title: Protein-disulfide isomerase-associated 3 (Pdia3) mediates the membrane response to 1,25-dihydroxyvitamin D3 in osteoblasts.
abstract: Protein-disulfide isomerase-associated 3 (Pdia3) is a multifunctional protein hypothesized to be a membrane receptor for 1,25(OH)(2)D(3). In intestinal epithelium and chondrocytes, 1,25(OH)(2)D(3) stimulates rapid membrane responses that are different from genomic effects via the vitamin D receptor (VDR). In this study, we show that 1,25(OH)(2)D(3) stimulates phospholipase A(2) (PLA(2))-dependent rapid release of prostaglandin E(2) (PGE(2)), activation of protein kinase C (PKC), and regulation of bone-related gene transcription and mineralization in osteoblast-like MC3T3-E1 cells (WT) via a mechanism involving Pdia3. Pdia3 was present in caveolae based on co-localization with lipid rafts and caveolin-1. In Pdia3-silenced (Sh-Pdia3) cells, 1,25(OH)(2)D(3) failed to stimulate PKC and PGE(2) responses; in Pdia3-overexpressing cells (Ov-Pdia3), responses to 1,25(OH)(2)D(3) were augmented. Downstream mediators of Pdia3, PLA(2)-activating protein (PLAA) and arachidonic acid, stimulated similar PKC activation in wild-type, Sh-Pdia3, and Ov-Pdia3 cells supporting the hypothesis that Pdia3 mediates the membrane action of 1,25(OH)(2)D(3). Treatment of MC3T3-E1 cells with 1,25(OH)(2)D(3) for 9 min stimulated rapid phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and increased expression of alkaline phosphatase, MMP-13, and osteopontin but decreased expression of osteocalcin, osteoprotegerin (mRNA and protein), and smad2. These effects were attenuated in Sh-Pdia3 cells. Sh-Pdia3 cells produced higher numbers of von Kossa-positive nodules and alizarin red-positive nodules compared with WT cells with or without 1,25(OH)(2)D(3) treatment whereas Ov-Pdia3 did not show any mineralization. Our data suggest Pdia3 is an important initiator of 1,25(OH)(2)D(3)-stimulated membrane signaling pathways, which have both genomic and non genomic effects during osteoblast maturation.
authors.affiliation: Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332-0363, USA.
authors.forename: Jiaxuan
authors.initials: J
authors.lastname: Chen
authors.forename: Rene
authors.initials: R
authors.lastname: Olivares-Navarrete
authors.forename: Yun
authors.initials: Y
authors.lastname: Wang
authors.forename: Tyler R
authors.initials: TR
authors.lastname: Herman
authors.forename: Barbara D
authors.initials: BD
authors.lastname: Boyan
authors.forename: Zvi
authors.initials: Z
authors.lastname: Schwartz
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002113:Calcification, Physiologic / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism*; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism*; D010766:Phosphorylation; D019704:Protein Disulfide-Isomerases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013347:Subcellular Fractions; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; C118756:dihydroxy-vitamin D3; D014807:Vitamin D; D011493:Protein Kinase C; C513195:Pdia3 protein, mouse; D019704:Protein Disulfide-Isomerases