Insulin Binding Properties of Normal and Transformed Human Epidermal Cultured Keratinocytes

Insulin binding to its receptors was studied in cultured normal and transformed (A431 line) human epidermal keratinocytes. The specific binding was a temperature-dependent, saturable process. Normal keratinocytes possess a mean value of about 80,000 receptors per cell. Fifteen hours exposure of the cells to insulin (2 × 10−7M) lowered their receptor number (about 65% loss in available sites); these reappeared when the hormone was removed from the culture medium (“down-regulation” process). In the A431 epidermoid carcinoma cell line, there is a net decrease in insulin binding (84% of the initial bound/free hormone ratio in comparison with normal cells) essentially related to a loss in receptor affinity for insulin. Thus, cultured human keratinocytes which express insulin receptors may be a useful tool in understanding skin pathology related to insulin disorders

Neprilysin, a Novel Target for Ultraviolet B Regulation of Melanogenesis Via Melanocortins

SummaryCompelling evidence suggest a role for melanocortins in the regulation of melanogenesis by ultraviolet radiation. Within the epidermis, melanocytes and keratinocytes produce α-melanocyte-stimulating hormone and adrenocorticotropic hormone. The persistence and the strength of the biologic signal delivered by these peptides depend on their local concentration, which is controlled by the rate of peptide production and by the rate of its degradation. In this study, we investigated the mechanism of melanocortin degradation by melanocytes and the effect of ultraviolet on this process. We have focused our attention on a neutral endopeptidase, neprilysin, which has been implicated in the ending of numerous peptidergic signals. We have shown that this enzyme is expressed at the surface of human melanocytes. Interestingly, its activity and its expression are dramatically downregulated by ultraviolet B treatment. Moreover, in the presence of phosphoramidon, a stable inhibitor of neprilysin, we observed an increased efficiency of α-melanocyte-stimulating hormone and adrenocorticotropic hormone to stimulate both tyrosinase activity and microphthalmia expression. Taken together, these data indicate that neprilysin expressed by melanocytes has a physiologic role in the regulation of melanogenesis by proopiomelanocortin peptide. Further, its downregulation by ultraviolet B irradiation shed light on a new and appealing mechanism of ultraviolet B induced melanogenesis via the control of melanocortins degradation

Role of Thiol Compounds in Mammalian Melanin Pigmentation: Part I. Reduced and Oxidized Glutathione

Evidence for the postulated role of glutathione reductase in melanin pigmentation has been obtained by determinations of the glutathione concentrations in Tortoiseshell guinea pig skin of different colors (black, yellow, red, and white). As expected, the lowest levels of reduced glutathione (GSH) were found associated with eumelanin type pigmentation, whereas the highest ones were found in the skin with phaeomelanin producing melanocytes. On the other hand, white skin of guinea pig having no active melanocytes showed GSH levels which were intermediate between those of the black and yellow areas.These results are consistent with the view that the activity of the enzyme glutathione reductase, though not primarily related to pigmentation, plays an important role in the regulation and control of the biosynthetic activity of melanocytes leading to various types of melanin pigments

Identification of a Lethal Form of Epidermolysis Bullosa Simplex Associated with a Homozygous Genetic Mutation in Plectin

Genetic mutations in plectin, a cytoskeleton linker protein expressed in a large variety of tissues including skin, muscle, and nerves, cause epidermolysis bullosa simplex with muscular dystrophy, a recessive inherited disease characterized by blistering of the skin and late onset of muscular dystrophy, and Ogna epidermolysis bullosa simplex, a rare dominant inherited form of epidermolysis bullosa simplex with no muscular involvement. Here we report a novel homozygous genetic mutation (2727del14) in the plectin gene (PLEC1) associated with a lethal form of recessive inherited epidermolysis bullosa in a consanguineous family with three affected offspring. This new clinical variant of epidermolysis bullosa is characterized by general skin blistering, aplasia cutis of the limbs, developmental complications, and rapid demise after birth. Mutation 2727del14 is the first genetic defect described in PLEC1 that disrupts the plakin domain of plectin. The severe phenotype of the patients may be linked to the role of the N-terminal domain in the function of plectin and develops the understanding of the genotype-phenotype correlations in the genodermatoses affecting the dermal-epidermal junction

Microphthalmia-associated transcription factor regulates RAB27A gene expression and controls melanosome transport.

Melanosomes are lysosome-related organelles specialized in melanin synthesis and transport. In this study, we show that microphthalmia-associated transcription factor (MITF) silencing induces melanosome gathering around the nucleus and causes the relocalization of Rab27A, Slac2a-Mlph, and Myo5a that control the transport of melanosomes on the actin network. In an attempt to elucidate the mechanism by which MITF controls melanosome distribution, we identify RAB27A as a new MITF target gene. Indeed, MITF silencing leads to a dramatic decrease in Rab27A expression and blocks the stimulation of Rab27A expression evoked by cAMP. Further, forced expression of MITF increases Rab27A expression, indicating that MITF is required and sufficient for Rab27A expression in melanoma cells. MITF binds to two E-boxes in the proximal region of the Rab27A promoter and stimulates its transcriptional activity. Finally, re-expression of Rab27A, in MITF-depleted cells, restores the transport of melanosomes to the cell periphery. These results show that RAB27A is a new direct transcriptional target of MITF and link MITF to melanosome transport, another key parameter of melanocyte differentiation and skin pigmentation. Interestingly, Rab27A is involved in other fundamental physiological functions, such as the transport of lytic granules and insulin secretion. Thus our results, deciphering the mechanism of Rab27A transcriptional regulation, have an interest that goes beyond the skin pigmentation field

Transcriptional Profiling Shows Altered Expression of Wnt Pathway– and Lipid Metabolism–Related Genes as Well as Melanogenesis-Related Genes in Melasma

Melasma is a commonly acquired hyperpigmentary disorder of the face, but its pathogenesis is poorly understood and its treatment remains challenging. We conducted a comparative histological study on lesional and perilesional normal skin to clarify the histological nature of melasma. Significantly, higher amounts of melanin and of melanogenesis-associated proteins were observed in the epidermis of lesional skin, and the mRNA level of tyrosinase-related protein 1 was higher in lesional skin, indicating regulation at the mRNA level. However, melanocyte numbers were comparable between lesional and perilesional skin. A transcriptomic study was undertaken to identify genes involved in the pathology of melasma. A total of 279 genes were found to be differentially expressed in lesional and perilesional skin. As was expected, the mRNA levels of a number of known melanogenesis-associated genes, such as tyrosinase, were found to be elevated in lesional skin. Bioinformatics analysis revealed that the most lipid metabolism-associated genes were downregulated in lesional skin, and this finding was supported by an impaired barrier function in melasma. Interestingly, a subset of Wnt signaling modulators, including Wnt inhibitory factor 1, secreted frizzled-related protein 2, and Wnt5a, were also found to be upregulated in lesional skin. Immunohistochemistry confirmed the higher expression of these factors in melasma lesions

Rab27a: A key to melanosome transport in human melanocytes

Normal pigmentation depends on the uniform distribution of melanin-containing vesicles, the melanosomes, in the epidermis. Griscelli syndrome (GS) is a rare autosomal recessive disease, characterized by an immune deficiency and a partial albinism that has been ascribed to an abnormal melanosome distribution. GS maps to 15q21 and was first associated with mutations in the myosin-V gene. However, it was demonstrated recently that GS can also be caused by a mutation in the Rab27a gene. These observations prompted us to investigate the role of Rab27a in melanosome transport. Using immunofluorescence and immunoelectron microscopy studies, we show that in normal melanocytes Rab27a colocalizes with melanosomes. In melanocytes isolated from a patient with GS, we show an abnormal melanosome distribution and a lack of Rab27a expression. Finally, reexpression of Rab27a in GS melanocytes restored melanosome transport to dendrite tips, leading to a phenotypic reversion of the diseased cells. These results identify Rab27a as a key component of vesicle transport machinery in melanocytes