Inhibition of 1,25-dihydroxyvitamin D3 stimulated osteocalcin gene transcription by tumor necrosis factor-alpha: structural determinants within the vitamin D response element

Abstract

Control of osteoblast function requires the coordinate activity of systemic and local regulatory factors. We have investigated the mechanism of interaction between the secosteroid 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and the cytokine tumor necrosis factor-alpha (TNF-alpha) by measuring their effects on two 1,25-(OH)2D3 responsive matrix protein genes, osteocalcin (OC) and osteopontin (OP). Our previous studies revealed that an inhibitory effect of TNF-alpha on 1,25-(OH)2D3-stimulated OC gene transcription is conferred by the same 25 base pair region of 5'-flanking DNA that confers a response to vitamin D (VDRE). Gel mobility shift studies of [32P]VDRE binding to ROS 17/2.8 cell nuclear extract revealed that TNF-alpha inhibits 1,25-(OH)2D3 stimulated formation of specific retinoid X receptor/vitamin D receptor (RXR/VDR)-DNA complexes in vitro. To determine if TNF-alpha was inhibiting nuclear protein-VDRE binding by modulation of VDR availability, we measured intranuclear VDR in cells treated with 1,25-(OH)2D3 (10(-8) M), TNF-alpha (100 ng/ml), or both, by western blot. 1,25-(OH)2D3 caused upregulation of the nuclear VDR. Treatment with TNF-alpha inhibited the 1,25-(OH)2D3-stimulated up-regulation of VDR nuclear protein content. However, down-regulation of VDR was unlikely to be the mechanism of TNF-alpha action because TNF-alpha had no effect on 1,25-(OH)2D3 stimulation of steady state OP messenger RNA or transcription of an OP-VDRE-chloramphenicol acetyl transferase reporter construct. These results suggest that decreased VDR alone does not explain the mechanism of TNF-alpha action. VDRE structural requirements for TNF-alpha action were characterized by comparing binding of mutant and hybrid forms of mouse (m)OP-, rat (r)OC-, and human (h)OC-VDRE probes to nuclear protein from cells treated with 1,25-(OH)2D3 and/or TNF-alpha. These homologous vitamin D response elements differ in that an AP-1 sequence is included in the rOC-VDRE and hOC-VDRE but not in the OP-VDRE. Gel mobility shift analysis revealed that TNF-alpha inhibited 1,25-(OH)2D3 stimulation of nuclear protein binding to rOC-VDRE and hOC-VDRE to 59% and 69% of control, respectively, but had no effect on 1,25-(OH)2D3 stimulation of nuclear protein binding to OP-VDRE. The effect of TNF-alpha could not be conferred in a mutant OP-VDRE in which the rOC-VDRE AP-1 sequence was inserted.