RESUMEN
Rationale: SOX10 (SRY-related HMG-box 10) and MITF-M (microphthalmia-associated transcription factor M) restrict the expression of melanogenic genes, such as TYR (tyrosinase), in melanocytes. DACE (diacetylcaffeic acid cyclohexyl ester) inhibits melanin production in α-MSH (α-melanocyte stimulating hormone)-activated B16-F0 melanoma cells. In this study, we evaluated the antimelanogenic activity of DACE in vivo and elucidated the molecular basis of its action. Methods: We employed melanocyte cultures and hyperpigmented skin samples for pigmentation assays, and applied chromatin immunoprecipitation, immunoblotting, RT-PCR or siRNA-based knockdown for mechanistic analyses. Results: Topical treatment with DACE mitigated UV-B-induced hyperpigmentation in the skin with attenuated expression of MITF-M and TYR. DACE also inhibited melanin production in α-MSH- or ET-1 (endothelin 1)-activated melanocyte cultures. As a mechanism, DACE blocked the nuclear import of CRTC1 (CREB-regulated co-activator 1) in melanocytes. DACE resultantly inhibited SOX10 induction, and suppressed the transcriptional abilities of CREB/CRTC1 heterodimer and SOX10 at MITF-M promoter, thereby ameliorating facultative melanogenesis. Furthermore, this study unveiled new issues in melanocyte biology that i) KPNA1 (Impα5) escorted CRTC1 as a cargo across the nuclear envelope, ii) SOX10 was inducible in the melanogenic process, and iii) CRTC1 could direct SOX10 induction at the transcription level. Conclusion: We propose the targeting of CRTC1 as a unique strategy in the treatment of acquired pigmentary disorders.
Asunto(s)
Núcleo Celular/metabolismo , Hiperpigmentación/tratamiento farmacológico , Melanocitos/efectos de los fármacos , Pigmentación/efectos de los fármacos , Factores de Transcripción/antagonistas & inhibidores , Animales , Línea Celular , Modelos Animales de Enfermedad , Ratones , Transporte de ProteínasRESUMEN
BACKGROUND AND PURPOSE: cAMP as a second messenger stimulates expression of microphthalmia-associated transcription factor (MITF) or the tyrosinase gene in UVB-induced skin pigmentation. Diphenylmethylene hydrazinecarbothioamide (QNT 3-80) inhibits α-melanocyte-stimulating hormone (α-MSH)-induced melanin production in B16 murine melanoma cells but its molecular basis remains to be defined. Here, we investigated the mechanism underlying the amelioration of skin hyperpigmentation by QNT 3-80. EXPERIMENTAL APPROACH: We used melanocyte cultures with raised levels of cAMP and UVB-irradiated dorsal skin of guinea pigs for pigmentation assays. Immunoprecipitation, kemptide phosphorylation, fluorescence analysis and docking simulation were applied to elucidate a molecular mechanism of QNT 3-80. KEY RESULTS: QNT 3-80 inhibited melanin production in melanocyte cultures with elevated levels of cAMP, including those from human foreskin. This compound also ameliorated hyperpigmentation in vivo in UVB-irradiated dorsal skin of guinea pigs. As a mechanism, QNT 3-80 directly antagonized cAMP binding to the regulatory subunit of PKA, nullified the dissociation and activation of inactive PKA holoenzyme in melanocytes and fitted into the cAMP-binding site on the crystal structure of human PKA under the most energetically favourable simulation. QNT 3-80 consequently inhibited cAMP- or UVB-induced phosphorylation (activation) of cAMP-responsive element-binding protein in vitro and in vivo, thus down-regulating expression of genes for MITF or tyrosinase in the melanogenic process. CONCLUSIONS AND IMPLICATIONS: Our data suggested that QNT 3-80 could contribute significantly to the treatment of skin disorders with hyperpigmented patches with the cAMP-binding site of PKA as its molecular target.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Pigmentación de la Piel/fisiología , Tiosemicarbazonas/farmacología , Animales , Línea Celular Tumoral , Células Cultivadas , Regulación hacia Abajo , Prepucio/citología , Cobayas , Humanos , Hiperpigmentación/tratamiento farmacológico , Masculino , Melaninas/metabolismo , Melanocitos/efectos de los fármacos , Melanocitos/metabolismo , Factor de Transcripción Asociado a Microftalmía/genética , Monofenol Monooxigenasa/genética , Piel/efectos de la radiación , Pigmentación de la Piel/efectos de los fármacos , Rayos UltravioletaRESUMEN
Ultraviolet (UV) radiation under sunlight stimulates skin pigmentation through immediately affecting the oxidative modification of existing melanin pigments and the spatial redistribution of pigmented melanosomes followed by the up-regulation of melanogenic genes in delayed kinetics. However, abnormal accumulation and synthesis of melanin biopolymers are responsible for skin disorders with more pigmented patches. Chemical-based regulation of the hyperpigmented disorders has been a long-standing goal for cosmetic and pharmaceutical applications. A large number of the chemicals with antimelanogenic activity have met with limited or no success in the treatment of skin patients, since they may not overcome the challenge of penetrating the skin barrier. Guinea pig skin displays similar kinetic parameters to human skin in the transdermal absorption of numerous chemicals, thus can serve as the surrogate for human skin. Here, we provide a concise review of our current understanding of the chemical-based therapy against skin hyperpigmentation in UV-irradiated guinea pig models, suggest molecular mechanisms of the action and emphasize the translation from preclinical outcomes to skin patients.