Thrombin-stimulated growth factor and cytokine expression in osteoblasts is mediated by protease-activated receptor-1 and prostanoids

Abstract

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 β, 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 E₂ 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 E₂ 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.

Introduction

As well as converting fibrinogen to fibrin during clot formation, the serine protease thrombin (EC 3.4.21.5) elicits a number of responses from many cell types and is believed to participate in many pathological processes, including platelet activation, inflammation, and tissue repair [1]. Many of the cellular effects of thrombin are achieved through the action of the thrombin-responsive members of the protease-activated receptor (PAR) family of seven transmembrane domain G-protein-coupled receptors, [reviewed by [2]]. Three of the four known members of the PAR family, PARs-1,-3 and -4, are activated by proteolytic cleavage of their N-terminal extracellular domains by thrombin [3], [4], [5], [6], whilst the fourth, PAR-2, is activated by a number of proteases such as trypsin, but not thrombin [7]. Protease activated receptors-1 and -4 can also be activated by synthetic peptides analogous to the new N-terminal “tethered ligands” created by thrombin cleavage [4], [5]. Synthetic activating peptides based upon the naturally occurring tethered ligand sequences are able to activate receptors with high specificity and potency; for example, the peptide TFLLRN (single letter amino acid sequence) specifically activates PAR-1, while the synthetic peptide AYPGKF specifically activates PAR-4. Furthermore, activation of PAR-4 by thrombin or activating peptides is antagonised by trans-cinnamoyl modified peptides [8].

Mice have been derived in which the PAR-1 gene has been targeted by homologous recombination [9]. Although 50% of PAR-1 null mice die in utero due to placental defects, development and haemostasis of the surviving animals are normal [9]. Haemostasis is unaffected in PAR-1 null mice as murine platelet activation is dependent upon the presence of functional PAR-3 and PAR-4 rather than functional PAR-1 [4].

Primary wildtype mouse osteoblast-like cells are known to express PARs-1, -2 and -4 but not PAR-3, whilst PAR-1 null mouse osteoblasts express only PAR-2 and PAR-4 [10]. In vitro, thrombin treatment of osteoblasts has been shown to stimulate proliferation, Ca_i²⁺ mobilisation, release of prostaglandin E₂ (PGE₂), interleukin-6 (IL-6) and plasminogen activator inhibitor, and to inhibit the differentiation and serum deprivation- or glucocorticoid-induced apoptosis of isolated osteoblasts and osteoblast-like cells [10], [11], [12], [13], [14], [15]. The use of peptide agonists of the PARs and cells isolated from PAR-1 null mice indicates that most, but not all of these effects are mediated by the activation of PAR-1 [10], [16], [17].

The current study was conducted to gain a better understanding of how thrombin controls osteoblast function by examining the regulation of expression of a number of secreted factors in response to thrombin treatment of undifferentiated primary mouse osteoblast-like cells. We hypothesised that PAR-1-dependent and PAR-1-independent signalling pathways activated by thrombin would result in an altered pattern of expression of genes encoding factors known to modulate osteoblast function in vivo and in vitro. The twelve candidate genes chosen for study were insulin-like growth factor (IGF)-1 and -2, transforming growth factor β1 (TGFβ), fibroblast growth factor (FGF)-1 and -2, fibronectin (FN), IL-6, connective tissue growth factor (CTGF), tenascin C (TnC), platelet-derived growth factor A (PDGF), cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF). All of the factors chosen, with the exception of FGF-1 have previously been shown to be expressed by osteoblasts [18], [19], [20], [21], [22], [23], [24], [25], and most of them have been shown to be induced by thrombin in other cell types (FGF-2, PDGF, TGFβ, CTGF, TnC, COX-2, FGF-1, VEGF and FN) or in osteoblast-like cells (IL-6) [14], [26], [27], [28], [29], [30], [31], [32], [33]. Furthermore, FGF-1 and -2, CTGF, TGFβ, TnC and IL-6 function as mitogens and stage-specific modulators of differentiation for osteoblast-like cells [34], [35], [36], [37], [38], while FGF-2 and VEGF stimulate bone repair in animal models [39], [40]; cyclooxygenases (which metabolise arachidonic acid to form prostaglandins) are required for normal bone repair [41], [42].

Having noted that expression by osteoblast-like cells of eight of the factors was thrombin-inducible, we observed that induction of all genes was PAR-1- and cyclooxygenase-dependent. Further experiments were conducted to study the mechanism by which thrombin induces IL-6 and PGE₂ expression. The results suggest that activation of PAR-1 on the surface of osteoblasts results in prostanoid release, which is responsible for activating intracellular signalling pathways that result in increased expression of a wide range of secreted factors with important functional effects on both isolated osteoblasts and bone healing generally.