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. Author manuscript; available in PMC: 2017 Jul 12.
Published in final edited form as: Arthritis Rheum. 2011 Dec;63(12):3768–3778. doi: 10.1002/art.30630

Cadherin-11 engagement modulates matrix metalloproteinase production by rheumatoid arthritis synovial fibroblasts

Erika H Noss 1, Sook Kyung Chang 1, Gerald FM Watts 1, Michael B Brenner 1
PMCID: PMC5507176  NIHMSID: NIHMS319231  PMID: 22127696

Abstract

Objective

Cadherin-11 is a homophilic cell-to-cell adhesion molecule expressed on joint synovial fibroblasts. Absence of cadherin-11 in a mouse rheumatoid arthritis (RA) model led to striking reductions in cartilage erosion. Matrix metalloproteinases (MMPs) are enzymes expressed by synovial fibroblasts important for cartilage erosion. The objective of this study was to determine if synovial fibroblast MMP production is regulated by cadherin-11.

Methods

To mimic cadherin-11 engagement, human RA synovial fibroblasts were stimulated with a chimeric construct consisting of the cadherin-11 extracellular domain linked to the human IgG1 Fc domain (cad-11-Fc). Effects on MMP production were measured by ELISA, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and immunoblotting assays.

Results

Human cad-11-Fc upregulated MMP-1 and MMP-3 protein production by RA synovial fibroblasts both alone and in synergy with tumor necrosis factor (TNF)-α. This upregulation required cell cadherin-11 engagement, as a mutant cad-11-Fc with reduced binding affinity stimulated significantly less MMP production. Also, short-hairpin (shRNA) cadherin-11 silencing almost completely inhibited cad-11-Fc-induced MMP expression. Cad-11-Fc stimulation increased RA synovial fibroblast MMP mRNA levels. It also increased mitogen-activated protein kinases (MAPKs) jun N-terminal kinase (JNK), extracellular signal-related kinase (ERK), and p38 kinase phosphorylation, nuclear factor-kappaB (NF-κB) p65 phosphorylation, and activated protein (AP)-1 transcription factor nuclear translocation. MAPK and NF-κB inhibitors partially blocked RA synovial fibroblast MMP expression.

Conclusions

Cadherin-11 engagement stimulates increased synthesis of several MMPs by RA synovial fibroblasts in a MAPK and NF-κB-dependent fashion. These results underscore a pathway by which cadherin-11 regulates MMP production, with important implications for joint destruction in RA.

INTRODUCTION

The cadherin family consists of at least six subfamilies of cell adhesion molecules characterized by the presence of 110 amino acid immunoglobulin-like extracellular cadherin domains(1). The classical cadherins are comprised of an extracellular N-terminal region consisting of five cadherin domains, a single-pass transmembrane domain, and a cytoplasmic tail(2). Cell-to-cell adhesion is mediated by homophilic binding of the extracellular domains, namely a cadherin on one cell binds a cadherin of the same type on an adjacent cell. On each cell, the cadherins organize into adherens junctions to link to and regulate the actin cytoskeleton through interactions at their cytoplasmic tails with the catenin molecules: β-catenin, α-catenin, and p120 catenin(3). Besides connecting with actin, cadherins and their associated catenins interact with variety of signaling molecules, including growth factor receptors, soluble kinases/phosphatases, and Rho family small GTPases(4, 5).

Cadherins play a critical role in embryogenesis, where temporospatial expression of specific cadherins results in cell aggregation, directing migration of cell sheets and tissue morphogenesis(2, 6). Cadherin expression continues to show tissue specificity important for the maintenance of adult tissue architecture. For example, expression of epithelial (E)-cadherin is a hallmark of epithelial layers, and loss of E-cadherin results in epithelial lining layer disruption(7).

Cadherin expression is also important in the synovium, the lining tissue of diarthrodial joints that provides lubrication for movement and nourishment for articular cartilage(8). The normal synovium consists of a cellular lining layer overlying a mainly connective tissue sublining layer. The synovial lining is generally one to three cells layers thick and consists of both fibroblast and macrophage cell types in close proximity. The sublining layer is mainly extracellular matrix, with scattered blood vessels, fibroblasts, and innate immune cells. A cloning technique identified cadherin-11 expression in synovial tissues, and further studies confirmed strong expression of this molecule by lining layer synovial fibroblasts(9). Furthermore, the presence of cadherin-11 appears essential for the development of normal synovial architecture, as mice deficient in cadherin-11 show a hypoplastic synovial lining characterized by loss of both lining cells and extracellular matrix(10).

The pathogenesis of RA involves infiltration of the synovial sublining by leukocytes and transformation of the normal synovial lining into a hyperplastic, invasive tissue pannus capable of eroding bone and cartilage(8). Cadherin-11 on synovial fibroblasts appears critical for the full development of this pathologic process. When inflammatory arthritis was induced in cadherin-11 null mice using the K/BxN serum transfer model, cadherin-11 null mice developed approximately 50% less joint inflammation compared to wildtype mice(10). Strikingly, cadherin-11 null mice were almost completely protected from cartilage erosions, despite ongoing bone erosion. These studies support a central role for synovial fibroblasts in cartilage invasion and damage in RA and reveal cadherin-11 as an important regulator of this synovial fibroblast behavior.

Key to cartilage erosion in RA are the matrix metalloproteinases (MMPs), a diverse family of zinc-dependent endopeptidases with broad specificity against extracellular matrix components(11). In particular, the collagenases (MMP-1, -8, -13, -14), belong to a very restricted subset of enzymes capable of cleaving intact fibrillar collagens, like the type II collagen matrix that provides tensile strength to cartilage(12). Several MMPs are upregulated in RA synovial fluid and tissues(13, 14), and MMP expression has been positively correlated with increased synovial fibroblast invasion(15–18). Furthermore, increased MMP-3 expression is a specific biomarker of joint damage in RA patients(19).

In this study, we show that cadherin-11 engagement on RA synovial fibroblasts by a fusion protein linking the cadherin-11 extracellular domain to the human IgG1 Fc domain (cad-11-Fc) induces the expression of several MMPs, both alone and strikingly in synergy with inflammatory cytokines such as TNF-α. Increased MMP production is dependent on activation of RA synovial fibroblast MAPK and NF-κB pathways. These results point to an unexpected pathway by which cadherin-11 regulates RA synovial fibroblast degradative and invasive behavior, suggesting that the formation and turnover of cadherin-11 contacts in pannus tissue may facilitate cartilage erosion through upreguation of MMP expression.

MATERIALS AND METHODS

Cell culture and media

Human RA and OA synovial tissues were obtained after synovectomy or joint replacement surgery performed as part of indicated clinical care at Brigham and Women’s Hospital. Synovial fibroblasts were released from synovial tissue by mincing followed by collagenase digestion, purified by serial passage as previously described(20), and used experimentally between passages 5 and 10. Purified normal human lung and skin fibroblast lines were obtained commercially (Lonza, Rockland, ME). Fibroblast cell lines were cultured at 37°C under 10% CO2 in 10% serum-containing media: DMEM supplemented with 10% fetal bovine serum (FBS; Gemini, West Sacromento, CA), 2 mM L-glutamine, 100 units/ml penicillin, 50 µM 2-mercaptoethanol (2-ME), and essential and nonessential amino acids (Gibco BRL, Gaithersburg, MD). RA synovial fibroblast cell lines were serum-starved prior to assays by incubation for 24–72 hours in media identical to that used for routine culture except that FBS content was reduced to 1%. All ELISA assays were performed on culture supernatants obtained from 15,000 RA synovial fibroblasts/well stimulated in 96-well plates.

Cadherin-11 fusion proteins, antibodies, and other reagents

Cad-11-Fc fusion proteins were stably expressed in HEK293 cells after cloning the cadherin-11 extracellular domain (residues 1–1827) into the pFUSE-human IgG1-Fc1 vector (InvivoGen, San Diego, CA)(21). Cad-11-Fc was purified from culture supernatants by protein A column (Bio X Cell, West Lebanon, NH). The antibodies used are as follows: human IgG1 isotype control (Sigma, St. Louis, MO); mouse IgG1 isotype control (clone MOPC-21, Bio X Cell); anti-cadherin-11 monoclonal antibody 23C6(10); donkey anti-mouse IgG phycoerythrin (PE) conjugate (Jackson ImmunoResearch, West Grove, PA); and anti-phosphorylated JNK (No. 4668), anti-total JNK (No. 9252), anti-phosphorylated ERK1/2 (No. 4370), anti-total ERK1/2 (No. 4695), anti-phosphorylated p38 kinase (No. 4115), anti-phosphorylated p65 (No. 3033), and anti-total p65 (No. 4764) (Cell Signaling Technology, Danvers, MA). The other reagents used are as follows: human MMP-1 and MMP-3 ELISAs (R&D Systems, Minneapolis, MN); human TNF-α and IL-1β (R&D Systems); PathScan® Phospho-NF-κB p65 (Ser536) ELISA (Cell Signaling Technology); lipopolysaccharide (LPS, from Samonella, Sigma); JNK inhibitors JNK I (HIV-TAT-JNK binding domain peptide construct) and SP600125 (EMD Chemicals, Gibbstown, NJ); ERK inhibitors U0126 and FR180204 (EMD Chemicals); p38 kinase inhibitor SB203580 (EMD Chemicals); NF-κB (IKK-2) inhibitors IKK-2 inhibitor IV and SC-514 (EMD Chemicals). Inhibitors SP600125, U0126, FR180204, SB203580, IKK-2 inhibitor IV, and SC-514 were first dissolved in dimethyl sulfoxide (DMSO) before dilution in DMEM-containing media, while the JNK I inhibitor was directly soluble in media.

Cell adhesion assay

RA synovial fibroblasts were released from culture flasks using 0.02% trypsin (Worthington Biochemical, Lakewood, NJ) in HBS Ca (20 mM Hepes, 137 mM NaCl, 3 mM KCl, 1 mM CaCl, pH 7.4) for 5 minutes at 37°C to minimize cadherin-11 proteolysis. After adding one volume 0.04% trypsin soybean inhibitor (Sigma) in HBS Ca, cells were washed and fluorescently labeled for one hour with 15 ug/ml calcium AM (Invitrogen, Carlsbad, CA) in 10% serum-containing media. Labeled RA synovial fibroblasts (30,000/well) resuspended in HBS Ca containing 0.1% bovine serum albumin (BSA, Sigma) were added to cad-11-Fc-coated 96 well microplates blocked with 1% BSA. Cells were incubated for 30 to 90 minutes at 37°C to allow adhesion. Percent adhesion was calculated as previously described(22) by dividing fluorescence remaining after sequential washing by the starting fluorescence and multiplying by 100.

Synovial fibroblast lentiviral transfection

Lentivirus containing short-hairpin (shRNA) against cadherin-11 (TRCN0000054334 and TRCN0000054335, Thermo Scientific Open Biosystems, Huntsville, AL) or a control sequence (MISSION® control, Sigma) were generated by transfecting 293T cells with a combination of packaging plasmid pCMV-dR8.91 (Broad Institute, Cambridge, MA), envelope plasmid VSV-G/pMD2.G (Broad Institute), and hairpin-pLKO.1 vector. RA synovial fibroblasts plated at confluency in 6-well plates were spun at 700 x g for 30 minutes in media containing equivalent viral concentrations and 6 µg/ml polybrene (Sigma). After 2 days, 3.5 µg/ml puromycin (Sigma) was added for 2 to 3 days to select for infected cells. Cells were then trypsinized (0.02% tryspin, 5 mM EDTA) and replated for 1 day in 10% serum-containing media before use in experiments. Cadherin-11 surface expression after infection was analyzed by flow cytometry (FACS Canto, BD Biosciences, Rockville, MD) as previously described(9) using anti-cadherin-11 23C6 or mouse IgG1 isotype control antibodies for primary staining and donkey-anti-mouse IgG-PE for secondary staining.

Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR)

Total cellular RNA was extracted from equivalent numbers of cultured RA synovial fibroblasts using RNeasy Micro columns (Qiagen, Valencia, CA) and reverse transcribed using QuantiTect Reverse Transcription Kit (Qiagen) per manufacturer’s instructions. Equivalent volumes of cDNA were analyzed by qPCR using Brilliant SYBR® Green QPCR Master Mix (Agilent Technologies, Santa Clara, CA) using the following primers (Integrated DNA Technologies, Coralville, IA): MMP-1 Forward 5’-TAAAGACAGATTCTACATGCGC-3’ Reverse 5’-GTATCCGTGTAGCACATTCTG-3’; MMP-3 Forward 5’-CTGCTGTTGAGAAAGCTCTG-3’ Reverse 5’-AATTGGTCCCTGTTGTATCCT-3’; MMP-13 Forward 5’-GACATTCTGGAAGGTTATCCC-3’ Reverse 5’-AGTATCATCATATCTCCAGACC-3’; MMP-14 Forward 5’-TCATGATCTTCTTTGCCGAG-3’ Reverse 5’-GATGTCATTTCCATTCAGATCC-3’; hypoxanthine-guanine phosphoribosyltransferase (HPRT): Forward 5’-GGGCTATAAATTCTTTGCTGAC-3’ Reverse 5’-CTGGTCATTACAATAGCTCTTCAG-3’.

Cell lysates and western blots

RA synovial fibroblasts (150,000 cells/well, 6-well plates) were lysed using the following buffer: 50 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM PMSF, and a mixture of protease inhibitors (Complete, Roche Applied Science, Indianapolis, IN). Lysates were cleared of insoluble material by centrifugation, electrophoresed though polyacrylamide-SDS gels and then transferred to Immobilon-P membranes (Bio-Rad, Hercules, CA). Membranes were blocked in TBS-T (25 mM Tris-HCl [pH 7.2], 150 mM NaCl, 0.05% Tween) supplemented with 5% nonfat skim milk or 5% BSA (for detecting phosphorylated proteins) and incubated with antibodies against the target proteins. After washing, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG before detection with a chemiluminscent substrate.

AP-1detection and NF-κB p65 ELISA

To assay for AP-1, RA synovial fibroblast nuclear extracts were isolated by lysing cells in hypotonic buffer (10 mM Hepes, pH 7.9, 1 mM Mg2Cl, 10 mM KCl, 0.1% Triton X-100, 20% glycerol, 1 mM dithiothreitol, and proteases inhibitors), resupsending the remaining pellet in hypertonic buffer (10 mM Hepes, pH 7.9, 0.1% Triton X-100, 400 mM NaCl, 1 mM EDTA, 20% glycerol, 1 mM dithiothreitol, and proteases inhibitor), and then collecting the supernatant after centrifugation. AP-1 was detected in 5 µg total protein/well using an anti-phosphorylated c-Jun antibody in the DNA-binding TransAM™ AP-1 ELISA (Active Motif, Carlsbad, CA) per manufacturer’s instructions. To assay for ELISA for phosphorylated NF-κB p65, RA synovial lysates were sonicated using the buffer provided by the PathScan® Phospho-NF-κB p65 (Ser536) ELISA kit (Cell Signaling). Phosphorylated p65 was detected in 5 µg total protein/well per manufacturer’s instructions.

RESULTS

Cad-11-Fc increases expression of MMP-1 and MMP-3, alone and synergistically with inflammatory cytokines

Given the in vivo findings that cadherin-11 deficiency protects mice with inflammatory arthritis from cartilage degradation, we hypothesized that cadherin-11 engagement might regulate synovial fibroblast production of MMPs that are important for cartilage erosion in RA. Initially, we focused on MMP-1 and MMP-3, two MMPs upregulated by RA synovial fibroblasts in response to inflammatory cytokines(23). MMP-1 is a collagenase capable of cleaving intact type II collagen fibrils found in cartilage, and retroviral MMP-1 silencing in RA synovial fibroblasts blocked cartilage invasion in an ex vivo assay(11, 16). MMP-3 is a stromelysin with activity against many noncollagen matrix proteins; it also cleaves other MMP zymogens, leading their activation(24). Furthermore, increased MMP-3 serum levels are a biomarker for joint damage in RA patient populations(19).

To model cadherin-11 engagement pharmacologically, we developed a soluble, recombinant cad-11-Fc fusion protein that binds to cell cadherin-11 in a manner like that of cadherin-11 interactions between adhering cells (Fig. 1A)(9, 25). These cadherin fusion proteins, when bound to latex beads, have been shown to interact with cell surface cadherins, recruiting catenins and linking to the actin cytoskeleton(21). Incubation of serum-starved human RA synovial fibroblasts with increasing concentrations of human cad-11-Fc increased production of MMP-1 and MMP-3 in a dose dependent manner, compared to no stimulation or stimulation with matched human isotype control (Fig. 1B). The ability of cadherin-11 to regulate MMP production was not unique to RA synovial fibroblasts. Cultured osteoarthritis (OA) synovial fibroblasts, normal skin fibroblasts, and normal lung fibroblasts also express cell surface cadherin-11 and upregulate MMP-3 expression after incubation with cad-11-Fc (Supplemental Fig. 1).

Figure 1. Cad-11-Fc increases RA synovial fibroblast MMP expression alone and synergistically with TNF-α.

Figure 1

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A. The cad-11-Fc fusion protein consists of the Fc domain of human IgG1 (Fc) linked to the five extracelluar domains of cadherin-11 (EC1-5). Individual fusion protein molecules dimerize through disulfide linkages in the Fc region(9). Cad-11-Fc binds to surface cadherin-11, which in turn binds to p120 catenin (p120), β-catenin (β), and α-catenin (α) at its cytoplasmic tail. Interactions between β-catenin and α-catenin help link cadherin-11 to the actin cytoskeleton. B. Serum-starved RA synovial fibroblasts (15,000 cells/well, 96-well plate) were incubated for 24 hours in 1% serum-containing media with or without increasing concentrations of cad-11-Fc or human IgG1 isotype control. Culture media was then assayed for MMP-1 or MMP-3 by ELISA. This figure shows one representative experiment from experiments performed with six separate cell lines. C. Culture media from serum-starved RA synovial fibroblasts incubated for 24 hours with isotype or cad-11-Fc (15ug/ml) alone or with 0.2ng/ml TNF-α was assayed for MMP-3 by ELISA. This figure shows one representative experiment chosen from eight separate experiments performed with three RA synovial fibroblast cell lines. All error bars represent the standard deviation of the mean from duplicate wells.

The inflammatory milieu in the RA joint contains cytokines like TNF-α and IL-1β that strongly induce expression of MMP-1 and -3 by RA synovial fibroblasts. To test if cadherin-11 stimulation augments cytokine-driven MMP expression, RA synovial fibroblasts were incubated with either cad-11-Fc or low dose TNF-α alone or in combination, and the effect on MMP-3 expression was measured by ELISA (Fig. 1C). The combination of cad-11-Fc and low-dose TNF-α induced more MMP-3 expression than the additive effects of each agent alone: cad-11-Fc alone 1164 pg/ml; TNF-α alone 735 pg/ml; predicted stimulation based on additive effect 1899 pg/ml; actual stimulation based on combination 4720 pg/ml. On average, there was 2.5 fold increased MMP production by combined stimulation with cad-11-Fc and TNF-α compared to the sum of each agent alone (n=8 experiments). Similar results were seen with IL-1β stimulation (data not shown). These results suggest that cadherin engagement acts in synergy with inflammatory cytokines to stimulate MMP expression, complementing the effects of inflammatory cytokines on RA synovial fibroblast activation.

Cad-11-Fc action depends on engagement of cell surface cadherin-11 molecules

Two approaches were taken to determine if cad-11-Fc acts specifically through engagement of cadherin-11, rather than from nonspecific mechanisms. In the first approach, we created a mutant cad-11-Fc by exchanging tryptophans 2 and 4 in the first extracellular domain for alanines. Structural studies have shown that insertion of these tryptophans into a hydrophobic pocket in the first extracellular domain on the cadherin from another cell bound in trans helps stabilize cadherin-to-cadherin adhesion(26, 27). In keeping with the known important role for these amino acids in increasing cadherin-11 binding affinity, RA synovial fibroblasts adhered substantially less well in a static adhesion assay to plates coated with the mutant cad-11-Fc (cad-11-Fc W2,4A) compared to plates coated with similar amounts of wildtype cad-11-Fc (Fig. 2A). Correspondingly, when RA synovial fibroblasts were stimulated with equivalent amounts of wildtype and mutant cad-11-Fc (Fig. 2B,C), the mutant cad-11-Fc induced significantly less MMP-1 and MMP-3, consistent with its reduced binding affinity for cellular cadherin-11 (average % decrease MMP expression by mutant cad-11-Fc stimulation compared to wildtype: MMP-1 59.8 +/− 13.4% (n=3 cell lines); MMP-3 60.3 +/− 25.3% (n=4 cells lines)).

Figure 2. A mutant cad-11-Fc construct with reduced cadherin binding affinity stimulates less MMP-1 and MMP-3 expression from RA synovial fibroblasts compared to wildtype cad-11-Fc.

Figure 2

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A. The ability of calcein-AM labeled RA synovial fibroblasts to adhere after a 90 minute incubation to 96-well plates coated with either wildtype or W2,4A cad-11-Fc was calculated by dividing the fluorescence remaining after sequential washing by the starting fluorescence and multiplying by 100. This representative experiment was chosen from three separate experiments done with three different cell lines. Error bars show the standard deviation of the mean from triplicate wells. B, C. Serum-starved RA synovial fibroblasts were stimulated for 24 hours either isotype control antibody, wildtype cad-11-Fc, or W2,4A cad-11-Fc, and the amount of MMP-1 (B) or MMP-3 (C) in the culture media was measured by ELISA. This representative experiment was chosen from four separate experiments done with four different cell lines. Error bars show the standard deviation of the mean from triplicate wells.

In addition, to confirm that cadherin-11 expression at the cell surface is required for cad-11-Fc activation, we tested the ability of cad-11-Fc to induce MMPs after lentiviral shRNA silencing of cellular cadherin-11. Lentiviral infection with a cadherin-11 specific virus decreased cadherin-11 expression by greater than 90% compared to control virus or uninfected cells, as shown by surface staining and flow cytometry for cadherin-11 (Fig. 3A) or by mRNA levels (data not shown). Stimulation of MMP-3 production by cad-11-Fc was almost completely lost in cadherin-11-silenced cells compared to control infected cells (Fig. 3B). In contrast, stimulation of MMP-3 production by either LPS or IL-1β was unaffected by cadherin-11 silencing, providing further evidence that cad-11-Fc induces MMP expression specifically through binding to RA synovial fibroblast cadherin-11.

Figure 3. RA synovial fibroblast cadherin-11-silencing blocks MMP-3 expression by cad-11-Fc but not IL-1 or LPS.

Figure 3

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A. RA synovial fibroblasts infected with control or cadherin-11 shRNA-containing lentivirus were stained with either an isotype control or anti-cadherin-11 primary antibody followed by donkey anti-mouse IgG phycoerythrin secondary antibody. Cadherin-11 surface staining was then analyzed by flow cytometry. B. Serum-starved RA synovial fibroblasts were incubated with media alone or with isotype control antibody (40 µg/ml), cad-11-Fc (40 µg/ml), LPS (20 ng/ml), or IL-1β (0.1 ng/ml) for 24 hours before the amount of MMP-3 in the culture media was determined by ELISA. The figures presented are generated from a single representative experiment. For the flow cytometry data (A) and the cad-11-Fc stimulation (B), the experiment was done seven times with five different cell lines. For the LPS and IL-1 stimulation, the experiment was done three times with three different cell lines. Error bars show the standard deviation of the mean from duplicate wells.

Cad-11-Fc increases mRNA expression of a subset of MMPs

MMP activity is tightly controlled by several mechanisms, including transcriptional regulation, zymogen activation, and production of endogeneous inhibitors(11, 28) . Since MMP-1 and -3 protein levels rose substantially after cad-11-Fc stimulation, qRT-PCR was used to test whether cad-11-Fc stimulated mRNA expression of a panel of MMPs. Cad-11-Fc induced a rise in MMP-1, MMP-3, and MMP-13 mRNA levels compared to appropriate isotype control (Fig. 4, two representative cell lines shown). These three MMPs are known to be cytokine-inducible and dependent on activation of the transcription factor AP-1(11). In contrast, the more constitutively expressed MMP-14 was not induced by cad-11-Fc, suggesting that cadherin-11 engagement regulates a subset, but not all, MMP transcription. As a control, cad-11-Fc did not alter cadherin-11 mRNA levels (data not shown).

Figure 4. Cad-11-Fc increases MMP-1, -3, and -13 but not MMP-14 mRNA levels in RA synovial fibroblasts.

Figure 4

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Two serum-starved RA synovial fibroblast cell lines (90,000 cells/well in 24-well plates) were stimulated for 24 hours with either cad-11-Fc (40 µg/ml) or appropriate isotype control before isolating total cellular RNA for qRT-PCR for MMP-1, MMP-3, MMP-13, and MMP-14. Expression of each MMP was normalized to the housekeeping gene HPRT. Error bars show the standard deviation of the mean from duplicate wells, and the experiments shown are representative of eight experiments done with four separate cells lines.

Cad-11-Fc increases MMP production by activating MAPKs and NF-κB

MMP-1, -3, and -13 belong to an inducible group of MMPs whose proximal promoter contains both a TATA box and AP-1 transcription factor consensus sequences (28). The constitutively expressed MMP-14, on the other hand, belongs to a subset of MMPs without either a TATA box or AP-1 consensus sequences in the proximal promoter. The ability of cad-11-Fc to induce the transcription of MMP-1, -3, and -13, but not MMP-14, suggests that it signals through AP-1 activation. This hypothesis is consistent with what has been described for MMP-1 and -3 transcription by inflammatory cytokines in RA synovial fibroblasts(12, 29, 30). In RA synovial fibroblasts and many other cell types, MMP-1 and -3 transcription by upstream stimuli is critically dependent on activation of the MAPK signaling cascade. Once phosphorylated, the MAPK family members ERK, JNK, and p38 kinase stimulate AP-1 activity, both directly through AP-1 phosphorylation and indirectly by increased synthesis of the Jun and Fos family members that comprise the AP-1 heterodimer(31).

To determine if cad-11-Fc also induces MMP expression by a MAPK and AP-1-dependent pathway, we first tested total cellular RA synovial fibroblast lysates made from unstimulated or cad-11-Fc-stimulated cells for MAPK phosphorylation. Cad-11-Fc increased ERK1/2, JNK, and p38 phosphorylation (Fig. 5A), compared to unstimulated controls. Next, we determined whether or not cad-11-Fc increased AP-1 activation by isolating nuclear fractions from control or cad-11-Fc-stimulated RA synovial fibroblasts. Cells incubated with cad-11-Fc increased phosphorylated AP-1 in their nucleus, suggesting that cad-11-Fc induces MMP transcription by accumulation of nuclear AP-1 upon MAPK activation (Fig. 5B).

Figure 5. Cad-11-Fc stimulation of RA synovial fibroblasts increases MAPK ERK1/2, JNK, and p38 kinase phosphorylation, NF-κB p65 phosphorylation and AP-1 activation.

Figure 5

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A. Serum-starved RA synovial fibroblasts were incubated with cad-11-Fc (20 µg/ml) for 0, 5, 15, or 30 minutes and then lysed. Samples were then assayed for phosphorylated ERK1/2, JNK, and p38 kinase. Total ERK1/2 and JNK served as loading controls. The chosen experiment is representative of four separate experiments with two cells lines. B. Nuclear lysates isolated from serum-starved RA synovial fibroblasts incubated with isotype control antibody (40 µg/ml), cad-11-Fc (40 µg/ml) or TNF-α (20 ng/ml) for 1 hour were tested for AP-1 using a DNA-binding ELISA. Chosen experiment is representative of three separate experiments with two cells lines. Error bars represent standard deviation of the mean from duplicate conditions. C. Serum-starved RA synovial fibroblasts were left unstimulated or incubated with either cad-11-Fc (40 µg/ml) for 30 minutes or TNF-α (10 ng/ml) for 15 minutes before lysis with sonication. The same samples were then assayed for phosphorylated p65 by two methods, western blot and ELISA. Total p65 western blot served as a loading control. Chosen experiment is representative of five separate experiments done with four cell lines.

MMP-1 and MMP-3 expression in synovial fibroblasts also depends on activation of the transcription factor NF-κB(32, 33). To determine whether cadherin-11 engagement increases NF-κB activity, total cellular lysates isolated from unstimulated, cad-11-Fc-stimulated or TNF-α-stimulated RA synovial fibroblasts were assayed for NF-κB p65 phosphorylation by either western blot or ELISA (Fig. 5C). As was seen for MAPK/AP-1, cad-11-Fc also induced NF-κB activation in RA synovial fibroblasts.

To determine if JNK, ERK1/2, p38 kinase, or NF-κB activation leads to MMP-3 expression, RA synovial fibroblasts were incubated with inhibitors specific for each of these pathways prior to stimulation with cad-11-Fc. Inhibition of ERK1/2, JNK, p38 kinase, or NF-κB activity all reduced MMP-3 expression by approximately 25–50% in multiple RA synovial fibroblast cell lines (Fig. 6A–D). These results confirm that both MAPKs and NF-κB play a role in translating the signal induced by cad-11-Fc engagement at the cell surface to MMP expression by RA synovial fibroblasts.

Figure 6. Blockade of ERK, JNK, p38 kinase, and NF-κB activation inhibits cad-11-Fc-induced MMP-3 production by RA synovial fibroblasts.

Figure 6

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Serum-starved RA synovial fibroblasts were preincubated for 30 minutes with indicated inhibitors against ERK1/2 (A), JNK (B), p38 kinase (C) or NF-κB (D) and then stimulated with cad-11-Fc for 24 hours before measuring MMP-3 expression by ELISA. To normalize experiments different cells lines, % inhibition was calculated by dividing the amount of cad-11-Fc-stimulated MMP-3 expression in the presence of DMSO alone or indicated inhibitor by the amount of cad-11-Fc-stimulated MMP-3 without inhibitor, subtracting from 1, and multiplying by 100. All inhibitor concentrations shown were statistically different from DMSO alone by student’s T test (p values < 0.05). All error bars display the standard error of the mean (for ERK1/2 n=6; for JNK n=5; for p38 n=6; for NF-κB n=5).

DISCUSSION

There is a growing appreciation that synovial fibroblasts are key participants in cartilage erosion in RA. Early histological studies showed close proximity of synovial fibroblasts to sites of cartilage erosion, indicating a role for these cells in mediating cartilage damage(34, 35). In vitro studies then demonstrated that synovial fibroblasts are invasive cells capable of secreting many MMPs important for cartilage degradation(13, 18, 29, 36), while ex vivo studies using cartilage implanted under the renal capsule in SCID mice showed that RA synovial fibroblasts, but not osteoarthritis or normal synovial fibroblasts, were capable of mediating cartilage damage in the absence of an adaptive immune system(37). More recently, in vivo studies genetically depleting a molecule dominantly expressed by synovial fibroblasts, cadherin-11, confirmed that synovial fibroblasts are critical drivers of cartilage erosion. The absence of cadherin-11 resulted in almost complete protection of cartilage from erosions after induction of serum transfer arthritis, even though the amount of inflammation was only partially reduced and bone erosions occurred(10).

Cartilage erosion by synovial fibroblasts likely involves several steps, including attachment to extracellular matrix, cell migration over and into cartilage, and digestion of the cartilage matrix. These steps are classically driven by integrin-matrix interactions. How cadherin cell-to-cell adhesion molecules influence cell migration, invasion, and matrix degradation is not fully understood, but there are growing examples of cross-talk between integrin and cadherin signaling pathways(4, 38). Furthermore, tumor models have shown that loss of epithelial E-cadherin with aberrant acquisition of mesenchymal cadherins such as N-cadherin and cadherin-11 leads to an epithelial to meschenchymal transition (EMT), increasing tumor cell invasion and metastasis, providing evidence that cadherins contribute to invasive processes by several mechanisms(39, 40).

Prior studies in our laboratory showed that absence or blockade of cadherin-11 on synovial fibroblasts reduced cell migration and invasion through basement membrane extracts(10, 41), while the expression of cadherin-11 in a cadherin null fibroblast cell line promoted invasion(42). Here, we suggest an additional, unique pathway by which cadherin-11 may influence cartilage erosion. Incubation of RA synovial fibroblasts with cad-11-Fc, a molecule that mimics cadherin-11 engagement, increased the synthesis of several inducible MMPs implicated in joint destruction in inflammatory arthritis. These MMPs include MMP-1, a collagenase shown to be major mediator of fibroblast-driven cartilage erosion in an ex vivo model system(16), and MMP-3, a stromelysin that has proved valuable as a joint damage biomarker in RA trials(19). Interestingly, not all MMPs were regulated by cadherin-11. RA synovial fibroblast expression of MMP-14, a membrane-bound MMP shown to be critical for synovial fibroblast cartilage invasion in two in vitro assay systems(15, 17), was not increased by cad-11-Fc.

Cartilage erosion in RA is a complex process. Current models suggest that degradative enzymes secreted by the synovium and synovial fluid inflammatory cells damage the cartilage surface, removing the protective proteoglycan layer(43, 44). This damage exposes the underlying cartilage matrix and allows deposition of immune complexes and fibrin(45, 46). The hyperplastic synovial lining then converts into pannus tissue, further perpetuating cartilage damage by attaching to, invading, and degrading the cartilage surface(47).

The overlapping expression and functions of the 23 human MMPs has made it challenging to define which MMPs play a role in the various steps leading to cartilage damage. Experiments using intact collagen matrices have shown that, once activated, soluble MMPs are capable of digesting broad areas of matrix, while membrane MMPs mediate more focal, pericellular cell degradation and invasion(48). Therefore, cadherin-11, by inducing expression of several soluble MMPs, may help promote early damage to the cartilage matrix that facilitates later synovial fibroblast attachment, allowing other MMP pathways to promote direct cell invasion into the cartilage matrix.

Formation of new cadherin contacts causes activation of cell signaling pathways, as measured by a transient increase in cell tyrosine phosphorylation(5). In vitro, we pharmacologically mimicked this process using cad-11-Fc and showed both increased phosphorylation of the MAPKs ERK, JNK, p38, and p65 and nuclear translocation of AP-1 (Fig. 5). However, individually, NF-κB and MAPK activation only contributed partially to MMP expression (Fig. 6), and combinations of MAPKs inhibitors or MAPK/NF-κB inhibitors were unable to completely block MMP production (data not shown), suggesting other signaling pathways are also important. In addition to AP-1 and NF-κB consensus sequences, various MMP promoters contain combinations of binding sites for many transcription factors, including ETS, STAT, and TCF/LEF family members(12, 28). The precise role other signaling pathways play in cadherin-11-stimulated MMP expression requires further study.

The upstream signals between cad-11-Fc engagement at the cell surface and MAPK and NF-κB signaling are not known. Cadherins at the cell surface associate with growth factor receptor tyrosine kinases, soluble kinases and phosphatases, catenins, and Rho family small GTPases(4, 5), which alone or in combination might help transmit signals upon cadherin-11 binding. This study makes it clear that cadherins are not just passive mediators of cell-to-cell adhesion. Rather, engagement of cadherins provides a mechanism to communicate extracellular signals inside the cell.

Although cell-to-cell contacts are maintained in normal tissues, these contacts are continually remodeled, with individual cadherin molecules recycling in and out of the cell contact site, breaking and reforming cadherin binding interactions(49). This remodeling process may provide a homeostatic level of cell signaling appropriate to help maintain normal cell function and tissue architecture, not just for synovial fibroblasts but for fibroblasts from other tissues when cadherin-11 is expressed. In a noninflammatory state, any MMPs stimulated by this process would likely contribute to basal extracellular matrix remodeling.

In the RA synovium, however, histologic evaluation has shown that the number of cadherin-11 contacts is increased(50). We propose that either increased numbers and/or increased turnover of cadherin-11 contacts in the compacted, hyperplastic RA synovial lining stimulates cadherin-11-mediated cell signaling, activating RA synovial fibroblasts and increasing their sensitivity to inflammatory cytokines such as TNF-α and IL-1β. This process results in increased in MMP production (shown in this study) and inflammatory mediator secretion(21). Consistent with this model, this study showed that cad-11-Fc acted synergistically with low dose TNF-α to promote marked MMP-3 production. These findings suggest a model in which formation and turnover of cadherin-11 contacts in pannus tissue acts together with the inflammatory cytokines to stimulate production of enzymes that degrade the cartilage matrix, leading to the eventual destruction of joint structure in RA.

Supplementary Material

Supp Figure S1
Supplementary Legends

Acknowledgments

Financial Support

Erika H. Noss - Funding for this project was provided by the American College of Rheumatology Research and Education Health Professional New Investigator Award and the Abbott Scholar Award for Rheumatology Research.

Michael B. Brenner - Funding for this project was provided by ACR-REF Within Our Reach grant, NIH R01 AR048114 “Synovial Cadherin in Rheumatoid Arthritis” grant, and NIH P01 AI065858 “Cadherin-11 Regulation of Murine Synoviocytes” grant.

Footnotes

Disclosures

Michael B. Brenner
  • Consultant, Stock/Stock Options - Synovex Corp.
  • Consultant – CalciMedica Inc.
  • Patents/Licensing Fees –Synovex Corp
Erika H. Noss, Sook Kyung Chang
  • Patents/Licensing Fees –Synovex Corp
Gerald F.M. Watts
  • No Disclosures

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Supplementary Materials

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Supplementary Legends
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