c-Src-dependent transactivation of PDGFR contributes to TNF-α-induced MMP-9 expression and functional impairment in osteoblasts

Highlights

• TNF-α-induced MMP-9 expression in osteoblasts

• TNF-α-induced MMP-9 expression is mediated through a c-Src-dependent transactivation of PDGFR linking to PI3K/Akt/MAPKs/AP-1 (c-Jun/ATF2) cascade in MC3T3-E1 cells.

• TNF-α leads to MMP-9-mediated type I collagen degradation and detaching osteoblasts.

Abstract

Matrix metalloproteinases (MMPs), MMP-9 especially, have been shown to be induced by cytokines, including tumor necrosis factor-α (TNF-α) and may contribute to bone inflammatory diseases and postnatal bone modeling and remodeling. However, the mechanisms underlying MMP-9 expression induced by TNF-α in osteoblasts remain unclear. Here, we showed that in MC3T3-E1 cells, TNF-α induced MMP-9 gene expression determined by real-time PCR, zymography, and promoter assay. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of protein tyrosine kinase (PTK; genistein), c-Src (PP1), PDGFR (AG1296), PI3K (LY294002), Akt (SH-5), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) and transfection with siRNA of c-Src, PDGFR, p85, Akt, c-Jun, or ATF2. Moreover, TNF-α also time-dependently stimulated phosphorylation of c-Src and PDGFR and c-Src/PDGFR complex formation, which were reduced by pretreatment with PP1 or AG1296. TNF-α-stimulated Akt phosphorylation was inhibited by genistein, PP1, AG1296, LY294002, or SH5. We further demonstrated that TNF-α stimulated ERK1/2, p38 MAPK, and JNK1/2 phosphorylation via a c-Src-dependent PDGFR/PI3K/Akt pathway. TNF-α stimulated AP-1 activation, including c-Jun and ATF2 phosphorylation and AP-1 transcription activity via MAPK-dependent pathways. In addition, TNF-α-induced MMP-9 promoter activity was mediated through an AP-1 binding domain of the MMP-9 promoter region. Finally, we found that up-regulation of MMP-9 contributes to MMP-mediated type I collagen degradation and osteoblasts detachment. These results suggested that TNF-α-induced MMP-9 expression is mediated through a c-Src-dependent PDGFR transactivation and PI3K/Akt cascade linking to MAPK-mediated activation of AP-1 (c-Jun/ATF2) and leading to functional impairment in osteoblasts.

Introduction

In bone remodeling cycle, osteoblasts play an important role in regulating consecutive stages of bone resorption, which include dissolution of extracellular matrix (ECM) and osteoclast recruitment. Matrix metalloproteinases (MMPs) have been shown to facilitate these bone cell interactions during bone remodeling and hydrolyze small and single chain peptide substrates in type I collagen [1]. Especially, MMP-2 and MMP-9 can degrade a variety of collagens, including basement membrane (type IV collagen), denatured fibrillar type I collagen (gelatin), and type V collagen in osteoarticular diseases [2]. MMP-9 has been implicated osteocytes and osteoblasts in both physiological and pathological processes of skeletal system, however, the potential influence of MMP-9 on the ECM (such as type I collagen) degradation and bone remodeling remains largely unknown.

MMPs, a major group of ECM-degrading enzymes, are essential for various normal biological processes and also play an important role in pathological processes including inflammation, arthritis, and tissue remodeling [3], [4], [5]. Several studies based on clinical research, animal model or cell culture model showed that bone resorption induced by inflammatory cytokines is associated with increased expression of MMP-9 [6], [7], [8]. Moreover, MMP-9 is highly expressed in bone cells, particularly in osteoclasts [9], [10], [11]. Given the role of osteoblasts, it is somewhat counter-intuitive that they also express many matrix degrading MMPs including MMP-9 [12]. Many studies demonstrated that TNF-α-induced MMP-9 up-regulation is involved in osteoclasts during differentiation and bone destruction [13], [14]. Moreover, previous studies have demonstrated that TNF-α induces MMP-9 expression in osteoblasts and bone marrow-derived osteoprogenitor cells [6].

Protein tyrosine kinases (PTKs) have been shown to be involved in TNF-α-stimulated signaling pathways [15], [16]. PTK family can be divided into the receptor tyrosine kinases (RTKs), such as epidermal growth factors receptor (EGFR) or platelet-derived growth factors receptor (PDGFR) and the non-receptor tyrosine kinases (non-RTKs), such as Src family kinases [17], [18], [19]. Previous reports suggest that Src tyrosine kinases promote inflammatory processes under various pathologic conditions [20]. Several reports have shown that TNF-α-activated c-Src might transactivate RTKs, including EGFR or PDGFR [21]. Moreover, TNF-α-induced MMP-9 gene expression is mediated through activation of alternative signaling molecules, including c-Src, EGFR, or PDGFR [22], [23], [24]. Yang et al. showed that association of c-Src with PDGFR brings c-Src into proximity with PI3K/Akt cascade [25]. Activation of PI3K appears to occur via phosphorylation of tyrosine residues in the Src homology 2 (SH2) domain of p85, which permits docking of PI3K to the plasma membrane and allows allosteric modifications that increase its catalytic activity [26], [27], [28]. Previous study has demonstrated that TNF-α induces MMP-9 expression via a c-Src-dependent RTKs/PI3K/Akt cascade leading to activation of NF-κB in human tracheal smooth muscle cells [22]. Moreover, recent evidence suggests that PI3K activation may regulate ERK1/2, JNK1/2, and p38 MAPK. Activation of MAPKs is essential for proteolytic cartilage degradation-divergence in pathways leading to MMP-mediated articular cartilage degradation [29]. However, in osteoblasts, the mechanisms underlying TNF-α-induced MMP-9 expression are not completely defined.

Several regulatory elements have been identified within MMP-9 promoter, including NF-κB and AP-1 [30]. AP-1 is a heterogeneous collection of dimeric transcription factors comprising c-Jun, c-Fos, and ATF2 subunits which are regulated by multiple mechanisms [31]. For example, v-Src activates the expression MMP-9 gene through AP-1 [32]. Growth factors activate ATF2 via phosphorylation through the Ras-MEK-ERK and Src-p38 pathways [33], [34]. In human trophoblastic cells, TNF-α activates ERK1/2 which in turn initiates NF-κB activation and JNK1/2 which activates AP-1 leading to MMP-9 expression [35]. However, the molecular mechanisms of AP-1 associated with MMP-9 expression by TNF-α in osteoblasts are not completely defined.

Here, we investigated the molecular mechanisms underlying TNF-α-induced MMP-9 expression in osteoblasts and its effects on type I collagen degradation. These findings suggested that TNF-α-induced MMP-9 expression is mediated through a c-Src-dependent transactivation of PDGFR linking to PI3K/Akt/MAPKs/AP-1 (c-Jun/ATF2) cascade and leads to MMP-9-mediated type I collagen degradation and detaching osteoblasts. These results provide new insights into the mechanisms by which TNF-α induced MMP-9 expression and then exaggerated bone injury or inflammatory responses.