We define previously unrecognized in vivo pathways of vitamin D ₃ (D3) metabolism generating novel D3‐hydroxyderivatives different from 25‐hydroxyvitamin D ₃ [25(OH)D3] and 1,25(OH) ₂ D3. Their novel products include 20‐hydroxyvitamin D ₃ [20(OH)D3], 22(OH)D3, 20,23(OH) ₂ D3, 20,22(OH) ₂ D3, 1,20(OH) ₂ D3,1,20,23(OH) ₃ D3, and 17,20,23(OH) ₃ D3 and were produced by placenta, adrenal glands, and epidermal keratinocytes. We detected the predominant metabolite [20(OH)D3] in human serum with a relative concentration ~20 times lower than 25(OH)D3. Use of inhibitors and studies performed with isolated mitochondria and purified enzymes demonstrated involvement of the steroidogenic enzyme cytochrome P450scc (CYP11A1) as well as CYP27B1 (1α‐hydroxylase). In placenta and adrenal glands with high CYP11A1 expression, the predominant pathway was D3 → 20(OH)D3 → 20,23(OH) ₂ D3 → 17,20,23(OH) ₃ D3 with further 1α‐hydroxylation, and minor pathways were D3 → 25(OH)D3 → 1,25(OH) ₂ D3 and D3 → 22(OH)D3 → 20,22(OH) ₂ D3. In epidermal keratinocytes, we observed higher proportions of 22(OH)D3 and 20,22(OH) ₂ D3. We also detected endogenous production of 20(OH)D3, 22(OH) D3, 20,23(OH) ₂ D3, 20,22(OH) ₂ D3, and 17,20,23(OH) ₃ D3 by immortalized human keratinocytes. Thus, we provide in vivo evidence for novel pathways of D3 metabolism initiated by CYP11A1, with the product profile showing organ/cell type specificity and being modified by CYP27B1 activity. These findings define the pathway intermediates as natural products/endogenous bioregulators and break the current dogma that vitamin D is solely activated through the sequence D3 → 25(OH)D3 → 1,25(OH) ₂ D3.—Slominski, A. T., Km, T.‐K., Shehabi, H. Z., Semak, I., Tang, E. K. Y., Nguyen, M. N., Benson, H. A. E., Korik, E., Janjetovic, Z., Chen, J., Yates, C. R., Postlethwaite, A. , Li, W., Tuckey, R. C. In vivo evidence for a novel pathway of vitamin D ₃ metabolism initiated by P450scc and modified by CYP27B1. FASEB J. 26, 3901–3915 (2012). www.fasebj.org

To investigate whether novel pathways of vitamin D3 (D3) and 7-dehydrocholesterol (7DHC) metabolism initiated by CYP11A1 and previously characterizedin vitro, occurin vivo, we analyzed samples of human serum and epidermis and pig adrenals for the presence of intermediates and products of these pathways. We extracted human epidermis from 13 individuals and sera from 13 individuals and analyzed them by LC/qTOF-MS alongside the corresponding standards. Pig adrenal glands were also analyzed for these steroids and secosteroids. Epidermal, serum and adrenal samples showed the presence of D3 hydroxy-derivatives corresponding to 20(OH)D3, 22(OH)D3, 25(OH)D3, 1,25(OH)₂D3, 20,22(OH)₂D3, 20,23(OH)₂D3, 20,24(OH)₂D3, 20,25(OH)₂D3, 20,26(OH)₂D3, 1,20,23(OH)₃D3 and 17,20,23(OH)₃D3, plus 1,20(OH)₂D3 which was detectable only in the epidermis. Serum concentrations of 20(OH)D3 and 22(OH)D3 were only 30- and 15-fold lower than 25(OH)D3, respectively and at levels above those required for biological activity as measuredin vitro. We also detected 1,20,24(OH)₃D3, 1,20,25(OH)₃D3 and 1,20,26(OH)₃D3 in the adrenals. Products of CYP11A1 action on 7DHC, namely 22(OH)7DHC, 20,22(OH)₂7DHC and 7-dehydropregnenolone were also detected in serum, epidermis and the adrenal. Thus, we have detected novel CYP11A1-derived secosteroids in the skin, serum and adrenal gland and based on their concentrations and biological activity suggest that they act as hormonesin vivo.

RORα and RORγ are expressed in human skin cells that produce the noncalcemic 20‐hydroxyvitamin D ₃ [20(OH)D ₃ ] and 20,23‐dihydroxyvitamin D ₃ [20,23(OH) ₂ D ₃ ]. Chinese hamster ovary (CHO) cells stably expressing a Tet‐on RORα or RORγ expression vector and a ROR‐responsive element (RORE)‐LUC reporter, and a mammalian 2‐hybrid model examining the interaction between the ligand binding domain (LBD) of RORα or RORγ with an LBD‐interacting LXXLL‐peptide, were used to study ROR‐antagonist activities. These assays revealed that 20(OH)D ₃ and 20,23(OH) ₂ D ₃ function as antagonists of RORα and RORγ. Moreover, 20(OH)D ₃ inhibited the activation of the promoter of the Bmal1 and G6pase genes, targets of RORα, and 20(OH)D ₃ and 20,23(OH) ₂ D ₃ inhibited Il17 promoter activity in Jurkat cells overexpressing RORα or RORγ. Molecular modeling using crystal structures of the LBDs of RORα and RORγ revealed docking scores for 20(OH)D ₃ , 20, 23(OH) ₂ D ₃ and 1,25(OH) ₂ D ₃ similar to those of the natural ligands, predicting good binding to the receptor. Notably, 20(OH)D ₃ , 20,23(OH) ₂ D ₃ , and 1,25(OH) ₂ D ₃ inhibited RORE‐mediated activation of a reporter in keratinocytes and melanoma cells and inhibited IL‐17 production by immune cells. Our study identifies a novel signaling pathway, in which 20(OH)D ₃ and 20,23(OH) ₂ D ₃ act as antagonists or inverse agonists of RORα and RORγ, that opens new possibilities for local (skin) or systemic regulation.—Slominski, A. T., Kim, T.‐K., Takeda, Y., Janjetovic, Z., Brożyna, A. A., Skobowiat, C., Wang, J., Postlethwaite, A., Li, W., Tuckey, R. C., Jetten, A. M. RORα and ROR γ are expressed in human skin and serve as receptors for endogenously produced noncalcemic 20‐hydroxy‐ and 20,23‐dihydroxyvitamin D. FASEB J . 28, 2775–2789 (2014). www.fasebj.org