Antagonistic effects of transforming growth factor-beta on vitamin D-3, enhancement of osteocalcin and osteopontin transcription: Reduced interactions of vitamin D receptor retinoid X receptor complexes with vitamin D response elements

Osteocalcin and osteopontin are noncollagenous proteins secreted by osteoblasts and regulated by a complex interplay of systemic and locally produced factors, including growth factors and steroid hormones. We investigated the mechanism by which transforming growth factor-beta (TGF beta) inhibits 1,25-dihydroxyvitamin D-3 (1,25-(OH)(2)D-3)-enhanced expression of the osteocalcin (OC) and osteopontin (OP) genes. ROS 17/2.8 cells, in which both genes are expressed, were transfected with reporter constructs driven by native (i.e. wild-type) rat OC and mouse OP promoters. TGF beta abrogated the 1,25-(OH)(2)D-3 enhanced transcription of both the OC and OP genes. The inhibitory TGF beta response for each requires vitamin D response element (VDRE) sequences, although there are additional contributions hom proximal basal regulatory elements. These transcriptional effects were further investigated for contribution of the trans-activating factors, which interact with OC and OP VDREs, involving the vitamin D receptor (VDR) and retinoid X receptor (RXR). Gel mobility shift assays show that TGF beta significantly reduces induction of the heterodimeric VDR/RXR complexes in 1,25-(OH)(2)D-3-treated ROS 17/2.8 cells. However, Western blot and ligand binding analyses reveal that TGF beta does not affect nuclear availability of the VDR. We also show that activator protein-1 activity is up-regulated by TGF beta; thus, activator protein-1 binding sites in the OC promoter may potentially contribute to inhibitory effects of TGF beta on basal transcription. Our studies demonstrate that the inhibitory action of TGF beta on the 1,25-(OH)(2)D-3 enhancement of OC and OP transcription in osteoblastic cells results from modulations of protein-DNA interactions at the OC and OP VDRE, which cannot be accounted for by changes in VDR protein levels. As OC and OP participate in bone turnover, our results provide insight into the contributions of TGF beta and 1,25-(OH)(2)D-3 to VDR-mediated gene regulatory mechanisms operative in bone formation and/or resorption events.