24,25-(OH)(2)D-3 REGULATION OF MATRIX VESICLE PROTEIN-KINASE-C OCCURSBOTH DURING BIOSYNTHESIS AND IN THE EXTRACELLULAR-MATRIX

Plasma membranes and matrix vesicles isolated from rat costochondral r esting zone chondrocyte cultures contain predominantly protein kinase C alpha (PKC alpha) and PKC zeta, respectively, and the level of PKC s pecific activity in these membrane fractions is regulated by 24,25-(OH )(2)D-3 [14]. In the present study, we examined whether the effect of 24,25-(OH)(2)D-3 on membrane PKC is via genomic mechanisms during biog enesis and through a nongenomic the matrix vesicles are resident in th e matrix. There was a dose-dependent decrease in matrix vesicle PKC sp ecific activity and a significant increase in plasma membrane enzyme a ctivity in cultures treated for 90 minutes with 10(-9)-10(-7) M 24,25- (OH)(2)D-3. However, at 12 hours, matrix vesicle PKC was stimulated, b ut no effect was seen in the plasma membranes, suggesting that the eff ect seen at 90 minutes was due to a direct action of the hormone on PK C activity in the membrane, and that the effect seen at 12 hours was d ue to new matrix vesicle production with altered PKC content. Neither actinomycin D nor cycloheximide inhibited matrix vesicle PKC at 30, 60 , or 90 minutes, but by 12 hours, these inhibitors blocked the effect of the hormone. 24,25-(OH)(2)D-3-dependent plasma membrane PKC was sen sitive to both actinomycin D and cycloheximide at early time points, b ut by 12 hours, no effect of the inhibitors was seen. Monensin did not alter basal plasma membrane PKC activity or the 24,25-(OH)(2)D-3-depe ndent increase, suggesting that this increase was due to translocation of cytosolic PKC rather than new membrane synthesis. Monensin did not affect matrix vesicle PKC at early time points, but it decreased 24,2 5-(OH)(2)D-3-dependent enzyme activity at later times, indicating that new matrix vesicle production was blocked. At least part of the effec t of 24,25-(OH)(2)D-3 on PKC involved phospholipase A(2) (PA(2)). Quin acrine (a PA(2) inhibitor) alone had no effect on matrix vesicle PKC, but in cultures treated for 12 hours with quinacrine and 24,25(OH)(2)D -3, a synergistic increase in matrix vesicle PKC was observed. Quinacr ine caused a time-dependent decrease in matrix vesicle PKC and a dose- and time-dependent increase in plasma membrane PKC when incubated dire ctly with the membranes, supporting the hypothesis that PA(2) plays a role in the nongenomic regulation of PKC by 24,25(OH)(2)D-3. Experimen ts using anti-isoform specific antibodies showed that 24,25-(OH)(2)D-3 modulated the distribution of PKC alpha, beta, and zeta between the p lasma membrane and matrix vesicle compartments via translocation and n ew PKC synthesis. Thus, the data support the hypothesis that 24,25(OH) (2)D-3 regulates matrix vesicles through two pathways: a genomic one a t the stage of biosynthesis and packaging, and a second nongenomic mec hanism acting directly upon matrix vesicles in the matrix. These data also indicate that matrix vesicle regulation consists of complex event s with several different points of regulation.