Slominski Andrzej T, Kim Tae-Kang, Hobrath Judith V, Oak Allen S W, Tang Edith K Y, Tieu Elaine W, Li Wei, Tuckey Robert C, Jetten Anton M
Department of Dermatology, USA; Comprehensive Cancer Center, Cancer Chemoprevention Program, University of Alabama at Birmingham, USA; Pathology and Laboratory Medicine Service, VA Medical Center, Birmingham, AL, 35249, USA.
Department of Dermatology, USA.
J Steroid Biochem Mol Biol. 2017 Oct;173:42-56. doi: 10.1016/j.jsbmb.2016.09.024. Epub 2016 Sep 28.
The classical pathway of vitamin D activation follows the sequence D3→25(OH)D3→1,25(OH)D3 with the final product acting on the receptor for vitamin D (VDR). An alternative pathway can be started by the action of CYP11A1 on the side chain of D3, primarily producing 20(OH)D3, 22(OH)D3, 20,23(OH)D3, 20,22(OH)D3 and 17,20,23(OH)D3. Some of these metabolites are hydroxylated by CYP27B1 at C1α, by CYP24A1 at C24 and C25, and by CYP27A1 at C25 and C26. The products of these pathways are biologically active. In the epidermis and/or serum or adrenals we detected 20(OH)D3, 22(OH)D3, 20,22(OH)D3, 20,23(OH)D3, 17,20,23(OH)D3, 1,20(OH)D3, 1,20,23(OH)D3, 1,20,22(OH)D3, 20,24(OH)D3, 1,20,24(OH)D3, 20,25(OH)D3, 1,20,25(OH)D3, 20,26(OH)D3 and 1,20,26(OH)D3. 20(OH)D3 and 20,23(OH)D3 are non-calcemic, while the addition of an OH at C1α confers some calcemic activity. Molecular modeling and functional assays show that the major products of the pathway can act as "biased" agonists for the VDR with high docking scores to the ligand binding domain (LBD), but lower than that of 1,25(OH)D3. Importantly, cell based functional receptor studies and molecular modeling have identified the novel secosteroids as inverse agonists of both RORα and RORγ receptors. Specifically, they have high docking scores using crystal structures of RORα and RORγ LBDs. Furthermore, 20(OH)D3 and 20,23(OH)D3 have been tested in a cell model that expresses a Tet-on RORα or RORγ vector and a RORE-LUC reporter (ROR-responsive element), and in a mammalian 2-hybrid model that test interactions between an LBD-interacting LXXLL-peptide and the LBD of RORα/γ. These assays demonstrated that the novel secosteroids have ROR-antagonist activities that were further confirmed by the inhibition of IL17 promoter activity in cells overexpressing RORα/γ. In conclusion, endogenously produced novel D3 hydroxy-derivatives can act both as "biased" agonists of the VDR and/or inverse agonists of RORα/γ. We suggest that the identification of large number of endogenously produced alternative hydroxy-metabolites of D3 that are biologically active, and of possible alternative receptors, may offer an explanation for the pleiotropic and diverse activities of vitamin D, previously assigned solely to 1,25(OH)D3 and VDR.
维生素D激活的经典途径遵循D3→25(OH)D3→1,25(OH)D3的顺序，最终产物作用于维生素D受体（VDR）。另一条途径可由CYP11A1作用于D3的侧链启动，主要产生20(OH)D3、22(OH)D3、20,23(OH)D3、20,22(OH)D3和17,20,23(OH)D3。这些代谢产物中的一些在C1α处被CYP27B1羟基化，在C24和C25处被CYP24A1羟基化，在C25和C26处被CYP27A1羟基化。这些途径的产物具有生物活性。在表皮和/或血清或肾上腺中，我们检测到了20(OH)D3、22(OH)D3、20,22(OH)D3、20,23(OH)D3、17,20,23(OH)D3、1,20(OH)D3、1,20,23(OH)D3、1,20,22(OH)D3、20,24(OH)D3、1,20,24(OH)D3、20,25(OH)D3、1,20,25(OH)D3、20,26(OH)D3和1,20,26(OH)D3。20(OH)D3和20,23(OH)D3无血钙活性，而在C1α处添加一个OH则赋予一定的血钙活性。分子建模和功能测定表明，该途径的主要产物可作为VDR的“偏向性”激动剂，与配体结合域（LBD）的对接分数较高，但低于1,25(OH)D3。重要的是，基于细胞的功能性受体研究和分子建模已将这些新型甾醇类化合物鉴定为RORα和RORγ受体的反向激动剂。具体而言，使用RORα和RORγ LBD的晶体结构时，它们具有较高的对接分数。此外，20(OH)D3和20,23(OH)D3已在表达Tet-on RORα或RORγ载体以及RORE-LUC报告基因（ROR反应元件）的细胞模型中，以及在测试LBD相互作用的LXXLL肽与RORα/γ的LBD之间相互作用的哺乳动物双杂交模型中进行了测试。这些测定表明，这些新型甾醇类化合物具有ROR拮抗剂活性，在过表达RORα/γ的细胞中对IL17启动子活性的抑制进一步证实了这一点。总之，内源性产生的新型D3羟基衍生物既可以作为VDR 的“偏向性”激动剂，也可以作为RORα/γ的反向激动剂。我们认为，大量内源性产生的具有生物活性的D3替代羟基代谢产物以及可能的替代受体的鉴定，可能为维生素D的多效性和多样活性提供一种解释，而此前这些活性仅归因于1,25(OH)D3和VDR。
