RESUMO
Thrombin exerts multiple effects upon osteoblasts including stimulating proliferation, and inhibiting osteoblast differentiation and apoptosis. Some of these effects are believed to be mediated by the synthesis and secretion of autocrine factors such as growth factors and cytokines. Many but not all cellular responses to thrombin are mediated by members of the protease-activated receptor (PAR) family of G protein-coupled receptors. The current study was undertaken to investigate the nature of thrombin's induction of autocrine factors by analysing the expression of twelve candidate genes in thrombin-stimulated primary mouse osteoblasts. Analysis by quantitative reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that thrombin induced transforming growth factor beta, cyclooxygenase-2, tenascin C, fibroblast growth factor-1 and -2, connective tissue growth factor and interleukin-6 expression in wild type osteoblasts, but not PAR-1 null mouse osteoblasts. Induction of all the thrombin-responsive genes was blocked by the presence of the non-selective cyclooxygenase inhibitor indomethacin. Further studies were conducted on interleukin-6, which was the gene that showed the greatest increase in expression following stimulation of osteoblast-like cells with thrombin. A PAR-1-specific activating peptide, but neither a PAR-4-activating peptide nor catalytically inactive thrombin induced release of interleukin-6 by osteoblasts. Furthermore, in the presence of the selective cyclooxygenase-1 and -2 inhibitors SC-560 and NS-398 thrombin-induced interleukin-6 release was prevented. Levels of both prostaglandin E(2) and interleukin-6 in medium conditioned by thrombin-stimulated osteoblast-like cells were found to be significantly increased compared to medium conditioned by non-stimulated cells, however release of prostaglandin E(2) was found to precede release of interleukin-6. Treatment of isolated osteoblast-like cells with a number of synthetic prostanoids stimulated secretion of interleukin-6 with differing potencies. These studies suggest that activation of PAR-1 on osteoblasts by thrombin induces cyclooxygenase activity, which in turn results in the increased expression of multiple secreted factors. The induction of these secreted factors may act in an autocrine fashion to alter osteoblast function, allowing these cells to participate in the earliest stages of bone healing by both autocrine and paracrine mechanisms.
Assuntos
Citocinas/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Prostaglandinas/metabolismo , Receptor PAR-1/fisiologia , Trombina/farmacologia , Animais , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo/genética , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/fisiologia , Inibidores de Ciclo-Oxigenase/farmacologia , Ensaio de Imunoadsorção Enzimática , Fator 1 de Crescimento de Fibroblastos/genética , Fibronectinas/genética , Indometacina/farmacologia , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like II/genética , Interleucina-6/genética , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Derivado de Plaquetas/genética , Receptor PAR-1/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta/genéticaRESUMO
Cells responsible for the formation and maintenance of bone express thrombin-responsive members of the protease-activated receptor family of G protein-coupled receptors. Thrombin has been shown to elicit a number of functional responses in these cells, including proliferation and cytokine production in osteoblasts. Many, but not all, of the effects of thrombin on bone cells are initiated by activation of protease-activated receptor-1. A combination of in vitro observations and results of in vivo studies in protease-activated receptor-1-null mice suggest that thrombin plays multiple roles in the early stages of bone healing.
