1,25-Dihydroxyvitamin D3 stimulated protein kinase C phosphorylation of type VI adenylyl cyclase inhibits parathyroid hormone signal transduction in rat osteoblastic UMR 106-01 cells

Abstract

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) treatment of osteoblastic cells was shown previously to attenuate Parathyroid hormone (PTH) response by inhibiting adenylyl cyclase (AC) activity. In this study, we have investigated the mechanism by which 1,25(OH)2D 3 inhibits AC in rat osteoblastic UMR 106-01 cells. 1,25(OH) 2D3 treatment inhibited both PTH and forskolin-stimulated AC activity by 25%-50% within 12 min in a concentration-dependent manner suggesting a direct inhibition of the AC enzyme. Treatment with 25(OH)D 3 had no effect on basal or stimulated AC activity. We determined the profile of AC subtypes expressed in UMR cells and found AC VI to be the dominant subtype accounting for 50% of AC mRNA. Since AC VI can be inhibited by protein kinase C (PKC) phosphorylation, we examined 1,25(OH)2D 3 activation of various PKC isoforms. 1,25(OH)2D 3 increased the membrane translocation of PKC-β1, -δ, and -ζ with a concomitant increase in PKC activity. The translocation of PKC-β1 and -δ was blocked by the PLC inhibitor U73122 whereas that of PKC-ζ was abolished by the PI-3 kinase inhibitor wortmannin. The attenuation of cAMP production by 1,25(OH)2D3 was antagonized by the PKC inhibitors Gö6850, calphostin C, and wortmannin, but not by a calmodulin kinase II (CaMKII) inhibitor. Treatment with 1,25(OH) 2D3 for 20 min increased AC VI phosphorylation by 10.8-fold and this was blocked partially by Gö6850 and partially by wortmannin but was unaffected by CaMKII inhibitor. These results demonstrate that 1,25(OH)2D3 activation of PKC isoforms leads to phosphorylation of AC VI and inhibition of PTH-activation of this pathway in osteoblasts. © 2004 Wiley-Liss, Inc.