1α,25(OH)2D3 regulates chondrocyte matrix vesicle protein kinase C (PKC) directly via G-protein-dependent mechanisms and indirectly via incorporation of PKC during matrix vesicle biogenesis

Abstract

Matrix vesicles are extracellular organelles involved in mineral formation that are regulated by 1α,25 (OH)2D3. Prior studies have shown that protein kinase C (PKC) activity is involved in mediating the effects of 1α,25(OH)2D3 in both matrix vesicles and plasma membranes. Here, we examined the regulation of matrix vesicle PKC by 1α,25(OH)2D3 during biogenesis and after deposition in the matrix. When growth zone costochondral chondrocytes were treated for 9 min with 1α,25 (OH)2D3, PKCζ in matrix vesicles was inhibited, while PKCα in plasma membranes was increased. In contrast, after treatment for 12 or 24 h, PKCζ in matrix vesicles was increased, while PKCα in plasma membranes was unchanged. The effect of 1α,25(OH)2D3 was stereospecific and metabolite-specific. Monensin blocked the increase in matrix vesicle PKC after 24 h, suggesting the secosteroid-regulated packaging of PKC. In addition, the 1α,25(OH)2D3 membrane vitamin D receptor (1,25-mVDR) was involved, since a specific antibody blocked the 1α,25(OH)2D3-dependent changes in PKC after both long and short treatment times. In contrast, antibodies to annexin II had no effect, and there was no evidence for the presence of the nuclear VDR on Western blots. To investigate the signaling pathways involved in regulating matrix vesicle PKC activity after biosynthesis, matrix vesicles were isolated and then treated for 9 min with 1α,25(OH)2D3 in the presence and absence of specific inhibitors. Inhibition of phosphatidylinositol-phospholipase C, phospholipase D, or Gi/Gs had no effect. However, inhibition of Gq blocked the effect of 1α,25(OH)2D3. The rapid effect of 1α,25(OH)2D3 also involved the 1,25-mVDR. Moreover, arachidonic acid was found to stimulate PKC when added directly to isolated matrix vesicles. These results indicate that matrix vesicle PKC is regulated by 1α,25(OH)2D3 at three levels: 1) during matrix vesicle biogenesis; 2) through direct action on the membrane; and 3) through production of other factors such as arachidonic acid.