Title: Cathepsin L2 Levels Inversely Correlate with Skin Color
Abstract: cathepsin L2 reverse transcriptase TO THE EDITOR While skin color is the most notable difference among ethnic skins, many other differences exist in the structure and function of ethnic skins. Unfortunately, the current knowledge of physiological and pathological properties of the skin is based mainly on Caucasian skin studies (reviewed in Taylor, 2002Taylor S.C. Skin of color: biology, structure, function, and implications for dermatologic disease.J Am Acad Dermatol. 2002; 46: S41-S62Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). Elucidating differences among ethnic skins is not only of great biological importance, but also of great interest in skin care improvement. Keratinocytes play an important role in skin physiology, affecting skin barrier function, immune responses and wound-healing processes (Williams and Kupper, 1996Williams I.R. Kupper T.S. Immunity at the surface: homeostatic mechanisms of the skin immune system.Life Sci. 1996; 58: 1485-1507Crossref PubMed Scopus (157) Google Scholar). Earlier studies documented the role of the keratinocyte receptor PAR-2 in the regulation of skin pigmentation (Seiberg et al., 2000Seiberg M. Paine C. Sharlow E. Andrade-Gordon P. Costanzo M. Eisinger M. et al.Inhibition of melanosome transfer results in skin lightening.J Invest Dermatol. 2000; 115: 162-167Crossref PubMed Scopus (184) Google Scholar, Seiberg et al., 2000Seiberg M. Paine C. Sharlow E. Andrade-Gordon P. Costanzo M. Eisinger M. et al.The protease-activated receptor 2 regulates pigmentation via keratinocyte-melanocyte interactions.Exp Cell Res. 2000; 254: 25-32Crossref PubMed Scopus (196) Google Scholar; Paine et al., 2001Paine C. Sharlow E. Liebel F. Eisinger M. Shapiro S. Seiberg M. An alternative approach to depigmentation by soybean extracts via inhibition of the PAR-2 pathway.J Invest Dermatol. 2001; 116: 587-595Crossref PubMed Scopus (134) Google Scholar), and the differential expression of the PAR-2 pathway in keratinocytes of different ethnic origins (Babiarz-Magee et al., 2004Babiarz-Magee L. Chen N. Seiberg M. Lin C.B. The expression and activation of protease-activated receptor-2 correlate with skin color.Pigment Cell Res. 2004; 17: 241-251Crossref PubMed Scopus (67) Google Scholar). In order to identify other differences in keratinocytes' expression profiles, we performed DNA chip analysis of keratinocytes obtained from different skin types (four individuals of either lightly (LK) or darkly (DK) pigmented skins) (Cascade Biologics, Portland, OR). When evaluating expression levels of cathepsins (chip results, Table S1), only cathepsin L2 (CTSL2) was found to be differentially expressed in relation to pigmentary skin backgrounds, and was about 7.5-folds higher in keratinocytes from lightly pigmented, relative to darkly pigmented skins (Table S1). CTSL2 is a lysosomal cysteine protease (Adachi et al., 1998Adachi W. Kawamoto S. Ohno I. Nishida K. Kinoshita S. Matsubara K. et al.Isolation and characterization of human cathepsin V: a major proteinase in corneal epithelium.Invest Ophthalmol Vis Sci. 1998; 39: 1789-1796PubMed Google Scholar; Santamaria et al., 1998Santamaria I. Velasco G. Cazorla M. Fueyo A. Campo E. Lopez-Otin C. Cathepsin L2, a novel human cysteine proteinase produced by breast and colorectal carcinomas.Cancer Res. 1998; 58: 1624-1630PubMed Google Scholar; Bromme et al., 1999Bromme D. Li Z. Barnes M. Mehler E. Human cathepsin V functional expression, tissue distribution, electrostatic surface potential, enzymatic characterization, and chromosomal localization.Biochemistry. 1999; 38: 2377-2385Crossref PubMed Scopus (186) Google Scholar). It is expressed in primary keratinocytes, melanocytes, and HaCaT keratinocytes (Figure 1a), but not in primary fibroblasts (Figure 1b). The differential expression of CTSL2 mRNA in keratinocytes (but not of other cathepsins and of cystatins, the natural inhibitors of cathepsins) was confirmed by reverse transcriptase (RT)-PCR (Figure 1c; Table S2 in Supplementary material for primers). Interestingly, melanocytes from different ethnic origins expressed the same levels of CTSL2 mRNA (Figure 1d). Download .pdf (.03 MB) Help with pdf files Table S1 and Table S2Arbitrary expression value of cathepsins selected from microarray analysis of keratinocytes derived from light and dark skins.PCR primers used in figure 1. Download .pdf (.09 MB) Help with pdf files Supplementary Materials and Methods Western blot analysis of keratinocytes using polyclonal antibodies specific to CTSL2 (Figure S1) showed that keratinocytes derived from lightly pigmented skins (LK) have about 3.5-fold increase in CTSL2 protein levels (normalized by β-actin) relative to those of darkly pigmented skins (DK) (Figure 2a). In addition, CTSL2 protein levels in human skins with different levels of pigment deposition (as shown by Fontana–Mason staining, Figure 2b and c) were analyzed by immunohistochemistry staining. In agreement with the CTSL2 mRNA levels in ethnic skins, CTSL2 protein levels were found to be higher in lighter skins, and strongly reduced in darker skins (Figure 2d and e). Download .jpg (.03 MB) Help with files Supplementary Figure S1The CTSL2 polyclonal antibodies are specific to CTSL2. To date, there are no specific inhibitors to CTSL2; therefore, we could not directly measure CTSL2 activity in keratinocyte's derived from skins of different colors. The total enzymatic activity of cathepsins was measured using substrate Z-Phe-Arg-AMC (Bachem, Torrance, CA). Substrate hydrolysis catalyzed by cathepsins was monitored by fluorescent emission (excitation at 360 nm and emission at 465 nm). Cell lysate of keratinocytes from lightly pigmented skins showed 1.5±0.16-fold higher cathepsin activity than keratinocytes derived from darker skins (data were pooled from three independent experiments). CA074, a specific cathepsin B inhibitor, reduced keratinocytes cathepsin B activity by ∼70%. Cathepsin activities of CA074-treated keratinocytes were about 2.2-fold higher in light-skin-derived keratinocytes versus dark ones (for Materials and Methods, see Supplementary materials). Since overall cathepsin expression (excluding CSTL2) is similar in both dark and light keratinocytes (Table S1 and Figure 1), the difference of the remaining cathepsin activity represents CTSL2. This suggests that the total CTSL2 enzymatic activity is higher in light-skin-derived keratinocytes. Cathepsins are a large family of lysosomal cysteine proteases, which are involved in numerous cellular processes. CTSL2 shares 80% protein sequence identity with CTSL, but its expression is mostly confined to the thymus, testis, and cornea (Adachi et al., 1998Adachi W. Kawamoto S. Ohno I. Nishida K. Kinoshita S. Matsubara K. et al.Isolation and characterization of human cathepsin V: a major proteinase in corneal epithelium.Invest Ophthalmol Vis Sci. 1998; 39: 1789-1796PubMed Google Scholar; Santamaria et al., 1998Santamaria I. Velasco G. Cazorla M. Fueyo A. Campo E. Lopez-Otin C. Cathepsin L2, a novel human cysteine proteinase produced by breast and colorectal carcinomas.Cancer Res. 1998; 58: 1624-1630PubMed Google Scholar; Bromme et al., 1999Bromme D. Li Z. Barnes M. Mehler E. Human cathepsin V functional expression, tissue distribution, electrostatic surface potential, enzymatic characterization, and chromosomal localization.Biochemistry. 1999; 38: 2377-2385Crossref PubMed Scopus (186) Google Scholar). In skin, CTSL2 was isolated from human stratum corneum (Watkinson, 1999Watkinson A. Stratum corneum thiol protease (SCTP): a novel cysteine protease of late epidermal differentiation.Arch Dermatol Res. 1999; 291: 260-268Crossref PubMed Scopus (46) Google Scholar; Bernard et al., 2003Bernard D. Mehul B. Thomas-Collignon A. Simonetti L. Remy V. Bernard M.A. et al.Analysis of proteins with caseinolytic activity in a human stratum corneum extract revealed a yet unidentified cysteine protease and identified the so-called "stratum corneum thiol protease" as cathepsin l2.J Invest Dermatol. 2003; 120: 592-600Crossref PubMed Scopus (70) Google Scholar) and was shown to have a high caseinolytic activity, which is distinct from that of CTSL. To date, no CTSL2 ortholog has been identified in mice, and the human CTSL2 protein is more homologous to the mouse CTSL than to the human CTSL sequence (Bromme et al., 1999Bromme D. Li Z. Barnes M. Mehler E. Human cathepsin V functional expression, tissue distribution, electrostatic surface potential, enzymatic characterization, and chromosomal localization.Biochemistry. 1999; 38: 2377-2385Crossref PubMed Scopus (186) Google Scholar). ctsl−/− mice exhibit a complex skin phenotype consisting of periodic hair loss and epidermal hyperplasia with hyperproliferation of basal epidermal keratinocytes, acanthosis, and hyperkeratosis, but no appreciable changes in hair pigmentation (Roth et al., 2000Roth W. Deussing J. Botchkarev V.A. Pauly-Evers M. Saftig P. Hafner A. et al.Cathepsin L deficiency as molecular defect of furless: hyperproliferation of keratinocytes and pertubation of hair follicle cycling.FASEB J. 2000; 14: 2075-2086Crossref PubMed Scopus (271) Google Scholar; Benavides et al., 2002Benavides F. Starost M.F. Flores M. Gimenez-Conti I.B. Guenet J.L. Conti C.J. Impaired hair follicle morphogenesis and cycling with abnormal epidermal differentiation in nackt mice, a cathepsin L-deficient mutation.Am J Pathol. 2002; 161: 693-703Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar; Tobin et al., 2002Tobin D.J. Foitzik K. Reinheckel T. Mecklenburg L. Botchkarev V.A. Peters C. et al.The lysosomal protease cathepsin L is an important regulator of keratinocyte and melanocyte differentiation during hair follicle morphogenesis and cycling.Am J Pathol. 2002; 160: 1807-1821Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). This defective skin and hair phenotype was largely rescued by human keratin 14 promoter driven CTSL2 (Hagemann et al., 2004Hagemann S. Gunther T. Dennemarker J. Lohmuller T. Bromme D. Schule R. et al.The human cysteine protease cathepsin V can compensate for murine cathepsin L in mouse epidermis and hair follicles.Eur J Cell Biol. 2004; 83: 775-780Crossref PubMed Scopus (42) Google Scholar). These data suggest that the CTSL2 proteolytic activities play a role in the maintenance of homeostasis of both the epidermis and the hair follicles. While the differential expression of CTSL2 was discovered in relation to skin color, we could not find evidence for the differential expression of CTSL2 in melanocytes, or for the regulation of melanogenesis via CTSL2. Our data demonstrate ethnic skin differences that are not directly associated with melanogenesis, expanding on ethnic skin differences beyond the pigmentary process. Ashy skin is a condition that affects many dark-skinned individuals. Ashiness is described as a common physiological skin condition, induced by environmental influence, and in particular by cold and dry weather. In dry conditions, light reflectance from dead stratum corneum cells of dark skins results in a dull, ashy look. Ashy skin has seldom been studied, but is known to be unrelated to inflammation or to skin pathology (Uhoda et al., 2003Uhoda E. Pierard-Franchimont C. Petit L. Pierard G. Skin weathering and ashiness in black Africans.Eur J Dermatol. 2003; 13: 574-578PubMed Google Scholar). The development of ashiness might result from defects in corneodesmolysis and desquamation (Sato et al., 1998Sato J. Denda M. Nakanishi J. Koyama J. Dry condition affects desquamation of stratum corneum in vivo.J Dermatol Sci. 1998; 18: 163-169Abstract Full Text PDF PubMed Scopus (53) Google Scholar), in which enzymatic activities of proteases, particularly serine- and cathepsin-like enzymes, might be altered. Here, we show that CTSL2 is differentially expressed in ethnic skins, with lower levels of mRNA, protein, and enzymatic activity in darkly pigmented skins. While the physiological function of CTSL2 in human skin remains to be explored, we hypothesize that reduced proteolytic activity within the stratum corneum of darker skins is involved, at least in part, in the creation of the "ashy skin phenotype". CTSL2 has been identified as a human stratum corneum desquamation processing-related enzyme (Bernard et al., 2003Bernard D. Mehul B. Thomas-Collignon A. Simonetti L. Remy V. Bernard M.A. et al.Analysis of proteins with caseinolytic activity in a human stratum corneum extract revealed a yet unidentified cysteine protease and identified the so-called "stratum corneum thiol protease" as cathepsin l2.J Invest Dermatol. 2003; 120: 592-600Crossref PubMed Scopus (70) Google Scholar), further supporting our hypothesis. This work highlights the importance of understanding ethnic skin at a deeper level than skin color. Johnson & Johnson CPPW, a division of Johnson & Johnson Consumer Companies Inc. approved all described studies. This research was supported and funded by Johnson & Johnson CPPW, a division of Johnson & Johnson Consumer Companies Inc. The authors are employed by the company. SUPPLEMENTARY MATERIAL We thank Drs Andrew Carmen and Xuejun Liu of Johnson & Johnson Pharmaceutical Research and Development (PRD), and Laura Babiarz-Magee of Johnson & Johnson CPPW, for DNA chip analysis. Dr N. Fusenig (Heidelberg, Germany) provided HaCaT Cells. Special thanks to Dr Robin Thurmond from The Johnson & Johnson PRD for providing purified recombinant CTSL and CTSL2 proteins. Tissue samples were provided by the Cooperative Human Tissue Network, which is funded by the National Cancer Institute.