740 PLAA is Required for 1α,25(OH)2D3-Induced Rapid-Activation of PKC in Osteoblasts

Friday, March 23, 2012: 10:45 a.m. - 12:15 p.m.
Presentation Type: Oral Session
M. DOROUDI1, B.D. BOYAN2, and Z. SCHWARTZ1, 1Georgia Institute of Technology, Atlanta, GA, 2Institute of Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA
Objective: 1α,25-dihydroxyvitamin D3 (1,25D3) regulates cells via two different mechanisms: VDR-dependent gene transcription and rapid membrane-signaling. In membrane-signaling, phospholipase-A2 (PLA2) is activated after 1,25D3 binds its membrane-associated receptor, protein disulfide isomerase family A, member 3 (Pdia3), leading to release of arachidonic acid, activation of phospholipase C (PLC), and activation of PLC-dependent protein kinase C (PKC). Caveolae are required for 1,25D3-dependent PKC activation. PLA2 activating protein (PLAA), which activates PLA2, exhibits homology with the G-protein beta subunit, and was considered to play a role in this process. The aim of the present study was to evaluate if and how PLAA mediates the membrane effects of 1,25D3.

Method: Subconfluent cultures of MC3T3-E1 cells immunostained against PLAA, Cav-1 and Pdia3 were imaged using confocal microscopy. Wild type and PLAA-silenced (shPLAA) MC3T3-E1 osteoblasts were treated with either vehicle or 1,25D3; activities of PLA2 and PKC as well as release of PGE2 were measured. Whole cell lysates were collected for immunoprecipitation and caveolae isolation and analyzed by Western blot.

Result: PLAA, Pdia3, and Cav-1 were detected in MC3T3-E1 cells, and in their plasma membranes and caveolae. Co-localization of cav-1 and Pdia3 was 1,25D3 independent whereas co-localization of PLAA and Pdia3 was 1,25D3-dependent. 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.  1,25D3 activated PLA2 and PKC and caused PGE2 release in wild type cells but failed to do so in shPLAA osteoblasts.

Conclusion: Pdia3 and PLAA are present in caveolae from MC3T3-E1 cells and interact with Cav-1. However, PLAA interacts with Pdia3 only in the presence of 1,25D3. 1,25D3 initiates conformational changes bringing Pdia3 into proximity with PLAA, which activates PLA2. These data indicate that PLAA mediates the membrane effect of 1,25D3.

Keywords: Bone, Hormones and growth factors, Molecular biology and Osteoblasts/osteoclasts