Antagonistic effects of transforming growth factor-beta on vitamin D3 enhancement of osteocalcin and osteopontin transcription: reduced interactions of vitamin D receptor/retinoid X receptor complexes with vitamin E 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 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. TGF beta abrogated the 1,25-(OH)2D3 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 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 TGF beta significantly reduces induction of the heterodimers VDR/RXR complexes in 1,25-(OH)2D3-treated ROS 17/2.8 cells. However, Western blot and ligand binding analysis 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)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 TGF beta and 1,25-(OH)2D3 to VDR-mediated gene regulatory mechanism operative in bone formation and/or resorption events

Pulsed Direct Current Electric Fields Enhance Osteogenesis in Adipose-Derived Stromal Cells

Adipose-derived stromal cells (ASCs) constitute a promising source of cells for regenerative medicine applications. Previous studies of osteogenic potential in ASCs have focused on chemicals, growth factors, and mechanical stimuli. Citing the demonstrated role electric fields play in enhancing healing in bone fractures and defects, we investigated the ability of pulsed direct current electric fields to drive osteogenic differentiation in mouse ASCs. Employing 50 Hz direct current electric fields in concert with and without osteogenic factors, we demonstrated increased early osteoblast-specific markers. We were also able to establish that commonly reported artifacts of electric field stimulation are not the primary mediators of the observed effects. The electric fields caused marked changes in the cytoskeleton. We used atomic force microscopy–based force spectroscopy to record an increase in the cytoskeletal tension after treatment with electric fields. We abolished the increased cytoskeletal stresses with the rho-associated protein kinase inhibitor, Y27632, and did not see any decrease in osteogenic gene expression, suggesting that the pro-osteogenic effects of the electric fields are not transduced via cytoskeletal tension. Electric fields may show promise as candidate enhancers of osteogenesis of ASCs and may be incorporated into cell-based strategies for skeletal regeneration