TUMOR-NECROSIS-FACTOR-ALPHA DECREASES 1,25-DIHYDROXYVITAMIN D(3) RECEPTORS IN OSTEOBLASTIC ROS 17 2.8 CELLS

Bone remodeling is a complex process regulated by systemic hormones, l ocal cytokines, and growth factors. One cytokine, tumor necrosis facto r alpha (TNF-alpha), is known to have potent inhibitory effects on ost eoblast matrix protein production and to stimulate osteoclast recruitm ent. We have previously shown that TNF-alpha inhibits 1,25-(OH)2D3-sti mulated synthesis of bone gla protein (BGP), an abundant and osteoblas t-specific matrix constituent. We hypothesized that the mechanism of T NF-alpha action included inhibition of intracellular 1,25-(OH)2D3 rece ptor (VDR) number or function. To test this, the osteoblastic cell lin e ROS 17/2.8 was cultured in the presence or absence of TNF-alpha (100 ng/ml), and binding of [H-3]1,25-(OH)2D3 to 0.3 M KCl extracts of cyt osol was measured by equilibrium assay. Specific [H-3]1,25-(OH)2D3 bin ding decreased 70%, 25 h after addition of TNF-alpha. The decrease in [H-3]1,25-(OH)2D3 binding was seen by 18 h, was sustained throughout t he 72 h culture period, and was greater in low-density cultures. Scatc hard analysis confirmed that TNF-alpha (100 ng/ml for 24 h) caused a d ecrease in the number of binding sites without change in VDR affinity. Northern analysis with a VDR fiboprobe revealed that the decrease in VDR occurred without a change in the 4.4 kb steady-state VDR mRNA [VDR /cyclophilin mRNA signal ratio: control, 2.25; TNF-alpha, 2.24 (24 h), 2.17 (40 h), n = 2 flasks/time point]. These results suggest that TNF- alpha action on osteoblastic cells includes an inhibitory effect on VD R number st a point distal to the synthesis of VDR mRNA.