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The human body follows a physiological rhythm in response to the day/night cycle which is synchronized with the circadian rhythm through internal clocks. Most cells in the human body, including skin cells, express autonomous clocks and the genes responsible for running those clocks. Melatonin, a ubiquitous small molecular weight hormone, is critical in regulating the sleep cycle and other functions in the body. Melatonin is present in the skin and, in this study, we showed that it has the ability to dose-dependently stimulate PER1 clock gene expression in normal human dermal fibroblasts and normal human epidermal keratinocytes. Then we further evaluated the role of MT-1 melatonin receptor in mediating melatonin actions on human skin using fibroblasts derived from young and old subjects. Using immunocytochemistry, Western blotting and RT-PCR, we confirmed the expression of MT-1 receptor in human skin fibroblasts and demonstrated a dramatic age-dependent decrease in its level in mature fibroblasts. We used siRNA technology to transiently knockdown MT-1 receptor in fibroblasts. In these MT-1 knockdown cells, UV-dependent oxidative stress (H 2 O 2 production) was enhanced and DNA damage was also increased, suggesting a critical role of MT-1 receptor in protecting skin cells from UV-induced DNA damage. These studies demonstrate that the melatonin pathway plays a pivotal role in skin aging and damage. Moreover, its correlation with skin circadian rhythm may offer new approaches for decelerating skin aging by modulating the expression of melatonin receptors in human skin.

SIRT6 is a member of the sirtuin family, which is involved in multiple cellular pathways related to aging, inflammation, epigenetics, and a variety of other cellular functions, including DNA repair (1). Multiple pathways involving different cellular functions are impacted by the deacetylase activity of SIRT6. Genomic integrity is maintained by the capacity of SIRT6 to modulate the accessibility of DNA repair proteins. Glucose metabolism is suppressed by SIRT6 via the deacetylation of histones located at the promoter regions of multiple glycolytic genes and the corepression of hypoxia-inducible factor-1α. SIRT6 is also a corepressor of nuclear factor (NF)-κB, silencing NF-κB target genes through the deacetylation of histones at their promoters’ regions. We used SIRT6 small-interfering RNA as a tool to modulate residual DNA damage and NF-κB expression in human dermal fibroblasts. We measured NF-κB levels in the presence or the absence of ultraviolet B (UVB). The impact of SIRT6 knockdown as shown by a decrease in SIRT6 messenger RNA levels resulted in residual DNA damage as evaluated by the comet assay. Our results show that NF-κB was increased significantly (up to 400%) due to SIRT6 silencing in the absence of UVB, illustrating the master regulatory function of SIRT6 in inflammation. We also found a significant increase in DNA damage without UV exposure as a result of SIRT6 silencing, indicating the importance of SIRT6 in DNA repair pathways in cultured human dermal fibroblasts.

To evaluate the effects of ultraviolet-induced environmental trauma on human skin cells, primary normal human epidermal keratinocytes were exposed to ultraviolet-B radiation (290-320 nm). We found that relatively low doses of ultraviolet-B (62.5-500 mJ per cm2) caused dose-dependent increases in 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a biomarker of oxidative DNA damage. Unirradiated normal human epidermal keratinocytes contained 1.49 (+/- 0.11) 8-oxo-dG per 10(6) 2'-deoxyguanosine (dG) residues in cellular DNA, which increased linearly to as high as 6.24 (+/- 0.85) 8-oxo-dG per 10(6) dG after irradiation with 500 mJ per cm2. Further, this oxidative damage was reduced by 60.7% when the cells were pretreated with 1 mM mannitol. As hydrogen peroxide (H2O2) is known to be generated during oxidative stress, its accumulation in ultraviolet-B-irradiated normal human epidermal keratinocytes was also assessed and correlated to 8-oxo-dG formation. An ultraviolet-B-induced increase in H2O2 was observed in normal human epidermal keratinocytes and its production was inhibited by the addition of catalase. Based on the ability of a neutral molecule like H2O2 to permeate membranes, our data indicate that, after ultraviolet-B irradiation, H2O2 migrates from the cytosol to the nucleus where it participates in a Fenton-like reaction that results in the production of hydroxyl radicals (OH*), which may then cause 8-oxo-dG formation in cellular DNA. This conclusion is supported by our data showing that OH* scavengers, such as mannitol, are effective inhibitors of oxidative DNA base damage. Although increased levels of 8-oxo-dG were previously found in immortalized mouse keratinocytes exposed to ultraviolet-B radiation, we now report the induction of 8-oxo-dG in normal human skin keratinocytes at ultraviolet-B doses relevant to human skin exposure.

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