The expression of melanopsin and clock genes in Xenopus laevis melanophores and their modulation by melatonin

Vertebrates have a central clock and also several peripheral clocks. Light responses might result from the integration of light signals by these clocks. The dermal melanophores of Xenopus laevis have a photoreceptor molecule denominated melanopsin (OPN4x). The mechanisms of the circadian clock involve positive and negative feedback. We hypothesize that these dermal melanophores also present peripheral clock characteristics. Using quantitative PCR, we analyzed the pattern of temporal expression of Opn4x and the clock genes Per1, Per2, Bmal1, and Clock in these cells, subjected to a 14-h light:10-h dark (14L:10D) regime or constant darkness (DD). Also, in view of the physiological role of melatonin in the dermal melanophores of X. laevis, we determined whether melatonin modulates the expression of these clock genes. These genes show a time-dependent expression pattern when these cells are exposed to 14L:10D, which differs from the pattern observed under DD. Cells kept in DD for 5 days exhibited overall increased mRNA expression for Opn4x and Clock, and a lower expression for Per1, Per2, and Bmal1. When the cells were kept in DD for 5 days and treated with melatonin for 1 h, 24 h before extraction, the mRNA levels tended to decrease for Opn4x and Clock, did not change for Bmal1, and increased for Per1 and Per2 at different Zeitgeber times (ZT). Although these data are limited to one-day data collection, and therefore preliminary, we suggest that the dermal melanophores of X. laevis might have some characteristics of a peripheral clock, and that melatonin modulates, to a certain extent, melanopsin and clock gene expression.

Biological activity and binding of estradiol to SK-Mel 23 human melanoma cells

Patients expressing estradiol receptors in melanoma cells have been reported to have a better prognosis. We therefore decided to investigate the in vitro effects of á-estradiol and tamoxifen on the growth and tyrosinase activity of SK-Mel 23 human melanoma cells. Twenty-four-hour treatment with 0.4 nM á-estradiol inhibited cell proliferation in 30 percent (0.70 ñ 0.03 x 10(5) cells) and increased tyrosinase activity in 50 percent (7130.5 ñ 376.5 cpm/10(5) cells), as compared to untreated cells (1.0 ñ 0.05 x 10(5) cells and 4769 ñ 25.5 cpm/10(5) cells, respectively). Both responses were completely (100 percent) blocked by 1 æM tamoxifen. Higher concentrations (up to 1.6 nM) or longer treatments (up to 72 h) did not result in a larger effect of the hormone on proliferation or tyrosinase activity. Competition binding assays demonstrated the presence of binding sites to [2,4,6,7- H]-á-estradiol, and that the tritiated analogue was displaced by the unlabeled hormone (1 nM to 100 æM, Kd = 0.14 æM, maximal displacement of 93 percent) or by 10 æM tamoxifen (displacement of 60 percent). á-estradiol also increased the phosphorylated state of two proteins of 16 and 46 kDa, after 4-h treatment, as determined by Western blot. The absorbance of each band was 1.9- and 4-fold the controls, respectively, as determined with Image-Pro Plus software. Shorter incubation periods with á-estradiol did not enhance phosporylation; after 6-h treatment with the hormone, the two proteins returned to the control phosphorylation levels. The growth inhibition promoted by estradiol may explain the better prognosis of melanoma-bearing women as compared to men, and open new perspectives for drug therapy.

The role of calcium, calcium-activated K+ channels, and tyrosine/kinase in psoralen-evoked responses in human melanoma cells

8-Methoxy psoralen (8-MOP) exerts a short-term (24 h) mitogenic action, and a long-term (48-72 h) anti-proliferative and melanogenic action on two human melanoma cell lines, SK-Mel 28 and C32TG. An increase of intracellular calcium concentration was observed by spectrofluorometry immediately after the addition of 0.1 mM 8-MOP to both cell lines, previously incubated with calcium probe fluo-3 AM (5 µM). The intracellular Ca2+ chelator BAPTA/AM (1 µM) blocked both early (mitogenic) and late (anti-proliferative and melanogenic) 8-MOP effects on both cell lines, thus revealing the importance of the calcium signal in both short- and long-term 8-MOP-evoked responses. Long-term biological assays with 5 and 10 mM tetraethylammonium chloride (TEA, an inhibitor of Ca2+-dependent K+ channels) did not affect the responses to psoralen; however, in 24-h assays 10 mM TEA blocked the proliferative peak, indicating a modulation of Ca2+-dependent K+ channels by 8-MOP. No alteration of cAMP basal levels or forskolin-stimulated cAMP levels was promoted by 8-MOP in SK-Mel 28 cells, as determined by radioimmunoassay. However, in C32TG cells forskolin-stimulated cAMP levels were further increased in the presence of 8-MOP. In addition, assays with 1 µM protein kinase C and calcium/calmodulin-dependent kinase inhibitors, Ro 31-8220 and KN-93, respectively, excluded the participation of these kinases in the responses evoked by 8-MOP. Western blot with antibodies anti-phosphotyrosine indicated a 92 percent increase of the phosphorylated state of a 43-kDa band, suggesting that the phosphorylation of this protein is a component of the cascade that leads to the increase of tyrosinase activity.

Topical application of a melanotropin analogue to vulgar vitiligo dermo-epidermal minigrafts

Human subjects with active vulgar vitiligo do not respond well to autologous dermo-epidermal minigrafting. Eighteen subjects were treated with the a-melanocyte-stimulating hormone (a-MSH) synthetic analogue [Nle4, D-Phe7]-a-MSH. The hormone (50 µl, 0.4 mM) was applied topically to 30-cm2 lesions in which 29-48 minigrafts had been made. The hormone did not improve the success of the minigrafting and no differences were observed in local or distant repigmentation in treated subjects as compared to the placebo group. Aliquots of 24-h urine concentrated by lyophilization irreversibly darkened toad skins, demonstrating the presence of the analogue. This is the first report of the transdermal delivery of a topically applied melanotropin in living human subjects

Cellular signalling in vertebrate pigment cells

Chromatophores are specialized integumental stellate cells that synthesize and store pigments. Pigment granules are translocated within chromatophores of poikilothermic vertebrates and crustaceans in response to photic, thermal and/or neurohormonal stimuli, allowing the animal to rapidly change color for thermoregulation, adaptation to light and background, and social behavior display. Birds and mammals do not show color changes, but may present slow long-term responses, such as melanocyte proliferation, melanin synthesis and melanin granule translocation into feathers, hair and surrounding keratinocytes. Pigment translocation in lower vertebrates as well as pigment production in all vertebrates are modulated by a variety of hormones and neurotransmitters acting on transmembrane receptors located on the cell surface. Alpha-melanocyte-stimulating hormone (alpha-MSH), melanin-concentrating hormone (MCH), melatonin and catecholamines are the most important pigment cell agonists in vertebrates. The major signalling pathway leading to pigment dispersion and melanin synthesis appears to involve stimulation of adenylate cyclase followed by an increase in the cAMP level and activation cAMP-dependent protein kinases (PKAs). Another melanogenesis related intracellular pathway involves the activation of protein kinase C (PKC) by diacylglycerol, and the increase in cytosolic Ca2+ by inositol triphosphate. Growth factors such as basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF) and mast cell growth factor (MGF or KIT tigand), and UV radiation modulate the melanogenic and mitogenic processes in vertebrates melanocytes as well.