RESUMO
In melanocytes and melanoma cells alpha-melanocyte stimulating hormone (alpha-MSH), via the cAMP pathway, elicits a large array of biological responses that control melanocyte differentiation and influence melanoma development or susceptibility. In this work, we show that cAMP transcriptionally activates Hif1a gene in a melanocyte cell-specific manner and increases the expression of a functional hypoxia-inducible factor 1alpha (HIF1alpha) protein resulting in a stimulation of Vegf expression. Interestingly, we report that the melanocyte-specific transcription factor, microphthalmia-associated transcription factor (MITF), binds to the Hif1a promoter and strongly stimulates its transcriptional activity. Further, MITF "silencing" abrogates the cAMP effect on Hif1a expression, and overexpression of MITF in human melanoma cells is sufficient to stimulate HIF1A mRNA. Our data demonstrate that Hif1a is a new MITF target gene and that MITF mediates the cAMP stimulation of Hif1a in melanocytes and melanoma cells. Importantly, we provide results demonstrating that HIF1 plays a pro-survival role in this cell system. We therefore conclude that the alpha-MSH/cAMP pathway, using MITF as a signal transducer and HIF1alpha as a target, might contribute to melanoma progression.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Melanócitos/metabolismo , Melanoma/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Proteínas de Ligação a DNA/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/genética , Genes Reguladores/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Melanócitos/citologia , Melanócitos/efeitos dos fármacos , Melanoma/genética , Camundongos , Fator de Transcrição Associado à Microftalmia , Células NIH 3T3 , Regiões Promotoras Genéticas/fisiologia , Interferência de RNA/efeitos dos fármacos , Interferência de RNA/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/fisiologia , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular/genética , alfa-MSH/metabolismoRESUMO
Prolidase is a ubiquitous enzyme that plays a major role in the metabolism of proline-rich proteins. Prolidase deficiency is a rare autosomal recessive inborn metabolic and multisystemic disease, characterized by a protean association of symptoms, namely intellectual disability, recurrent infections, splenomegaly, skin lesions, auto-immune disorders and cytopenia. To our knowledge, no published review has assembled the different clinical data and research studies over prolidase deficiency. The aim of this study is to summarize the actual state of the art from the descriptions of all the patients with a molecular diagnosis of prolidase deficiency reported to date regarding the clinical, biological, histopathological features, therapeutic options and functional studies.
RESUMO
Epidermal wound repair is a complex process involving the fine orchestrated regulation of crucial cell functions, such as proliferation, adhesion and migration. Using an in vitro model that recapitulates central aspects of epidermal wound healing, we demonstrate that the transcription factor HIF1 is strongly stimulated in keratinocyte cultures submitted to mechanical injury. Signals generated by scratch wounding stabilise the HIF1alpha protein, which requires activation of the PI3K pathway independently of oxygen availability. We further show that upregulation of HIF1alpha plays an essential role in keratinocyte migration during the in vitro healing process, because HIF1alpha inhibition dramatically delays the wound closure. In this context, we demonstrate that HIF1 controls the expression of laminin-332, one of the major epithelial cell adhesion ligands involved in cell migration and invasion. Indeed, silencing of HIF1alpha abrogates injury-induced laminin-332 expression, and we provide evidence that HIF1 directly regulates the promoter activity of the laminin alpha3 chain. Our results suggest that HIF1 contributes to keratinocyte migration and thus to the re-epithelialisation process by regulating laminin-332.
Assuntos
Moléculas de Adesão Celular/metabolismo , Movimento Celular/fisiologia , Fator 1 Induzível por Hipóxia/metabolismo , Queratinócitos/fisiologia , Animais , Sequência de Bases , Moléculas de Adesão Celular/genética , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Queratinócitos/citologia , Laminina/genética , Laminina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais/fisiologia , Cicatrização , CalininaRESUMO
Covering denuded dermal surfaces after injury requires migration, proliferation, and differentiation of skin keratinocytes. To clarify the major traits controlling these intermingled biological events, we surveyed the genomic modifications occurring during the course of a scratch wound closure of cultured human keratinocytes. Using a DNA microarray approach, we report the identification of 161 new markers of epidermal repair. Expression data, combined with functional analysis performed with specific inhibitors of ERK, p38(MAPK) and phosphatidylinositol 3-kinase (PI3K), demonstrate that kinase pathways exert very selective functions by precisely controlling the expression of specific genes. Inhibition of the ERK pathway totally blocks the wound closure and inactivates many early transcription factors and EGF-type growth factors. p38(MAPK) inhibition only delays "healing," probably in line with the control of genes involved in the propagation of injury-initiated signaling. In contrast, PI3K inhibition accelerates the scratch closure and potentiates the scratch-dependent stimulation of three genes related to epithelial cell transformation, namely HAS3, HBEGF, and ETS1. Our results define in vitro human keratinocyte wound closure as a repair process resulting from a fine balance between positive signals controlled by ERK and p38(MAPK) and negative ones triggered by PI3K. The perturbation of any of these pathways might lead to dysfunction in the healing process, similar to those observed in pathological wounding phenotypes, such as hypertrophic scars or keloids.