Connie B. Lin

The role of keratinocyte growth factor in melanogenesis: a possible mechanism for the initiation of solar lentigines

Please cite this paper as: The role of keratinocyte growth factor in melanogenesis: a possible mechanism for the initiation of solar lentigines. Experimental Dermatology 2010; 19: 865–872.

Immuno-histochemical evaluation of solar lentigines: The association of KGF/KGFR and other factors with lesion development

BACKGROUND Solar lentigines (SLs) are macular hyperpigmented lesions associated with sun exposure and age. Histopathologically, SLs are defined by a hyperpigmented basal layer and elongated rete ridges. The molecular mechanisms involved in the formation and the development of SLs are not completely understood. Our earlier data show that keratinocyte growth factor (KGF) induces hyperpigmentary lesions with histological resemblance to SLs. OBJECTIVE To investigate the association of KGF/KGF receptor (KGFR) and other pigmentary genes with the progression of SL development. To better understand the possible role of KGF in the pathology of SLs. METHODS Archived human skin biopsies (24 SLs and 14 healthy skins) were studied using immunohistochemistry for KGF/KGFR, proliferation marker Ki67, stem cell marker keratin-15 (K15), tyrosinase (TYR), stem cell factor (SCF), and protease-activated receptor-2 (PAR-2). RESULTS An increase in TYR-positive cells and expression was found throughout SL progression, as compared to normal skin. The levels of KGF, KGFR, SCF, Ki67 and PAR-2 varied during SL progression. Ki67, K15 and KGF/KGFR were significantly upregulated at early-mid SL stages. The latest-stage SLs expressed the lowest levels of KGF, KGFR, SCF, Ki67 and PAR-2. CONCLUSIONS The upregulation of KGF/KGFR might induce the formation of rete ridges and hyperpigmentation. The reduced levels of all examined proteins (except TYR and K15) suggest a possible inactive status (dormancy or quiescence) of advanced lesions.

LIGR, a protease‐activated receptor‐2‐derived peptide, enhances skin pigmentation without inducing inflammatory processes

The protease‐activated receptor‐2 (PAR‐2) is a seven transmembrane G‐protein‐coupled receptor that could be activated by serine protease cleavage or by synthetic peptide agonists. We showed earlier that activation of PAR‐2 with Ser‐Leu‐Ile‐Gly‐Arg‐Leu‐NH2 (SLIGRL), a known PAR‐2 activating peptide, induces keratinocyte phagocytosis and increases skin pigmentation, indicating that PAR‐2 regulates pigmentation by controlling phagocytosis of melanosomes. Here, we show that Leu‐Ile‐Gly‐Arg‐NH2 (LIGR) can also induce skin pigmentation. Both SLIGRL and LIGR increased melanin deposition in vitro and in vivo, and visibly darkened human skins grafted onto severe combined immuno‐deficient (SCID) mice. Both SLIGRL and LIGR stimulated Rho‐GTP activation resulting in keratinocyte phagocytosis. Interestingly, LIGR activates only a subset of the PAR‐2 signaling pathways, and unlike SLIGRL, it does not induce inflammatory processes. LIGR did not affect many PAR‐2 signaling pathways, including [Ca2+] mobilization, cAMP induction, the induction of cyclooxgenase‐2 (COX‐2) expression and the secretion of prostaglandin E2, interleukin‐6 and ‐8. PAR‐2 siRNA inhibited LIGR‐induced phagocytosis, indicating that LIGR signals via PAR‐2. Our data suggest that LIGR is a more specific regulator of PAR‐2‐induced pigmentation relative to SLIGRL. Therefore, enhancing skin pigmentation by topical applications of LIGR may result in a desired tanned‐like skin color, without enhancing inflammatory processes, and without the need of UV exposure.

Aldo‐keto reductase 1C subfamily genes in skin are UV‐inducible: possible role in keratinocytes survival

Abstract:  Human skin is endowed with the capacity to synthesize and metabolize steroid hormones, a function of importance in skin physiology and pathology. It is the hormone‐regulatory enzymes, including the aldo‐keto reductase 1C subfamily (AKR1Cs) that are largely responsible for the local levels of active steroid hormones. AKR1C1 and AKR1C2 inactivate progesterone and 5α‐dihydrotestosterone, respectively, whereas AKR1C3 activates oestradiol and testosterone. Here, we show that AKR1C1‐3 are expressed in keratinocytes and fibroblasts, with marginal expression in melanocytes. In human primary keratinocytes, AKR1C1 and ‐2 were UVB‐inducible in a dose‐dependent manner, as shown by quantitative PCR and Western blot analyses. The induction of AKR1C1 by UVB was concomitant with the presence of an apoptotic marker, the cleavage product of poly‐ADP ribose polymerase. Similarly, the activation of AKR1C1 and ‐2 upon UVB exposure was demonstrated in swine skin in vivo and in human skin explants. As expected, hydrogen peroxide‐derived reactive oxygen species also induced AKR1C1 and ‐2 mRNA and protein levels in keratinocytes in a dose‐dependent manner. Furthermore, down‐regulation of AKR1Cs by small interfering ribonucleic acid led to significantly reduced cell viability. Based on the combined evidence of the presence of an apoptotic marker in the UVB‐exposed keratinocytes with increased AKR1Cs expression and reduced cell viability in down‐regulated AKR1Cs, we suggest that AKR1C subfamily genes are stress‐inducible and might function as survival factors in keratinocytes.

Nondenatured soy extracts reduce UVB-induced skin damage via multiple mechanisms.

UV irradiation results in DNA damage, inflammation and immunosuppression, leading to the development of basal and squamous cell carcinomas. Earlier data show that topical treatment with nondenatured soy extracts reduced the incidence and delayed the development/progression of already‐initiated skin tumors in high‐risk hairless mice. Here we show that pretreatment with nondenatured soy extracts reduced UVB‐induced Thymine‐Thymine (TT) dimer formation. In vitro, nondenatured soy extracts enhanced UVB‐induced checkpoint kinase‐1 (Chk1) activation, suggesting a delay in cell cycle progression that enables longer time for DNA repair. Soy also reduced UVB‐induced cyclo‐oxygenase‐2 (COX‐2) expression and prostaglandin E2 secretion, and inhibited p38 MAP kinase activation, suggesting its anti‐inflammatory activity. Mice pretreated topically with nondenatured soy extracts had reduced levels of UVB‐induced TT dimers and COX‐2 expression in their skins compared to UVB alone. The nondenatured soy extracts also inhibited vascular endothelial growth factor‐induced endothelial tube formation in Matrigel, suggesting a possible inhibitory effect on angiogenesis and tumor progression. Taken together, nondenatured soy extracts could prevent or reduce UVB‐induced skin damage via multiple mechanisms, affecting both the initiation and the progression of skin cancer. These data suggest that topical application of nondenatured soy extracts could potentially reduce the incidence of skin cancer.

Inhibitory effect of topical applications of nondenatured soymilk on the formation and growth of UVB-induced skin tumors.

Treatment of female SKH-1 hairless mice with ultraviolet B light twice a week for 20 weeks resulted in a population of tumor-free mice with a high risk of developing skin tumors during the next several months in the absence of additional UVB treatment (high-risk mice). Topical applications of nondenatured soymilk but not heat-denatured soymilk once a day, 5 days a week to these high-risk mice inhibited the formation and growth of skin tumors. Similar topical applications of soybean trypsin inhibitor or Bowman-Birk inhibitor also inhibited the formation and growth of skin tumors, but these agents were less active than nondenatured soymilk. Treatment of miniswine skin with nondenatured soymilk once a day for 5 days prior to UVB irradiation reduced or completely eliminated UVB-induced formation of thymine dimers and apoptotic cells in the epidermis. These data suggest that nondenatured soymilk could be applied to humans to prevent sunlight-induced skin damage and to reduce the risk of skin tumor formation and progression.

Differential levels of elastin fibers and TGF-β signaling in the skin of Caucasians and African Americans.

BACKGROUND While skin color is the most notable difference among ethnic skins the current knowledge on skin physiological and aging properties are based mainly on Caucasian skin studies. OBJECTIVE To evaluate histological differences in elastin fiber network and differential responsiveness to TGF-β in skin of Caucasians and African Americans. METHODS These studies were undertaken using human skin biopsies, primary dermal fibroblasts, Western blot analyses, immunofluorescence microscopy, cDNA array and quantitative real-time PCR. RESULTS In Caucasian subjects, tropoelastin expression and elastin fibers in photoprotected skin was substantially less than in age-matched African American skin. Expression of tropoelastin in photoexposed skin of African American was similar to their photoprotected skin, suggesting that photoexposure did not affect elastin fibers in African American skin to the same extent as Caucasian skin. An elevated level of TGF-β1 present in media from dermal fibroblasts derived from African American skins correlated well with the higher levels of TGF-β mRNA in African American skins analyzed by cDNA array. Treatment with TGF-β1 resulted in a considerably higher induction of elastin mRNA in dermal fibroblasts from African Americans than from Caucasian fibroblasts, indicative of enhanced TGF-β signaling in African American skins. Furthermore, UVA exposure decreased levels of elastin mRNA in Caucasian fibroblasts compared to African Americans fibroblasts. CONCLUSION These results suggest that there are ethnic differences in the elastin fiber network and in TGF-β signaling in African American and Caucasian skin, and that African American have less UV dependent loss of elastin than Caucasian which may contribute to the different perceived aging phenotypes.

Galvanic zinc–copper microparticles inhibit melanogenesis via multiple pigmentary pathways

The endogenous electrical field of human skin plays an important role in many skin functions. However, the biological effects and mechanism of action of externally applied electrical stimulation on skin remain unclear. Recent study showed that galvanic zinc–copper microparticles produce electrical stimulation and reduce inflammatory and immune responses in intact skin, suggesting the important role of electrical stimulation in non-wounded skin. The objective of this study is to investigate the biological effect of galvanic zinc–copper microparticles on skin pigmentation. Our findings showed that galvanic zinc–copper microparticles inhibited melanogenesis in a human melanoma cell line (MNT-1), human keratinocytes and melanoma cells co-cultures, and in pigmented epidermal equivalents. Treatment of galvanic zinc–copper microparticles inhibited melanogenesis by reducing the promoter transactivation of tyrosinase and tyrosinase-related protein-1 in human melanoma cells. In a co-culture Transwell system of keratinocytes and melanoma cells, galvanic zinc–copper microparticles reduced melanin production via downregulation of endothelin-1 secretion from keratinocytes and reduced tyrosinase gene expression in melanoma cells. In addition, exposure of pigmented epidermal equivalents to galvanic zinc–copper microparticles resulted in reduced melanin deposition. In conclusion, our data demonstrated for the first time that galvanic zinc–copper microparticles reduced melanogenesis in melanoma cells and melanin deposition in pigmented epidermal equivalents by affecting multiple pigmentary pathways.