Skin acts as a barrier between the environment and internal organs and performs functions that are critical for the preservation of body homeostasis. In mammals, a complex network of circadian clocks and oscillators adapts physiology and behavior to environmental changes by generating circadian rhythms. These rhythms are induced in the central pacemaker and peripheral tissues by similar transcriptional-translational feedback loops involving clock genes. In this work, we investigated the presence of functional oscillators in the human skin by studying kinetics of clock gene expression in epidermal and dermal cells originating from the same donor and compared their characteristics. Primary cultures of fibroblasts, keratinocytes, and melanocytes were established from an abdominal biopsy and expression of clock genes following dexamethasone synchronization was assessed by qPCR. An original mathematical method was developed to analyze simultaneously up to nine clock genes. By fitting the oscillations to a common period, the phase relationships of the genes could be determined accurately. We thereby show the presence of functional circadian machinery in each cell type. These clockworks display specific periods and phase relationships between clock genes, suggesting regulatory mechanisms that are particular to each cell type. Taken together, our data demonstrate that skin has a complex circadian organization. Oscillators are present not only in fibroblasts but also in epidermal keratinocytes and melanocytes and are likely to act in coordination to drive rhythmic functions within the skin.

Although the induction of pigmentation following exposure of melanocytes to ultraviolet light in vivo and in vitro is well documented, the intracellular mechanisms involved in this response are not yet fully understood. Exposure to UV-B radiation leads to the production of DNA damage, mainly cyclobutane pyrimidine dimers, and it was recently suggested that the thymidine dinucleotide pTpT, mimicking small DNA fragments released in the course of excision repair mechanisms, could trigger melanin synthesis. We now report that the thymidine dinucleotide pTpT induces melanogenesis both in human normal adult melanocytes and in human melanoma cells. Thus, the SOS-like response suggested by Gilchrest's work to be evolutionary conserved, based primarily on work in murine cells and guinea pigs, is also apparently present in the human. Thymidine dinucleotide is nontoxic to melanoma cells and does not induce apoptosis in these cells, but induces S phase cell cycle arrest and a proliferation slow down. Because thymidine excess in culture medium leads to the synchronization of cells in S phase, we investigated whether this phenomenon was involved in the increase in melanin synthesis. We show that melanin synthesis is specifically triggered by the dimeric form of the thymidine and not by the monomeric form pT. Thus, our data strongly support that thymidine dinucleotides pTpT mimic at least part of the effects of ultraviolet irradiation, and may hence represent an invaluable model in the study of the molecular events involved in melanogenesis induction triggered through DNA damage.

However, CYP4A11 may participate in the defence mechanism against UVA-induced oxidative damage.

The effects of calcium D-pantetheine-S-sulfonate (PaSSO3Ca) on human pigmentation were examined by in vitro assays using two types of human melanocytes: normal adult melanocytes (HNM) and M4Be melanoma cells. The compound, when added to a culture medium at doses indicating no cytotoxicity, causes a visually recognizable, reversible loss of pigment in both types of cells. Determination of melanin content, incorporation of 14C-DOPA into melanins and tyrosinase activities demonstrated that treatment of these cells with PaSSO3Ca resulted in a marked decrease in all three areas. When homogenates of these cells were assayed with lectins, the glycosylation pattern was modified, as tyrosinase activities were reduced in the cells treated with the compound. Immunoprecipitation of tyrosinase and tyrosinase-related protein 1 (Tyrp1 or TRP1) in cells incubated with radioactive glucosamine disclosed that glucosamine uptake by these enzymes was apparently increased, suggesting structural alterations in their sugar moieties. It is also noted that PaSSO3Ca is analogous in its chemical structure to Coenzyme A (CoA), which plays an important role in the intracellular transport of proteins. Based on these findings, it is likely that the compound exerts its depigmenting effects in human pigment cells through the modification of glycosylation of tyrosinase and TRP1, which are key enzymes for melanogenesis.