Plasma membrane requirements for 1α,25(OH)2D3 dependent PKC signaling in chondrocytes and osteoblasts

1,25-Dihydroxyvitamin D-3 [1 alpha,25(OH)(2)D-3] acts on chondrocytes and osteoblasts through traditional nuclear Vitamin D receptor (VDR) mechanisms as well as through rapid actions on plasma membranes that initiate intracellular signaling pathways. We have investigated the mechanisms involved in activation of protein kinase C (PKC) and downstream biological responses that depend on the latter pathway. These studies show that PKC activation depends on presence of a membrane receptor ERp60 and rapid increases in phospholipase A(2) (PLA(2)) activity. Cells that are responsive to 1 alpha,25(OH)(2)D-3 express PLA2 activating protein (PLAA), suggesting a link between ERp60 and PLA2. Increased PLA2 results in increased arachidonic acid release and formation of lysophospholipid, which then activates phospholipase C beta (PLC beta), leading to rapid formation of inositol-trisphosphate (IP3) and diacylglycerol (DAG). PLA(2), PLC, and DAG are all associated with lipid rafts including caveolae in many cells, suggesting that the caveolar environment may be an important mediator of PKC activation by 1 alpha,25(OH)(2)D-3. Here, we use the VDR-/- mouse costochondral cartilage growth plate to examine the expression of ERp60 and PLAA in vivo in 1 alpha,25(OH)(2)D-3-responsive hypertrophic chondrocytes (growth zone cells) and in resting zone cells that do not respond to this Vitamin D metabolite in vitro. In addition, we determined if intact lipid rafts are required for the response of rat costochondral cartilage growth zone cells to 1 alpha,25(OH)(2)D-3. The results show that ERp60 and PLAA are localized to 1 alpha,25 (OH)(2)D-3-responsive growth zone cells and metaphyseal osteoblasts, even in VDR-/- mice. Disruption of lipid rafts using beta-cyclodextrin blocks the activation of PKC by 1 alpha,25(OH)(2)D-3 and reduces the ability of 1 alpha,25(OH)(2)D-3 to regulate [S-35]-sulfate incorporation. @ 2005 Elsevier Inc. All rights reserved.