Abstract
Fibronectin with IIICS region is present in rheumatoid synovium, and fibronectin fragments are increased in rheumatoid joints. We investigated the ability of COOH-terminal heparin-binding fibronectin fragment (COOH-HBFN-f) containing IIICS to induce matrix metalloproteinase (MMP) production and the role of mitogen-activated protein kinase (MAPK) pathway and CS-1 sequence that can bind α4β1 integrin in MMP induction by COOH-HBFN-f in rheumatoid synovial fibroblasts (RSF). When RSF in monolayer culture were incubated with COOH-HBFN-f, COOH-HBFN-f stimulated the production of MMP-1, MMP-3, and MMP-13 by RSF in association with activation of extracellular signal-regulated kinase, p38 MAPK, and c-Jun NH2-terminal kinase. Immunoprecipitation of cell lysates demonstrated the presence of α4 integrin in cultured RSF. Similar to COOH-HBFN-f, treatment with CS-1 synthetic peptide derived from IIICS resulted in increased MMP production and activation of the kinases, although the MMP levels were low. Preincubation of RSF with anti-α4 integrin antibody resulted in partial suppression of the COOH-HBFN-f–stimulated MMP production. Inhibition studies using protein kinase inhibitors (PD98059 and SB203580) showed that those MAPK pathways contributed to MMP up-regulation by COOH-HBFN-f and CS-1. Thus, the present results have clearly shown that COOH-HBFN-f and CS-1 stimulate MMP production in association with activation of MAPK pathways in RSF. Integrin α4β1 may be partially involved in the MMP induction by COOH-HBFN-f.
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Introduction
Fibronectin (FN) is a family of glycoproteins present in body fluids and extracellular matrices. FN has various biologic functions, including cell attachment, cell migration, wound healing, and oncogenic transformation (Hynes, 1990). FN consists predominantly of three types of homologous repeating segments (designated I, II, and III). FN contains NH2-terminal heparin-, gelatin-, cell-, and COOH-terminal heparin-binding domains. Multiple isoforms of FN are produced from a single gene by alternative splicing at three distinct sites, EDA, EDB, and IIICS (Hynes, 1985). FN is ubiquitous within active rheumatoid synovium. Enhanced accumulation of FN is found on the inflamed synovial and pannus surfaces in the knee joints of patients with rheumatoid arthritis (RA) (Scott et al, 1981; Shiozawa and Ziff, 1983).
Rheumatoid synovial fibroblasts (RSF), found at the site of invasion of rheumatoid synovium into the adjacent cartilage and bone, play important roles in joint destruction at the cartilage-pannus contact. RSF extend out over the articular surface of cartilage, including the point of cartilage-pannus junction, where they express elevated collagenase levels (Wooley et al, 1977). Matrix metalloproteinases (MMPs) are thought to be critical players in rheumatoid joint destruction (Firestein et al, 1991). MMP-1 (collagenase 1) and MMP-13 (collagenase 3), found at elevated levels in RA (Vincenti, 2001), act at a committed step in the progression of RA, degrading type II collagen in cartilage (Firestein et al, 2001; Nagase and Woessner, 1999). MMP-3 (stromelysin 1) has activity against other components of cartilage matrix, such as proteoglycan and FN, and can activate proMMP-1 and proMMP-13 (Nagase, 1997). Activator protein-1 (AP-1), which includes members of the Jun and Fos families, is a pivotal transcriptional factor that regulates the production of cytokines and MMPs. The upstream regulatory regions of MMP genes including MMP-1 (Rutter et al, 1997) and MMP-13 (Pendas et al, 1997) contain the AP-1 recognition site. AP-1 can be activated by protein kinases that phosphorylate specific amino acid residues, especially by mitogen-activated protein kinase (MAPK) families (Karin et al, 1997). Three major MAPK families have been identified: extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun NH2-terminal kinase (JNK) (Garrington and Johnston, 1999; Segar and Krebs, 1995). All of the three MAPK pathways are involved in the transcriptional regulation of Fos and Jun family genes. AP-1 components Jun and Fos have been identified in RA synovial tissues (Han et al, 1998).
Integrins are heterodimeric transmembrane proteins that consist of α and β subunits. Integrins bind extracellular matrix molecules and mediate cell adhesion, migration, and invasion during development, tissue repair, tumor invasion, and metastasis. In concert with growth factor or cytokine receptors, integrins regulate cell proliferation, differentiation, and survival (Giancotti and Mainiero, 1994; Hynes, 1992). Integrins also serve as cell surface receptors that transduce intracellular signals (Clark and Brugge, 1995; Defilippi et al, 1997; Giancotti and Ruoslahti, 1999; Schwarz et al, 1995). Although the cytoplasmic domains of the integrin α and β subunits have no intrinsic enzymatic activity, integrin signaling is achieved by coupling signaling molecules to the cytoplasmic and transmembrane domains of the integrin subunits (Yamada and Miyamoto, 1995). Integrins activate signaling pathways that are either common to all integrins or heterodimer-specific (Giancotti, 1997). The cytoplasmic domains of α subunits may trigger signaling events that are specific for each individual integrin heterodimers (Sastry et al, 1996; Zhang et al, 1995). In addition, coupling of integrin receptors to MAPK pathways has been reported (Chen et al, 1994). RSF at the cartilage-pannus junction express integrin subunits α4, α5, and β1 (Ishikawa et al, 1994). Integrin α4β1 recognizes CS-1 in alternatively spliced IIICS domain of fibronectin (Guan and Hynes, 1990; Wayner et al, 1989), whereas the arginine-glycine-aspartate (RGD) sequence within the central cell-binding domain of fibronectin is recognized by integrins α5β1 and αvβ1 (Damsky and Werb, 1992; Yamada, 1991).
Proteolytic fragments of FN are increased in RA synovial fluid (Carsons et al, 1985). Degradation products of FN are of interest as amplifiers or catalysts in diseased joints including RA and osteoarthritis (OA) (Homandberg, 1999) because of the ability to stimulate MMP induction (Homandberg et al, 1992; Werb et al, 1989; Yasuda and Poole, 2002). Although rabbit synovial fibroblasts plated on the central RGD-containing fragments of FN have been shown to induce expression of MMP-1 and MMP-3 (Werb et al, 1989), MMP induction by fibronectin fragment (FN-f) in RSF remains to be clarified. Previous studies have shown that FN isoform with IIICS is present in RSF in synovial lining layer (Müller-Ladner et al, 1997). Recently, we have demonstrated that exogenous addition of 40 kd COOH-terminal heparin-binding FN fragment (COOH-HBFN-f), which contains type III12 to 14 segments and IIICS, induces MMP-3 and MMP-13 in bovine articular cartilage culture (Yasuda and Poole, 2002). However, it is still unclear whether CS-1 sequence in IIICS of COOH-HBFN-f, the recognition site for α4β1 integrin, plays a role in MMP induction. Thus, we attempted to analyze the ability of COOH-HBFN-f to induce MMP production in RSF and the role of MAPK pathway in MMP production by COOH-HBFN-f. We further investigated the involvement of α4β1 integrin ligation with CS-1 on RSF in the MMP production. The present study showed herein that COOH-HBFN-f and the α4β1 integrin-binding synthetic peptide stimulated the production of MMP-1, MMP-3, and MMP-13 in association with MAPK activation in RSF.
Results
MMP Production by COOH-HBFN-f in RSF
Our initial study was attempted to test the ability of COOH-HBFN-f to induce MMP production in RSF. When 1 to 100 nm COOH-HBFN-f was added to RSF cultures, we found that 100 nm COOH-HBFN-f caused the production of MMP-1, MMP-3, and MMP-13 by RSF, in contrast to the barely detectable secretion of the enzymes in control or intact FN-treated cultures (Fig. 1A). Higher concentrations (~1 μm) of COOH-HBFN-f also produced a stimulatory effect on MMP induction in RSF (data not shown). Interleukin (IL)-1β–induced MMPs verified the specificity of immunoreactive proteins induced by COOH-HBFN-f (Fig. 1B). Compared with the levels induced by 2 ng/ml IL-1β (calculated protein band density as 100%), the secreted levels of MMP-1, MMP-3, and MMP-13 in association with stimulation with 100 nm COOH-HBFN-f were 80 ± 15, 60 ± 12, and 40 ± 5%, respectively (mean ± sd, n = 4). In contrast to COOH-HBFN-f, NH2-terminal heparin-binding fibronectin fragment (NH2-HBFN-f) without any known recognition site for integrins including α4β1, served as a negative control and failed to stimulate MMP production by RSF (Fig. 1B).
MMP Production by COOH-HBFN-f Is Not via an IL-1–Mediated Pathway
Whereas FN-f has been shown to stimulate the release of proinflammatory cytokines including IL-1 from cartilage (Homandberg et al, 1997), FN-f has been shown to increase MMP expression via an IL-1 autocrine loop in several studies. RGD integrin-binding sequence of FN enhances MMP-3 synthesis by chondrocytes in an IL-1–dependent manner (Arner and Tortorella, 1995). FN-f–induced MMP-3 production by human cartilage in culture can be partially blocked by neutralizing antibodies against IL-1 (Homandberg et al, 1997). Thus, the involvement of IL-1 in MMP production by COOH-HBFN-f–stimulated RSF was investigated using IL-1 receptor antagonist (IL-1ra) to competitively inhibit IL-1 interaction with its receptor. When RSF were incubated with IL-1ra at 100 ng/ml in the presence of 2 ng/ml IL-1β, IL-1ra significantly blocked IL-1β–stimulated production of MMP-1, MMP-3, and MMP-13 (Fig. 2). In contrast, IL-1ra at 100 ng/ml failed to suppress the production of these MMPs by RSF stimulated with 100 nm COOH-HBFN-f (Fig. 2), indicating that COOH-HBFN-f could induce the MMPs via an IL-1–independent pathway. Similar IL-1–independent effects of FN-f have been demonstrated in the previous studies on FN-f–induced nitric oxide synthesis in human chondrocytes (Gemba et al, 2002) and on FN-f–stimulated production of MMP-3 and MMP-13 in pig cartilage (Stanton et al, 2002).
Activation of MAPK by COOH-HBFN-f in RSF
We next examined whether treatment with COOH-HBFN-f results in activation of ERK, p38 MAPK, and JNK in RSF. After stimulation with COOH-HBFN-f at 100 nm, cell lysates were subjected to immunoblot analysis (Fig. 3). We used RSF stimulated with 2 ng/ml IL-1β as a positive control because the proinflammatory cytokine has been shown to activate the three MAPK pathways in RSF (Schett et al, 2000). Enhanced phosphorylation of ERK1/2 by COOH-HBFN-f was observed from 10 to 30 minutes that peaked at 30 minutes, followed by the decline by 120 minutes. IL-1β also caused enhanced activation of ERK1/2 from 10 to 30 minutes with maximal activation at 30 minutes. COOH-HBFN-f caused significant activation of p38 MAPK at 120 minutes, whereas activation of p38 was observed from 10 minutes with the maximal activation at 120 minutes in IL-1β–stimulated RSF. COOH-HBFN-f also induced JNK activation in RSF. Treatment with COOH-HBFN-f resulted in phosphorylation of 54 kd JNK (p54) at 120 minutes. Activation of p54 JNK by IL-1β was evident from 10 to 30 minutes and thereafter faded into control levels. The protein levels of ERK1/2, p38, and JNK (p54) were unchanged during the whole period of experiments.
Intracellular Signaling Cascades Stimulated by COOH-HBFN-f Involved in MMP Induction in RSF
To investigate which intracellular signaling cascades were involved in MMP induction by COOH-HBFN-f in RSF, we used the selective protein kinase inhibitors: PD98059, a specific MAPK, ERK-kinase (MEK) inhibitor that inhibits the ERK signaling cascade (Dudley et al, 1995), and SB203580, which selectively inhibits p38 MAPK at low concentrations (≤1 μm) but which inhibits both p38 and JNK at high concentrations (≥10 μm) (Whitmarsh et al, 1997) (Fig. 4). After preincubation with one of those inhibitors for 1 hour, RSF were stimulated for 24 hours with COOH-HBFN-f at 100 nm. The MEK inhibitor at 10 μm significantly blocked COOH-HBFN-f–stimulated production of MMP-1, MMP-3, and MMP-13. SB203580 at 1 μm partially suppressed COOH-HBFN-f–induced MMP-1 production; approximately 50% decrease in protein band intensity compared with COOH-HBFN-f–induced MMP-1 levels (the calculated protein band intensity as 100%). The inhibitor at 10 μm completely inhibited the MMP-1 production. SB203580, even at 1 μm, completely blocked COOH-HBFN-f–induced production of MMP-3. However, SB203580 only at 10 μm was capable of inhibiting COOH-HBFN-f–stimulated MMP-13 production in RSF.
Identification of α4 Integrin in RSF
Under the guidance of Jurkat cells as a positive control, immunoblot analysis with immunoprecipitated samples of RSF cell lysates with anti-integrin α4 antibodies, sc-6589 (Fig. 5A), and sc-6590 (data not shown) that recognize COOH- and NH2-terminal regions of α4 integrin, respectively, demonstrated the presence of α4 integrin subunit in RSF. Integrin α4 subunit was also found in RSF derived from three other patients with RA (Fig. 5B). These observations are consistent with the previous finding that cultured RSF express α4 integrin subunit (Müller-Ladner et al, 1997).
MMP Production and MAPK Activation by CS-1 in RSF
Because RSF expressed α4 integrin, we further investigated MMP induction with α4β1 integrin ligation with CS-1 peptide derived from IIICS in COOH-HBFN-f. Treatment with 10 μm CS-1 for 48 hours resulted in an increase in secreted levels of MMP-1, MMP-3, and MMP-13 in conditioned media detected by immunoblot analysis (Fig. 6A). Similar to CS-1, in line with the previous reports using RGD-containing peptide derived from III10 of central cell-binding domain in FN that can bind α5β1 and αvβ1 integrins (Werb et al, 1989), 10 μm glycine-arginine-glycine-aspartate-serine (GRGDS) as a positive control stimulated the MMP production by RSF (Fig. 6A). However, the levels of MMPs induced by CS-1 were low, compared with the levels by COOH-HBFN-f (Fig. 6B). The protein band intensities of MMP-1, MMP-3, and MMP-13 with CS-1 stimulation were 35 ± 14, 29 ± 10, and 25 ± 15% (mean ± sd, n = 4), respectively, when those by COOH-HBFN-f were calculated as 100%. In contrast to CS-1, 10 μm peptide V derived from the III14 repeat of COOH-HBFN-f as a negative control failed to induce those MMPs in RSF (Fig. 6B).
We also investigated MAPK activation in association with α4β1 integrin ligation with CS-1 at 10 μm (Fig. 7). Stimulation of α4β1 integrin with CS-1 resulted in enhanced activation of ERK1/2 from 10 to 60 minutes that peaked at 30 minutes. CS-1 induced significant phosphorylation of p38 from 60 to 120 minutes in RSF. Stimulation of α4β1 integrin with CS-1 caused slight activation of p54 JNK from 60 to 120 minutes in RSF. Total protein levels of ERK1/2, p38, and p54 JNK were unchanged during the period of experiments. Pretreatment with PD98059 at 10 μm resulted in complete inhibition of CS-1–stimulated production of MMP-1, MMP-3, and MMP-13 in RSF (Fig. 8). SB203580 at 10 μm completely blocked collagenase (MMP-1 and MMP-13) production by CS-1–stimulated RSF, whereas 1 μm of the inhibitor failed to inhibit it (Fig. 8). SB203580 even at 1 μm suppressed MMP-3 production by CS-1–stimulated RSF (Fig. 8).
Inhibition of COOH-HBFN-f–Stimulated MMP Induction by Anti-α4 Integrin Antibody
To determine the role of α4 integrin in COOH-HBFN-f–stimulated MMP production, we co-incubated RSF with 100 nm COOH-HBFN-f after preincubation for 1 hour with 10 μg/ml anti-α4 integrin antibody (sc-6590), which recognizes an extracellular NH2-terminal region of α4 integrin of human origin. Compared with the protein band intensities of MMPs induced by COOH-HBFN-f calculated as 100%, the antibody blocked the enhanced production of MMP-1, MMP-3, and MMP-13 by approximately 40, 60, and 50%, respectively (Fig. 9). In contrast, nonspecific goat immunoglobulin G (IgG) at 10 μg/ml had no significant effect on the COOH-HBFN-f–stimulated MMP induction.
Discussion
In synovial joints with RA, invasion of synovial tissue into the adjacent cartilage matrix is observed in the form of a pannus, with proteolytic degradation of articular cartilage and bone as a consequence (Shiozawa and Tokushima, 1992). Pannus invasion occurs at the junction between synovial tissue and cartilage. Pannus destroys cartilage by extension over the cartilage surface or direct penetration into the bare zone between cartilage and synovial insertion. The observation that cartilage degradation by pannus takes place in the absence of vascular supply (Shiozawa and Ziff, 1983) suggests that local factors at the cartilage-pannus junction are involved in the degradation processes. IL-1, found in the elevated levels in RA synovium (Wang et al, 1997) and synovial fluid (Kahle et al, 1992), is considered to be one of those factors. Up-regulation and overexpression of MMPs are commonly found in rheumatoid joint destruction (Firestein et al, 1991), and increased levels of collagenase are found in RSF at the cartilage-pannus junction (Wooley et al, 1977). Of these enzymes, particular attention has focused on MMP-1 (collagenase 1) and MMP-13 (collagenase 3) because they are induced in response to IL-1 and tumor necrosis factor (Vincenti, 2001). Indeed, collagenase activity has been shown to be required for cartilage invasion by IL-1β–stimulated RSF (Wang et al, 1997). In the present study, we have demonstrated for the first time that COOH-HBFN-f containing both III12 to 14 and IIICS domains stimulates the production of collagenases (MMP-1 and MMP-13) and stromelysin 1 (MMP-3) by RSF in contrast to intact FN or NH2-HBFN-f (Fig. 1). Because FN-f concentrations found in RA joints are more than 1 μm (Xie et al, 1992), the concentration (100 nm) used in the current study is within a concentration range that may be found in RA joints. In articular cartilage explant culture, COOH-HBFN-f at 100 nm causes type II collagen cleavage by collagenase after proteoglycan degradation in association with MMP-3 and MMP-13 production (Yasuda and Poole, 2002). Furthermore, significant proteoglycan release from cultured cartilage is found at FN-f concentrations as low as 20 nm (Homandberg et al, 1992). As shown in the role in chondrocyte activation, specific FN-f, such as COOH-HBFN-f, in RA synovial fluid may be another local factor for cartilage destruction by RSF at the cartilage-pannus junction through collagenase induction in addition to proinflammatory cytokines.
Specific sequences have been found in the promoters of MMP genes that mediate the increase in gene expression, including AP-1 site that binds the Fos and Jun families of transcription factors (Auble and Brinckerhoff, 1991). RA synovium contains AP-1 with elevated activities compared with OA synovium (Han et al, 1998). Activation of MAPK families ERK, p38, and JNK results in the production of phosphorylated active AP-1 transcription factor (Eder, 1997; Whitmarsh and Davis, 1996). The three mammalian MAPK pathways have been characterized in detail: ERK1/2 pathway (Raf/MEK1, 2/ERK1, 2), JNK/stress-activated protein kinase pathway (MEK kinase-1–4/MKK4, 7/JNK1–3), and p38 pathway (MAPK kinase kinase/MKK3, 6/p38α, β) (Westermarck and Kahari, 1999). Although recent studies have shown that NH2-HBFN-f can activate those MAPKs in OA chondrocytes (Gemba et al, 2002) and COOH-HBFN-f activates MAPK cascade in human natural killer cells (Mainiero et al, 1998), the ability of FN-f to stimulate MAPK pathways in RSF has not been investigated. The present study has clearly demonstrated that exogenous addition of COOH-HBFN-f causes phosphorylation of ERK1/2, JNK, and p38 in RSF. Increased FN-f in RA synovial fluid could contribute to the enhanced activation of ERK, p38, and JNK found in synovial tissues from patients with RA (Schett et al, 2000). Upstream events in activation of MAPK cascades in association with COOH-HBFN-f stimulation remain to be clarified.
The role of individual MAPK pathways in individual MMP expressions seems to vary with cell types and stimulus. All of the pathways of ERK, p38, and JNK contribute to MMP-1 expression in rabbit synovial fibroblasts (Barchowsky et al, 2000). Although IL-1 activation of MMP-13 in rabbit chondrocytes is primarily p38-dependent because it is blocked with p38 inhibitor SB203580 (Mengshol et al, 2000), p38 pathway plays no significant role in MMP-13 expression by synovial fibroblasts in rat adjuvant arthritis (Han et al, 2001). IL-1 as well as tumor necrosis factor-α and IL-6 is a potent activator of all of the three MAPK pathways in RSF (Schett et al, 2000), as shown in the present study (Fig. 3). In IL-1–stimulated RSF, the recently developed specific JNK inhibitor significantly inhibits expression of MMP-1 and MMP-3 in association with AP-1 suppression. In the cells, ERK/MEK inhibitor (PD98059) weakly inhibits IL-1–induced MMP-1 expression with modest suppression of AP-1 binding. In contrast, p38 inhibitor (SB203580) has no significant effect on MMP-1 expression or AP-1 binding (Han et al, 2001). The present results suggest different roles of individual MAPK pathways in the production of individual MMPs by RSF in association with COOH-HBFN-f stimulation. In RSF monolayer culture, ERK seems to be involved in MMP-1, MMP-3, and MMP-13 production with COOH-HBFN-f stimulation because PD98059 effectively blocked the MMP production (Fig. 4). Because low concentration (1 μm) of SB203580 inhibited MMP-3 production (Fig. 4), p38 MAPK may contribute to MMP-3 induction by COOH-HBFN-f. In contrast, because only high levels (10 μm) of SB203580 were able to block collagenase induction (Fig. 4), JNK seems to promote the production of MMP-1 and MMP-13 by COOH-HBFN-f. However, high concentrations of the inhibitor may inhibit p38 and JNK as well as other kinases that could alter cellular function. The precise role of JNK in the collagenase induction with COOH-HBFN-f stimulation remains to be determined, ideally by using a selective JNK inhibitor.
There is an increasing body of evidence that cell-matrix interactions regulate MMP induction through integrins (Ivaska and Heino, 2000). At the cartilage-pannus junction, RSF are found to express integrin subunits α4, α5, and β1 (Ishikawa et al, 1994). Normal synoviocytes are unable to adhere to the cartilage surface, whereas in RA, at the cartilage-pannus junction, RSF adhere directly to the cartilage surface (Ishikawa et al, 1991). FN in the superficial region of articular cartilage is likely to serve as a local factor for pannus extension on RA cartilage. RGD-containing synthetic peptide inhibits synovial tissue extension on articular cartilage, indicating an important role of α5β1 and αvβ1 integrins at the cartilage-pannus junction in pannus attachment to cartilage, probably through the binding of integrins to FN (Shiozawa et al, 1992). However, although FN isoform containing IIICS are found to be present in rheumatoid synovium (Müller-Ladner et al, 1997), inhibition studies using synthetic peptides have shown that RSF is unlikely to adhere FN on cartilage via α4β1 integrin (Hino et al, 1996). Similar to COOH-HBFN-f (Figs. 1 and 3), the present study demonstrated that exogenous addition of CS-1 peptide derived from IIICS of COOH-HBFN-f stimulates MMP induction (Fig. 6) in association with MAPK activation (Fig. 7) in RSF. In addition, the MAPK inhibitors (PD98059 and SB203580) provided similar inhibitory effects on MMP production by RSF stimulated with CS-1 (Fig. 8) and COOH-HBFN-f (Fig. 4). From the observation that anti-α4 integrin antibody can partially block COOH-HBFN-f–stimulated MMP production (Fig. 9), it is likely that COOH-HBFN-f uses α4β1 integrin expressed on RSF as one of the signaling receptors for MMP induction. Compared with CS-1 peptide, COOH-HBFN-f can induce greater levels of MMPs (Fig. 6B). Previous studies have shown similar findings that excessive amounts (1 mm) of RGD peptide are required to induce proteoglycan release in cartilage explant culture, whereas FN-f with RGD sequence at 1 μm causes stronger release of proteoglycan (Homandberg et al, 1992). Thus, FN-f could activate integrins more effectively than synthetic peptides. Alternatively, MMP induction by COOH-HBFN-f–stimulated RSF may involve other mechanisms. Because cell surface heparan sulfate and chondroitin sulfate are implicated in cell adhesion to several sites in the III12 to 14 domains of COOH-HBFN-f (Drake et al, 1992; Iida et al, 1992), binding of COOH-HBFN-f with cell surface proteoglycans may contribute to MMP induction, which is under investigation.
Materials and Methods
Materials
Anti-human integrin α4 antibodies (C-20: sc-6589; N-19: sc-6590) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, California). Anti-human MMP-1 (M4177), MMP-3 (M4802), and MMP-13 (M4052) antibodies were obtained from Sigma. Anti-ERK (K-23; sc-94) and -p38 MAPK (C-20; sc-535) were purchased from Santa Cruz Biotechnology, Inc. Anti-phospho-ERK, -phospho-p38 kinase, -JNK, and -phospho-JNK were obtained from Cell Signaling Technology (Beverly, Massachusetts). Alkaline phosphatase-conjugated anti-rabbit IgG was purchased from Southern Biotechnology Associates, Inc. (Birmingham, Alabama). Alkaline phosphatase-conjugated anti-goat IgG and nonspecific goat IgG were obtained from Zymed Laboratories, Inc. (South San Francisco, California). Synthetic peptides peptide V (WQPPRARI), CS-1 (DELPQLVTLPHPNLHGPEILDVPST), and GRGDS were purchased from Takara Shuzo Company (Kusatsu, Japan). Human plasma FN and COOH-HBFN-f prepared from human plasma FN with α-chymotrypsin digestion were obtained from GIBCO BRL (Rockville, Maryland). The 40-kd COOH-HBFN-f contains a heparin-binding region with type III12 to 14 segments and IIICS. NH2-HBFN-f, which is a proteolytic 30-kd fragment of human plasma FN with the first type I repeats, was purchased from Sigma. The purity of protein preparation has been confirmed as shown in our previous studies (Yasuda and Poole, 2002). PD98059 and SB203580 were from Wako Pure Chemical Industries (Osaka, Japan). Recombinant human IL-1β and IL-1ra were purchased from R&D Systems (Minneapolis, Minnesota).
Rheumatoid Synovial Cell Culture and Preparation of Cell Lysate
Synovial tissues were obtained at knee joint replacement surgery from four patients who fulfilled the American College of Rheumatology (1987) revised criteria. Samples were immediately prepared for culture of RSF. The tissue was minced into small pieces, digested enzymatically, and filtered through a nylon mesh. The released cells were grown on 75 cm2 dishes (Corning, New York) in Dulbecco's modified Eagle's medium containing 100 units/ml penicillin, 100 units/ml streptomycin, and 10 mm HEPES (DMEM; all from GIBCO BRL) in the presence of 10% heat-inactivated fetal bovine serum (ICN Biomedicals, Inc., Aurora, Ohio) in a humidified 5% CO2 atmosphere at 37°C. At confluence, cells were trypsinized, split at a 1:3 ratio, and cultured in DMEM with 10% fetal bovine serum. After 3 to 5 passages, RSF were plated on 35-mm six-well plates (Corning). At confluence, the cells were used for experiments. After extensive wash with PBS, all experiments were done under serum-free conditions. RSF were incubated with peptide V, CS-1, GRGDS, FN, HBFN-f, or IL-1β. In some experiments, IL-1ra was also added together with COOH-HBFN-f or IL-1β. In other experiments, after pretreatment with one of the MAPK-specific inhibitors for 1 hour, RSF were stimulated with COOH-HBFN-f, CS-1, or IL-1β. In another set of experiments, after preincubation with anti-α4 integrin antibody (sc-6590) or nonspecific IgG for 1 hour, RSF were co-incubated with COOH-HBFN-f. Assays of endotoxin levels in 100 nm COOH- and NH2-HBFN-f solutions before use showed minimal levels of detection of 6 to 8 and ~20 ng/ml, respectively.
After incubation for various periods of time at 37°C, cell lysates were prepared after the harvest of conditioned media for assays. Briefly, cells were washed twice with cold PBS and then lysed in a lysis buffer containing 50 mm Tris (pH 7.5), 150 mm NaCl, 5 mm EDTA, 10 mm NaF, 2 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 2 mm N-ethylmaleimide, and 1% Triton X-100 at 4°C. Total cell lysates were cleared by centrifugation at 16,000 g for 10 minutes at 4°C.
Immunoblot Analysis
Total cell lysates and conditioned media were heated with SDS-PAGE sample buffer at 80°C for 20 minutes. Proteins were separated by SDS-PAGE under reducing conditions and thereafter transferred onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Membranes were blocked in Tris-buffered saline (50 mm Tris [pH 7.5], 150 mm NaCl) containing 5% nonfat dry milk and 0.1% Tween 20 and incubated with the first antibody (concentration 1/1000) overnight at 4°C. After incubation with the alkaline phosphatase-conjugated second antibody (concentration 1/1000) for 3 hours at room temperature, immunoreactive bands were visualized using 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium. Protein band intensity was evaluated by densitometry using National Institutes of Health image.
Immunoprecipitation
At confluence after three to four passages, after being wash with cold PBS, RSF were lysed on 75-cm2 dishes as described above. Total cell lysates were first incubated overnight at 4°C with or without the antibody recognizing α4 integrin subunit, followed by the addition of protein G-Sepharose, and then incubated for additional 3 hours at 4°C. The precipitates were washed five times with the lysis buffer and thereafter boiled with SDS-PAGE sample buffer for 5 minutes to dissociate the proteins. The immunoprecipitated proteins were detected using the first antibody against integrin α4 (concentration 1/1000) and the alkaline phosphatase-conjugated second antibody (concentration 1/1000) by immunoblot analysis under reducing conditions, as described above.
DNA Assay
After collection of conditioned media, cultured RSF were digested with 0.5 mg/ml proteinase K in 50 mm Tris-HCl (pH 7.5) for 6 hours at 37°C. DNA content in proteinase K digests was determined as described previously (Labarca and Paigen, 1980). We found no significant difference of DNA content in RSF between any treatment groups (data not shown).
References
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This work was supported by Grants-in-Aid for Scientific Research (No. 14571375) from the Japan Society for the Promotion of Science
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Yasuda, T., Shimizu, M., Nakagawa, T. et al. Matrix Metalloproteinase Production by COOH-Terminal Heparin-Binding Fibronectin Fragment in Rheumatoid Synovial Cells. Lab Invest 83, 153–162 (2003). https://doi.org/10.1097/01.LAB.0000056999.08437.B2
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DOI: https://doi.org/10.1097/01.LAB.0000056999.08437.B2