Matrix-Degrading Metalloproteinases in Photoaging

Transcription

1 REVIEW Taihao Quan 1, Zhaoping Qin 1,2, Wei Xia 1,2, Yuan Shao 1, John J. Voorhees 1 and Gary J. Fisher 1 radiation from the sun impacts skin health adversely through complex, multiple molecular pathways. Premature skin aging (photoaging) is among the most widely appreciated harmful effects of chronic exposure to solar radiation. Extensive damage to the dermal connective tissue is a hallmark of photoaged skin. Disruption of the normal architecture of skin connective tissue impairs skin function and causes it to look aged. irradiation induces expression of certain members of the matrix metalloproteinase (MMP) family, which degrade collagen and other extracellular matrix proteins that comprise the dermal connective tissue. Although the critical role of MMPs in photoaging is undeniable, important questions remain. This article summarizes our current understanding of the role of MMPs in the photoaging process and presents new data that (1) describe the expression and regulation by irradiation of all members of the MMP family in human skin in vivo and (2) quantify the relative contributions of epidermis and dermis to the expression of irradiationinduced MMPs in human skin in vivo. Journal of Investigative Dermatology Symposium Proceedings (29) 14, 2 24; doi:1.138/jidsymp.29.8 INTRODUCTION Human skin is the only organ directly exposed to radiation from the sun. Solar radiation is a wellrecognized environmental hazard. Acute exposure of human skin to radiation causes sunburn, altered pigmentation, inflammation, immune suppression, and dermal connective tissue damage (Kripke, 1984; Gilchrest and Yaar, 1992; Kaminer, 1995; Fisher et al., 1996, 1997, 22). Chronic irradiation over many years disrupts normal architecture of the skin and ultimately causes premature skin aging (photoaging) and skin cancer. Clinically, photoaging is recognizable by fine and coarse wrinkles, blotchy dyspigmentation, increased fragility, and rough skin texture (Kligman and Kligman, 1986; Berneberg et al., 2; Scharffetter-Kochanek et al., 2). Histological and ultrastructural studies have revealed minimal epidermal changes. In contrast, major alterations are seen in dermal connective tissue, characterized by damaged and disorganized collagen fibrils, which constitute the bulk (9% dry weight) of skin connective tissue, and massive accumulation of aberrant elastic material, referred to as solar elastosis. These observations indicate that radiation causes qualitative alterations in extracellular matrix (ECM) proteins and suggest that matrixdegrading proteases participate in this process. Type I collagen is the major structural protein in the dermal ECM (Nimni, 1983; Uitto and Bernstein, 1998). Collagen precursor molecules (procollagen) are synthesized by dermal fibroblasts. Procollagen is secreted into extracellular spaces, where it is enzymatically processed to mature collagen. Mature collagen spontaneously forms fibrils, which are stabilized by cross-links. Collagen fibrils are largely responsible for the strength and resilience of skin. Collagen fibrils have an estimated half-life of 17 years. Therefore, fragmented collagen fibrils accumulate with the passage of time and have longlasting consequences on skin structure and function. irradiation causes alternations in dermal collagen through two primary pathways: (1) stimulation of collagen breakdown, resulting in fragmented, disorganized collagen and (2) inhibition of procollagen biosynthesis, resulting in a loss of collagen content. A single exposure to radiation (2MED) causes near-complete loss of procollagen synthesis, which persists for 24 hours, followed by recovery hours later (Fisher et al., 2). This reduction in procollagen synthesis is most likely mediated by impairment of transforming growth factor-b pathways, which control procollagen expression (Quan et al., 21, 22, 24, 25). Matrixdegrading metalloproteinases (MMPs) are key mediators of collagen degradation that is observed in photoaged skin (Fisher et al., 1996, 1997, 1998, 22; Fisher and Voorhees, 1998). This review article summarizes matrix MMP expression and regulation by irradiation in human skin in vivo. MMP GENE EXPRESSION IN NON-IRRADIATED AND -IRRADIATED HUMAN SKIN IN VIVO MMPs comprise a family of zinc-containing proteinases that are responsible for degrading ECM proteins, which form skin dermal connective tissue (Nelson et al., 2; Sternlicht and Werb, 21). To date, the MMP gene family consists of 25 1 Department of Dermatology, University of Michigan Medical School, Ann Arbor, Michigan, USA 2 Authors contributed equally to this manuscript Correspondence: Dr Gary J. Fisher, Department of Dermatology, University of Michigan Medical School, 131 Catherine, Medical Science I, Room 6447, Ann Arbor, Michigan , USA. dianemch@umich.edu Abbreviations: ECM, extracellular matrix; MMP, matrix metalloproteinase; RT-PCR, reverse transcriptase PCR Received 14 November 28; accepted 8 January 29 2 Journal of Investigative Dermatology Symposium Proceedings (29), Volume 14 & 29 The Society for Investigative Dermatology

2 members, 24 of which are expressed in mammals (Sternlicht and Werb, 21; Egeblad and Werb, 22; Fu et al., 28). MMPs are classified as collagenases, gelatinases, stromelysins, and membrane-type MMPs according to their substrate specificities and depending on whether they are secreted as soluble proteins or bound to cell surface membranes (Fu et al., 28). MMPs are involved in a wide range of proteolytic events in physiological and pathological circumstances, including embryogenesis, wound healing, inflammation, angiogenesis, and cancer (Birkedal-Hansen et al., 1993; Sternlicht and Werb, 21; Egeblad and Werb, 22; Kerkela and Saarialho-Kere, 23). Studies conducted by us and by others over the past several years have revealed that radiation elevates at least three different MMPs in human skin in vivo, that is interstitial collagenase (MMP-1), stromelysin-1 (MMP-3), and 92-kDa gelatinase (MMP-9) (Fisher et al., 1996, 1997, 22; Fisher and Voorhees, 1998; Brenneisen et al., 22; Wang et al., 28). These three MMPs are strongly regulated by transcription factor activator protein-1, which is rapidly induced and activated by radiation in human skin in vivo (Gutman and Wasylyk, 199; Birkedal-Hansen et al., 1993; Mauviel, 1993; Fisher and Voorhees, 1998). The combined actions of MMP- 1, -3, and -9 have the capacity to degrade most of the proteins that comprise the dermal ECM. However, whether irradiation induces expression of other members of the MMP family in human skin has not been fully determined. To address this question, we investigated basal and radiation-induced gene expression of each mammalian MMP in human skin in vivo. With the exception of MMP-14, basal mrna expression levels of the MMP family members were extremely low in normal healthy, sun-protected, adult human skin (Figure 1). Transcripts for MMP-8, -1, -12, -2, and -26 were not detected. Transcripts for remaining MMPs were near the level of detection, approximately 1,-fold lower than the internal control, housekeeping gene 36B4. Basal expression of MMP-14 mrna was approximately 35-fold higher than that of other detectable MMPs. Interestingly, among the 19 MMPs expressed in normal human skin, only three were significantly induced in response to irradiation: MMP-1 (collagenase), MMP-3 (stromelysin-1), and MMP-9 (92-kDa gelatinase) (Figure 2). MMP-1 and MMP-3 mrna levels were induced several 1,-fold at 24 hours post- irradiation, whereas MMP-9 was induced modestly (sixfold). MMP-1 initiates cleavage of fibrillar collagen, typically type I and III in skin, at a single site within its central triple helix. Once cleaved by MMP-1, collagen can be further degraded by elevated levels of MMP- 3 and MMP-9. These data confirm and extend our earlier findings (Fisher et al., 1996, 1997, 22; Fisher and Voorhees, 1998; Wang et al., 28). Interestingly, in contrast to -inducible MMPs, MMP-14 was reduced nearly 8% at 8 hours post- irradiation, and remained decreased for at least 24 hours (data not shown). The physiological function of MMP-14 in human skin remains to be determined. We have earlier reported that MMP-8 (neutrophil collagenase) (Fisher et al., 21) and MMP-12 (macrophage MMPs mrna expression (Relative levels 1-2 ) MMPs Figure 1. Basal gene expression of MMP family members in human skin in vivo. Full-thickness (4 mm) sun-protected buttock human skin was obtained from healthy adult human volunteers (eight subjects, average 36 years of age), as described earlier (Fisher et al., 1991; Quan et al., 24). Total RNA was extracted using a commercial kit (RNeasy Midi Kit; Qiagen, Chatsworth, CA) according to the manufacturer s protocol. Reverse transcription was performed using Taqman Reverse Transcription kit (Applied Biosystems, Foster City, CA). Real-time RT-PCR was performed using a Taqman Universal PCR Master Mix kit (Applied Biosystems) and 73 Real-Time PCR System (Applied Biosystems). All primers and probes were purchased from Applied Biosystems (Assays-on-Demand Gene Expression Products). Results are means±sem of MMP mrna normalized to 36B4 (internal control) mrna. All procedures involving human subjects were conducted in accord with the regulations set forth by the University of Michigan Institutional Review Board, and all subjects provided written informed consent. MMP, matrix metalloproteinase; RT-PCR, reverse transcriptase PCR. elastase) (Chung et al., 22) proteins are present in human skin 24 hours after irradiation, as a result of an influx of neutrophils and macrophages from the circulation. Skin neutrophils and macrophages are terminally differentiated cells that no longer transcribe new mrna. Residual MMP-8 and MMP-12 mrna was below the limit of detection. We have earlier reported that the majority of MMP-8 protein remained in the inactive precursor form (Fisher et al., 21) and accounted for little, if any, collagenolytic activity in skin following irradiation (Brennan et al., 23). Furthermore, the exposure of sun-protected human skin to purified human MMP-1 in organ culture causes collagen fragmentation and alterations in the structure and organization of collagen fibrils in the dermis that resemble those observed in photoaged skin (Varani et al., 21, 28; Fligiel et al., 23). Taken together, these studies suggest that MMP-1, MMP-3, and MMP-9 are primary -inducible collagenolytic enzymes, and MMP-1 is the major protease capable of initiating the degradation of native fibrillar collagens in human skin in vivo. EPIDERMISISTHEMAJORSOURCEOF IRRADIATION-INDUCED MMPs IN HUMAN SKIN IN VIVO On the basis of in situ hybridization and immunohistology, we have earlier reported that keratinocytes are the major cellular source of MMPs in -irradiated human skin in vivo (Fisher et al., 1997; Fisher and Voorhees, 1998). To quantify the relative contributions of epidermis and dermis to 21

3 MMP-1 mrna MMP-3 mrna 8, 7, 6, 5, 4, 3, 2, 1, 1,2 1, Ctrl 8 24 Hours post- Before LCM Seperate epidermis and dermis Ctrl 8 24 MMP-9 mrna Hours post- irradiation-induced MMP expression, epidermis and dermis were analyzed separately by laser capture microdissection, coupled with real-time reverse transcriptase PCR. Figure 3 shows human skin sections before and after laser capture microdissection of epidermis and dermis. Total RNA was extracted from microdissected epidermis and dermis and irradiation-induced MMP mrna levels were determined by real-time reverse transcriptase PCR. In agreement with our earlier data, all three irradiation-inducible MMPs were produced primarily in the epidermis, rather than in the dermis (Figure 4). It should be noted that dermal samples contained Ctrl 8 24 Hours post- Figure 2. irradiation induces MMP-1, MMP-3, and MMP-9 in human skin in vivo. Sun-protected buttock skin of healthy adult human volunteers was exposed to twice the minimum erythema dose of solar-simulated irradiation (SPEC 45 W xenon arc solar simulator). Full-thickness (4 mm) biopsies were obtained 8 and 24 hours post-irradiation. Total RNA was extracted and MMP-1, MMP-3, and MMP-9 mrna levels were determined by real-time RT-PCR, as described in Figure 1 legend. Results are means±sem of MMP mrna normalized to 36B4 (internal control) mrna. N ¼ 8, Po.5. MMP, matrix metalloproteinase; RT-PCR, reverse transcriptase PCR. Rn 1 1 Amplification - IFN gamma MP Cycle After LCM MMP real-time PCR Figure 3. Laser capture microdissection (LCM)-coupled real-time RT-PCR. Human skin punch biopsies were embedded in OCT, sectioned, and stained with hematoxylin and eosin. and dermis were captured using LCM (Leica ASLMD System; Leica Microsystems, Germany). Arrows indicate locations where epidermis and dermis were separated by laser. Total RNA was extracted from captured epidermis and dermis, and quantitative real-time RT-PCR was performed as described in Figure 1 legend. Scale bar ¼.1 mm. RT-PCR, reverse transcriptase PCR. some epidermal contamination (approximately 1%), based on measurement of keratin 14 mrna, which is expressed exclusively by keratinocytes. Therefore, dermal contributions to irradiation-induced MMP expression cannot be greater than those shown in Figure 4. In contrast to the results presented above, cell culture and skin equivalent model studies have concluded that dermal fibroblasts are the major source of MMPs that are expressed in response to irradiation (Fagot et al., 22, 24). The reasons for the discrepancies between responses of human 22 Journal of Investigative Dermatology Symposium Proceedings (29), Volume 14

4 MMP-1 mrna MMP-3 mrna MMP-9 mrna No Figure 5. Collagenase activity induced by solar-simulated irradiation in human skin in vivo. Sun-protected buttock skin of healthy adult human volunteers was exposed to twice the minimum erythema dose of solarsimulated radiation (SPEC 45 W xenon arc solar simulator). Fullthickness (4 mm) biopsies were obtained 24 hours post-irradiation as described in Figure 1 legend. Collagenase activity was detected by in situ zymography, using FITC-labeled type I collagen as the substrate (green fluorescence). Collagenase-catalyzed collagen cleavage causes loss of green fluorescence, resulting in darkened areas, which are most noticeable in the epidermis and upper dermis of -irradiated human skin. Images are representative of five experiments. Scale bar ¼.3 mm. direct binding of MMPs to the collagenous ECM (Windsor et al., 1991; Knauper et al., 1997; Overall, 21). Taken together with earlier findings, the data presented above indicate that epidermal keratinocytes are the major cellular source of MMPs that are produced in response to exposure of human skin to solar radiation. However, it is possible that dermal cells may also play a role in epidermal production of MMPs, through indirect paracrine mechanisms, by release of growth factors or cytokines, which in turn modulate MMP production by epidermal keratinocytes. CONFLICT OF INTEREST The authors state no conflict of interest. Figure 4. irradiation induces gene expression of MMP-1, MMP-3, and MMP-9 primarily in human epidermis in vivo. Sun-protected buttock skin of healthy adult human volunteers was exposed to twice the minimum erythema dose of solar-simulated radiation (SPEC 45 W xenon arc solar simulator). Full-thickness (4 mm) biopsies were obtained 24 hours after irradiation as described in Figure 1 legend. Total RNA was extracted from epidermis and dermis, which were obtained by laser capture microdissection. MMP-1, MMP-3, and MMP-9 mrna levels were determined by real-time RT-PCR, as described in Figure 1 legend. Results are means±sem of MMPs mrna normalized to 36B4 (internal control) mrna. N ¼ 6, Po.5. MMP, matrix metalloproteinase; RT-PCR, reverse transcriptase PCR. skin cells in vivo and responses of cultured skin cells in vitro are not known. However, there are many other examples of cultured skin cells failing to mimic the behavior of cells in vivo. Obviously, it is important to compare, whenever possible, results obtained from simple model systems with those obtained from direct in vivo observations. Although collagen-degrading MMP-1, -3, and -9 are primarily induced in the epidermis, the secreted enzymes diffuse into the dermis and degrade collagen, as shown by in situ zymography (Figure 5). This diffusion may be aided by REFERENCES Berneberg M, Plettenberg H, Krutmann J (2) Photoaging of human skin. Photodermatol Photoimmunol Photomed 16: Birkedal-Hansen H, Moore W, Bodden M, Windsor L, Birkedal-Hansen B, DeCarlo A et al. (1993) Matrix metalloproteinases: a review. Crit Rev Oral Biol Med 4: Brennan M, Bhatti H, Nerusu K, Bhagavathula N, Kang S, Fisher G et al. (23) Matrix mettalloproteinase-1 is the collagenolytic enzyme responsible for collagen damage in -irradiated human skin. Photochem Photobiol 78:43 8 Brenneisen P, Sies H, Scharffetter-Kochanek K (22) Ultraviolet-B irradiation and matrix metalloproteinases: from induction via signaling to initial events. Ann N Y Acad Sci 973:31 43 Chung JH, Seo JY, Lee MK, Eun HC, Lee JH, Kang S et al. (22) Ultraviolet modulation of human macrophage metalloelastase in human skin in vivo. J Invest Dermatol 119:57 12 Egeblad M, Werb Z (22) New functions for the matrix metalloproteinases in cancer progression. Nat Rev 2: Fagot D, Asselineau D, Bernerd F (22) Direct role of human dermal fibroblasts and indirect participation of epidermal keratinocytes in MMP- 1 production after -B irradiation. Arch Dermatol Res 293: Fagot D, Asselineau D, Bernerd F (24) Matrix metalloproteinase-1 production observed after solar-simulated radiation exposure is assumed by dermal fibroblasts but involves a paracrine activation through epidermal keratinocytes. Photochem Photobiol 79:

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