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Human Skin Aging Is Associated with Reduced Expression of the Stem Cell Markers β1 Integrin and MCSP

2009; Elsevier BV; Volume: 130; Issue: 2 Linguagem: Inglês

10.1038/jid.2009.297

1523-1747

Adam Giangreco, Stephen J. Goldie, Virgilio Failla, Gaëlle Saintigny, Fiona M. Watt,

melanin and skin pigmentation

melanoma chondroitin sulfate proteoglycan TO THE EDITOR Intrinsic and UV-induced human skin aging involves a number of changes, including reduced epidermal proliferation, impaired melanocyte function, and decreased collagen biosynthesis (Yaar et al., 2002Yaar M. Eller M.S. Gilchrest B.A. Fifty years of skin aging.J Investig Dermatol Symp Proc. 2002; 7: 51-58Google Scholar; McCullough and Kelly, 2006McCullough J.L. Kelly K.M. Prevention and treatment of skin aging.Ann N Y Acad Sci. 2006; 1067: 323-331Google Scholar). To ameliorate or inhibit these effects it is important to identify the principal factors that influence skin aging. Reduced stem cell abundance or self-renewal ability is a feature of aging in a number of different tissues (Flores et al., 2005Flores I. Cayuela M.L. Blasco M.A. Effects of telomerase and telomere length on epidermal stem cell behavior.Science (NY). 2005; 309: 1253-1256Google Scholar; Rando, 2006Rando T.A. Stem cells, ageing and the quest for immortality.Nature. 2006; 441: 1080-1086Google Scholar; Sharpless and DePinho, 2007Sharpless N.E. DePinho R.A. How stem cells age and why this makes us grow old.Nat Rev. 2007; 8: 703-713Google Scholar; Rossi et al., 2008Rossi D.J. Jamieson C.H. Weissman I.L. Stems cells and the pathways to aging and cancer.Cell. 2008; 132: 681-696Google Scholar). Nevertheless, when we examined young and intrinsically aged murine skin (Giangreco et al., 2008Giangreco A. Qin M. Pintar J.E. Watt F.M. Epidermal stem cells are retained in vivo throughout skin aging.Aging Cell. 2008; 7: 250-259Google Scholar), we found that despite alterations in epidermal proliferation, dermal thickness, and peripheral T cells, the abundance of CD34-positive epidermal stem cells did not decline with increasing age. Furthermore, young and aged murine epidermal stem cells have similar in vitro growth and differentiation potential (Stern and Bickenbach, 2007Stern M.M. Bickenbach J.R. Epidermal stem cells are resistant to cellular aging.Aging Cell. 2007; 6: 439-452Google Scholar). These findings suggest that murine epidermal stem cells may be unaffected by intrinsic skin aging (Giangreco et al., 2008Giangreco A. Qin M. Pintar J.E. Watt F.M. Epidermal stem cells are retained in vivo throughout skin aging.Aging Cell. 2008; 7: 250-259Google Scholar). We have now examined whether expression of epidermal stem cell markers changes during aging of human skin. We examined frozen sections of abdominal skin from 23 Caucasian donors (22 female and 1 male), three of whom were recently deceased. Tissue was obtained in compliance with the relevant European Union and United Kingdom regulations. Cadaveric skin samples were obtained with the approval of the Anatomy Department, University of Glasgow, UK. Other samples were obtained from Biopredic International, Rennes, France. Helsinki principles were adhered to and participants gave written, informed consent to provide samples for research. The ages of the donors were non-uniformly distributed and instead clustered into three distinct groups, as shown in Figure 1a. The youngest age group ranged from 21 to 33 years (average age 30.5±1.1 years; n=10). The intermediate group was aged 51–59 years (average 53.1±1.0 years; n=7). The oldest group was over 60 years (average 68.5±2.8 years; n=6). Immunostaining revealed a low level of epidermal p53 expression, indicating that the skin had not been subject to significant sun exposure (Tyrrell, 1996Tyrrell R.M. Activation of mammalian gene expression by the UV component of sunlight – from models to reality.BioEssays. 1996; 18: 139-148Google Scholar), except for two skin samples in the youngest age group (data not shown). We measured rete ridge height, epidermal thickness, and basal cell density in haematoxylin and eosin stained sections (Figure 1b–e). Consistent with earlier reports of skin aging (Makrantonaki and Zouboulis, 2007Makrantonaki E. Zouboulis C.C. Molecular mechanisms of skin aging.Ann N Y Acad Sci. 2007; 1119: 40-50Google Scholar), subjects within over 60-year-old group exhibited increased inter-individual variability in all measurements relative to both 21- to 33- and 51- to 59-year-old subjects (see Figures 1f–h and 2m). There were no statistically significant differences in epidermal thickness between the different age groups (Figure 1f). However, both the 51- to 59-year-old group and the over 60-year-old group exhibited significantly reduced rete ridge height relative to the youngest group, as assessed by both unpaired t-test (P<0.005) and one-way ANOVA post-test trend analyses (P<0.0001; Figure 1g). There was also a statistically significant reduction in basal cell density when the oldest group was compared with the other two groups (P<0.005; Figure 1h). Two markers of human interfollicular epidermal stem cells are melanoma chondroitin sulfate proteoglycan (MCSP; NG2; Legg et al., 2003Legg J. Jensen U.B. Broad S. Leigh I. Watt F.M. Role of melanoma chondroitin sulphate proteoglycan in patterning stem cells in human interfollicular epidermis.Development. 2003; 130: 6049-6063Google Scholar) and high levels of β1 integrins (Jones and Watt, 1993Jones P.H. Watt F.M. Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression.Cell. 1993; 73: 713-724Google Scholar; Jones et al., 1995Jones P.H. Harper S. Watt F.M. Stem cell patterning and fate in human epidermis.Cell. 1995; 80: 83-93Google Scholar; Jensen et al., 1999Jensen U.B. Lowell S. Watt F.M. The spatial relationship between stem cells and their progeny in the basal layer of human epidermis: a new view based on whole-mount labelling and lineage analysis.Development. 1999; 126: 2409-2418Google Scholar). Frozen skin sections were labeled with anti-β1 integrin antibodies, and the fluorescence pixel intensity of lateral cell borders in the basal epidermal layer was quantified (Jones et al., 1995Jones P.H. Harper S. Watt F.M. Stem cell patterning and fate in human epidermis.Cell. 1995; 80: 83-93Google Scholar; Molès and Watt, 1997Molès J.P. Watt F.M. The epidermal stem cell compartment: variation in expression levels of e-cadherin and catenins within the basal layer of human epidermis.J Histochem Cytochem. 1997; 45: 867-874Google Scholar) (Figure 2a–e). In the skin samples of 21- to 33-year-old subjects, the cells with highest β1 integrin levels tended to lie at the tops of the rete ridges (Figure 2a), as reported previously (Jones et al., 1995Jones P.H. Harper S. Watt F.M. Stem cell patterning and fate in human epidermis.Cell. 1995; 80: 83-93Google Scholar). Although the modal pixel intensity values for all age groups were similar, the mean and median values decreased with age (Figure 2e). In the 21- to 33-year-old group, the mean was 143 arbitrary units and the median was 132. The 51- to 59-year-old group had a mean of 139 and median of 120. In the over 60-year-old group the mean was 103 and the median was 102. In addition, the lowest pixel intensity values were only found in the over 60-year-old group (Figure 2e). A similar reduction in integrin expression has been observed in chronically UV-exposed skin (Bosset et al., 2003Bosset S. Bonnet-Duquennoy M. Barre P. Chalon A. Lazou K. Kurfurst R. et al.Decreased expression of keratinocyte beta1 integrins in chronically sun-exposed skin in vivo.Br J Dermatol. 2003; 148: 770-778Google Scholar). MCSP expression is confined to discrete clusters of cells, corresponding to the cells that express the highest β1 integrin levels (Legg et al., 2003Legg J. Jensen U.B. Broad S. Leigh I. Watt F.M. Role of melanoma chondroitin sulphate proteoglycan in patterning stem cells in human interfollicular epidermis.Development. 2003; 130: 6049-6063Google Scholar) (Figure 2f). MCSP-positive cluster size was similar in the 21- to 33- and 51- to 59-year-old groups; however, one-way ANOVA analysis indicated a significant decline in the oldest sample group (P<0.0001; Figure 2f–i). We also stained frozen skin sections with antibodies to Ki67 as a marker of proliferation (Figure 2j–l). There was a statistically significant increase in Ki67-positive cells in the 51- to 59-year-old group relative to the 21- to 33-year-old group (P<0.05; Figure 2m), and a reduced tendency for Ki67-positive cells to cluster in the rete ridges (Figure 2j and k). There was no statistically significant difference in Ki67 abundance between the oldest samples and the other groups; however, there was a large spread in values within the over 60-year-old samples (Figure 2m). We have previously reported that cells with the highest in vitro self-renewal capacity express MCSP and high β1 integrin levels (Jones and Watt, 1993Jones P.H. Watt F.M. Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression.Cell. 1993; 73: 713-724Google Scholar; Jones et al., 1995Jones P.H. Harper S. Watt F.M. Stem cell patterning and fate in human epidermis.Cell. 1995; 80: 83-93Google Scholar; Legg et al., 2003Legg J. Jensen U.B. Broad S. Leigh I. Watt F.M. Role of melanoma chondroitin sulphate proteoglycan in patterning stem cells in human interfollicular epidermis.Development. 2003; 130: 6049-6063Google Scholar). Our current findings are therefore consistent with the earlier report that significantly aged human epidermal cells exhibit reduced in vitro self-renewal ability (Barrandon and Green, 1987Barrandon Y. Green H. Three clonal types of keratinocyte with different capacities for multiplication.Proc Natl Acad Sci USA. 1987; 84: 2302-2306Google Scholar). The reduction in β1 integrin levels and MCSP expression with advancing age may also contribute to age-associated changes in dermal thickness and skin vascularization. Integrins collaborate with growth factor receptors to influence growth factor production and responsiveness (Legate et al., 2009Legate K.R. Wickstrom S.A. Fassler R. Genetic and cell biological analysis of integrin outside-in signaling.Genes Dev. 2009; 23: 397-418Google Scholar; Streuli and Akhtar, 2009Streuli C.H. Akhtar N. Signal co-operation between integrins and other receptor systems.Biochem J. 2009; 418: 491-506Google Scholar), and MCSP can enhance integrin-dependent signaling (Yang et al., 2004Yang J. Price M.A. Neudauer C.L. Wilson C. Ferrone S. Xia H. et al.Melanoma chondroitin sulfate proteoglycan enhances FAK and ERK activation by distinct mechanisms.J Cell Biol. 2004; 165: 881-891Google Scholar). It will now be of interest to determine whether restoration of β1 integrin levels might reverse some aspects of normal human skin aging. Overall, our studies provide evidence of previously unappreciated molecular changes that occur before the skin alterations most typically seen after 70 years of age (Makrantonaki and Zouboulis, 2007Makrantonaki E. Zouboulis C.C. Molecular mechanisms of skin aging.Ann N Y Acad Sci. 2007; 1119: 40-50Google Scholar). We are most grateful to Will Howat for excellent technical support. This work was supported by fellowships from the NIH (AG) and MRC (SJG), and funds from Chanel Parfums Beauté (FMW) and Cancer Research UK (AG, SJG, VF, FMW). We gratefully acknowledge the support of the University of Cambridge and Hutchison Whampoa.