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

Methods
Data were analyzed with Ingenuity Pathway Analysis (Ingenuity® Systems, www.ingenuity.com). For generating networks, a data set containing gene identifiers and corresponding expression values was uploaded into the application. Each identifier was mapped to its corresponding object in Ingenuity’s Knowledge Base. A fold change cutoff of +/-2 was set to identify molecules whose expression was significantly differentially regulated. These molecules, called Network Eligible molecules, were overlaid onto a global molecular network developed from information contained in Ingenuity’s Knowledge Base. Networks of Network Eligible Molecules were then algorithmically generated based on their connectivity. The Functional Analysis identified the biological functions and/or diseases that were most significant to the entire data set. Molecules from the dataset that met the fold change cutoff of +/-2 and were associated with biological functions and/or diseases in Ingenuity’s Knowledge Base were considered for the analysis. Right-tailed Fisher’s exact test was used to calculate the p-value determining the probability that each biological function and/or disease assigned to that data set is due to chance alone.

既往研究表明，细胞色素P450 11A1（CYP11A1）作用于维生素D3所生成的非血钙活性20-羟基维生素D3（20(OH)D3），在人皮肤成纤维细胞及博莱霉素诱导的硬皮病小鼠模型中展现出抗纤维化活性。本研究针对皮肤表达的视黄酸相关孤儿受体γ（RORγ），探究其在CYP11A1来源开环甾体类化合物（secosteroids）生物学效应中的作用；实验所用小鼠成纤维细胞分离自野生型（RORg+/+）、基因敲除型（RORg-/-）及杂合子型（RORg+/-）小鼠皮肤组织。
CYP11A1衍生的20(OH)D3、20,23-二羟基维生素D3（20,23(OH)2D3）、1,20-二羟基维生素D3（1,20(OH)2D3）及1,20,23-三羟基维生素D3（1,20,23(OH)3D3）可通过剂量依赖性方式抑制RORγ+/+成纤维细胞的增殖，其效能与1,25-二羟基维生素D3（1,25(OH)2D3）相当。令人意外的是，该效应在RORg+/-及RORg-/-成纤维细胞中发生逆转，其中RORg-/-成纤维细胞中观察到最为显著的促增殖效应。
所有受试类似物均可抑制RORγ+/+成纤维细胞中转化生长因子β1（TGF-β1）诱导的胶原合成及其他纤维化相关基因的表达，但该效应在RORg-/-成纤维细胞中被削弱甚至逆转。上述结果证实，维生素D羟基衍生物的抗增殖与抗纤维化活性依赖于功能完整的RORγ。经20(OH)D3或1,20(OH)2D3处理后，RORg-/-小鼠与野生型小鼠成纤维细胞的转录组（transcriptomes）出现显著变化，该现象可为解释所观察到的表型差异提供至少部分分子层面的依据。

方法
本研究数据采用Ingenuity经典通路分析（Ingenuity Pathway Analysis，Ingenuity® Systems，www.ingenuity.com）进行分析。构建调控网络时，将包含基因标识符及对应表达值的数据集上传至该分析软件，每个标识符均匹配至Ingenuity知识库中的对应分子。设置±2倍表达变化阈值以筛选出表达显著差异的分子，此类分子被称为符合网络分析条件的分子（Network Eligible molecules），随后将其叠加至基于Ingenuity知识库信息构建的全局分子网络中。基于分子间的连接关系，通过算法生成符合网络分析条件的分子所构成的调控网络。
功能富集分析可识别与整个数据集关联最显著的生物学功能及/或疾病。本分析纳入满足±2倍表达变化阈值，且与Ingenuity知识库中生物学功能及/或疾病相关的数据集分子。采用右侧费舍尔精确检验计算P值，以判断每个关联的生物学功能及/或疾病的显著性是否仅由随机误差导致。