Vitamin A and its derivatives in experimental photocarcinogenesis: preventive effects and relevance to humans.

The 1925 classical observation that vitamin A deficiency leads to squamous metaplasia and epithelial keratinization, coupled with the later finding that excess vitamin A inhibits keratinization of chick embryo skin, set the foundation for the potential therapeutic use of retinoids in cutaneous conditions of keratinization. Significant progress has since been made understanding the molecular biology, biochemistry, pharmacology, and toxicology of vitamin A and its derivatives, collectively named retinoids. Natural and synthetic retinoids are now routinely used to treat acne, psoriasis, skin keratinization disorders, and photodamage. Retinoids also inhibit tumor formation and skin cancer development in experimental systems and in humans. Retinol and retinyl palmitate (RP) are found in cosmetic products and in foods and dietary supplements, which are all considered safe, by inclusion in the Generally Recognized as Safe Substances Database. However, the safety of topical retinoids was questioned in one publication and in a recent National Toxicology Program report of RP-containing topical preparations, suggesting the possible earlier onset of ultraviolet-induced squamous cell carcinomas in the hairless mouse photocarcinogenesis model. This suggestion contradicts a large body of data indicating that topical retinoids are chemoprotective in humans, and it was immediately challenged by new reviews on the safety of RP in general and within sunscreens. This paper will review the preclinical and clinical data supporting the safety and chemopreventive activity of retinoids, with an emphasis on RP, and will examine the experimental systems used to evaluate the safety of topical vitamin A preparations in order to provide perspective relative to human skin.

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

Solar lentigines (SLs) are hyperpigmentary lesions presented on sun-exposed areas of the skin and associated with ageing. The molecular mechanism of SL initiation is not completely understood. Ultraviolet B (UVB) stimulates keratinocytes to produce interlukin-1 alpha (IL-1α), which then induces keratinocyte growth factor (KGF) secretion; therefore, we examined their possible roles in the induction of SLs. We found that KGF increases pigment production in both pigmented epidermal equivalents and human skin explants. In addition, UVB exposure increases KGF expression, and KGF treatment induces tyrosinase (TYR) expression in primary melanocytes. The KGF-induced pigmentary changes were confirmed using pigmented Yucatan swine, and human skins grafted onto immuno-deficient mice. In both model systems, the topical treatment with KGF, alone or in combination with IL-1α, resulted in the in vivo formation of hyperpigmentary lesions with increased pigment deposition and elongated rete ridges, which resemble the histological features of human SLs. Preliminary immunohistochemical analysis of human skins showed a moderate increase in KGF, and a strong induction in KGF receptor (KGFR) in SL lesions. In summary, KGF increases pigment production and deposition in vitro and in vivo. Moreover, we show for the first time the in vivo generation of hyperpigmentary lesions with histological resemblance to human SLs and indicate the involvement of KGF/KGFR in the molecular pathology of human SLs.

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

Please cite this paper as: Aldo-keto reductase 1C subfamily genes in skin are UV-inducible: possible role in keratinocytes survival. Experimental Dermatology 2009; 18: 611-618.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 5alpha-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.

Extracts from Glycine max (soybean) induce elastin synthesis and inhibit elastase activity.

Elastic fibres are essential extracellular matrix components of the skin, contributing to its resilience and elasticity. In the course of skin ageing, elastin synthesis is reduced, and elastase activity is accelerated, resulting in skin sagging and reduced skin elasticity. Our studies show that non-denatured Glycine max (soybean) extracts induced elastin promoter activity, inhibited elastase activity and protected elastic fibres from degradation by exogenous elastases in vitro. Mouse and swine skins topically treated with soybean extracts showed enhanced elastic fibre network and increased desmosine content. Elastin expression was also augmented in human skin transplanted onto SCID mice in response to soy treatment. These data suggest that non-denatured soybean extracts may be used as skin care agents to reduce the signs of skin ageing.

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.

Proteinase-activated receptor-2 stimulates prostaglandin production in keratinocytes: analysis of prostaglandin receptors on human melanocytes and effects of PGE2 and PGF2alpha on melanocyte dendricity.

Prostaglandins (PG) are key mediators of diverse functions in the skin and several reports suggest that PG mediate post-inflammatory pigmentary changes through modulation of melanocyte dendricity and melanin synthesis. The proteinase-activated receptor 2 (PAR-2) is important for skin pigmentation because activation of keratinocyte PAR-2 stimulates uptake of melanosomes through phagocytosis in a Rho-dependent manner. In this report, we show that activation of keratinocyte PAR-2 stimulates release of PGE(2) and PGF(2alpha) and that PGE(2) and PGF(2alpha) act as paracrine factors that stimulate melanocyte dendricity. We characterized the expression of the EP and FP receptors in human melanocytes and show that human melanocytes express EP1 and EP3, and the FP receptor, but not EP2 and EP4. Treatment of melanocytes with EP1 and EP3 receptor agonists resulted in increased melanocyte dendricity, indicating that both EP1 and EP3 receptor signaling contribute to PGE(2)-mediated melanocyte dendricity. Certain EP3 receptor subtypes have been shown to increase adenosine 3',5'-cyclic monophosphate (cAMP) through coupling to Gs, whereas EP1 is known to couple to Gq to activate phospholipase C with elevation in Ca(2+). The cAMP/protein kinase A system is known to modulate melanocyte dendrite formation through modulation of Rac and Rho activity. Neither PGF(2alpha) or PGE(2) elevated cAMP in human melanocytes showing that dendricity observed in response to PGE(2) and PGF(2alpha) is cAMP-independent. Our data suggest that PAR-2 mediates cutaneous pigmentation both through increased uptake of melanosomes by keratinocytes, as well as by release of PGE(2) and PGF(2alpha) that stimulate melanocyte dendricity through EP1, EP3, and FP receptors.

The proteinase-activated receptor-2 mediates phagocytosis in a Rho-dependent manner in human keratinocytes.

Recent work shows that the G-protein-coupled receptor proteinase activated receptor-2 activates signals that stimulate melanosome uptake in keratinocytes in vivo and in vitro. The Rho family of GTP-binding proteins is involved in cytoskeletal remodeling during phagocytosis. We show that proteinase-activated receptor-2 mediated phagocytosis in human keratinocytes is Rho dependent and that proteinase-activated receptor-2 signals to activate Rho. In contrast, Rho activity did not affect either proteinase-activated receptor-2 activity or mRNA and protein levels. We explored the signaling mechanisms of proteinase-activated receptor-2 mediated Rho activation in human keratinocytes and show that activation of proteinase-activated receptor-2, either through specific proteinase-activated receptor-2 activating peptides or through trypsinization, elevates cAMP in keratinocytes. Proteinase-activated receptor-2 mediated Rho activation was pertussis toxin insensitive and independent of the protein kinase A signaling pathway. These data are the first to show that proteinase-activated receptor-2 mediated phagocytosis is Rho dependent and that proteinase-activated receptor-2 signals to Rho and cAMP in keratinocytes. Because phagocytosis of melanosomes is recognized as an important mechanism for melanosome transfer to keratinocytes, these results suggest that Rho is a critical signaling intermediate in melanosome uptake in keratinocytes.

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.

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.

In vitro 3D full-thickness skin-equivalent tissue model using silk and collagen biomaterials.

Current approaches to skin equivalents often only include the epidermis and dermis. Here, a full-thickness skin equivalent is described including epidermis, dermis, and hypodermis, that could serve as an in vitro model for studying skin biology or as a platform for consumer product testing. The construct is easy to handle and is maintained for >14 d while expressing physiological morphologies of the epidermis and dermis, seen by keratin 10, collagens I and IV expression. The skin equivalent produces glycerol and leptin, markers of adipose metabolism. This work serves as a foundation for understanding a few necessary factors needed to develop a stable, functional model of full-thickness skin.

Melanocortin-5 receptor and sebogenesis.

The melanocortins (α-MSH, ß-MSH, γ-MSH, and ACTH) bind to the melanocortin receptors and signal through increases in cyclic adenosine monophosphate to induce biological effects. The melanocortin MC(5) and MC(1) receptors are expressed in human sebaceous glands, which produce sebum, a lipid mixture of squalene, wax esters, triglycerides, cholesterol esters, and free fatty acids that is secreted onto the skin. Excessive sebum production is one of the major factors in the pathogenesis of acne. The expression of melanocortin MC(5) receptor has been associated with sebocyte differentiation and sebum production. Sebaceous lipids are down-regulated in melanocortin MC(5) receptor-deficient mice, consistent with the observation that α-MSH acts as a sebotropic hormone in rodents. These findings, which suggest that melanocortins stimulate sebaceous lipid production through the MC(5) receptor, led to our search for MC(5) receptor antagonists as potential sebum-suppressive agents. As predicted, an antagonist was shown to inhibit sebocyte differentiation in vitro, and to reduce sebum production in human skin transplanted onto immunodeficient mice. The melanocortin MC(5) receptor antagonists may prove to be clinically useful for the treatment of sebaceous disorders with excessive sebum production, such as acne.

A melanocortin receptor 1 and 5 antagonist inhibits sebaceous gland differentiation and the production of sebum-specific lipids.

BACKGROUND: The melanocortin receptor-5 (MC5R) is present in human sebaceous glands, where it is expressed in differentiated sebocytes only. The targeted disruption of MC5R in mice resulted in reduced sebaceous lipid production and a severe defect in water repulsion. OBJECTIVE: To investigate the physiological function of MC5R in human sebaceous glands. METHODS: A novel MC1R and MC5R antagonist (JNJ-10229570) was used to treat primary human sebaceous cells or human skins grafted onto severe combined immunodeficient (SCID) mice. Transcription profiling, lipid analyses, and histological and immunohistochemical staining were used to analyze the effect of MC5R inhibition on sebaceous gland differentiation and sebum production. RESULTS: JNJ-10229570 dose dependently inhibited the production of sebaceous lipids in cultured primary human sebocytes. Topical treatment with JNJ-10229570 of human skins transplanted onto SCID mice resulted in a marked decrease in sebum-specific lipid production, sebaceous gland's size and the expression of the sebaceous differentiation marker epithelial-membrane antigen (EMA). Treatment with flutamide, a known inhibitor of sebum production, gave similar results, validating the human skin/SCID mouse experimental system for sebaceous secretion studies. CONCLUSION: Our data suggest that antagonists of MC1R and MC5R could be effective sebum suppressive agents and might have a potential for the treatment of acne and other sebaceous gland pathologies.

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-NH(2) (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-NH(2) (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 [Ca(2+)] 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.

The expression and activation of protease-activated receptor-2 correlate with skin color.

Skin color results from the production and distribution of melanin in the epidermis. The protease-activated receptor-2 (PAR-2), expressed on keratinocytes but not on melanocytes, is involved in melanosome uptake via phagocytosis, and modulation of PAR-2 activation affects skin color. The pattern of melanosome distribution within the epidermis is skin color-dependent. In vitro, this distribution pattern is regulated by the ethnic origin of the keratinocytes, not the melanocytes. Therefore, we hypothesized that PAR-2 may play a role in the modulation of pigmentation in a skin type-dependent manner. We examined the expression of PAR-2 and its activator, trypsin, in human skins with different pigmentary levels. Here we show that PAR-2 and trypsin are expressed in higher levels, and are differentially localized in highly pigmented, relative to lightly pigmented skins. Moreover, highly pigmented skins exhibit an increase in PAR-2-specific protease cleavage ability. Microsphere phagocytosis was more efficient in keratinocytes from highly pigmented skins, and PAR-2 induced phagocytosis resulted in more efficient microsphere ingestion and more compacted microsphere organization in dark skin-derived keratinocytes. These results demonstrate that PAR-2 expression and activity correlate with skin color, suggesting the involvement of PAR-2 in ethnic skin color phenotypes.