Sylvia V L, Schwartz Z, Ellis E B, Helm S H, Gomez R, Dean D D, Boyan B D
Department of Orthopaedics, University of Texas Health Science Center at San Antonio 78284, USA.
J Cell Physiol. 1996 Jun;167(3):380-93. doi: 10.1002/(SICI)1097-4652(199606)167:3<380::AID-JCP2>3.0.CO;2-L.
Prior studies have shown that vitamin D regulation of protein kinase C activity (PKC) in the cell layer of chondrocyte cultures is cell maturation-dependent. In the present study, we examined the membrane distribution of PKC and whether 1 alpha,25-(OH)2D3 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-1 and inhibited by a PKC-specific pseudosubstrate inhibitor peptide. Depletion of membrane PKC activity using isoform-specific anti-PKC antibodies suggested that PKC alpha is the major isoform in cell layer lysates as well as in plasma membranes isolated from both cell types; PKC zeta 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 PKC zeta-specific pseudosubstrate inhibitor peptide. The presence of PKC zeta 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 membrane-specific 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, PKC zeta may be important in nongenomic, autocrine signal transduction at sites distal from the cell.
先前的研究表明，维生素D对软骨细胞培养物细胞层中蛋白激酶C活性（PKC）的调节是细胞成熟依赖性的。在本研究中，我们检测了PKC的膜分布，以及1α,25-(OH)2D3和24R,25-(OH)2D3是否能直接调节分离的质膜和细胞外基质小泡中的酶活性。基质小泡PKC被苔藓抑素-1激活，并被PKC特异性假底物抑制剂肽抑制。使用亚型特异性抗PKC抗体耗尽膜PKC活性表明，PKCα是细胞层裂解物以及从两种细胞类型分离的质膜中的主要亚型；PKCζ是基质小泡中的主要形式。这在免疫沉淀的蛋白质印迹以及使用对照肽阻断亚型特异性抗体与酶的结合和使用PKCζ特异性假底物抑制剂肽的研究中得到了证实。免疫电子显微镜进一步证实了基质小泡中存在PKCζ。基质小泡中的酶活性对外源脂质不敏感，而质膜中的酶活性需要脂质才能完全发挥活性。当直接添加到分离的膜组分中时，1,25-(OH)2D3和24,25-(OH)2D3抑制基质小泡PKC，但刺激质膜PKC。基质小泡中的PKC活性不依赖钙，而质膜中的PKC活性需要钙。此外，基质小泡中对维生素D敏感的PKC不依赖钙，而质膜中对维生素D敏感的酶依赖钙。结论是，PKC亚型在基质小泡和质膜之间存在差异分布，并且酶活性以膜特异性方式调节。这表明存在一种非基因组机制，可以通过PKC介导1,25-(OH)2D3和24,25-(OH)2D3的作用。此外，PKCζ可能在远离细胞的部位的非基因组自分泌信号转导中起重要作用。
