title: Membrane actions of 1α,25(OH)2D3 are mediated by Ca(2+)/calmodulin-dependent protein kinase II in bone and cartilage cells.
abstract: 1α,25(OH)2D3 regulates osteoblasts and chondrocytes via its membrane-associated receptor, protein disulfide isomerase A3 (Pdia3) in caveolae. 1α,25(OH)2D3 binding to Pdia3 leads to phospholipase-A2 (PLA2)-activating protein (PLAA) activation, stimulating cytosolic PLA2 and resulting in prostaglandin E2 (PGE2) release and PKCα activation, subsequently stimulating differentiation. However, how PLAA transmits the signal to cPLA2 is unknown. Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) activation is required for PLA2 activation in vascular smooth muscle cells, suggesting a similar role in 1α,25(OH)2D3-dependent signaling. The aim of the present study is to evaluate the roles of CaM and CaMKII as mediators of 1α,25(OH)2D3-stimulated PLAA-dependent activation of cPLA2 and PKCα, and downstream biological effects. The results indicated that 1α,25(OH)2D3 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 1α,25(OH)2D3-dependent CaMKII. Caveolae disruption abolished activation of CaMKII by 1α,25(OH)2D3 or PLAA. CaMKII-specific and CaM-specific inhibitors reduced cPLA2 and PKC activities, PGE2 release and osteoblast maturation markers in response to 1α,25(OH)2D3. Camk2a-silenced but not Camk2b-silenced osteoblasts showed comparable effects. Immunoprecipitation showed increased interaction of CaM and PLAA in response to 1α,25(OH)2D3. The results indicate that membrane actions of 1α,25(OH)2D3 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 1α,25(OH)2D3.
authors.affiliation: School of Biology, Georgia Institute of Technology, 310 Ferst Drive NW, Atlanta, GA 30332, USA.
authors.forename: Maryam
authors.initials: M
authors.lastname: Doroudi
authors.affiliation: Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, 313 Ferst Drive NW, Atlanta, GA 30332, USA.
authors.forename: Marc C
authors.initials: MC
authors.lastname: Plaisance
authors.affiliation: School of Biology, Georgia Institute of Technology, 310 Ferst Drive NW, Atlanta, GA 30332, USA; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, 313 Ferst Drive NW, Atlanta, GA 30332, USA; Department of Biomedical Engineering, School of Engineering, Virginia Commonwealth University, 601 West Main Street, Richmond, VA 23284, USA. Electronic address: bboyan@vcu.edu.
authors.forename: Barbara D
authors.initials: BD
authors.lastname: Boyan
authors.affiliation: Department of Biomedical Engineering, School of Engineering, Virginia Commonwealth University, 601 West Main Street, Richmond, VA 23284, USA.
authors.forename: Zvi
authors.initials: Z
authors.lastname: Schwartz
mesh_terms: D016475:3T3 Cells; D000469:Alkaline Phosphatase / Q000378:metabolism; D000527:Alprostadil / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001842:Bone and Bones / Q000378:metabolism*; D002117:Calcitriol / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D002356:Cartilage / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D019902:Chondrocytes / Q000166:cytology; D005786:Gene Expression Regulation; D015971:Gene Expression Regulation, Enzymologic; D008297:Male; D051379:Mice; D010006:Osteoblasts / Q000166:cytology; D053495:Osteopontin / Q000378:metabolism; D054467:Phospholipases A2 / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D014807:Vitamin D / Q000378:metabolism
chemical_list: D053495:Osteopontin; D014807:Vitamin D; C033246:prostaglandin E3; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D054467:Phospholipases A2; D000469:Alkaline Phosphatase; C513195:Pdia3 protein, mouse; D019704:Protein Disulfide-Isomerases; D000527:Alprostadil; D002117:Calcitriol
keywords: CaMKII; Calmodulin; Chondrocytes; Osteoblasts; Pdia3; Vitamin D