Phospholipase A2 activating protein (PLAA) is required for 1α,25(OH)2D3 signaling in growth plate chondrocytes

Abstract

Phospholipase A2 (PLA2) is pivotal in the rapid membrane-mediated actions of 1,25-dihydroxyvitamin D3 [1α,25(OH) 2D3]. Microarray analysis indicated that PLA2 activating protein (PLAA) mRNA is upregulated 6-fold before rat growth plate cells exhibit 1α,25(OH)2D3-dependent protein kinase C (PKC) increases, suggesting that it plays an important role in 1α,25(OH)2D3's mechanism of action. PLAA mRNA was confirmed in 1α,25(OH)2D3-responsive growth zone (prehypertrophic and upper hypertrophic cell zones) chondrocytes by RT-PCR and Northern blot in vitro and by in situ hybridization in vivo. PLAA protein was shown by Western blot and immunohistochemistry. PLAAs role in 1α,25(OH)2D3 signaling was evaluated in growth zone cell cultures using PLAA peptide. Arachidonic acid release was increased as was PLA2-specific activity in plasma membranes and matrix vesicles. PKCα, but not PKCβ, PKCε, or PKCζ, was increased. PLAAs effect was comparable to that of 1α,25(OH)2D3 and was additive with 1α,25(OH)2D3. PLA2 inhibitors quinacrine and AACOCF3, and cyclooxygenase inhibitor indomethacin blocked the effect of PLAA peptide on PKC, indicating arachidonic acid and its metabolites were involved. This was confirmed using exogenous arachidonic acid. Prostaglandin acted via EP1 based on inhibition by SC19220 and not via EP2 since AH6809 had no effect. Like 1α,25(OH)2D 3, PLAA peptide also increased activity of phospholipase C-specific activity via beta-1 and beta-3 isoforms, but not delta-1 or gamma-1; the effect of PLAA was via lysophospholipid but not via arachidonic acid. PLAA peptide decreased [3H]-thymidine incorporation to 50% of the decrease caused by 1α,25(OH)2D3. In contrast, PLAA peptide increased alkaline phosphatase-specific activity and proteoglycan production in a manner similar to 1α,25(OH)2D3. This indicates that PLAA is a specific activator of PLA2 in growth plate chondrocytes, and suggests that it mediates the membrane effect of 1α,25(OH) 2D3, thereby modulating physiological response. © 2004 Wiley-Liss, Inc.