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 os teoblasts and regulated by a complex interplay of systemic and locally produced factors, including growth factors and steroid hormones. We i nvestigated the mechanism by which transforming growth factor-beta (TG F 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 re porter constructs driven by native (i.e. wild-type) rat OC and mouse O P promoters. TGF beta abrogated the 1,25-(OH)(2)D-3 enhanced transcrip tion of both the OC and OP genes. The inhibitory TGF beta response for each requires vitamin D response element (VDRE) sequences, although t here are additional contributions hom proximal basal regulatory elemen ts. These transcriptional effects were further investigated for contri bution 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 redu ces 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 a nalyses reveal that TGF beta does not affect nuclear availability of t he VDR. We also show that activator protein-1 activity is up-regulated by TGF beta; thus, activator protein-1 binding sites in the OC promot er may potentially contribute to inhibitory effects of TGF beta on bas al transcription. Our studies demonstrate that the inhibitory action o f TGF beta on the 1,25-(OH)(2)D-3 enhancement of OC and OP transcripti on in osteoblastic cells results from modulations of protein-DNA inter actions at the OC and OP VDRE, which cannot be accounted for by change s in VDR protein levels. As OC and OP participate in bone turnover, ou r results provide insight into the contributions of TGF beta and 1,25- (OH)(2)D-3 to VDR-mediated gene regulatory mechanisms operative in bon e formation and/or resorption events.