Membrane actions of 1α,25(OH)2D3 are mediated by Ca2+/calmodulin-dependent protein kinase II in bone and cartilage cells

1 alpha,25(OH)(2)D-3 regulates osteoblasts and chondrocytes via its membrane-associated receptor, protein disulfide isomerase A3 (Pdia3) in caveolae. 1 alpha,25(OH)(2)D-3 binding to Pdia3 leads to phospholipase-A(2) (PLA(2))-activating protein (PLAA) activation, stimulating cytosolic PLA2 and resulting in prostaglandin E2 (PGE(2)) release and PKCa activation, subsequently stimulating differentiation. However, how PLAA transmits the signal to cPLA(2) is unknown. Ca2+/Vcalmodulin (CaM)-dependent protein kinase II (CaMKII) activation is required for PLA2 activation in vascular smooth muscle cells, suggesting a similar role in 1 alpha,25(OH)(2)D-3-dependent signaling. The aim of the present study is to evaluate the roles of CaM and CaMKII as mediators of 1 alpha,25(OH)(2)D-3-stimulated PLAA-dependent activation of cPLA2 and PKCa, and downstream biological effects. The results indicated that 1 alpha,25(OH)(2)D-3 and PLAA-peptide increased CaMKII activity within 9 min. Silencing Cav-1, Pdia3 or Plaa in osteoblasts suppressed this effect. Similarly, antibodies against Plaa or Pdia3 blocked 1 alpha,25(OH)(2)D-3-dependent CaMKII. Caveolae disruption abolished activation of CaMKII by 1 alpha,25(OH)(2)D-3 or PLAA. CaMKII-specific and CaM-specific inhibitors reduced cPLA2 and PKC activities, PGE2 release and osteoblast maturation markers in response to 1 alpha,25(OH)(2)D-3. Camk2 alpha-silenced but not Camk2b-silenced osteoblasts showed comparable effects. Immunoprecipitation showed increased interaction of CaM and PLAA in response to 1 alpha,25(OH)(2)D-3. The results indicate that membrane actions of 1 alpha,25(OH)(2)D-3 via Pdia3 triggered the interaction between PLAA and CaM, leading to dissociation of CaM from caveolae, activation of CaMKII, and downstream PLA2 activation, and suggest that CaMKII plays a major role in membrane-mediated actions of 1 alpha,25(OH)(2)D-3. (C) 2014 Elsevier Ltd. All rights reserved.