Prostaglandin E2 stimulates fibronectin expression through EP1 receptor, phospholipase C, protein kinase Cα, and c-Src pathway in primary cultured rat osteoblasts

Abstract

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 PGE2 enhanced extracellular Fn assembly in rat primary osteoblasts, as shown by immunofluorescence staining and enzyme-linked immunosorbent assay. PGE2 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 EP1 receptor but not other PGE receptors is involved in PGE2-mediated up-regulation of Fn. At the mechanistic level, Ca2+ 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 PGE2-induced Fn expression. Protein kinase C (PKC) inhibitor (GF109203X) also inhibited the potentiating action of PGE2. Furthermore, treatment with antisense oligonucleotides of various PKC isoforms, including α, β, ε, and δ, demonstrated that α isozyme plays an important role in the enhancement action of PGE2 on Fn assembly. Flow cytometry and reverse transcription-PCR showed that PGE 2 and 17-phenyl trinor PGE2 (EP1/EP3 agonist) increased the surface expression and mRNA level of α5 or β1 integrals. Fn promoter activity was enhanced by PGE2 and 17-phenyl trinor PGE2 in cells transfected with pGL2F1900-Luc. Co-transfection with dominant negative mutants of PKCβ or c-Src inhibited the potentiating action of PGE2 on Fn promoter activity. Local administration of PGE2 or 17-phenyl trinor PGE2 into the metaphysis of the tibia via the implantation of a needle cannula significantly increased the Fn and α5β1 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 EP1/phospholipase C/PKCα/c-Src signaling pathway. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.