Prior studies have shown that vitamin D regulation of protein kinase C activity (PKC) in the cell layer of chondrocyte cultures is cell matu ration-dependent, In the present study, we examined the membrane distribution of PKC and whether 1α,25-(OH),D, and 24R,25-(OH)2D3 can directly regulate enzyme activity in isolated plasma membranes and extracellular matrix vesicles. Matrix vesicle PKC was activated by bryostatin-l and inhibited by a PKC-specific pseudosubstrate inhibitor peptide. Depletion of membrane PKC activity using isoform-specific anti-PKC antibodies suggested that PKCα is the major isoform in cell layer lysates as well as in plasma membranes isolated from both cell types; PKCζ is the predominant form in matrix vesicles. This was confirmed in Western blots of immunoprecipitates as well as in studies using control peptides to block binding of the isoform specific antibody to the enzyme and using a PKCζ-specific pseudosubstrate inhibitor peptide. The presence of PKCζ in matrix vesicles was further verified by immunoelectron microscopy. Enzyme activity in the matrix vesicle was insensitive to exogenous lipid, whereas that in the plasma membrane required lipid for full activity. 1,25-(OH)2D3 and 24,25-(OH)2D3 inhibited matrix vesicle PKC, but stimulated plasma membrane PKC when added directly to the isolated membrane fractions. PKC activity in the matrix vesicle was calcium-independent, whereas that in the plasma membrane required calcium. Moreover, the vitamin D-sensitive PKC in matrix vesicles was not dependent on calcium, whereas the vitamin D-sensitive enzyme in plasma membranes was calcium-dependent. It is concluded that PKC isoforms are differentially distributed between matrix vesicles and plasma membranes and that enzyme activity is regulated in a membranespecific manner. This suggests the existence of a nongenomic mechanism whereby the effects of 1,25-(OH)2D3 and 24,25-(OH)2D3 may be mediated via PKC. Further, PKCζ may be important in nongenomic, autocrine signal transduction at sites distal from the cell.
|Original language||English (US)|
|Number of pages||14|
|Journal||Journal of Cellular Physiology|
|State||Published - Jun 1 1996|
ASJC Scopus subject areas
- Clinical Biochemistry
- Cell Biology