Plasma membrane requirements for 1alpha,25(OH)2D3 dependent PKC signaling in chondrocytes and osteoblasts.
1,25-Dihydroxyvitamin D(3) [1alpha,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 1alpha,25(OH)(2)D(3) express PLA(2) activating protein (PLAA), suggesting a link between ERp60 and PLA(2). Increased PLA(2) results in increased arachidonic acid release and formation of lysophospholipid, which then activates phospholipase C beta (PLCbeta), 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 1alpha,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 1alpha,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 1alpha,25(OH)(2)D(3). The results show that ERp60 and PLAA are localized to 1alpha,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 1alpha,25(OH)(2)D(3) and reduces the ability of 1alpha,25(OH)(2)D(3) to regulate [(35)S]-sulfate incorporation.
Boyan
Barbara D
BD
Wallace H. Coulter Department of Biomedical Engineering at Georgia, Tech and Emory University, Georgia Institute of Technology, Atlanta, 30332-0363, USA. barbara.boyan@bme.gatech.edu
Wang
Liping
L
Wong
Kevin L
KL
Jo
Hanjoong
H
Schwartz
Zvi
Z
D000818:Animals; D002117:Calcitriol / Q000502:physiology*; D002462:Cell Membrane / Q000201:enzymology / Q000502:physiology*; D019902:Chondrocytes / Q000201:enzymology / Q000502:physiology*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D010006:Osteoblasts / Q000201:enzymology / Q000502:physiology*; D011493:Protein Kinase C / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D018167:Receptors, Calcitriol / Q000172:deficiency / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*
D018167:Receptors, Calcitriol; D011493:Protein Kinase C; D002117:Calcitriol