¹The Johnson & Johnson Skin Research Center, CPPW, a division of Johnson & Johnson Consumer Companies Inc., Skillman, New Jersey, USA. E-mail: blin1@cpcus.jnj.com

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, 2002). 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, 1996). Earlier studies documented the role of the keratinocyte receptor PAR-2 in the regulation of skin pigmentation (Seiberg et al., 2000a, 2000b; Paine et al., 2001), and the differential expression of the PAR-2 pathway in keratinocytes of different ethnic origins (Babiarz-Magee et al., 2004). 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., 1998; Santamaria et al., 1998; Bromme et al., 1999). 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).

Figure 1.

CTSL2 mRNA expression is higher in keratinocytes from lighter-colored skins. (a) RNA was isolated from primary neonatal keratinocytes (K), melanocytes (M), and from HaCaT cells (H). CTSL2 expression was analyzed by RT-PCR. (b) RNA was isolated from primary fibroblasts and analyzed by RT-PCR for CTSL2, CTSL, and CTSB expression. (c) Neonatal keratinocytes from lightly and darkly pigmented skins (LK and DK, respectively) were analyzed by RT-PCR for expression levels of CTSL, CTSB, CTSL2, and cystatins A, B, and C (the endogenous physiological inhibitors of cathepsins). Only CTSL2 was differentially expressed in keratinocytes of different skin colors. The experiment was repeated three times with different lots of keratinocytes. Representative data are shown. (d) Neonatal melanocytes from lightly and darkly pigmented skins (LM and DM, respectively) were analyzed by RT-PCR for expression levels of CTSL, CTSB, and CTSL2. No differential expression was identified in melanocytes of different skin colors. The experiment was repeated three times with different lots of melanocytes. Representative data are shown. Primers used in this figure are listed in Table S2.

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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 -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).

Figure 2.

CTSL2 protein levels inversely correlate with skin color. (a) Keratinocytes obtained from lightly pigmented skin (LK) have higher levels of CTSL2 protein than those of darkly pigmented skins (DK). The CTSL2 polyclonal antibodies recognize three bands by Western blot analysis, corresponding to the three processed forms of CTSL2 protein in cells. Protein lysates (15 g) of dark or light keratinocytes were used to detect CTSL2, and 2 g of the same lysates were used to detect -actin. The experiment was carried out twice with four different lots of keratinocytes. Representative data are shown. (b–e) Human skin biopsies were obtained from the Cooperative Human Tissue Network Eastern Division (Philadelphia, PA). Skin pigment deposition levels were documented by Fontana–Mason staining. (b and c) Examples of lightly and darkly pigmented skins are presented. (d and e) Immunohistochemistry was performed with anti-CTSL2 antibodies using skin sections of same individuals (b and d, c and e, are each from the same individual). Sections were counterstained with hematoxylin (Vector Labs, Burlingame, CA). CTSL2 protein levels are higher in (d) lighter skins, and are strongly reduced in (e) darker skins. Immunohistochemistry was repeated twice, with total of 16 skin sections, eight each from lightly and darkly pigmented skins. Representative pictures are shown. Bar=0.05 mm.

Full figure and legend (149K)

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 1.50.16-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., 1998; Santamaria et al., 1998; Bromme et al., 1999). In skin, CTSL2 was isolated from human stratum corneum (Watkinson, 1999; Bernard et al., 2003) 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., 1999). 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., 2000; Benavides et al., 2002; Tobin et al., 2002). This defective skin and hair phenotype was largely rescued by human keratin 14 promoter driven CTSL2 (Hagemann et al., 2004). 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., 2003). The development of ashiness might result from defects in corneodesmolysis and desquamation (Sato et al., 1998), 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., 2003), 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.