Blue light entrains circadian rhythm via peropsin in keratinocytes of skin organotypic cultures

Summary: <br /> The epidermis possesses its own independent circadian rhythm (CR), whose primary environmental cue is light. Various opsins, proteins responsible for light detection, have been observed in different differentiation states of keratinocytes. Peropsin (RRH), in particular, detects near-ultraviolet blue light and is highly expressed in differentiated keratinocytes. Here, we use a differentiated skin organotypic culture to examine the effect of blue light in keratinocyte circadian rhythm, and investigate its effect on inflammatory cytokines such as IL-13. To examine CR, we established N/TERT cell cultures and maintained 2D and 3D skin equivalent in dark-dark (DD) cycle. One group was moved under 12h:12h light-dark (LD) conditions to an incubator equipped with a lighting panel. Both groups were treated with all-trans-retinal (an opsin chromophore), and mRNA was analyzed. A 3D full-thickness skin equivalent culture model for atopic dermatitis (AD) was developed using IL-13 treatment for 21 days. Finally, to examine BMAL1/CLOCK binding to IL-13 recognition sites, promoter pulldown assays and electrophoretic mobility shift assays (EMSA) were performed. The skin organotypic cultures exhibited typical clock gene oscillations when exposed to LD cycles. Deletion of RRH in keratinocytes abolished light-induced CR, indicating that peripheral light induced CR, and Bmal1 (a key CR player) relies on RRH activation and blue light. We also demonstrated that the pro-inflammatory cytokine IL-13 is expressed in keratinocytes, and IL-13 expression underwent a circadian pattern when exposed to LD cycle. Finally, we demonstrated BMAL1/CLOCK binding to IL-13 gene promoters. These results show that peropsin is a functional photoreceptor in keratinocytes and visible light alone may entrain CR in the epidermis. Our preliminary data also suggests that activation of RRH triggers an inflammatory state in the epidermis, allowing for a better understanding of diurnal symptoms in atopic dermatitis. Sydney Lo^{1, 2}, Elena Bigliardi², Mei Bigliardi-Qi^{1, 2} 1. Dermatology, University of Minnesota Twin Cities, Minneapolis, MN, United States. 2. McGuire Translational Research Facility, University of Minnesota Twin Cities, Minneapolis, MN, United States. Translational Studies: Cell and Molecular Biology