Differential gene regulation by a synthetic vitamin D receptor ligand and active vitamin D in human cells
Vitamin D (VD) exerts a wide variety of biological functions including calcemic activity. VD nutritional status is closely associated with the onset and development of chronic diseases. To develop a VD analog with the desired VD activity but without calcemic activity, we screened synthetic VDR antagonists. We identified DLAM-2b  as a VDR ligand in a competitive VDR binding assay for 1a,25(OH)2 vitamin D3 (1,25D3), and DLAM-2b  showed an antagonistic effect on 1,25D3-induced cell differentiation in HL60 cells. In a luciferase reporter assay in which human VDR was exogenously expressed in cultured COS1 cells, DLAM-2b acted as a transcriptional antagonist. Consistently, DLAM-2b had an antagonistic effect on the 1,25D3-induced expression of a known VD target gene (CYP24A1), and VDR-bound DLAM-2b was recruited to an endogenous VD response element in chromatin in human keratinocytes (HaCaT) and human colon cancer cells (HCT116) endogenously expressing VDR. In an ATAC-seq assay, the effects of 1,25D3 and DLAM-2b on chromatin reorganization were undetectable in HCT116 cells, while the effect of an androgen receptor (AR) antagonist (bicalutamide) was confirmed in prostate cancer cells (LNCaP) expressing endogenous AR. However, whole genome analysis using RNA-seq and ATAC-seq revealed differential gene expression profiles regulated by DLAM-2b versus 1,25D3. The upregulated and downregulated genes only partially overlapped between cells treated with 1,25D3 and those treated with DLAM-2b. Thus, the present findings illustrate a novel VDR ligand with gene regulatory activity differing from that of 1,25D3. Overall design: Examination of 3 different method in 1 cell types.