conjugated SNA and immunoblotted for the β1 integrin. As shown in Fig.4B, β1 integrins from Par and EV cells exhibited high levels of α2 6 sialylation (as measured by SNA reactivity), and sialylation was markedly reduced following PMA treatment. In contrast, mature β1 integrins from both the control and PMA-treated ST6 cells displayed high levels of SNA reactivity. These data indicate that

67 Figure 4 Forced ST6Gal-I expression blocks PMA-induced β1 integrin hyposialylation and VCAM-1 binding. (A) Immunoblot analysis of β1expression in control and PMA treated parental U937 cells (Par), empty vector-transduced U937 cells (EV) and U937 cells with constitutive overexpression of ST6Gal-I (ST6). (B) α2 6 sialylated proteins were precipitated by SNA-agarose from control and PMA treated Par, EV and ST6 cell lysates. Sialylated proteins were then immunoblotted for the β1 integrin. (C) Analysis of PMA-dependent VCAM-1 binding. Data represent the means and SEMs from three independent experiments performed in triplicate. *P<0.05 (D) Immunoblot analysis of phosphoerk and total ERK. 52

68 53 the constitutive expression of ST6Gal-I prevents PMA-induced expression of hyposialylated β1 integrins. To determine whether blocking integrin hyposialylation affects α4β1 integrin function, we compared Par, EV and ST6 cell adhesion to VCAM-1. As shown in Fig.4C, VCAM-1 binding is significantly increased by PMA in Par and EV cells. However, ST6 cells did not exhibit any increased binding to VCAM-1 following PMA treatment. Importantly, this loss in PMA-induced VCAM-1 binding was not due to a nonspecific disruption in signaling mechanisms, because PMA still activates ERK in ST6 cells, as evidenced by results from immunoblots for phosphorylated forms of ERK (Fig.4D). Thus, preventing the expression of hyposialylated α4β1 integrin receptors eliminates PMA-dependent VCAM-1 binding, despite the presence of robust ERK signaling. These results suggest that differential sialylation plays a crucial role in regulating α4β1 function. Preventing integrin hyposialylation blocks the PMA-induced expression of an activationassociated epitope on the β1 subunit. Changes in integrin activation state are commonly measured by evaluating the degree of exposure of specific activation-dependent epitopes, as reported by the binding of conformation-specific anti-integrin antibodies. To investigate whether PMA treatment causes conformational changes in the β1 integrin subunit and whether ST6Gal-I downregulation is necessary for these changes, we performed flow cytometric analyses using an antibody (12G10) that reportedly exhibits enhanced binding to β1 when the integrin is in a more activated conformation (38). As shown in Fig.5A, 12G10 displays

69 Figure 5 Forced ST6Gal-I expression blocks PMA-induced conformational changes of the β1 integrin. (A) Flow cytometric analysis of control and PMA treated Par, EV and ST6 cells incubated with the conformation specific anti-β1 antibody, 12G10. Graphical data represent the mean fluorescent intensities and SEMs from three independent experiments. *P<0.05 (B) Flow cytometric analysis of control and PMA treated Par, EV and ST6 cells incubated with the conformation insensitive anti-β1 antibody, CBL481F. Graphical data represent the mean fluorescent intensities and SEMs from three independent experiments. 54

70 55 markedly enhanced binding to PMA-treated Par and EV cells. However, PMA-treated ST6 cells failed to show enhanced 12G10 reactivity, suggesting that PMA-induced ST6Gal-I downregulation, and therefore hyposialylation of integrins, is important for conformational changes associated with integrin activation. The graphical data in Fig.5A depicts mean fluorescent intensities for 3 independent experiments. To confirm equivalent levels of β1 integrin cell surface expression on Par, EV and ST6 cells, we performed flow cytometric analysis using the conformation-insensitive antibody, CBL481F. As shown in Fig.5B, cell surface expression of β1 integrin does not change upon treatment with PMA in Par, EV or ST6 cells. These results support the hypothesis that PMA-induced binding of the 12G10 antibody to Par and EV cells is due to β1 activation, and that β1 activation can be blocked by eliminating hyposialylation. ST6Gal-I downregulation results from BACE1-mediated proteolytic cleavage. Given our results suggesting that differential α2 6 sialylation regulates α4β1 function, we were interested in the mechanisms directing ST6Gal-I downregulation. It has been previously reported that BACE1 cleaves ST6Gal-I, both in vitro (27) and in vivo (26). Therefore, we evaluated BACE1 expression in control and PMA-treated U937 cells. Western blot analyses revealed that, not only is BACE1 expressed in U937 cells, its expression is markedly upregulated upon treatment with PMA (Fig.6A). We hypothesized that if BACE1 is responsible for ST6Gal-I downregulation, then the expression of these two enzymes should be regulated by the same signaling cascade. Accordingly, we evaluated BACE1 expression in cells treated with pharmacologic inhibitors of PKC, ras and MEK, as well as in cells expressing activated MEK. As

71 56 shown in Fig.6A, PMA-induced BACE1 expression is blocked by pretreatment with R , manumycin A, and PD98059 but not wortmannin. In addition, BACE1 expression is significantly increased by constitutively active MEK (Fig.6B). Collectively these data indicate that BACE1 expression is upregulated by a PKC/ras/ERK signaling pathway, which we previously reported is the same pathway that induces ST6Gal-I downregulation and integrin hyposialylation (48). In order to evaluate the kinetics of BACE1 expression and ST6Gal-I downregulation, U937 cells were treated with PMA for varying time points, and the expression of these enzymes was examined by immunoblotting. BACE1 upregulation Figure 6 BACE1 expression is upregulated by a PKC/ras/ERK signaling cascade. Immunoblot analysis of BACE1 from: (A) control cells; PMA treated cells; cells incubated with pharmacologic inhibitors prior to PMA treatment, and (B) cells that express constitutively active MEK. begins to occur as early as 2 hours following PMA treatment (Fig.7A), which correlates well with the time course required for loss of ST6Gal-I from cellular homogenates (Fig.7B). Equivalent lane loading was confirmed by immunoblotting for β-actin (Fig.7C). BACE1 cleaves ST6Gal-I very near the transmembrane domain, releasing most of the protein from the cell (27). Therefore, to determine whether ST6Gal-I is actively cleaved in PMA-treated U937 cells, conditioned media from U937 cells treated with PMA for 2 or 4 hours was collected and immunoblotted for the secreted form of ST6Gal-

72 57 I. As shown in Fig.7D, cleaved ST6Gal-I begins to accumulate in the cell culture supernatant at approximately 4 hours following the initiation of PMA treatment. Figure 7 Upregulation of BACE1 expression temporally correlates with ST6Gal-I downregulation and secretion. (A) Immunoblot analysis of BACE1 expression. (B) Immunoblot analysis of cellular ST6Gal-I expression. (C) Immunoblot analysis of β- actin. (D) Conditioned media was collected from PMA treated cells and immunoblotted for secreted ST6Gal-I.

73 58 BACE1 activity is required for ST6Gal-I downregulation and PMA-dependent VCAM-1 binding. To establish a functional role for BACE1 in PMA-dependent VCAM-1 binding, cells were treated with a BACE1 inhibitor (β-secretase Inhibitor IV; Calbiochem) for 24 hours prior to overnight PMA treatment and then monitored for ST6Gal-I shedding and integrin activity. Activity assays confirmed that our treatment conditions blocked 99.5% of BACE1 activity (data not shown). As shown in Fig.8A, PMA treatment significantly increased U937 cell binding to VCAM-1, however VCAM-1 binding was completely blocked by the BACE1 inhibitor. To verify that the BACE1 inhibitor was effective in preventing BACE1-mediated ST6Gal-I downregulation, cells were lysed and immunoblotted for cellular ST6Gal-I. These experiments revealed that BACE1 inhibition attenuated the PMA-induced decrease in ST6Gal-I (Fig.8B). Furthermore, BACE1 inhibition prevented the PMA-induced cleavage and secretion of ST6Gal-I, as shown by western blot analysis of conditioned media (Fig.8C). Collectively these results suggest that BACE1 activity is required for PMA-induced ST6Gal-I shedding, as well as α4β1-dependent VCAM-1 binding.

74 Figure 8 BACE1 inhibition prevents PMA-dependent ST6Gal-I downregulation and secretion, as well as VCAM-1 binding. Cells were treated with the BACE1 inhibitor, β Secretase Inhibitor IV, for 24 hours prior to PMA treatment. (A) PMA dependent VCAM-1 adhesion in the presence or absence of a BACE1 inhibitor. Data represent the means and SEMs from four independent experiments performed in triplicate. * denotes significant difference from control cells (P<0.05) (B) Immunoblot analysis of cellular ST6Gal-I. (C) Immunoblot analysis of secreted ST6Gal-I in cell culture supernatant. 59

75 60 Differentiation of primary CD14 + monocytes along the macrophage lineage induces BACE1 upregulation, ST6Gal-I downregulation, and enhanced binding to VCAM-1. The U937 cell line is a well-established model system for dissecting mechanisms regulating monocyte activation and differentiation. However, it is of obvious importance to establish that such mechanisms are operative in primary human monocytes. Thus, we repeated some of our experiments using primary human CD14 + monocytes, which can be in vitro differentiated into macrophages. Specifically, CD14 + cells were differentiated by exposure to 30% human serum according to standard protocols (45), and then evaluated for expression of BACE1 and ST6Gal-I, and also monitored for VCAM-1 binding. As shown (Figs.9A and B), in vitro differentiation of primary CD14 + monocytes induced a dramatic increase in BACE1 expression, along with a coordinate loss in ST6Gal-I. We then monitored primary CD14+ cell adhesion to VCAM-1 (Fig.9C), and found that in vitro-differentiated cells were more adherent to VCAM-1, consistent with the activation of α4β1 integrins. Although further experiments will be needed to confirm our model in primary cells, these studies strongly support the hypothesis that BACE1-mediated ST6Gal-I cleavage plays a role in regulating monocyte α4β1 activation.

76 61 Figure 9 Differentiation of CD14 + monocytes induces increased VCAM-1 binding, ST6Gal-I downregulation and BACE1 upregulation. (A) Immunoblot analysis of BACE1 expression. (B) Immunoblot analysis of cellular ST6Gal-I expression. (C) VCAM-1 adhesion of undifferentiated and in vitro-differentiated primary CD14 + monocytes. Data are representative of two independent experiments performed in triplicate with cells from two individual donors.

77 62 Discussion The binding of α4β1 integrins to VCAM-1 plays a crucial role in many physiologic and pathologic processes, and therapeutic blockade of this interaction is effective in ameliorating symptoms associated with several immune-related disorders (60, 61). Much of the research aimed at defining α4β1 regulatory mechanisms has focused on inside-out signaling pathways, which direct very rapid changes in integrin affinity and/or avidity (42). In the current study, the activation of α4β1 integrins was induced by incubating U937 promonocytic cells with PMA, a treatment which simultaneously activates monocytic cells and causes differentiation along the macrophage lineage. Consistent with other studies (6, 7, 13, 17), we find that α4β1 integrins are rapidly activated by PMA, most probably through traditional inside-out signaling mechanisms. However, this rapid activation is a transient response, which is subsequently followed by a substantially larger, and more prolonged, increase in α4β1 binding activity that temporally correlates with the acquisition of a differentiated monocyte/macrophage phenotype. Importantly, sustained activation of α4β1 receptors is consistent with the kinetics of monocyte infiltration in vivo. While monocytes typically leave their marginal pools more slowly than neutrophils, they become the predominant cell type at sites of inflammation after 12 hours because they continue to migrate into these areas after neutrophil migration has ceased (22). Moreover, monocytes display a higher binding affinity for endothelium than other circulating leukocytes (12). These observations highlight the biological importance of sustained α4β1 activation. However, the molecular mechanisms contributing to this process are poorly understood. Our studies suggest that the sustained α4β1 activation associated with long-term phenotypic changes in differentiating monocytes occurs as a

78 63 consequence of a change in integrin structure, namely a loss in α2 6 sialylation on the β1 subunit. As a regulatory mechanism, differential sialylation may be particularly wellsuited for directing prolonged changes in integrin activity, given that sialic acids are added during integrin synthesis, and remain on the integrin for the lifetime of the molecule. There is growing appreciation for the importance of differential sialylation in regulating immune cell function. Sialylation has been reported to modulate cell signaling, activation, differentiation, as well as responsiveness and tolerance in the immune system (3, 10, 23, 55). In particular, the α2 6 sialic acid linkage, directed by ST6Gal-I, modulates the function of several specific glycoproteins. For example, α2 6 sialylation on the Fc region of immunoglobulin G (IgG) is a critical determinant in whether IgG elicits a pro- vs. anti-inflammatory response (24). Additionally, α2 6 sialylation of CD45 on T cells prevents the binding of this molecule to galectin-1, and thereby blocks galectin-1 induced apoptosis (1). Our studies add to this body of literature by showing that differential α2 6 sialylation regulates the function of α4β1 integrins, and by further defining the molecular mechanisms controlling the dynamic changes in ST6Gal-I expression during monocyte differentiation. Specifically we report that differentiation of monocytes along the macrophage lineage is associated with a downregulation in ST6Gal- I protein levels due to BACE1-directed shedding. Importantly, the activation of α4β1 integrins, as measured by VCAM-1 binding capability, can be completely blocked by constitutive overexpression of ST6Gal-I, which in turn prevents the synthesis of the hyposialylated β1 glycoform. Consistent with these results, forced expression of ST6Gal-I blocks the expression of a PMA-induced activation epitope on the β1 subunit.

79 64 Finally, computer modeling studies from our group (31) suggest that α2 6 sialic acids modulate the conformation and accessibility of specific regions within the β1 integrin I- like domain, a domain known to be involved in ligand binding. Collectively these results provide key evidence that sialylation directly regulates α4β1 function. Given the importance of α2 6 sialylation in regulating the function of immunerelated glycoproteins, there is a compelling need to elucidate the molecular mechanisms that control ST6Gal-I protein levels and activity. ST6Gal-I expression is known to be regulated by multiple mechanisms including oligomerization and glycosylation (5, 35), however the majority of studies have focused on transcriptional regulation (9). Recently, Kitazume et al. reported that ST6Gal-I is a cleavage substrate for the β-secretase BACE1 (27), which raises the possibility that ST6Gal-I levels are also controlled by shedding. BACE1 is highly enriched in the brain, and in fact, little is known regarding BACE1 function outside of the brain. Low levels of BACE1 mrna have been observed in most peripheral tissues, including a pooled leukocytic population (30, 56), however information regarding BACE1 protein levels in leukocytes has been lacking. BACE1 expression in U937 cells was inferred (though not directly shown) from data indicating that these cells exhibit a basal level of shedding of P-selectin glycoprotein ligand-1, another BACE1 substrate (29). However, in contrast to this study, Sinha et al. reported that cells of monocytic origin do not have detectable BACE1 activity (52). Results from our study provide important new insights regarding BACE1 expression in monocytes; specifically we find that BACE1 protein levels are low in monocytes, but are dramatically upregulated upon macrophage differentiation.

80 65 The physiologic importance of BACE1 in the immune system remains largely unexplored; however there is a wealth of circumstantial evidence suggesting that BACE1 is well-positioned to serve as an immunoregulator. Studies of neuronal and glial cells have shown that BACE1 expression is upregulated by proinflammatory stimuli including interferon-γ (8), tumor necrosis factor-α (59), and cholesterol (14), whereas cytokineinduced BACE1 upregulation is blocked by nonsteroidal anti-inflammatory drugs (44). Similarly BACE1 transcription is increased in response to nuclear factor κb activation (43) (although this is controversial (43)) but reduced by the anti-inflammatory transcription factor, peroxisome proliferator-activated receptor-γ (43). While these findings remain to be confirmed in immune cell types, they clearly point to a potential role for BACE1 in inflammation. The idea that BACE1 might modulate immune responsiveness was initially raised by Dominguez et al (11). These investigators observed that BACE1-null mice exhibited a high rate of perinatal death, and surviving mice were smaller in size. Based on these results, it was speculated that the loss of BACE1 might have contributed to some unidentified immunodeficiency in these mice. However, other groups observed no overt phenotypic changes as a result of bace1 gene ablation (4, 33, 34, 41). In the current study, we report that BACE1 is markedly upregulated during macrophage differentiation as a consequence of signaling through a PKC/ras/ERK pathway, a pathway known to be associated with the phenotypic changes induced by differentiation. Additionally, we determined that the upregulation of BACE1 causes cleavage and shedding of the ST6Gal-I sialyltransferase, which in turn leads to the expression of β1 integrins that lack α2 6 sialylation. Finally, the expression of a

81 66 hyposialylated β1 subunit promotes activation of the α4β1 receptor. In sum, the results of this study describe a novel mechanism for regulation of the α4β1 integrin, and further implicate BACE1 as a potential mediator of immune cell function. References 1. Amano M, Galvan M, He J, and Baum LG. The ST6Gal I sialyltransferase selectively modifies N-glycans on CD45 to negatively regulate galectin-1-induced CD45 clustering, phosphatase modulation, and T cell death. J Biol Chem 278: , Bellis SL. Variant glycosylation: an underappreciated regulatory mechanism for beta1 integrins. Biochim Biophys Acta 1663: 52-60, Brennan PJ, Saouaf SJ, Van Dyken S, Marth JD, Li B, Bhandoola A, and Greene MI. Sialylation regulates peripheral tolerance in CD4+ T cells. Int Immunol 18: , Cai H, Wang Y, McCarthy D, Wen H, Borchelt DR, Price DL, and Wong PC. BACE1 is the major beta-secretase for generation of Abeta peptides by neurons. Nat Neurosci 4: , Chen C and Colley KJ. Minimal structural and glycosylation requirements for ST6Gal I activity and trafficking /glycob/ Glycobiology 10: , Chen C, Mobley JL, Dwir O, Shimron F, Grabovsky V, Lobb RR, Shimizu Y, and Alon R. High affinity very late antigen-4 subsets expressed on T cells are mandatory for spontaneous adhesion strengthening but not for rolling on VCAM-1 in shear flow. J Immunol 162: , Chigaev A, Blenc AM, Braaten JV, Kumaraswamy N, Kepley CL, Andrews RP, Oliver JM, Edwards BS, Prossnitz ER, Larson RS, and Sklar LA. Real time analysis of the affinity regulation of alpha 4-integrin. The physiologically activated receptor is intermediate in affinity between resting and Mn(2+) or antibody activation. J Biol Chem 276: , Cho HJ, Kim SK, Jin SM, Hwang EM, Kim YS, Huh K, and Mook-Jung I. IFN-gamma-induced BACE1 expression is mediated by activation of JAK2 and ERK1/2 signaling pathways and direct binding of STAT1 to BACE1 promoter in astrocytes. Glia 55: , Dall'Olio F. The sialyl-alpha2,6-lactosaminyl-structure: biosynthesis and functional role. Glycoconj J 17: , 2000.

82 Daniels MA, Hogquist KA, and Jameson SC. Sweet 'n' sour: the impact of differential glycosylation on T cell responses. Nat Immunol 3: , Dominguez D, Tournoy J, Hartmann D, Huth T, Cryns K, Deforce S, Serneels L, Camacho IE, Marjaux E, Craessaerts K, Roebroek AJM, Schwake M, D'Hooge R, Bach P, Kalinke U, Moechars D, Alzheimer C, Reiss K, Saftig P, and De Strooper B. Phenotypic and Biochemical Analyses of BACE1- and BACE2-deficient Mice. J Biol Chem 280: , Fehr J and Jacob HS. In vitro granulocyte adherence and in vivo margination: two associated complement-dependent functions. Studies based on the acute neutropenia of filtration leukophoresis. J Exp Med 146: , Feigelson SW, Grabovsky V, Winter E, Chen LL, Pepinsky RB, Yednock T, Yablonski D, Lobb R, and Alon R. The Src kinase p56(lck) up-regulates VLA-4 integrin affinity. Implications for rapid spontaneous and chemokine-triggered T cell adhesion to VCAM-1 and fibronectin. J Biol Chem 276: , Ghribi O, Larsen B, Schrag M, and Herman MM. High cholesterol content in neurons increases BACE, beta-amyloid, and phosphorylated tau levels in rabbit hippocampus. Exp Neurol 200: , Gilbert HS, Rayfield EJ, Smith H, Jr., and Keusch GT. Effects of acute endotoxemia and glucose administration on circulating leukocyte populations in normal and diabetic subjects. Metabolism 27: , Ginsberg MH, Partridge A, and Shattil SJ. Integrin regulation. Current Opinion in Cell Biology 17: , Grabovsky V, Feigelson S, Chen C, Bleijs DA, Peled A, Cinamon G, Baleux F, Arenzana-Seisdedos F, Lapidot T, van Kooyk Y, Lobb RR, and Alon R. Subsecond induction of alpha4 integrin clustering by immobilized chemokines stimulates leukocyte tethering and rolling on endothelial vascular cell adhesion molecule 1 under flow conditions. J Exp Med 192: , Gu J and Taniguchi N. Regulation of integrin functions by N-glycans. Glycoconj J 21: 9-15, He H, Wang X, Gorospe M, Holbrook NJ, and Trush MA. Phorbol esterinduced mononuclear cell differentiation is blocked by the mitogen-activated protein kinase kinase (MEK) inhibitor PD Cell Growth Differ 10: , Hu X, Moscinski LC, Valkov NI, Fisher AB, Hill BJ, and Zuckerman KS. Prolonged activation of the mitogen-activated protein kinase pathway is required for macrophage-like differentiation of a human myeloid leukemic cell line. Cell Growth Differ 11: , 2000.

83 Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell 110: , Issekutz TB, Issekutz AC, and Movat HZ. The in vivo quantitation and kinetics of monocyte migration into acute inflammatory tissue. Am J Pathol 103: 47-55, Jenner J, Kerst G, Handgretinger R, and Muller I. Increased alpha2,6- sialylation of surface proteins on tolerogenic, immature dendritic cells and regulatory T cells. Exp Hematol 34: , Kaneko Y, Nimmerjahn F, and Ravetch JV. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation. Science 313: , Kharbanda S, Saleem A, Emoto Y, Stone R, Rapp U, and Kufe D. Activation of Raf-1 and mitogen-activated protein kinases during monocytic differentiation of human myeloid leukemia cells. J Biol Chem 269: , Kitazume S, Nakagawa K, Oka R, Tachida Y, Ogawa K, Luo Y, Citron M, Shitara H, Taya C, Yonekawa H, Paulson JC, Miyoshi E, Taniguchi N, and Hashimoto Y. In vivo cleavage of alpha2,6-sialyltransferase by Alzheimer betasecretase. J Biol Chem 280: , Kitazume S, Suzuki M, Saido TC, and Hashimoto Y. Involvement of proteases in glycosyltransferase secretion: Alzheimer's beta-secretase-dependent cleavage and a following processing by an aminopeptidase. Glycoconj J 21: 25-29, Ley K. Molecular mechanisms of leukocyte recruitment in the inflammatory process. Cardiovasc Res 32: , Lichtenthaler SF, Dominguez DI, Westmeyer GG, Reiss K, Haass C, Saftig P, De Strooper B, and Seed B. The cell adhesion protein P-selectin glycoprotein ligand-1 is a substrate for the aspartyl protease BACE1. J Biol Chem 278: , Lin X, Koelsch G, Wu S, Downs D, Dashti A, and Tang J. Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein. Proc Natl Acad Sci U S A 97: , Liu Y, Pan D, Bellis SL, and Song Y. Effect of altered glycosylation on the structure of the I-like domain of beta1 integrin: A molecular dynamics study. Proteins, Luo BH, Carman CV, and Springer TA. Structural basis of integrin regulation and signaling. Annu Rev Immunol 25: , Luo Y, Bolon B, Damore MA, Fitzpatrick D, Liu H, Zhang J, Yan Q, Vassar R, and Citron M. BACE1 (beta-secretase) knockout mice do not acquire compensatory gene expression changes or develop neural lesions over time. Neurobiol Dis 14: 81-88, 2003.

84 Luo Y, Bolon B, Kahn S, Bennett BD, Babu-Khan S, Denis P, Fan W, Kha H, Zhang J, Gong Y, Martin L, Louis JC, Yan Q, Richards WG, Citron M, and Vassar R. Mice deficient in BACE1, the Alzheimer's beta-secretase, have normal phenotype and abolished beta-amyloid generation. Nat Neurosci 4: , Ma J and Colley KJ. A disulfide-bonded dimer of the Golgi beta-galactoside alpha2,6-sialyltransferase is catalytically inactive yet still retains the ability to bind galactose. J Biol Chem 271: , McIntyre TM, Prescott SM, Weyrich AS, and Zimmerman GA. Cell-cell interactions: leukocyte-endothelial interactions. Curr Opin Hematol 10: , Miranda MB, McGuire TF, and Johnson DE. Importance of MEK-1/-2 signaling in monocytic and granulocytic differentiation of myeloid cell lines. Leukemia 16: , Mould AP, Garratt AN, Askari JA, Akiyama SK, and Humphries MJ. Identification of a novel anti-integrin monoclonal antibody that recognises a ligandinduced binding site epitope on the beta 1 subunit. FEBS Lett 363: , Pawlowski NA, Kaplan G, Abraham E, and Cohn ZA. The selective binding and transmigration of monocytes through the junctional complexes of human endothelium. J Exp Med 168: , Pretzlaff RK, Xue VW, and Rowin ME. Sialidase treatment exposes the beta1- integrin active ligand binding site on HL60 cells and increases binding to fibronectin. Cell Adhes Commun 7: , Roberds SL, Anderson J, Basi G, Bienkowski MJ, Branstetter DG, Chen KS, Freedman SB, Frigon NL, Games D, Hu K, Johnson-Wood K, Kappenman KE, Kawabe TT, Kola I, Kuehn R, Lee M, Liu W, Motter R, Nichols NF, Power M, Robertson DW, Schenk D, Schoor M, Shopp GM, Shuck ME, Sinha S, Svensson KA, Tatsuno G, Tintrup H, Wijsman J, Wright S, and McConlogue L. BACE knockout mice are healthy despite lacking the primary beta-secretase activity in brain: implications for Alzheimer's disease therapeutics. Hum Mol Genet 10: , Rose DM, Alon R, and Ginsberg MH. Integrin modulation and signaling in leukocyte adhesion and migration. Immunological Reviews 218: , Rossner S, Sastre M, Bourne K, and Lichtenthaler SF. Transcriptional and translational regulation of BACE1 expression--implications for Alzheimer's disease. Prog Neurobiol 79: , Sastre M, Dewachter I, Landreth GE, Willson TM, Klockgether T, van Leuven F, and Heneka MT. Nonsteroidal anti-inflammatory drugs and peroxisome proliferator-activated receptor-gamma agonists modulate immunostimulated processing of amyloid precursor protein through regulation of beta-secretase. J Neurosci 23: , 2003.

85 Seager Danciger J, Lutz M, Hama S, Cruz D, Castrillo A, Lazaro J, Phillips R, Premack B, and Berliner J. Method for large scale isolation, culture and cryopreservation of human monocytes suitable for chemotaxis, cellular adhesion assays, macrophage and dendritic cell differentiation. J Immunol Methods 288: , Seales EC, Jurado GA, Brunson BA, Wakefield JK, Frost AR, and Bellis SL. Hypersialylation of beta1 integrins, observed in colon adenocarcinoma, may contribute to cancer progression by up-regulating cell motility. Cancer Res 65: , Seales EC, Jurado GA, Singhal A, and Bellis SL. Ras oncogene directs expression of a differentially sialylated, functionally altered β1 integrin. Oncogene 22: , Seales EC, Shaikh FM, Woodard-Grice AV, Aggarwal P, McBrayer AC, Hennessy KM, and Bellis SL. A protein kinase C/Ras/ERK signaling pathway activates myeloid fibronectin receptors by altering β1 integrin sialylation. J Biol Chem 280: , Seales EC, Shaikh FM, Woodard-Grice AV, Aggarwal P, McBrayer AC, Hennessy KM, and Bellis SL. A protein kinase C/Ras/ERK signaling pathway activates myeloid fibronectin receptors by altering beta1 integrin sialylation. J Biol Chem 280: , Semel AC, Seales EC, Singhal A, Eklund EA, Colley KJ, and Bellis SL. Hyposialylation of integrins stimulates the activity of myeloid fibronectin receptors. J Biol Chem 277: , Shimizu Y, Rose DM, and Ginsberg MH. Integrins in the immune system. Adv Immunol 72: , Sinha S, Anderson JP, Barbour R, Basi GS, Caccavello R, Davis D, Doan M, Dovey HF, Frigon N, Hong J, Jacobson-Croak K, Jewett N, Keim P, Knops J, Lieberburg I, Power M, Tan H, Tatsuno G, Tung J, Schenk D, Seubert P, Suomensaari SM, Wang S, Walker D, Zhao J, McConlogue L, and John V. Purification and cloning of amyloid precursor protein beta-secretase from human brain. Nature 402: , Smith JW. Allostery and proteolysis: two novel modes of regulating integrin function. Matrix Biol 16: , Tan SL and Parker PJ. Emerging and diverse roles of protein kinase C in immune cell signalling. Biochem J 376: , Toscano MA, Bianco GA, Ilarregui JM, Croci DO, Correale J, Hernandez JD, Zwirner NW, Poirier F, Riley EM, Baum LG, and Rabinovich GA. Differential glycosylation of T(H)1, T(H)2 and T(H)-17 effector cells selectively regulates susceptibility to cell death. Nat Immunol 8: , 2007.

86 Vassar R, Bennett BD, Babu-Khan S, Kahn S, Mendiaz EA, Denis P, Teplow DB, Ross S, Amarante P, Loeloff R, Luo Y, Fisher S, Fuller J, Edenson S, Lile J, Jarosinski MA, Biere AL, Curran E, Burgess T, Louis JC, Collins F, Treanor J, Rogers G, and Citron M. Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE. Science 286: , Villavicencio-Lorini P, Laabs S, Danker K, Reutter W, and Horstkorte R. Biochemical engineering of the acyl side chain of sialic acids stimulates integrindependent adhesion of HL60 cells to fibronectin. J Mol Med 80: , Weber C. Novel mechanistic concepts for the control of leukocyte transmigration: specialization of integrins, chemokines, and junctional molecules. J Mol Med 81: 4-19, Yamamoto M, Kiyota T, Horiba M, Buescher JL, Walsh SM, Gendelman HE, and Ikezu T. Interferon-gamma and tumor necrosis factor-alpha regulate amyloidbeta plaque deposition and beta-secretase expression in Swedish mutant APP transgenic mice. Am J Pathol 170: , Yang GX and Hagmann WK. VLA-4 antagonists: potent inhibitors of lymphocyte migration. Med Res Rev 23: , Yusuf-Makagiansar H, Anderson ME, Yakovleva TV, Murray JS, and Siahaan TJ. Inhibition of LFA-1/ICAM-1 and VLA-4/VCAM-1 as a therapeutic approach to inflammation and autoimmune diseases. Med Res Rev 22: , Acknowledgements This work was supported by NIH grant R01CA84248, as well as grants from the American Heart Association and the Mizutani Foundation for Glycoscience (S.L.B.). Mrs. Woodard-Grice was supported by a Ruth L. Kirschstein National Research Service Award (NRSA) Predoctoral Fellowship.

87 72 A POTENTIAL ROLE FOR BACE IN IMMUNOLOGY ALENCIA V. WOODARD-GRICE and SUSAN L. BELLIS In preparation for Journal of Biological Chemistry Format adapted for dissertation

88 73 Introduction β site APP cleaving enzyme 1 (BACE1) is a type I membrane aspartyl protease that was initially identified by Vassar and colleagues to be the β-secretase enzyme responsible for the pathological cleavage of amyloid precursor protein (APP) in Alzheimer s Disease (AD) (50). BACE1-mediated cleavage of APP is followed by a secondary cleavage of APP by γ-secretase leading to the pathogenic secretion and accumulation of the misfolded amyloid β (Aβ) peptide (15). Early studies determined that BACE1 requires an acidic environment and is localized to both endosomes and the secretory pathway, particularly the trans-golgi network (14, 26). Later studies concluded that the BACE1 gene, located on human chromosome 11 (46), encodes a 501 amino acid protein which contains an N-terminal signal peptide domain followed by a prodomain, a luminal catalytic domain with two catalytic aspartic residues, a transmembrane domain and a short cytoplasmic tail (43). APP is the most widely studied BACE1 ligand, however additional BACE1 substrates have been identified in recent years, suggesting a broader physiological role for BACE1 activity. Supporting this concept, BACE1 mrna is expressed in most peripheral tissues, including leukocytes, although expression is known to be highest in the brain and pancreas (32, 50). Little is known regarding BACE1 function outside of the brain, however mounting evidence suggests that BACE1 may serve as an immunological regulator.

89 74 BACE1 cleaves several inflammatory associated ligands. Multiple substrates have been identified during the last decade. In addition to APP-like protein 1 & 2 (30, 39), the β subunit of voltage-gated sodium channels (52) and low density lipoprotein (LDL)-receptor related protein (51), BACE1 has been reported to cleave several proteins involved in the immune system. The identification of immune related BACE1 substrates supports a theory initially suggested by Roβner and colleagues. Since BACE1 is only found in vertebrates, these authors propose that, in addition to its role in the pathogenesis of AD, BACE1 may play a role in cellular processes developed later in evolution, such as the immune system (43). Specifically, BACE1 cleaves both P-selectin glycoprotein ligand-1 (PSGL-1) (31) and the B cell cytokine receptor, interleukin-1 type II receptor (IL-1R2) (27). PSGL-1 plays a critical role in the rolling and tethering of leukocytes to activated endothelium prior to transmigration (36). BACE1-mediated PSGL-1 cleavage may serve as an important control of leukocyte migration during acute inflammation. Interestingly, binding of the proinflammatory cytokine interleukin-1 (IL-1) to IL-1R2, prevents typical IL-1/nuclear factor-κ B (NF-κB) signaling (27). Furthermore, secreted IL-1R2 acts as a sink for IL-1 (27). BACE1 mediated shedding of IL-1R2 may serve as a mechanism for limiting detrimental effects of increased IL-1 during inflammation. More recently another BACE1 substrate has been identified. Kitazume et al report that BACE1 also cleaves the Golgi resident glycosyltransferase, ST6Gal-I, both in vivo and in vitro (23-25). As glycoproteins are trafficked through the trans-golgi, ST6Gal-I catalyzes the addition of α2-6 linked sialic acids to the N-linked glycans of specific substrates. Unlike the other BACE1 substrates, cleavage of ST6Gal-I does not

90 75 directly regulate the abundance of a particular receptor or ligand. Instead, loss of ST6Gal-I from the Golgi causes certain glycoproteins to acquire a different glycan composition. In fact, changes in sialylation have been shown to differentially regulate the function of several glycoproteins, including the immune related molecules, ICAM-1, VCAM-1, CD22 ligands and β1 integrins (16, 47). Moreover, ST6Gal-I has been reported to be directly involved in the immune response. Recently, studies of deficient mice have demonstrated a role for ST6Gal-I in regulating inflammation, circulating neutrophil homeostasis, and myeloid differentiation (38). ST6Gal-I activity is necessary for the expansion, activation and function of B cells (20). Specifically, α2,6 sialylated structures such as CD75w and CD22 regulate B cell maturation (10) and homing (13), respectively. ST6Gal-I may also govern the release of myeloid cells from the bone marrow into the peripheral blood (29). Futhermore, the secretion of liver ST6Gal-I is a hallmark of the acute phase response. Although the function of soluble ST6Gal-I remains unclear, BACE1 is reportedly responsible for this cleavage of ST6Gal-I from the liver (23). Taken together, the growing list of immune related BACE1 substrates strongly suggests a crucial role for BACE1 in the immune system. BACE1 mrna, protein and activity are present in leukocytes. Due to the pathogenesis of AD associated with BACE1 activity in the brain, most studies regarding the expression and function of BACE1 have been focused primarily on neurons and astrocytes. However, low levels of BACE1 mrna have been observed in several immune cell populations, including a pooled leukocytic population (32, 50),

91 76 MOLT-4 T-cells, HL-60 promyeloid cells and peripheral blood mononuclear cells (45). Although, the literature lacks information regarding the expression of BACE1 protein in cells outside of the brain, our laboratory recently demonstrated the expression of BACE1 protein in leukocytes (53). Specifically, we show low level detection of BACE1 protein in both the human promonocytic cell line U937 and in primary human CD14 + monocytes. Interestingly, BACE1 expression is dramatically elevated during macrophage differentiation of these cells (53). Although the physiological relevance for this increase is not currently clear, we speculate that enhanced BACE1 expression facilitates the inflammation induced downregulation of ST6Gal-I in immune cells. In fact, ST6Gal-I downregulation seems to be of importance in the differentiation and trafficking of T cells, dendritic cells and monocytes (2, 9, 49). For example, our results show that BACE1- mediated cleavage of ST6Gal-I leads to the hyposialylation of the β1 subunit, expression of a more active α4β1 integrin and increased binding to VCAM-1 (53). Increases in α4β1/vcam-1 interactions are crucial for monocytic transmigration through the vascular wall during an immune response (12). In addition to screening for BACE1 mrna and protein, several investigators have monitored BACE1 activity in various cell types. Lichtenthaler et al inferred BACE1 activity in U937 cells based on the basal cleavage of transfected PSGL-1 (31). However, Sinha et al. failed to detect BACE1 activity in cells of monocytic or lymphocytic origin (48). We speculate that these variable results may be due to the fact that the expression of BACE1 varies dependent on the cell type or differentiation state, as exemplified by the greater BACE1 expression in macrophages. Overall, these studies propose that BACE1 expression is present in specific leukocyte subsets.

92 77 BACE1 is regulated by inflammatory cytokines and transcription factors. Interestingly, there is a correlation between the development of Alzheimer s Disease and increased inflammation (1, 6, 17). AD involves activation of both the classical and alternative complement pathways, the upregulation of several cytokines and chemokines and the increased expression of the leukocytic β2 integrins (1, 6) Although the initial inflammatory response associated with AD may be protective, failure to clear Aβ peptide results in a chronic inflammatory response (54). Inflammatory stimulation, including IFN-γ and TNF-α, upregulates BACE1 expression in both neurons and astrocytes, which serves to exacerbate the pathogenesis of this disease (8, 21, 35, 42, 55). IFN-γ-mediated BACE1 regulation occurs as a result of Janus kinase (JAK)/ extracellular signal regulated kinase (ERK)/ signal transducer and activator of trabscription (STAT) signaling leading to the binding of STAT1 to the BACE1 promoter (8). In addition to STAT1, the BACE1 promoter includes several putative transcription factor binding sites, including those for the immune related transcription factors, NF-κB and peroxisome proliferators-activated receptor-γ (PPAR-γ). Data from luciferase reporter assays suggest that NF-κB displays a suppressor role in BACE1 transcription in neurons, but acts as a stimulator in activated astrocytes (5, 28). However, embryonic fibroblasts isolated from mice deficient in PPAR-γ exhibited an upregulation in BACE1 transcription and promoter activity, suggesting that PPAR-γ could serve as a repressor of BACE1. Moreover, the gene transcription of PPAR-γ is extremely reduced by inflammatory cytokines, a process that can be prevented by incubation with ibuprofen (3, 44). Additionally, neuroblastoma cells treated with nonsteroidal anti-inflammatory drugs

93 78 (NSAIDs) displayed decreased BACE1 mrna levels, protein expression and enzymatic activity (19, 44). Collectively, these studies describe a regulatory mechanism for BACE1 expression in the brain that involves several inflammatory mediators. Furthermore, these results combined with the discoveries of peripheral BACE1 expression and activity suggest that BACE1 is positioned to be regulated by inflammatory stimuli outside the brain as well. Therefore additional studies are necessary to confirm these results in leukocytes. In vivo studies demonstrate effects of BACE1 activity on the immune system. Several animal models in which BACE1 expression has been knocked out or overexpressed have been developed. Most of these studies have only focused on the function of BACE1 in neurons and the potential therapeutic benefit of BACE1 inhibition on the treatment and prevention of AD (4, 18, 37). Interestingly, results from several in vivo studies support the concept that BACE1 may play a role in the immune system. BACE1 knockout mice display a complex phenotype. Although other groups observed no obvious phenotypic changes associated with bace1 gene ablation (7, 33, 34, 41), Dominguez et al. report an increase in the mortality rate of BACE1 knockout offspring in the first weeks after birth (11). Additionally, the surviving mice maintained a lower body weight than their wild type littermates. The authors speculated that these findings may be the result of a deficient immune response in a non-pathogen free environment, however, the experiments performed in this study failed to expose any defect in the capacity of BACE1 knockout mice to mount an efficient immune response (11).

94 79 However, Kitazume et al previously used the Long-Evans Cinnamon (LEC) rat, a model of Wilson s disease, to investigate the role of increased BACE1 in a chronic inflammatory model. Similar to Wilson s disease patients, LEC rats undergo toxic copper accumulation in the liver and ultimately develop hepatitis. These authors show an increase in BACE1 mrna and liver secretion of ST6Gal-I in the LEC mice at 6 weeks of age, and concluded that BACE1 is responsible for the inflammation induced increase in soluble ST6Gal-I (22, 23, 40). Since many studies are currently focused on the development of a BACE1 inhibitor to treat AD, these studies provide sufficient cause for further investigation on the effects of BACE1 in the immune system. Conclusion As a potential therapeutic target for AD, many studies have been devoted to understanding the molecular biology of BACE1. Being located in the trans-golgi, BACE1 may theoretically co-localize with a multitude of potential targets as they traffic through the Golgi. However, this aspartyl protease displays a rather restricted substrate specificity reducing the possibility that it may cleave any given protein. Of the few substrates that have been identified in addition to APP, several have significant immunological relevance. Interestingly, BACE1 seems to be differentially expressed in leukocyte populations. Work from our laboratory suggests that BACE1 upregulation may be a vital component of monocyte/macrophage differentiation. In addition to cleaving immunologically relevant proteins and being expressed in leukocytes, BACE1 expression is regulated by several inflammatory mediators. Data from in vivo studies suggest that BACE1 activity may contribute to the inflammation induced secretion of

95 80 ST6Gal-I, a hallmark of the acute phase response. Overall, there exists a substantial amount of evidence indicating that, in addition to its famous role in AD, BACE1 activity may play a unique role in inflammation. References 1. Akiyama H, Barger S, Barnum S, Bradt B, Bauer J, Cole GM, Cooper NR, Eikelenboom P, Emmerling M, Fiebich BL, Finch CE, Frautschy S, Griffin WST, Hampel H, Hull M, Landreth G, Lue L-F, Mrak R, Mackenzie IR, McGeer PL, O'Banion MK, Pachter J, Pasinetti G, Plata-Salaman C, Rogers J, Rydel R, Shen Y, Streit W, Strohmeyer R, Tooyoma I, Van Muiswinkel FL, Veerhuis R, Walker D, Webster S, Wegrzyniak B, Wenk G, and Wyss-Coray T. Inflammation and Alzheimer's disease. Neurobiology of Aging 21: , Bax M, Garcia-Vallejo JJ, Jang-Lee J, North SJ, Gilmartin TJ, Hernandez G, Crocker PR, Leffler H, Head SR, Haslam SM, Dell A, and van Kooyk Y. Dendritic Cell Maturation Results in Pronounced Changes in Glycan Expression Affecting Recognition by Siglecs and Galectins. J Immunol 179: , Blasko I, Apochal A, Boeck G, Hartmann T, Grubeck-Loebenstein B, and Ransmayr G. Ibuprofen decreases cytokine-induced amyloid beta production in neuronal cells. Neurobiol Dis 8: , Bodendorf U, Danner S, Fischer F, Stefani M, Sturchler-Pierrat C, Wiederhold K-H, Staufenbiel M, and Paganetti P. Expression of human β- secretase in the mouse brain increases the steady-state level of β-amyloid doi: /j x. Journal of Neurochemistry 80: , Bourne KZ, Ferrari DC, Lange-Dohna C, Rossner S, Wood TG, and Perez- Polo JR. Differential regulation of BACE1 promoter activity by nuclear factor-kappab in neurons and glia upon exposure to beta-amyloid peptides. J Neurosci Res 85: , Bradt BM, Kolb WP, and Cooper NR. Complement-dependent proinflammatory properties of the Alzheimer's disease beta-peptide. J Exp Med 188: , Cai H, Wang Y, McCarthy D, Wen H, Borchelt DR, Price DL, and Wong PC. BACE1 is the major beta-secretase for generation of Abeta peptides by neurons. Nat Neurosci 4: , Cho HJ, Kim SK, Jin SM, Hwang EM, Kim YS, Huh K, and Mook-Jung I. IFN-gamma-induced BACE1 expression is mediated by activation of JAK2 and ERK1/2

96 81 signaling pathways and direct binding of STAT1 to BACE1 promoter in astrocytes. Glia 55: , Comelli EM, Sutton-Smith M, Yan Q, Amado M, Panico M, Gilmartin T, Whisenant T, Lanigan CM, Head SR, Goldberg D, Morris HR, Dell A, and Paulson JC. Activation of murine CD4+ and CD8+ T lymphocytes leads to dramatic remodeling of N-linked glycans. J Immunol 177: , Dall'Olio F. The sialyl-alpha2,6-lactosaminyl-structure: biosynthesis and functional role. Glycoconj J 17: , Dominguez D, Tournoy J, Hartmann D, Huth T, Cryns K, Deforce S, Serneels L, Camacho IE, Marjaux E, Craessaerts K, Roebroek AJM, Schwake M, D'Hooge R, Bach P, Kalinke U, Moechars D, Alzheimer C, Reiss K, Saftig P, and De Strooper B. Phenotypic and Biochemical Analyses of BACE1- and BACE2-deficient Mice. J Biol Chem 280: , Elices MJ, Osborn L, Takada Y, Crouse C, Luhowskyj S, Hemler ME, and Lobb RR. VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA- 4 at a site distinct from the VLA-4/fibronectin binding site. Cell 60: , Ghosh S, Bandulet C, and Nitschke L. Regulation of B cell development and B cell signalling by CD22 and its ligands {alpha}2,6-linked sialic acids /intimm/dxh402. Int Immunol 18: , Haass C, Lemere CA, Capell A, Citron M, Seubert P, Schenk D, Lannfelt L, and Selkoe DJ. The Swedish mutation causes early-onset Alzheimer's disease by betasecretase cleavage within the secretory pathway. Nat Med 1: , Haass C and Steiner H. Alzheimer disease gamma-secretase: a complex story of GxGD-type presenilin proteases. Trends Cell Biol 12: , Hanasaki K, Varki A, Stamenkovic I, and Bevilacqua M. Cytokine-induced beta-galactoside alpha-2,6-sialyltransferase in human endothelial cells mediates alpha 2,6-sialylation of adhesion molecules and CD22 ligands. J Biol Chem 269: , Hansson GK, Libby P, Schonbeck U, and Yan ZQ. Innate and adaptive immunity in the pathogenesis of atherosclerosis. Circ Res 91: , Harrison SM, Harper AJ, Hawkins J, Duddy G, Grau E, Pugh PL, Winter PH, Shilliam CS, Hughes ZA, Dawson LA, Gonzalez MI, Upton N, Pangalos MN, and Dingwall C. BACE1 ([beta]-secretase) transgenic and knockout mice: identification of neurochemical deficits and behavioral changes. Molecular and Cellular Neuroscience 24: , Heneka MT, Sastre M, Dumitrescu-Ozimek L, Hanke A, Dewachter I, Kuiperi C, O'Banion K, Klockgether T, Van Leuven F, and Landreth GE. Acute

97 82 treatment with the PPARgamma agonist pioglitazone and ibuprofen reduces glial inflammation and Abeta1-42 levels in APPV717I transgenic mice. Brain 128: , Hennet T, Chui D, Paulson JC, and Marth JD. Immune regulation by the ST6Gal sialyltransferase. Proc Natl Acad Sci U S A 95: , Hong HS, Hwang EM, Sim HJ, Cho HJ, Boo JH, Oh SS, Kim SU, and Mook- Jung I. Interferon gamma stimulates beta-secretase expression and sappbeta production in astrocytes. Biochem Biophys Res Commun 307: , Kaplan HA, Woloski BM, Hellman M, and Jamieson JC. Studies on the effect of inflammation on rat liver and serum sialyltransferase. Evidence that inflammation causes release of Gal beta 1 leads to 4GlcNAc alpha 2 leads to 6 sialyltransferase from liver. J Biol Chem 258: , Kitazume S, Nakagawa K, Oka R, Tachida Y, Ogawa K, Luo Y, Citron M, Shitara H, Taya C, Yonekawa H, Paulson JC, Miyoshi E, Taniguchi N, and Hashimoto Y. In vivo cleavage of alpha2,6-sialyltransferase by Alzheimer betasecretase. J Biol Chem 280: , Kitazume S, Saido TC, and Hashimoto Y. Alzheimer's beta-secretase cleaves a glycosyltransferase as a physiological substrate. Glycoconj J 20: 59-62, Kitazume S, Tachida Y, Oka R, Kotani N, Ogawa K, Suzuki M, Dohmae N, Takio K, Saido TC, and Hashimoto Y. Characterization of alpha 2,6-sialyltransferase cleavage by Alzheimer's beta -secretase (BACE1). J Biol Chem 278: , Koo EH and Squazzo SL. Evidence that production and release of amyloid betaprotein involves the endocytic pathway. J Biol Chem 269: , Kuhn PH, Marjaux E, Imhof A, De Strooper B, Haass C, and Lichtenthaler SF. Regulated intramembrane proteolysis of the interleukin-1 receptor II by alpha-, beta-, and gamma-secretase. J Biol Chem 282: , Lange-Dohna C, Zeitschel U, Gaunitz F, Perez-Polo JR, Bigl V, and Rossner S. Cloning and expression of the rat BACE1 promoter. J Neurosci Res 73: 73-80, Le Marer N and Skacel PO. Up-regulation of alpha2,6 sialylation during myeloid maturation: a potential role in myeloid cell release from the bone marrow. J Cell Physiol 179: , Li Q and Sudhof TC. Cleavage of amyloid-beta precursor protein and amyloidbeta precursor-like protein by BACE 1. J Biol Chem 279: , Lichtenthaler SF, Dominguez DI, Westmeyer GG, Reiss K, Haass C, Saftig P, De Strooper B, and Seed B. The cell adhesion protein P-selectin glycoprotein ligand-1 is a substrate for the aspartyl protease BACE1. J Biol Chem 278: , 2003.

98 Lin X, Koelsch G, Wu S, Downs D, Dashti A, and Tang J. Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein. Proc Natl Acad Sci U S A 97: , Luo Y, Bolon B, Damore MA, Fitzpatrick D, Liu H, Zhang J, Yan Q, Vassar R, and Citron M. BACE1 (beta-secretase) knockout mice do not acquire compensatory gene expression changes or develop neural lesions over time. Neurobiol Dis 14: 81-88, Luo Y, Bolon B, Kahn S, Bennett BD, Babu-Khan S, Denis P, Fan W, Kha H, Zhang J, Gong Y, Martin L, Louis JC, Yan Q, Richards WG, Citron M, and Vassar R. Mice deficient in BACE1, the Alzheimer's beta-secretase, have normal phenotype and abolished beta-amyloid generation. Nat Neurosci 4: , Maike Hartlage-Rübsamen UZ, Jenny Apelt, Ulrich Gärtner, Heike Franke, Tobias Stahl, Albrecht Günther, Reinhard Schliebs, Milena Penkowa, Volker Bigl, Steffen Roßner,. Astrocytic expression of the Alzheimer's disease β-secretase (BACE1) is stimulus-dependent. Glia 41: , McEver RP and Cummings RD. Perspectives series: cell adhesion in vascular biology. Role of PSGL-1 binding to selectins in leukocyte recruitment. J Clin Invest 100: , Mohajeri MH, Saini KD, and Nitsch RM. Transgenic BACE expression in mouse neurons accelerates amyloid plaque pathology. J Neural Transm 111: , Nasirikenari M, Segal BH, Ostberg JR, Urbasic A, and Lau JT. Altered granulopoietic profile and exaggerated acute neutrophilic inflammation in mice with targeted deficiency in the sialyltransferase, ST6Gal I /blood Blood: blood , Pastorino L, Ikin AF, Lamprianou S, Vacaresse N, Revelli JP, Platt K, Paganetti P, Mathews PM, Harroch S, and Buxbaum JD. BACE (beta-secretase) modulates the processing of APLP2 in vivo. Mol Cell Neurosci 25: , Paulson JC and Colley KJ. Glycosyltransferases. Structure, localization, and control of cell type-specific glycosylation. J Biol Chem 264: , Roberds SL, Anderson J, Basi G, Bienkowski MJ, Branstetter DG, Chen KS, Freedman SB, Frigon NL, Games D, Hu K, Johnson-Wood K, Kappenman KE, Kawabe TT, Kola I, Kuehn R, Lee M, Liu W, Motter R, Nichols NF, Power M, Robertson DW, Schenk D, Schoor M, Shopp GM, Shuck ME, Sinha S, Svensson KA, Tatsuno G, Tintrup H, Wijsman J, Wright S, and McConlogue L. BACE knockout mice are healthy despite lacking the primary beta-secretase activity in brain: implications for Alzheimer's disease therapeutics. Hum Mol Genet 10: , 2001.

99 Rossner S, Lange-Dohna C, Zeitschel U, and Perez-Polo JR. Alzheimer's disease β-secretase BACE1 is not a neuron-specific enzyme. Journal of Neurochemistry 92: , Rossner S, Sastre M, Bourne K, and Lichtenthaler SF. Transcriptional and translational regulation of BACE1 expression--implications for Alzheimer's disease. Prog Neurobiol 79: , Sastre M, Dewachter I, Landreth GE, Willson TM, Klockgether T, van Leuven F, and Heneka MT. Nonsteroidal anti-inflammatory drugs and peroxisome proliferator-activated receptor-gamma agonists modulate immunostimulated processing of amyloid precursor protein through regulation of beta-secretase. J Neurosci 23: , Satoh J and Kuroda Y. Amyloid precursor protein beta-secretase (BACE) mrna expression in human neural cell lines following induction of neuronal differentiation and exposure to cytokines and growth factors. Neuropathology 20: , Saunders AJ, Kim T-W, Tanzi; RE, Fan W, Bennett BD, Babu-Kahn S, Luo Y, Louis J-C, McCaleb M, Citron M, Vassar R, and Richards; WG. BACE Maps to Chromosome 11 and a BACE Homolog, BACE2, Reside in the Obligate Down Syndrome Region of Chromosome /science a. Science 286: 1255a-, Semel AC, Seales EC, Singhal A, Eklund EA, Colley KJ, and Bellis SL. Hyposialylation of integrins stimulates the activity of myeloid fibronectin receptors. J Biol Chem 277: , Sinha S, Anderson JP, Barbour R, Basi GS, Caccavello R, Davis D, Doan M, Dovey HF, Frigon N, Hong J, Jacobson-Croak K, Jewett N, Keim P, Knops J, Lieberburg I, Power M, Tan H, Tatsuno G, Tung J, Schenk D, Seubert P, Suomensaari SM, Wang S, Walker D, Zhao J, McConlogue L, and John V. Purification and cloning of amyloid precursor protein beta-secretase from human brain. Nature 402: , Taniguchi A, Higai K, Hasegawa Y, Utsumi K, and Matsumoto K. Differentiation elicits negative regulation of human beta-galactoside alpha2,6- sialyltransferase at the mrna level in the HL-60 cell line. FEBS Lett 441: , Vassar R, Bennett BD, Babu-Khan S, Kahn S, Mendiaz EA, Denis P, Teplow DB, Ross S, Amarante P, Loeloff R, Luo Y, Fisher S, Fuller J, Edenson S, Lile J, Jarosinski MA, Biere AL, Curran E, Burgess T, Louis JC, Collins F, Treanor J, Rogers G, and Citron M. Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE. Science 286: , 1999.

100 von Arnim CA, Kinoshita A, Peltan ID, Tangredi MM, Herl L, Lee BM, Spoelgen R, Hshieh TT, Ranganathan S, Battey FD, Liu CX, Bacskai BJ, Sever S, Irizarry MC, Strickland DK, and Hyman BT. The low density lipoprotein receptorrelated protein (LRP) is a novel beta-secretase (BACE1) substrate. J Biol Chem 280: , Wong HK, Sakurai T, Oyama F, Kaneko K, Wada K, Miyazaki H, Kurosawa M, De Strooper B, Saftig P, and Nukina N. beta Subunits of voltage-gated sodium channels are novel substrates of beta-site amyloid precursor protein-cleaving enzyme (BACE1) and gamma-secretase. J Biol Chem 280: , Woodard-Grice AV, McBrayer AC, Zhuo Y, Wakefield JK, and Bellis SL. Proteolytic Shedding of ST6Gal-I by BACE1 Regulates the Glycosylation and Function of α4β1 Integrins. J Biol Chem In Press, Wyss-Coray T, Yan F, Lin AH, Lambris JD, Alexander JJ, Quigg RJ, and Masliah E. Prominent neurodegeneration and increased plaque formation in complement-inhibited Alzheimer's mice. Proc Natl Acad Sci U S A 99: , Yamamoto M, Kiyota T, Horiba M, Buescher JL, Walsh SM, Gendelman HE, and Ikezu T. Interferon-gamma and tumor necrosis factor-alpha regulate amyloidbeta plaque deposition and beta-secretase expression in Swedish mutant APP transgenic mice. Am J Pathol 170: , 2007.

101 86 CONCLUSIONS In order to prevent unwanted attachment of circulating myeloid/promonocytic cells to the vascular wall, cell adhesion receptors are kept in an inactive state. However, differentiation of these cells along the monocytic/macrophage lineage occurs upon presentation of an inflammatory stimulus and is accompanied by a dramatic increase in cell adhesiveness. Increased adhesiveness serves to facilitate transmigration through the endothelium and tethering at sites of inflammation. It has been well established that increased monocytic VCAM-1 binding occurs via enhanced activity of α4β1 integrins. In fact, the α4β1/vcam-1 interaction is the target of several therapeutics aimed at ameliorating symptoms associated with numerous immune related disorders (226, 229). However, the mechanisms by which these increases in integrin activity occur have not been fully elucidated. Most studies aimed at elucidating the regulatory mechanisms governing α4β1 integrin function have primarily focused on inside-out signaling, which mediates rapid changes in integrin function. Like other studies (28, 30, 58, 68), our work demonstrates that PMA-induced macrophage differentiation causes rapid activation of α4β1 integrins (219). However, this rapid activation only accounts for an initial transient phase of PMA-dependent VCAM-1 binding. This first phase is followed by a more pronounced sustained increase in integrin activity initiated approximately 9 hours post PMA treatment (219). Kinetically, this later phase of increased adhesion temporally correlates

102 87 with monocyte infiltration in vivo (93). In fact, we have also shown that PMA-treatment induces two distinct phases of α5β1 mediated myeloid adhesion to FN as well (178). Interestingly, PMA treatment causes the mature β1 isoform to migrate faster upon SDS-PAGE analysis (178, 179, 219), suggesting that the subunit has a smaller molecular weight. We therefore hypothesized that a change in β1 structure was required for PMAdependent increases in integrin function. In fact, for 20 years, it has been observed that changes in cell phenotype are often associated with an altered electrophoretic mobility of the cell s β1 integrin isoform (13, 48, 106, 123, 151, 155, 162, 175, 176, 178, 179, 195, 206, 211, 213, 225). It was subsequently discovered that the mobility shifts were due to variation in the N-linked oligosaccharides on the β1 integrin as these changes can be prevented by removing the N-glycans prior to electrophoresis. As a result, altered β1 integrin glycosylation has been associated with several cellular processes, including myeloid differentiation (179, 206). By completely preventing N-glycan addition or processing, initial studies demonstrated that N-glycosylation was necessary for integrin activity. Results from these studies suggested that integrin glycosylation is required for proper folding, αβ subunit pairing, cell surface expression and normal cell adhesion to integrin ligands (13, 69). Although complete ablation of N-glycosylation impairs integrin function, there is no evidence that this type of change in integrin structure occurs biologically. Instead natural changes in integrin glycosylation involve more terminal carbohydrate structures added by enzymes in the trans-golgi. These terminal modifications directly modulate integrin function instead of expression or maturation. Furthermore, prior to work from our group, it was unknown what specific sugar residue modifications contributed to these

103 88 alterations in β1 integrin structure. Through lectin screening, our laboratory demonstrated that this second phase of PMA-dependent FN and VCAM-1 binding was associated with differential α2,6 sialylation. Specifically, our results suggested that this second phase is due to the expression of a β1 integrin glycoform that lacked α2,6 sialic acid (179, 219). In addition to reporting temporal correlation between delayed enhanced adhesion and hyposialylated β1 integrin expression, we show that this latter phase requires the expression of a new integrin species on the cell surface rather than alterations of integrins presently on the surface. By disrupting the Golgi, and thereby blocking the expression of newly synthesized integrins we were able to inhibit the delayed phase of PMA-dependent cell adhesion to FN (179). Importantly, Golgi disruption did not affect the initial phase of PMA-dependent FN binding, suggesting that rapid activation of α4β1 integrins is due to traditional inside-out signaling mechanisms leading to alterations of integrins already present on the cell surface, such as clustering or conformational changes. We, therefore, speculated that delayed PMA-dependent α4β1 mediated binding to VCAM-1 requires the expression of a new hyposialylated β1 integrin glycoform. Interestingly, a role for differential sialylation in regulating many aspects of immune cell function is emerging. Specifically, studies have shown that sialylation modulates cellular signaling, activation and differentiation (21, 44, 96, 205). Variant sialylation has been reported to mediate immunological responsiveness and tolerance. In particular, the α2 6 sialic acid linkage, directed by the trans-golgi enzyme ST6Gal-I, modulates the function of several specific glycoproteins. For example, α2 6 sialylation on the Fc region of immunoglobulin G (IgG) is a critical determinant in whether IgG

104 89 elicits a pro- vs. anti-inflammatory response (101). Additionally, α2 6 sialylation of CD45 on T cells prevents the binding of this molecule to galectin-1, and thereby blocks galectin-1 induced apoptosis (5). HL-60 myeloid cells bind greater to FN following enzymatic removal of cell surface sialic acids (159). In a separate study, in which an unnatural sialic acid variant was introduced into HL-60 cells, α5β1 and α4β1 integrins expressed by these cells exhibited enhanced ligand binding (212). We have added to this literature by showing that myeloid differentiation induces the downregulation of ST6Gal- I. Moreover, we identified β1 (176, 178, 179, 219), but not β3, β5 (176), α4 (219), and α5 (unpublished data), as a substrate for ST6Gal-I activity. We have successfully demonstrated temporal correlation between PMA mediated ST6Gal-I downregulation, integrin hyposialylation and increased FN and VCAM-1 binding. Collectively, our data suggested that monocyte/macrophage differentiation induced ST6Gal-I downregulation leads to the hyposialylation of β1 integrins with enhanced activity. However, to support our conclusions, we needed to establish a causal role for variant sialylation in regulating integrin function. To accomplish this, we directly manipulated ST6Gal-I expression and monitored integrin sialylation and function (219). Specifically, blocking ST6Gal-I downregulation prevents PMA-dependent integrin hyposialylation and VCAM-1 binding. To further establish that α2,6 linked sialic acids play a direct role in regulating integrin function, we previously manipulated the sialylation of purified α5β1 integrins and monitored integrin binding to FN. Enzymatic desialylation stimulated α5β1 integrin binding to FN, an increase that could be reversed by the addition of α2,6 sialic acid using recombinant ST6Gal-I (178). These data provide compelling evidence that α2,6 linked sialic acids directly regulate β1

105 90 integrin ligand binding activity. Therefore, we concluded that monocytic differentiation induced ST6Gal-I downregulation leads to the expression of hyposialylated β1 integrins which display enhanced binding to FN and VCAM-1. Nevertheless, we sought to identify mechanisms by which differential sialylation affects integrin activity. It has been long speculated that enhanced integrin affinity involved a conformational change in integrin extracellular domains which is triggered by cytoplasmic signaling pathways after cellular activation. Albeit not much attention has been given to the role of differential glycosylation in regulating conformational changes. Interestingly, to investigate alternative types of conformational movement in the ligandbinding headpiece, Springer s group mutationally introduced an N-glycosylation site into the hybrid I-like domain of the β3 and β1 integrin subunits. Both β3 and β1 integrins expressing this glycan wedge exhibited enhanced binding to physiological substrates (135). Like Springer, we also show that changes in N-glycosylation regulate integrin conformation. However, unlike Springer s work, which is an artificial model, we demonstrate a physiological change in global integrin conformation that is associated with enhanced binding. Specifically, we show that PMA-induced myeloid differentiation increases U937 cell binding of an antibody that specifically recognizes β1 integrins in an active/open conformation (219). Moreover, this recognition is completely ablated when ST6Gal-I downregulation is prevented and integrins remain in a sialylated state (219), suggesting that hyposialylated β1 integrins are in a more active/open conformation than the wild type glycoform. It is also possible that sialic acids may mask important functional domains within the integrin receptor. In fact, differential glycosylation controls critical immune

106 91 processes, including T-cell activation, homing and survival by generating or masking ligands for endogenous lectins (44). Epitope masking is thought to be an important function of sialic acids. However, the relationship between integrin function and epitope masking by sialic acid is multifaceted. We previously reported that unlike α5β1 and α3β1, de-sialylation of α1β1 inhibited ligand binding (176). Interestingly, unlike the α1 subunit, α5, α3 and α4 subunits lack an I-domain, which directly binds integrins (86, 198). It is hypothesized that in integrin receptors in which the α subunit lacks an I- domain, the β1 I-like domain is used for ligand binding (198). Therefore, the ligand binding mechanisms for α1β1 may be different which may explain the different effect of sialic acid on α1β1 function. In fact, our laboratory successfully mapped the N- glycosylation sites on the β1 integrin and identified three N-glycosylation sites within the β1 I-like domain (178). Additionally, computational modeling from our group suggest that α2,6 sialic acids regulate β1 integrin conformation and accessibility of specific regions within the I-like domain (131). The primary focus of this dissertation has been on the cellular and molecular biology mediating the effects of sialylation on integrin function. However, previous in vivo studies demonstrate the importance of ST6Gal-I activity in immunology. Therefore, there is clearly a need for to elucidate the effects of sialylation on immune cell behavior. Although there is not much known about the mechanisms by which sialylation directly affects integrin mediated monocyte/macrophage trafficking, there is some data to support our model. While ST6Gal-I null mice exhibit humoral immunodeficiency (78), P1 promoter specific ST6Gal-I deficient mice demonstrate an enhanced inflammatory response when challenged with a bacterial pathogen (7).

107 92 However, the molecular mechanisms by which decreased ST6Gal-I expression alters inflammatory cell homeostasis has yet to be elucidated. One possibility is that ST6Gal-I may act to divert the precursor substrate away from ligand synthesis for selectin-mediated adhesion. Additionally, earlier reports associated ST6Gal-I with the maintenance of hematologic homeostasis. ST6Gal-I upregulation accompanies myeloid maturation, suggesting that ST6Gal-I may govern the release of myeloid cells from the bone marrow (122). Increased ST6Gal-I expression has also been reported in activated endothelial cells (71). This increase in endothelial ST6Gal-I and α2,6 linked sialic acid has been shown to inhibit VCAM-1 dependent adhesion under flow conditions (2). Another possibility is that ST6Gal-I may mask pertinent structures from receptor recognition. In fact, differential sialylation of galectin ligands affects cellular responses to both galectin-1 and galectin-3, such as apoptosis, adhesion, and inflammatory responses (5, 128). For example, T H 2-differentiated cells are protected from galectin-1 mediated apoptosis through enhanced ST6Gal-I expression and the resultant α2,6 sialylation of cell surface glycoproteins (205), such as CD45 (5). Specifically, α2,6 sialylation of CD45 reduces CD45/galectin-1 binding and abrogates CD45 signaling (5). Additionally, data from our laboratory show that galectin-3 binds directly to the β1 subunit only when it is unsialylated (232). Interestingly, both galectin-1 and galectin-3 expression are enhanced in monocytes in response to inflammatory stimuli. Conceptually, differentiation induced ST6Gal-I downregulation and enhanced galectin-1 and -3 expression may cooperate to regulate monocyte/macrophage apoptosis and cell adhesion via β1 integrin receptors. Moreover, α2,6 linked sialic acid is considered necessary for the α2,6 specific siglec, Siglec-2, also known as CD22. The B cell specific

108 93 Siglec-2 is known to dampen immune signaling (36, 37, 52, 157, 204). Interestingly, CD22 is masked by sialic acid on the surface of resting cells, but is unmasked in B cells of ST6Gal-I deficient mice (34), which may play a role in the immunosuppression of B cells in these mice (78). Nevertheless, downregulation of ST6Gal-I seems to be of importance in the differentiation and trafficking of immune cells. Activation of CD4 and CD8 T cells, dendritic cell maturation and HL-60 myeloid cell differentiation involve a dramatic reduction in ST6Gal-I mrna (12, 35, 202). Peripheral blood mononuclear cells from patients with systemic sclerosis exhibit lower levels of ST6Gal-I mrna (121). In addition, our lab has reported decreases in protein levels of ST6Gal-I in differentiating U937, THP-1 and primary monocytes (177, 179, 219). Importantly, we have elucidated another mechanism by which monocytic differentiation induces ST6Gal-I downregulation. We are the first to report leukocytic protein expression of the BACE1 secretase, a neuronal enzyme that was previously reported to cleave ST6Gal-I (219). Monocyte/macrophage differentiation induces a dramatic upregulation in BACE1 expression and activity in both U937 cells and primary human monocytes (219). This dramatic upregulation suggests that BACE1 is positioned to play a critical role in macrophage differentiation. However, there has been minimal attention on BACE1 in immune cells. The majority of the studies concerning this enzyme have been targeted to the brain. However, many of these studies have investigated the regulation of BACE1 expression and activity in microglia (16, 77, 118, 172, 224), which are considered to be brain-resident macrophages.

109 94 Interestingly, there exist several findings that also implicate BACE1 in having a role in immunology. In addition to ST6Gal-I, BACE1 also cleaves the inflammatory molecules PSGL-I and IL-1R type 2. Dominguez et al reported that ablation of the bace1 gene caused an increase in the rate of perinatal death and smaller size of surviving mice, factors they speculated were due to an unidentified immunodeficiency (51). Furthermore, BACE1 expression is regulated by various inflammatory cytokines. BACE1 expression and activity can be reduced by nonsteroidal anti-inflammatory drugs (NSAIDs). Taken together, these findings suggest a potentially important role for BACE1 in mediating leukocyte trafficking. Our work suggests that in both cell lines and primary monocytes, BACE1 is upregulated by PKC/ras/ERK signaling, a cascade known to be involved in macrophage differentiation. Moreover, BACE1 expression temporally correlates with ST6Gal-I downregulation and secretion. In fact, inhibition of BACE1 activity prevents PMAinduced ST6Gal-I downregulation and secretion and VCAM-1 binding, suggesting that BACE1 activity is required for PMA-induced changes in integrin activity. Our data demonstrate that in the first 15 to 18 hours following differentiation, ST6Gal-I downregulation is driven primarily by BACE1 cleavage. However, preliminary results suggest that changes in ST6Gal-I mrna may be involved at later time points. Based on our findings, we conclude that monocyte/macrophage differentiation induces the downregulation of ST6Gal-I via BACE1 mediated cleavage through PKC/ras/ERK signaling. As a result, hyposialylated β1 integrins are expressed by differentiating monocytes. These hyposialylated integrins are expressed in a more open conformation, suggesting that these integrins are in a higher binding affinity state than

110 95 wild type integrins. Furthermore, we have shown that integrin hyposialylation is necessary for differentiation dependent enhanced binding to VCAM-1 and FN. We plan to utilize this working model for future studies to further elucidate the mechanisms by which differential sialylation regulates integrin function (Figure 1). Figure 1 Working model.