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-β (TGFβ) inhibits 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-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. TGFβ abrogated the 1,25-(OH)2D3 enhanced transcription of both the OC and OP genes. The inhibitory TGFβ response for each requires vitamin D response element (VDRE) sequences, although there are additional contributions from 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 TGFβ significantly reduces induction of the heterodimeric VDR/RXR complexes in 1,25-(OH)2D3-treated ROS 17/2.8 cells. However, Western blot and ligand binding analyses reveal that TGFβ does not affect nuclear availability of the VDR. We also show that activator protein-1 activity is up-regulated by TGFβ; thus, activator protein-1 binding sites in the OC promoter may potentially contribute to inhibitory effects of TGFβ on basal transcription. Our studies demonstrate that the inhibitory action of TGFβ on the 1,25-(OH)2D3 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 TGFβ and 1,25-(OH)2D3 to VDR-mediated gene regulatory mechanisms operative in bone formation and/or resorption events.
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