Ultraviolet B both damages the skin and is required for photochemical transformation of 7-dehydrocholesterol
to vitamin D3 (D3). Its sequential hydroxylation at C25 and C1 generates biologically active 1,25(OH)2D3 that
displays a variety of pleiotropic activities. It was believed that all of these effects are mediated by single
molecule, 1,25(OH)2D3, and single receptor, VDR. Discovery of an alternative pathway in which CYP11A1
oxidizes the side chain of D3 producing 20(OH)D3 with its further metabolism to other downstream-derivatives,
(OH)nD3, challenged this dogma. 20(OH)D3 is detectable in the human epidermis at concentration higher than
25(OH)D3, and in human serum at ~3nM. 20(OH)D3 shows biological activities suggestive that it can act as an
endogenous regulator of epidermal barrier, while its presence in circulation suggests hormonal functions.
20(OH)D3 is noncalcemic at pharmacological doses (30-60µg/kg). Initial data and computer modeling indicate
that it can act as biased agonist on VDR and reverse agonist on RORa and ROR¿. Hypothesis: 20(OH)D3
and/or its metabolites acting directly on VDR and/or RORa and ROR¿ stimulate keratinocyte differentiation
program and photoprotective and repair mechanisms that protect epidermis against UVB-induced pathology.
These effects would not require its hydroxylation in position C1a, in contrast to 1,25(OH)2D3. The hypothesis
will be tested as follows: Aim 1. To test the relative role of VDR, RORa, or ROR¿ in 20(OH)D3 mediated
regulation of the proliferation and differentiation programs in epidermal keratinocytes. Subaim 1: Through in
vitro binding to the ligand-binding domain of recombinant VDR and RORs we will define relative interactions of
20(OH)D3 and its downstream metabolites with the receptors in comparison to their native ligands. Then we
will apply complex cell-based models to measure ligands modulated transcriptional activities. These will be
supplemented by molecular modeling analyses. Subaim 2: The relative roles of VDR and RORs in regulation
of keratinocytes proliferation and differentiation will be tested using gene silencing technology. This will be
complemented by tests on keratinocytes isolated from VDR-/-, RORa-/- or ROR¿-/- mice. Subaim 3: RNA-Seq
for gene expression and ChIP-Seq for receptor enrichment on target genes followed by bioinformatics
analyses to identify alternative targets. Aim 2. To define protective role of 20(OH)D3 against UVB radiation in
the human epidermis. Subaim 1: Testing the role of 20(OH)D3 as a survival factor. Subaim 2: Testing whether
20(OH)D3 attenuates UVB induced apoptosis. Subaim 3: Testing whether 20(OH)D3 can act as an
antigenotoxic/antimutagenic agent. Subaim 4: Testing the role of RORa or ROR¿ in skin responses to the
UVB using mice with genotype -/-,-/+ and +/+ for these receptors and comparing with mice with defective or intact
VDR. Aim 3. To evaluate whether the phenotypic effects of 20(OH)D3 and its metabolites require 1a
hydroxylation as described for 1,25(OH)2D3. We will use cells with silenced CYP27B1 gene. This will be
complemented by pharmacological inhibition of the CYP27B1 activity in keratinocytes.