Supplemental tables from: Antifibrogenic activities of CYP11A1-derived vitamin D3-hydroxyderivatives are dependent on RORγ

Previous studies showed that non-calcemic 20(OH)D3, a product of CYP11A1 action on vitamin D3, has antifibrotic activity in human dermal fibroblasts and in a bleomycin mouse model of scleroderma. In this study we tested the role of RORγ, which is expressed in skin, in the action of CYP11A1-derived secosteroids using murine fibroblasts isolated from the skin of wild type (RORg^+/+), knock out (RORg^-/-) and heterozygote (RORg^+/-) mice. CYP11A1-derived 20(OH)D3, 20,23(OH)₂D3, 1,20(OH)₂D3, and 1,20,23(OH)₃D3 inhibited proliferation of RORγ^+/+ fibroblasts in a dose-dependent manner with a similar potency to 1,25(OH)₂D3. Surprisingly, this effect was reversed in RORγ^+/- and RORγ^-/- fibroblasts with the most pronounced stimulatory effect seen in RORγ^-/- fibroblasts. All of the analogs tested inhibited TGF-β1-induced collagen synthesis in RORγ^+/+ fibroblasts and the expression of other fibrosis-related genes. This effect was curtailed or reversed in RORγ^-/- fibroblasts. These results show that the antiproliferative and antifibrotic activities of the vitamin D hydroxy-derivatives are dependent on a functional RORγ. The dramatic changes in the transcriptomes of fibroblasts of RORg^-/- versus wild type mice following treatment with 20(OH)D3 or 1,20(OH)₂D3 provide a molecular basis to explain, at least in part, the observed phenotypic differences