Prostaglandin E2 stimulates fibronectin expression through EP1 receptor, phospholipase C, protein kinase Calpha, and c-Src pathway in primary cultured rat osteoblasts.
Fibronectin (Fn) is involved in the early stages of bone formation, and prostaglandin E (PGE) is an important factor regulating osteogenesis. Here we found that PGE(2) enhanced extracellular Fn assembly in rat primary osteoblasts, as shown by immunofluorescence staining and enzyme-linked immunosorbent assay. PGE(2) also increased the protein levels of Fn by using Western blotting analysis. By using pharmacological inhibitors or activators or genetic inhibition by the EP receptor, antisense oligonucleotides revealed that the EP(1) receptor but not other PGE receptors is involved in PGE(2)-mediated up-regulation of Fn. At the mechanistic level, Ca(2+) chelator (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester)), phosphatidylinositol-phospholipase C inhibitor (U73122), or Src inhibitor (PP2) attenuated the PGE(2)-induced Fn expression. Protein kinase C (PKC) inhibitor (GF109203X) also inhibited the potentiating action of PGE(2). Furthermore, treatment with antisense oligonucleotides of various PKC isoforms, including alpha, beta, epsilon, and delta, demonstrated that alpha isozyme plays an important role in the enhancement action of PGE(2) on Fn assembly. Flow cytometry and reverse transcription-PCR showed that PGE(2) and 17-phenyl trinor PGE(2) (EP(1)/EP(3) agonist) increased the surface expression and mRNA level of alpha5 or beta1 integrins. Fn promoter activity was enhanced by PGE(2) and 17-phenyl trinor PGE(2) in cells transfected with pGL2F1900-Luc. Cotransfection with dominant negative mutants of PKCalpha or c-Src inhibited the potentiating action of PGE(2) on Fn promoter activity. Local administration of PGE(2) or 17-phenyl trinor PGE(2) into the metaphysis of the tibia via the implantation of a needle cannula significantly increased the Fn and alpha5beta1 integrin immunostaining and bone volume of secondary spongiosa in tibia. Taken together, our results provided evidence that PGE(2) increased Fn and promoted bone formation in rat osteoblasts via the EP(1)/phospholipase C/PKCalpha/c-Src signaling pathway.
Tang
Chih-Hsin
CH
Departments of Pharmacology and Orthopaedics, College of Medicine, National Taiwan University, Taipei, Taiwan.
Yang
Rong-Sen
RS
Fu
Wen-Mei
WM
D000818:Animals; D015153:Blotting, Western; D015519:Bone Density; D001842:Bone and Bones / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D015232:Dinoprostone / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D004963:Estrenes / Q000494:pharmacology; D005353:Fibronectins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D005799:Genes, Dominant; D017930:Genes, Reporter; D015151:Immunoblotting; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D007211:Indoles / Q000494:pharmacology; D039482:Integrin alpha5 / Q000378:metabolism; D019012:Integrin beta1 / Q000378:metabolism; D016023:Integrins; D008301:Maleimides / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000737:chemistry; D011743:Pyrimidines / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D018078:Receptors, Prostaglandin E / Q000378:metabolism*; D058306:Receptors, Prostaglandin E, EP1 Subtype; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*; D015854:Up-Regulation; D019061:src-Family Kinases / Q000378:metabolism*
C412373:AG 1879; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D004963:Estrenes; D005353:Fibronectins; D007211:Indoles; D039482:Integrin alpha5; D019012:Integrin beta1; D016023:Integrins; D008301:Maleimides; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D020033:Protein Isoforms; C545637:Ptger1 protein, rat; D011743:Pyrimidines; D011760:Pyrrolidinones; D012333:RNA, Messenger; D018078:Receptors, Prostaglandin E; D058306:Receptors, Prostaglandin E, EP1 Subtype; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D019061:src-Family Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D010738:Type C Phospholipases; D015232:Dinoprostone; C070515:bisindolylmaleimide I; D002118:Calcium