Regulation of circadian clock genes by UVB radiation and vitamin D : a pilot study in human epidermal keratinocytes during different stages of skin photocarcinogenicity.

Abstract

Several cellular processes are constantly under regulation of a timekeeping system, known as the circadian clock (CC), with the main regulatory genes involved referred to as the circadian clock genes (CCGs). In spite of a master pacemaker existing in the suprachiasmatic nucleus (SCN) of the hypothalamus, several clock systems have been shown to operate independently in peripheral tissues, including the skin. CCGs interact with cellular processes by both regulating them and being regulated by them, a prime example of this being the bilateral relationship between CC and ultraviolet B radiation (UV-B): On one hand, UV-radiation regulates expression of CCGs in many cell types, and on the other, it has recently been shown that expression of CCGs modulates susceptibility for UV-B-induced cellular damage, including the formation of pyrimidine dimers and other DNA-lesions that are a hallmark of photocarcinogenesis. It was the aim of this study to gain further insights into the CCGs’ putative role for UVB-induced skin photocarcinogenesis. In particular, we aimed to investigate UV-effects on CC and whether these may at least in part be mediated by 1,25(OH)2D3 (D3), the active form of vitamin D, that both depends on UV-B for its synthesis and is known to protect the skin from UV-B’s damaging properties. We treated cells with D3, UV-B and a combination of the two and measured expression of two core clock genes, brain and muscle ARNT-like 1 (BMAL1) and Period-2 (Per2), both over several time points and in cells representing: normal (Normal Human Epidermal Keratinocytes – NHEK; p53 wild type), precancerous (HaCaT keratinocytes; mutated p53 status) and cancerous keratinocytes (Squamous Cell Carcinoma SCL-1; p53 null status). We also assessed the role of vitamin D receptor (VDR) and aryl hydrocarbon receptor (AhR) pathways by measuring UVB-induced damage, repair and cellular toxicity after treatment with D3 and UVB and under chemical antagonization of either/both VDR and/or AhR. Untreated HaCaT cells showed circadian rhythmicity (length of individual periods approximately 24 hours) for expression of BMAL1 and Per2, that were induced by UVB (p<0,001), which also resulted in significant increase in cellular toxicity 24h after treatment (p<0,05). In contrast, treatment with 1,25-dihydroxyvitamin D had only marginal effects (no visual effect on expression of BMAL1, trend for a marginal reduction of expression of Per2). While UV-B induced expression of Per2 in NHEK, HaCaT and SCL-1 cells, UVB-induced upregulation of BMAL1 was only detected in NHEKs and HaCaT, but not in SCL-1 cells. In regards to direct effects and interaction of UVB and D3 and the roles of VDR and AhR on DNA damage and repair, no safe conclusions can be drawn from this study. HaCaT keratinocytes express BMAL1 and Per2 with circadian rhythmicity. Our findings do not support the hypothesis that the UVB-induced upregulation of these CCGs may be mediated via UVB-induced synthesis of vitamin D. Comparing epidermal keratinocytes representing different stages of skin photocarcinogenesis, we provide further evidence for an independently operating timekeeping system in human skin cells, that is physiologically regulated by UVB. The destruction of this mechanism in malignant keratinocytes points to a contribution of CCGs for skin photocarcinogenesis.