Phospholipase A₂ activating protein is required for 1¦Á,25-dihydroxyvitamin D₃ dependent rapid activation of protein kinase C via Pdia3

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• 作者：Maryam Doroudi ; Zvi Schwartz ; Barbara D. Boyan
• 关键词：PLAA ; phospholipase A2 activating protein ; Pdia3 ; protein-disulfide isomerase-associated 3 ; PKC ; protein kinase C ; PLA2 ; phospholipase A2 ; PGE2 ; prostaglandin E2 ; AA ; arachidonic acid ; PLC ; phospholipase C ; BS3 ; bis(sulfosuccinimidyl) substrate
• 刊名：The Journal of Steroid Biochemistry and Molecular Biology
• 出版年：2012
• 出版时间：October, 2012
• 年：2012
• 卷：132
• 期：1-2
• 页码：48-56
• 全文大小：1056 K

文摘

1¦Á,25-Dihydroxyvitamin D₃ (1,25D3) regulates musculoskeletal cells via two different mechanisms: vitamin D receptor (VDR)-dependent gene transcription and rapid membrane-signaling via VDR as well as protein disulfide isomerase, family A, member 3 (Pdia3). In chondrocytes from the costochondral cartilage growth zone (GC), ligand binding to Pdia3 causes a rapid increase in phospholipase A₂ (PLA₂) activity leading to release of arachidonic acid and formation of lysophospholipid (LPL). LPL activates phospholipase C (PLC), and resulting inositol trisphosphate (IP₃) and diacylglycerol contribute to PKC¦Á activation and downstream activation of ERK1/2. PLA₂ activating protein (PLAA) is increased in the growth zone of rat growth plates suggesting that it mediates the 1,25D3-dependent pathway. This study examined the role of PLAA in mediating 1,25D3-dependent PKC activation using GC cells and MC3T3-E1 wild-type and PLAA-silenced osteoblasts as models. PLAA, Pdia3, and caveolin-1 (Cav-1) were detected in plasma membranes and caveolae of GC and MC3T3-E1 cells. Pdia3-immunoprecipitated samples were positive for PLAA only after 1,25D3 treatment. Cav-1 was detected when immunoprecipitated with anti-Pdia3 and anti-PLAA in both vehicle and 1,25D3 treated cells. These observations were confirmed by immunohistochemistry. 1,25D3 failed to activate PLA₂ and PKC or cause PGE₂ release in PLAA-silenced cells. PLAA-antibody successfully blocked the PLAA protein and consequently suppressed PKC activity in GC and MC3T3-E1 cells. Crosslinking studies confirmed the localization of PLAA on the extracellular face on the plasma membrane in untreated MC3T3-E1 cells. Taken together, our results suggest that PLAA is an important mediator of 1¦Á,25(OH)₂D₃ rapid membrane mediated signaling. 1¦Á,25(OH)₂D₃ likely causes conformational changes bringing Pdia3 into proximity with PLAA, and aiding in transducing the signal from caveolae to the plasma membrane.