Cell signalling pathways in the HSC niche. Dr. Abdullah Aljedai

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1 Cell signalling pathways in the HSC niche Dr. Abdullah Aljedai

2 Learning objectives & resources 1- To introduce the concept of cell signaling process in the haemopoietic system. 2- To outline some of the main signal transduction pathways that are involved in regulation of haemopoietic stem cell behaviors. 3- To describe the Notch, Wnt, and Sonic hedgehog pathwyas in term of their basic components, signal transduction mechanisms, and how they are involved in HSC cell fate decisions (self renewal Vs differentiation). References : 1-Rizo, A., Vellenga, E., de Haan, G. and Schuringa, J. J. (2006) Signaling pathways in self-renewing hematopoietic and leukemic stem cells: do all stem cells need a niche? Hum Mol Genet 15 Spec No 2, R Zhang, C. C. and Lodish, H. F. (2008) Cytokines regulating hematopoietic stem cell function. Curr Opin Hematol 15,

3 Cell signalling Cell signalling is part of a complex system of communication that governs basic cellular activities and coordinates cell actions. The ability of cells to perceive and correctly respond to their microenvironment is vital of development, and dictation of cell fate decisions. Errors in cellular information processing are responsible for diseases such as cancer. signal transduction refers to any process by which a cell converts one kind of signal or stimulus into another. Most processes of signal transduction involve ordered sequences of biochemical reactions inside the cell, resulting in a signal transduction pathway. Such processes are usually rapid, lasting on the order of milliseconds in the case of ion flux, or minutes for the activation of protein- and lipid-mediated kinase cascades, but some can take hours, and even days (as is the case with gene expression),

4 Examples of cell signalling pathways Cell membrane Nucleus

5 Regulation of HSC by signaling pathways The interaction of the stem cell with specific elements in the microenvironment is a key regulatory mechanism in maintenance of its selfrenewal and differentiation capacities. A number of different types of signaling and adhesion molecules that have a role in the regulation of stem cell quiescence, self-renewal and cell fate decisions have been reported. stem cell self-renewal divisions should be tightly controlled and deregulation of self-renewal of stem cells is the main cause for cancer. Stem and cancer cells share certain signaling pathways that regulate their self-renewal (Rizo et al, 2006)

6 Signaling pathways involved in the HSC fate (Rizo et al, 2006)

7 Important key players in the niche Adhesion molecules are essential for the physical association of HSC and their niche (eg, N-cadherin and Integrins such as VLA-4 and VLA-5). Growth factors and cytokines: - Secreted cytokines and growth factors can dictate stem cell fate by initiating specific signal transduction within the HSC (eg. Stem cell factor, Thrombopoietin, Flt3L, IL-3, and IL-6). - Transforming growth factor-b (TGF-b) is one of the few known negative regulators of HSCs (it maintains HSC in quiescent state, in vitro, by blocking the cell surface expression of cytokine receptors like c-kit, FLT3, MPL and IL-6R).

8 Intrinsic control of HSC Transcription factors play a vital role in the differentiation of HSCs and progenitor cells. Bmi-1, which is a member of the polycomb group (PcG) family of genes, has been shown to be highly expressed in HSCs and declines during haemopoietic development. Competitive repopulation studies have demonstrated that Bmi- 1 is crucial for HSCs self-renewal (Stein et al, 2004). The homebox (Hox) genes exhibit distinct pattern of expression with haemopoietic differentiation (Stein et al, 2004). HoxB4 for example is abundantly expressed in HSCs, but declines with lineage differentiation, which underlines a possible central role of this transcription factor in early haemopoiesis

9 Notch signaling pathway Notch signalling is an evolutionary conserved mechanism that controls cell fate decisions in various sites in the body, in both vertebrates and invertebrates (Ohishi et al. 2003). Notch signalling involves binding between a Notch receptor on one cell and a ligand on the neighbouring cell. This triggers the cleavage of the intra-cellular domain of Notch from its membrane-bound tether and a subsequent translocation to the nucleus, where it activates transcription RBP-J. This activation leads to increased transcription of certain target genes, such as the Hairy and Enhancer of Split (HES)-1 gene. The phenotype of the Notch gene of Drosophila was discovered by Morgan in 1916 in a mutant fly with notches in its wings, and the gene was found to be required for the wing outgrowth. The Drosophila Notch gene was cloned in Four mammalian Notch genes were cloned, known as Notch 1-4.

10 Notch mutation in Drosophila

11 Notch receptors Mammalian Notch genes encode four Notch receptors, Notch 1-4. Notch receptors consist of two domains, which remain noncovalently bound together by a calcium-dependent interaction. The extra-cellular domain (ECN) consists of tandem epidermal growth factor (EGF-like) repeats that bind Notch ligands, and three Lin 12/ Notch repeats which are crucial for maintaining Notch in a resting conformation before ligand binding.

12 Notch receptors The intracellular Notch domain (ICN) contains a RAM domain, a cdc 10/ ankyrin-like repeats flanked by two nuclear localisation signal sequences (NLS), and a c-terminal prolineglutamate-serine-threonine-rich (PEST) domain, which is important for regulating protein stability. ICN has a Notch cytokine response domain (NCR), which may be involved in cytokine signalling. The RAM domain and ANK repeats are binding sites for the downstream transcription factor CBF-1/RBPJ.

13 Notch receptor Proteolytic sites S1 S2 S3 ICN EGF like repeats LNR HD HD RAM ANK NCR TAD PEST TM Notch extracellular domain Notch transmembrane domain (NTM)

14 Notch ligands Two Notch ligands (Delta and Serrate) have been identified in Drosophila and five ligands have been identified in mammals (Jagged1, Jagged2, Delta-like1, Delta-like3, and Delta-like4). Notch ligands are trans-membrane proteins, which are composed of an extra-cellular domain, transmembrane domain and a relatively short cytoplasmic tail. The NT and DSL domains have been found to be indispensable for proper interaction with Notch expressing cells.

15 Notch ligands Extracellular region Intracellular region NT DSL EGF-like repeats Cys- rich Jagged/ Serrate NT DSL EGF-like repeats Delta EBD TM

16 Molecular mechanisms of Notch signalling Physical interaction between specific EGF repeats in the DSL domain of a ligand and the EGF repeats of the ECN receptor protein triggers two successive cleavages, resulting in the release of ICN and its subsequent translocation into the nucleus. Once in the nucleus, the ICN binds directly through its RAM and ANK domains to the CSL transcription factor (CBF1 in vertebrates) and converts it from a transcriptional repressor into a transcriptional activator. Binding of ICN to CSL displaces co-repressor complexes from CSL (such as histone deacetylase (HDAC)) and recruits, through ANK and TAD domains of ICN, different transcriptional co-activators such (MAML1 in mammals) to convert CSL into transcriptional activator. This results in transcription of Notch downstream target genes (e.g. HES) which eventually control specific developmental decisions in different cellular contexts. The most widely characterised mammalian Notch target gene is Hes1, although Hes5,C-myc, cycline D1, and deltex have been recently shown to be involved in Notch signalling. (Aster et al. 2008).

17 Mechanism of Notch signalling Sending cell Receiving cell GSI S1 S2 S3 Nucleus

18 Notch signalling in haemopoiesis Notch receptors (N1-4) have been identified in haemopoietic progenitors. Notch-1 has been shown to be expressed in a wide range of haemopoietic cells at different levels of maturation including CD34+ lin- precursors and CD34+lin+ precursors, as well as lymphoid, myeloid, and erythroid precursors. Notch1 hase been also detected in peripheral blood T and B lymphocytes, monocytes and neutrophils (Milner and Bigas, 1999). The expression patterns of Notch-1 and 2 in different haemopoietic lineages are distinct, ranging from low levels in CD34+ precursors to high levels in monocytes. Notch ligands have also been found in haemopoietic tissue including foetal liver, BM and thymus, and in populations of haemopoietic cells. Jagged-1, Delta-1 and Delta -4 have been detected in bone marrow stromal cells, whereas Jagged-1 is expressed in haematopoietic cells such as macrophages, megakaryocytes and mast cells.

19 Notch and haemopoietic stem cell (HSC) fate decisions Constitutive Notch-1 signalling in haemopoietic stem cells and progenitors allows the establishment of immortalised cell lines, which retain the capacity to generate either lymphoid or myeloid cells both in vitro and in vivo (Varnum- Finney et al. 2000). Inhibition of Notch signalling in mice caused accelerated differentiation of HSCs in vitro and depletion of HSCs in vivo. In RAG-1 deficient mouse stem cells, over-expression of Notch-1 promoted stem cell self-renewal over differentiation (Stier et al. 2002). Constitutively active Notch-4 promotes HSCs self-renewal, while inhibiting differentiation and altering lymphoid development (Vercauteren and Sutherland, 2004; Ye et al. 2004). Notch signalling activity has been demonstrated to be high in HSC and progenitor cells in the bone marrow niche (Duncan et al. 2005). When cultured with human Jagged-1, human HSCs showed increased survival and expansion potential in vivo (Karanu et al. 2000).

20 Notch expression of receptor genes in CD34+ Bone Marrow and CML populations

21 Expression of Notch1 receptor in CD34+ CD38- cells from CML patient Notch1 Notch1

22 Wnt signaling The secreted Wnt glycoproteins are signalling molecule involved in diverse roles in embryonic development, cell fate specification, polarity and proliferation. Abnormalities in the Wnt signalling pathway have been associated with embryonic lethality and various diseases, such as cancer (reviewed by Nusse, 2005). The Wnt signalling pathway has been implicated in the homeostasis of adult stem cells from various tissues, including the epidermis, intestinal epidermis and haematopoietic system (reviewed by Reya and Clevers, 2005). Aberrant Wnt signalling has been associated with cancers of various tissues. The Wnt canonical pathway involves the stabilisation of β-catenin to activate gene transcription in association with the T-cell factor/lymphoid enhancer factor-1 (Tcf/LEF-1) transcription factors (reviewed by Nusse, 2005).

23 Wnt signaling components There are 20 human Wnt genes that encode for 20 Wnt proteins (ligands). Wnt ligands are expressed either by cells in the niche or by HSC themselves. The Frizzled family of seven-transmembrane proteins are the primary Wnt receptors. In addition to the Wnt and Frizzled interaction, the presence of a single-pass transmembrane protein from the LRP family is required for Wnt signal transduction.

24 Wnt signaling pathway Reya & Clevers (2005) In the absence of Wnt signalling (left panel), b-catenin is in a complex with axin, APC and GSK3-b, and gets phosphorylated and targeted for degradation. - β-catenin also exists in a cadherin bound form and regulates cell cell adhesion. - In the presence of Wnt signalling (right panel), β -catenin is uncoupled from the degradation complex and translocates to the nucleus, where its binds Lef/Tcf transcription factors, thus activating target genes.

25 Wnt signaling pathway LRP Frizzled Hoyle (2009)

26 Murine studies: Wnt signaling and HSC Functional studies have shown that in vitro, soluble Wnt proteins synergize with stem cell factor) to promote the growth and inhibit the differentiation of murine haematopoietic progenitors. b-catenin as well as purified Wnt3A protein can promote self renewal of murine HSCs in vitro and enhance their ability to reconstitute the haematopoietic system of lethally irradiated mice in vivo. Human studies: Wnt5A treatment of human haematopoietic progenitors in the presence of stromal cell contact promotes the expansion of undifferentiated progenitors in vitro. Treatment of mice with Wnt5A-conditioned medium results in increased human HSC repopulation in a NOD-SCID xenotransplant model. Upon transplantation of human Lin BM cells in NOD/SCID mice, an increased repopulating activity and percentage of CD45+CD34+ were observed following in vivo treatment with GSK3β inhibitors. These studies suggest that Wnt signalling can contribute to HSC and progenitor cell selfrenewal.

27 Wnt signaling and HSC Wnt signalling is important in the growth and differentiation of osteoblasts. The lack of a functional LRP in mice results in osteopenia with a decreased osteoblast proliferation. Theses results suggest that Wnt signaling has direct and indirect control on HSC self renewal. Interestingly, it has been shown that Wnt signalling contributes to the differential expression of known Notch targets in HSCs and that Wnt and Notch may work together to promote self-renewal of HSCs (Duncan et al, 2005).

28 Dysregulated wnt signaling

29 Hedgehog signalling The Hedgehog signalling pathway has been identified as being crucial during many developmental processes in D. Melanogaster and vertebrates. The Hedgehog phenotype was first identified in D. Melanogaster during the study of segment polarity genes. It was discovered that the cuticles of larvae with this mutation were covered in hairy spikes, giving the gene its name. There are three mammalian hedgehog homologues and Shh has been shown to have the strongest signalling ability. In the mammalian system, the hedgehog pathway is involved in bone morphogenesis. The Hedgehog signalling pathway has been shown to be deregulated in prostate Cancers, brain tumors and basal cell carcinoma.

30 Hedgehog signalling Ingham & Placzek (2006)

31 Hedgehog signalling Fennessy (2008)

32 Hedgehog signalling mechanism (A) When hedgehog signalling is inactive, membrane bound patched (ptc) inhibits smoothened (smo). This inhibition allows a complex bound to the microtubules made up of fused (fu), costal2 (cos2) and suppressor of fused (su(fu) to bind the full length Gli3, allowing it to be phosphorylated by protein kinase A (PKA), resulting in the cleavage of Gli3 and the formation of repressor peptide (R). This R peptide translocates to the nucleus and represses the transcription of hedgehog responsive genes. (B) When hedgehog signalling is active, hedgehog (hh) binds to ptc and releases its inhibition of smo. Through mechanisms unidentified as of yet, possibly involving small molecules, cos2 and su(fu) are phosphorylated by fu, causing them to be unable to bind Gli3. At the same time, PKA is inactivated by phosphorylation. The complete Gli3 translocates to the nucleus and activates the transcription of Gli1. Gli2 can also activate the transcription of Gli1, the main mediator of Hedgehog signalling. Signalling can be inhibited by cyclopamine.

33 The Hedgehog Pathway and Stem Cell Maintenance Human CD34+ CD38- Lin- HSC expressed Shh, Ptch1, Smo as well as Gli1, Gli2 and Gli3, indicating that an active Hedgehog signalling pathway was present in these cells. Bhardwaj et al. (2001). They found that CD34+ CD38- Lin- HSC were capable of producing endogenous Shh and inhibition of this cytokine with anti-shh antibodies resulted in a 20-30% reduction in total CD34+ CD38- cell proliferation after 7 to 12 days. The addition of rshh to CD34+ CD38- Lin- resulted in an increase in the percentage of CD34+ CD38- cells after 7 days. CD34+ CD38- Lin- cells treated with Shh were more capable of engraftment in NOD/SCID mice after 8 weeks. Constitutive activation of the Hedgehog signalling pathway in the mouse Ptch1+/- model resulted in the expansion of the primitive mouse Lin- Sca- 1+ c-kit+ haematopoietic population. (Trowbridge et al, 2006).