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The Journal of Immunology, 2008, 181, 2813 -2820
Copyright © 2008 by The American Association of Immunologists, Inc.
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Annexin-1 Mediates TNF-{alpha}-Stimulated Matrix Metalloproteinase Secretion from Rheumatoid Arthritis Synovial Fibroblasts1

Clement E. Tagoe2,3,*,§, Nada Marjanovic2,*,{ddagger}, Jean Y. Park*,{ddagger}, Edwin S. Chan*,{dagger}, Aryeh M. Abeles*,{ddagger}, Mukundan Attur*, Steven B. Abramson* and Michael H. Pillinger*,{ddagger}

* Division of Rheumatology and {dagger} Division of Clinical Pharmacology, New York University School of Medicine/Hospital for Joint Diseases, New York, NY 10003; {ddagger} New York Harbor Veterans Administration Health Care System, New York, NY 10010; and § Division of Rheumatology, Albert Einstein College of Medicine, Bronx, NY 10467


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Annexins are intracellular molecules implicated in the down-regulation of inflammation. Recently, annexin-1 has also been identified as a secreted molecule, suggesting it may have more complex effects on inflammation than previously appreciated. We studied the role of annexin-1 in mediating MMP-1 secretion from rheumatoid arthritis (RA) synovial fibroblasts (SF) stimulated with TNF-{alpha}. TNF-{alpha} induced a biphasic secretion of annexin-1 from RA SF. Early (≤60 min), cycloheximide-independent secretion from preformed intracellular pools was followed by late (24 h) cycloheximide-inhibitable secretion requiring new protein synthesis. Exogenous annexin-1 N-terminal peptide Ac2-26 stimulated MMP-1 secretion in a dose- (EC50 {approx} 25 µM) and time- (8–24 h) dependent manner; full-length annexin-1 had a similar effect. Down-regulation of annexin-1 using small interfering RNA resulted in decreased secretion of both annexin-1 and MMP-1, confirming that annexin-1 mediates TNF-{alpha}-stimulated MMP-1 secretion. Erk, Jnk, and NF-{kappa}B have been implicated in MMP-1 secretion. Erk, Jnk, and NF-{kappa}B inhibitors had no effect on annexin-1 secretion stimulated by TNF-{alpha} but inhibited MMP-1 secretion in response to Ac2-26, indicating that these molecules signal downstream of annexin-1. Annexin-1 stimulation of MMP-1 secretion was inhibited by both a formyl peptide receptor antagonist and pertussis toxin, suggesting that secreted annexin-1 acts via formyl peptide family receptors, most likely FPLR-1. In contrast to its commonly appreciated anti-inflammatory roles, our data indicate that annexin-1 is secreted by RA SF in response to TNF-{alpha} and acts in an autacoid manner to engage FPRL-1, activate Erk, Jnk, and NF-{kappa}B, and stimulate MMP-1 secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Rheumatoid arthritis (RA)4 joint destruction is mediated in part by synovial fibroblasts (SF) that secrete matrix metalloproteinases (MMPs), aggrecanases, and other enzymes to degrade cartilage and bone (1, 2). MMPs secreted by RA SF include MMP-1, as well as MMP-3 and -13 (3). Multiple stimuli, including TNF-{alpha} and IL-1β, stimulate RA SF MMP secretion (3, 4). Intracellular signaling molecules implicated in regulating MMP synthesis/secretion include MAPKs Erk (4) and Jnk (5, 6), as well as NF-{kappa}B (7, 8). MAPKs participate in related but distinct signaling cascades, leading to both cellular activation and transcriptional regulation (9). In contrast, NF-{kappa}B is a direct transcription factor (10). Inhibition of Erk, Jnk, and/or NF-{kappa}B attenuates TNF-{alpha}-stimulated MMP-1 secretion, as well as multiple other inflammatory responses. Although the importance of MAPK and NF-{kappa}B in mediating RA SF responses to cytokines is well established, the complete pathways through which TNF-{alpha} and other cytokines stimulate MMP secretion remain incompletely elucidated.

Annexins are highly conserved calcium- and phospholipid-binding proteins that share common structures but exert diverse functions (11). Annexins have been variously implicated in cell differentiation and proliferation, extracellular processes such as coagulation (12), and membrane fusion events including endocytosis and exocytosis (12). The common calcium- and phospholipid-binding activities of annexins reside in the highly conserved core domain, whereas the unique properties of each annexin are mediated by divergent N termini (11). Complex interactions of annexins with cellular components such as membranes, cytoskeletal elements, and subcellular signaling assemblies result in multiple effects. For example, annexin-6 may be an important negative regulator of the Ras signaling pathway (13). Annexin-6 has also been implicated in the formation of lipid rafts and membrane signaling events, as well as endocytosis and membrane trafficking (14, 15).

Annexin-1 (previously lipocortin-1) is a potent inhibitor of inflammation both in vitro and in vivo. Induced in some cell types in response to glucocorticoids (16), annexin-1 was initially proposed to act intracellularly to inhibit cytosolic phospholipase A2, reducing prostaglandin biosynthesis (17). Studies indicate that annexin-1 can also be secreted (18), and subsequent reports have shown that secreted annexin-1 participates in additional anti-inflammatory effects including detachment of neutrophils from vascular endothelium (19, 20). The effects of secreted annexin-1 appear to derive from engagement of specific cell surface receptors by both the intact secreted protein and by various N-terminal-derived peptide fragments. Annexin-1 fragments are generated proteolytically in vivo at sites of inflammation and include a peptide consisting of amino acids 2-26 (Ac2-26). Ac2-26 and similar fragments reproduce the extracellular effects of intact annexin-1, implicating the annexin-1 amino terminus in extracellular signaling (21). Annexin-1 knockout (Anx-1(–/–)) mice develop more severe Ag-induced arthritis than wild-type littermates and respond poorly to dexamethasone (22).

In contrast to its better-appreciated anti-inflammatory effects, extracellular annexin-1 has been shown to exert a prosecretory role in some cell types through as yet uncharacterized receptors (23, 24) and to enhance chemotaxis and cellular polarization in neutrophils by its actions on members of the formyl peptide receptor (FPR) family (25). FPR-like 1 receptors (FPRL-1) are expressed on the surface of RA SF (26, 27) and are ligated not only by formyl peptides but also by serum amyloid A and lipoxin A4. Whereas serum amyloid A acts via FPRL-1 to stimulate MMP-1 and MMP-3 secretion from SF (26), engagement of FPRL-1 by lipoxin A4 mediates anti-inflammatory effects (28). Thus, diverse agents induce either pro- or anti-inflammatory effects through the FPRL-1 receptor, in a ligand- and possibly context-specific manner. The mechanisms of these effects have not been fully elucidated, and the effects of annexin-1 on RA SF via FPRs have also not been established. However, studies using macrophage and smooth muscle cell lines indicate that annexin-1 may share with other FPR ligands the ability to induce Erk signaling, suggesting a possible role in the regulation of RA SF proliferative and/or secretory functions (29, 30).

Given its importance in regulating inflammation and secretion (31), we tested whether annexin-1 or its peptides might regulate the signaling and/or secretory activity of TNF-{alpha}-stimulated RA SF, specifically the secretion of MMP-1. In contrast to the anti-inflammatory effects of annexin-1, we observed that annexin-1 mediates TNF-{alpha}-stimulated secretion of MMP-1 from RA SF. Specifically, annexin-1 is rapidly secreted from preformed intracellular pools in response to TNF-{alpha} stimulation, and autoengages FPRL-1 to stimulate Erk, Jnk, and NF-{kappa}B activation, resulting in MMP-1 secretion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials

Unless otherwise stated, all materials were purchased from Sigma-Aldrich. Annexin-1 peptide (Ac2-26) was obtained from Phoenix Pharmaceuticals. Anti-annexin-1 antiserum was purchased from Zymed Laboratories. Anti-phospho-Erk, anti-Erk 1, and anti-Erk 2 antisera, HRP-conjugated anti-rabbit antiserum, and annexin-1 small interfering RNA (siRNA) mixture (cat no. sc-29198: duplex 1 sense strand CAGACGUAAACGUGUUCAAtt; duplex 2 sense strand CACCAGAAGCUAUCCACAAtt; duplex 3 sense strand GCUUCAUCAAGCCAUGAAAtt; duplex 4 sense strand CAAG CCAUCCUGGAUGAAAtt; mRNA accession number NM_000700) and control siRNA (cat no. sc-37007: duplex sense strand UUCUCCGAACGUGUCACGUtt; proprietary) were obtained from Santa Cruz Biotechnology. Full-length annexin-1 (LC1) was purchased from Biodesign International. Anti-phospho- and total Jnk Abs, as well as anti-phospho- and total p38 Abs were obtained from Cell Signaling Technology. Anti-MMP-1 Abs were purchased from Chemicon International. Anti-MMP-3 and -13 Abs were obtained from Millipore. The 10% Tris-glycine/polyacrylamide gels were purchased from NOVEX/Invitrogen. Lipofectamine 2000 was purchased from Invitrogen. NF-{kappa}B p65 DNA-binding assay kit was purchased from ActiveMotif. UO126, SB203580, and SP600125 were obtained from BIOMOL. Pertussis toxin was obtained from Calbiochem. Boc peptide was purchased from MP Biomedical. Centricon centrifugal filter devices were obtained from Millipore. RPMI 1640 was obtained from Invitrogen. DMEM medium and FBS were purchased from BioWhittaker. The 2.5% trypsin/EDTA and penicillin G sodium (10,000 U/ml)/streptomycin sulfate (10,000 µg/ml) in 0.85% NaCl were obtained from Invitrogen. ECL detection kit was purchased from Amersham Biosciences. Pyrollidine dithiocarbamate (PDTC) was obtained from BIOMOL. HIG-82 rabbit SF cell line was purchased from American Type Culture Collection.

Preparation of synovial fibroblasts

Human RA SF were prepared from synovial tissue obtained at the time of total joint arthroplasty, as previously described (32). The collection and use of human SF was reviewed and approved by the Institutional Board of Research Associates, New York University School of Medicine. In brief, the acquired synovia were minced and incubated with 1 mg/ml collagenase type VIII in serum-free RPMI 1640 for 1 h at 37°C, filtered, extensively washed, and cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. After overnight incubation, non-adherent cells were cultured in DMEM with 10% FBS to 70–80% confluence, then split at 1:3 ratio and recultured and/or frozen for future use. For the reported experiments, RA SF were used between passages 3 and 6. Osteoarthritis (OA) SF were similarly prepared.

Cell culture and treatment

RA SF, grown to near confluence in DMEM medium containing 10% FBS and penicillin/streptomycin, were serum-starved for 24 h and equilibrated with fresh medium for 30–60 min before further treatment. Unless otherwise specified, the following concentrations of reagents were used: UO126 and SB203580, 10 µM; SP600125 and PDTC, 20 µM; TNF-{alpha}, IL6 and IL-1β, 20 ng/ml; cycloheximide, 50 µg/ml; Ac2-26, 50 µg/ml; pertussis toxin, 1 µg/ml; fMLP 10 nM; Boc peptide, 10 µM; PMA 200 nM; LTB4, 300 nM.

Measurement of annexin-1 and MMP-1 secretion

MMP-1 secretion was determined as previously described (4). In brief, RA or OA SF grown to near confluence were serum starved for 24 h, treated with Erk (UO126), p38 (SB203580), Jnk (SP600125), or NF-{kappa}B (PDTC) inhibitors for 30 min, pertusssis toxin (Calbiochem) for 2 h, anti-annexin-1 antiserum for 3 h, or Ac2-26, Boc or fMLP for 30 min, then stimulated with TNF-{alpha} or IL-1β for 24 h. Supernatants were collected and concentrated (Centricon centrifugal filter devices, MW cutoff 30,000 kDa) for 35 min x 5000 rpm at 4°C. Aliquots of concentrated supernants were analyzed by 10% Tris-glycine SDS-PAGE and electrophoretic transfer to nitrocellulose, followed by exposure to anti-annexin-1 (1/500 dilution) or anti-MMP-1 Ab (1/500 dilution) in TBS-T overnight at 4°C, and 1 h incubation with HRP-conjugated goat anti-rabbit Ab (1/2000 dilution). Blots were washed in TBST, developed with an ECL detection kit, and imaged by autoradiography. Autoradiograms were quantitated by scanning and determining pixel counts using Quantity One software, (version 4.0.3; Bio-Rad).

Measurement of MAPK activation

Erk, Jnk, and p38 activation were measured by immunoblotting as described (4). In brief, RA SF were serum-starved for 24 h, incubated ± UO126, SB203580, SP600125, or PDTC for 30 min, then stimulated with TNF-{alpha} or IL-1β (15 min at 37°C). Cells were lysed (20 mM Tris, pH 7.4, 1 mM EGTA, 2 mM sodium vanadate, 25 mM sodium fluoride, 0.5% (v/v) Triton X-100, 2 mM PMSF, and 1% of protease inhibitors mixture) for 10 min at 4°C, followed by centrifugation (12,000 rpm in a tabletop microfuge at 4°C) and analysis of the resultant supernatants by 10% Tris-glycine SDS-PAGE, transfer to nitrocellulose, and immunoblotting with anti-phospho-Erk (1/200), anti-phospho-Jnk (1/2000) or anti-phospho-p38 (1/1000) antisera in TBST overnight at 4°C, followed by incubation with HRP-conjugated anti-rabbit Ab, and imaging and quantitation as described for MMP-1 above. Blots were stripped and reblotted using Abs recognizing total Erk 1 and 2 (each at 1/800), total Jnk (1/1000), or total p38 (1/1000), respectively. Erk, Jnk, and p38 activation were calculated as the ratio of phosphorylated/total enzyme expression for each protein.

Measurement of NF-{kappa}B activation

NF-{kappa}B activation was measured as the in vitro ability of p65 NF-{kappa}B subunits in cell lysates to bind to DNA NF-{kappa}B response elements, using a commercially available kit according to the manufacturer’s instructions. NF-{kappa}B activation was also measured in vitro as the activation of a NF-{kappa}B luciferase reporter construct (gift of S. Ghosh, Yale University School of Medicine, New Haven, CT), transfected into rabbit synovial fibroblasts (HIG-82 cell line), as previously described (32).

siRNA transfection

Cells were grown in DMEM with 10% FBS, 1% penicillin/streptomycin until ~60% confluent. The day before transfection, medium was replaced with DMEM 10% FBS but without antibiotics. 60 pmol of annexin-1 or control siRNA were transfected using Lipofectamine 2000. After 5 h, the transfection medium was replaced with serum-free DMEM overnight. Transfected cells were then stimulated ± TNF-{alpha} and analyzed for annexin-1 and MMP-1 secretion.

Microarray analysis of FPRL and annexin-1 expression in normal and RA synovium

Synovium was obtained from patients undergoing knee replacement surgery for advanced RA or from autopsy patients within 24 h (National Diabetes Research Interchange). The use of discarded human synovium was approved by appropriate institutional review boards.

The synovium was homogenized (tissue homogenizer), and RNA extracted using TRIzol (Invitrogen). Total RNA was purified using micro RNeasy column (Qiagen) as recommended by the manufacturer. One µg of total RNA from each of 10 normal or RA samples were pooled, and 5 µg were used for ds cDNA synthesis (Life Technologies Superscript choice system). Biotin-labeled cRNA was synthesized (ENZO BioArray High-yield RNA transcript labeling kit, Affymetrix). The cRNA was purified using Qiagen RNeasy kit, fragmented at 95°C for 35 min for target preparation, and hybridized against U133A gene-chip array as suggested by manufacturer (Affymetrix).

Affymetrix Microarray Suite Version 5.0 (MAS 5.0) was used to stain, wash, scan, and quantitate each gene-chip, computing summary intensities for each probe. DNA Chip Analyzer (dChip) Version 1.3 was used to normalize and to generate model-based estimates of expression for each probe set (33). The "invariant set normalization" method was used to normalize all gene chips to a baseline chip.

Statistics

Statistical analyses were performed using Sigmastat, version 3.0.1 (SPSS). Experiments were analyzed by one way ANOVA with Dunnett’s method for pairwise comparisons, or by Student’s t test, as appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
TNF-{alpha} stimulates annexin-1 secretion from RA SF

We first determined the effect of TNF-{alpha} on annexin-1 secretion from RA SF. TNF-{alpha} (20 ng/ml) induced annexin-1 secretion, as measured by SDS-PAGE and immunoblotting of RA SF supernatants. Annexin-1 secretion was biphasic, with an initial, rapid (15–60 min) secretory event (Fig. 1, A and B), followed by a decline in extracellular annexin-1 (trough at 8 h), and a second, later secretory event (24 h) (Fig. 1, A and D). In contrast, lysates of cells stimulated with TNF-{alpha} showed an initial depletion in intracellular annexin-1, followed by a later (24 h) recovery of the intracellular protein (Fig. 1C). These data suggested that TNF-{alpha} stimulates an initial release of pre-formed annexin-1 pools, followed by reuptake and/or degradation and a later resynthesis/secretion event. Consistent with this interpretation, treating RA SF with cycloheximide to inhibit protein synthesis reduced the late but not early phase of TNF-{alpha}-stimulated annexin-1 secretion (Fig. 1D). In contrast to annexin-1, annexin-2 secretion was not stimulated by TNF-{alpha}, either at 1 or 24 h (n = 3, data not shown). Although other cytokines and growth factors (IL-1β, IL-6, EGF) also stimulated annexin-1 secretion, they did not uniformly stimulate both the early and late phases. Whereas IL-1β stimulated only late-phase annexin-1 secretion, EGF stimulated only the early response, and IL-6 stimulated both, but predominantly the late-phase response (Fig. 1E). Thus, early and late secretion of annexin-1 appears to be distinct, dissociable, and independently regulated events, though both are induced by TNF-{alpha}.


Figure 1
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  		FIGURE 1. TNF-{alpha} and other cytokines stimulate annexin-1 secretion from RA SF. A, RA SF were incubated with TNF-{alpha} for 0–24 h, followed by measurement of annexin-1 levels in the supernatants. B, RA SF were incubated with TNF-{alpha} for 0–60 min, and supernatant annexin-1 levels measured. C, RA SF from the experiment described in A were lysed, and the lysates analyzed for cellular annexin-1. D, RA SF were treated ± cycloheximide, stimulated with TNF-{alpha} for 1 h (filled bars) or 24 h (open bars), and the supernatants analyzed for annexin-1. E, RA SF were stimulated for the indicated times with IL-1β, IL-6, or EGF, followed by determination of supernatant annexin-1. Data shown are the mean ±SEM for three (A, C, and D) or four (B and E) experiments. (*, p < 0.05, relative to T = 0).

 
Annexin-1 mediates MMP-1 secretion from RA SF

TNF-{alpha} stimulation of MMP-1 secretion is a delayed response (Fig. 2A), indicating that intermediate events must occur before secretion of MMP-1. To determine whether secreted annexin-1 could mediate TNF-{alpha}-stimulated MMP-1 secretion, we first tested the effects of the annexin-1 N-terminal peptide Ac2-26 on the secretion of MMP-1. Ac2-26 induced MMP-1 secretion more rapidly (8 h) than TNF-{alpha} (Fig. 2A); supernatant levels of MMP-1 continued to increase for as long as 24 h in response to either agent. Ac2-26 stimulation of MMP-1 secretion was dose dependent (ED50 ~25 µg/ml), with maximal response at [Ac2-26] ≥50 µg/ml (Fig. 2B). MMP-1 secretion in response to both TNF-{alpha} and Ac2-26 was inhibited by cycloheximide, consistent with a requirement for de novo MMP-1 synthesis (data not shown).


Figure 2
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  		FIGURE 2. Annexin-1 stimulates MMP-1 secretion from rheumatoid and OA SF. A, RA SF were stimulated for indicated times ± Ac2-26 (50 µg/ml) or TNF-{alpha} (20 ng/ml), followed by analysis of supernatants for MMP-1. B, RA SF were incubated with the indicated concentrations of Ac2-26 for 24 h, and MMP-1 levels in the supernatants determined. C, RA SF were incubated for 24 h ± Ac2-26, in the absence or presence of IL-1β (filled bars) or TNF-{alpha} (open bars), and the supernatants analyzed for MMP-1 secretion, expressed as percentage of stimulated condition in the absence of Ac2-26. D, RA SF were stimulated with the indicated concentrations of full-length annexin-1 (An-1) for 24 h, and the supernatants analyzed for MMP-1 secretion. E, RA SF were incubated with the indicated dilutions of an anti-annexin-1 Ab for 3 h, stimulated with TNF-{alpha}, and analyzed for supernatant concentrations for MMP-1. F, RA SF were transfected with siRNA for annexin-1, or a control siRNA, and stimulated with TNF-{alpha}; annexin-1 secretion after 1 h (top panel) and MMP-1 secretion after 24 h (bottom panel) were determined by SDS-PAGE and immunblot of the supernatants. G, OA SF were stimulated with IL-1β and TNF-{alpha} (20 ng/ml)), and the supernatants assayed by SDS-PAGE and immunoblot for annexin-1 secretion. H, Supernatants from OA SF stimulated for 24 h with Ac2-26 were analyzed by SDS-PAGE and immunoblot for MMP-1 secretion. I, Supernatants from RA SF stimulated with Ac2-26 or annexin-1 (An-1) were analyzed for the presence of MMP-3. Data shown are the mean±SEM, or representative of two (E and H), three (B, D, F, and I), or four (A, C, and G) experiments. (*, p ≤ 0.05, relative to T = 0 (A and G) or [Ac2-26] = 0 (B)).

 
Because annexin-1 secreted in response to TNF-{alpha} would likely encounter cellular target(s) in the context of ongoing inflammatory stimulation (e.g., the high concentrations of TNF-{alpha} and other cytokines found in RA joints (1)), we also tested the effect of Ac2-26 in the presence of TNF-{alpha}. Incubation of RA SF with Ac2-26 enhanced TNF-{alpha}-stimulated MMP-1 secretion, indicating that annexin-1 can stimulate MMP-1 secretion even in highly inflammatory settings. A trend toward Ac2-26 enhancement of MMP-1 secretion in the presence of IL-1β was also observed (Fig. 2C).

To determine whether the effects of Ac2-26 reproduced those of the complete annexin-1 protein, we incubated RA SF with full-length annexin-1 protein. Like Ac2-26, full-length annexin-1 stimulated MMP-1 secretion in a dose-dependent manner (Fig. 2D).

To confirm a role for secreted annexin-1 as a mediator of TNF-{alpha}-stimulated MMP-1 secretion, we first tested the effects of an anti-annexin-1 Ab on MMP-1 secretion. Pre-incubation with the anti-annexin-1 Ab (1/5000 dilution) inhibited MMP-1 secretion in response to TNF-{alpha} (Fig. 2E); a control Ab had no effect (not shown). Higher concentrations of Ab resulted in increased MMP-1 secretion, indicating a secondary or artifactual effect. To further examine the role of annexin-1 during TNF-{alpha}-stimulated MMP-1 secretion, we depleted annexin-1 expression through the use of siRNA. Transfection with annexin-1 siRNA, but not a control siRNA, caused a marked reduction in TNF-{alpha}-stimulated annexin- 1 secretion (Fig. 2F, top). Transfection with the annexin-1 siRNA, but not the control siRNA, also inhibited MMP-1 secretion in response to TNF-{alpha} (Fig. 2F, bottom). These data confirm that annexin-1 plays a necessary role in TNF-{alpha}-stimulated MMP-1 secretion.

To determine whether these observations were unique to RA SF, or whether annexin-1 could also mediate MMP-1 secretion in other cell types, we tested the ability of TNF-{alpha}/IL-1β to stimulate annexin-1 secretion, and the ability of Ac2-26 to stimulate MMP-1 secretion, from primary human OA SF. TNF-{alpha}/IL-1β stimulated early, but not late annexin-1 secretion from OA SF (Fig. 2G). Moreover, the Ac2-26 peptide stimulated MMP-1 secretion from OA cells (Fig. 2H). In contrast, Ac2-26 did not stimulate MMP-1 secretion from AGS cells, a gastric epithelial cell line that secretes MMP-1 in response to cytokines (data not shown) (34, 35). These data suggest that the ability to secrete and respond to annexin-1 is a characteristic feature of synovial fibroblasts, in RA as well as other types of arthritis, but may not be universally shared by other cell types.

Both Ac2-26 and intact annexin-1 protein also stimulated secretion of MMP-3 (Fig. 2I), but not MMP-13 (data not shown), from RA SF. These data are consistent with prior reports that MMP-1 and 3, but not MMP-13, are concordantly regulated (4, 8).

Erk, Jnk, and NF-{kappa}B regulate MMP-1 secretion in response to Ac2-26

Because Erk, Jnk, and NF-{kappa}B have been reported to mediate TNF-{alpha}-stimulated MMP-1 secretion, we next asked whether these molecules regulate the secretion of annexin-1, or act downstream of annexin-1 to regulate MMP-1 release. As expected, specific inhibitors of the MAPKs Erk (UO126) and Jnk (SP600125) as well as an inhibitor of NF-{kappa}B (PDTC), inhibited MMP-1 secretion stimulated by TNF-{alpha} (Fig. 3A). The alternative MAPK p38 has been reported not to regulate RA SF MMP-1 secretion (4, 5). Consistent with those reports, the p38 inhibitor SB203580 did not inhibit TNF-{alpha}-stimulated MMP-1 secretion in our experiments. We next tested the effects of UO126, SP600125, SB203580, and PDTC on TNF-{alpha}-stimulated annexin-1 secretion. None of these agents inhibited annexin-1 secretion, either at 1 h or 24 h of stimulation (Fig. 3B). These agents also had no effect on intracellular annexin-1 levels (not shown). Thus, TNF-{alpha}-stimulation of annexin-1 secretion does not depend on MAPK or NF-{kappa}B activity.


Figure 3
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  		FIGURE 3. MMP-1 secretion in response to Ac2-26 is regulated by Erk, Jnk, and NF-{kappa}B. A, RA SF were incubated ± UO126, SP600125, SB203580, or PDTC, stimulated for 24 h with TNF-{alpha}, and the supernatants analyzed for MMP-1 secretion. B, RA SF were incubated ± UO126, SP600125, SB203580, or PDTC, stimulated for 1 (filled bars) or 24 h (open bars) with TNF-{alpha}, and the supernatants analyzed for annexin-1 secretion. C, RA SF were incubated ± UO126, SP600125, SB203580, or PDTC, stimulated for 24 h with Ac2-26, and the supernatants analyzed for MMP-1. Data shown are the mean±SEM of four (A and C), five (B, open bars), or nine (B, filled bars) experiments.

 
In contrast, UO126, SP600125, and PDTC each inhibited MMP-1 secretion stimulated by Ac2-26 (Fig. 3C), suggesting that MMP-1 secretion in response to annexin-1 depends on Erk, Jnk, and NF-{kappa}B activation. We therefore tested the ability of Ac2-26 to stimulate the activity of these molecules. Ac2-26 stimulated activation of both Erk (Fig. 4A) and Jnk (Fig. 4B). In contrast, p38 was not activated by Ac2-26 (Fig. 4C). Although UO126 and SP600125 are specific for their respective targets (36, 37), each agent inhibited both Erk and Jnk activation in response to Ac2-26, indicating cross-talk between the two pathways in this cell type (Fig. 4, A and B). Ac2-26 also stimulated the activation of NF-{kappa}B (measured in cell lysates as binding of the NF-{kappa}B p65 subunit to a target DNA, and in vivo as activation of a transfected luciferase NF-{kappa}B reporter construct), a process that was inhibited by PDTC (Fig. 4D). Activation of the NF-{kappa}B luciferase reporter was also observed in response to intact annexin-1 protein (not shown). Taken together, these data suggest that TNF-{alpha} acts independently of MAPK or NF-{kappa}B to stimulate annexin-1 secretion. Annexin-1 subsequently acts in an autacoid manner to activate Erk, Jnk, and NF-{kappa}B, providing signals to induce MMP-1 synthesis and secretion.


Figure 4
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  		FIGURE 4. Annexin-1 stimulates Erk, Jnk, and NF-{kappa}B activation in RA SF. RA SF were incubated for 30 min ± UO126, SP600125, SB203589, or PDTC as indicated, stimulated for 30 min ± TNF-{alpha}, and analyzed for Erk (A), Jnk (B), or p38 (C) activation as described in Materials and Methods. NF-{kappa}B activation in TNF-{alpha}-stimulated RA SF was determined as in vitro binding of the p65 NF-{kappa}B subunit to an NF-{kappa}B DNA binding sequence (D, filled bars). NF-{kappa}B was also assayed as the activation of an NF-{kappa}B luciferase reporter construct, transfected into rabbit (HIG-82) synoviocytes (D, open bars). Data shown are the mean ±SEM of three (B and D, open bars), four (A and C), or seven (D, filled bars) experiments. (*, p < 0.05 vs Ac2-26).

 
Annexin-1 regulates MMP-1 secretion via engagement of a G-protein-coupled FPR family

That annexin-1 acts extracellularly to activate RA SF suggested the involvement of a receptor-mediated event. We therefore tested whether annexin-1 stimulates MMP-1 secretion via engagement of FPR, a family that includes three receptor subtypes (FPR-1, FPRL-1, FPRL-2). A specific FPR antagonist, boc-peptide, inhibited Ac2-26 stimulation of MMP-1 secretion (Fig. 5A), supporting a role for FPR in annexin responses. Because FPR are pertussis toxin-sensitive, Gi protein-coupled receptors, we also tested the effect of pertussis toxin on Ac2-26-stimulated MMP-1 secretion. Pertussis toxin inhibited MMP-1 secretion stimulated by Ac2-26 (Fig. 5B). Pertussis toxin also inhibited MMP-1 secretion from RA SF stimulated with leukotriene B4, a proinflammatory eicosanoid whose mode of action is known to be G protein-dependent (54 ± 23% inhibition, p = 0.03). In contrast, neither boc-peptide nor pertussis toxin inhibited MMP-1 secretion induced by PMA, which bypasses surface receptors to directly activate protein kinase C (Fig. 5, C and D) (P for boc peptide and pertussis toxin inhibition of PMA-stimulated MMP-1 secretion = 0.23 and 0.28, respectively). Thus, annexin-1 stimulates MMP-1 secretion, at least in part, via engagement of a G protein-coupled FPR. In contrast to Ac2-26, the canonical formyl peptide, fMLP, at concentrations (10 nM) that selectively engage only one of the three members of the FPR family (FPR-1) (38), failed to stimulate MMP-1 secretion (P for no stimulation vs fMLP-stimulated MMP-1 secretion = 0.42) (Fig. 5E).


Figure 5
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  		FIGURE 5. Annexin-1 stimulates MMP-1 secretion from RA SF via engagement of a G protein-coupled FPR. RA SF were pretreated with boc peptide for 30 min (A), or with pertussis toxin for 2 h (B), before stimulation with Ac2-26 (24 h) and measurement of MMP-1 secretion. RA SF were treated with boc peptide (C) or pertussis toxin (D) before stimulation with PMA (24 h) and measurement of MMP-1 secretion. E, RA SF incubated ± boc peptide were stimulated with fMLP (10 nM, 24 h), and MMP-1 secretion was determined. Data shown are the mean ± SEM of four (A and B), five (E), or seven (C and D) experiments.

 
Expression of annexin-1 and FPRL-1 and -2 in intact normal and rheumatoid synovium

To confirm that annexin-1 and the formyl peptide receptors FPRL-1 and FPRL-2 are present in intact human synovium, we performed microarray analysis on synovial tissue from normal and RA joints. mRNA for both FPRL-1 and FPRL-2 were expressed constitutively in both normal and RA synovium (Fig. 6A). FPR-1 mRNA was similarly expressed (not shown). Annexin-1 mRNA also was present in both normal and RA synovium (Fig. 6B), suggesting that a pre-existing pool of intracellular annexin-1 may be present in both normal and RA SF, and situated to undergo secretion in response to cytokine stimulation such as may occur in RA.


Figure 6
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  		FIGURE 6. Expression of FPRL and Annexin-1 mRNA in synovium from normal and RA joints. Gene expression analysis of pooled RNA samples from 10 normal and 10 RA synovial specimens were analyzed for expression of FPRL-1 and -2 (A), and annexin-1 (B) message using Affymetrix U133A microarray, as described in Materials and Methods.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
MMPs are synthesized and secreted by SF in response to TNF-{alpha} and other cytokine signals (39). In RA, SF invade adjacent tissues through the actions of secreted MMPs, as well as aggrecanases and cathepsins (40, 41). MMP secretion in response to cytokines is mediated via activation of Erk, Jnk, and their effectors, including transcriptional regulators. NF-{kappa}B activation is also important for MMP synthesis, and cross-talk exists between MAPK and NF-{kappa}B signaling pathways (1). Multiple other signaling molecules are likely involved, but are as yet unidentified. Although disease-modifying antirheumatic drugs typically reduce inflammation, inflammation and joint destruction are linked but separable events, and tight control of inflammation alone does not universally prevent joint destruction (42). The relative efficacy of so-called biologic therapies for RA derives in part from their ability to more specifically limit cytokine signaling in, and protease release from, RA SF. However, significant numbers of RA patients fail to derive benefit from their use (43). A clearer understanding of the regulatory events surrounding RA SF-mediated tissue destruction is a prerequisite for the development of improved anti-erosive strategies.

The ability of some annexins to regulate secretion, as well as membrane trafficking and fusion events (9), led us to test the role of annexins in RA SF MMP secretion. We observed that annexin-1 was constitutively expressed in RA SF, and that TNF-{alpha}, as well as other cytokines, stimulated annexin-1 secretion. These data are similar to prior reports indicating that annexin-1 is constitutively expressed in human neutrophils and secreted in response to cell activation (44, 45). Annexin-1 (mRNA) expression was also observed in intact synovium, from both normal and RA patients, again suggesting that in contrast to annexin-1 expression which is constitutive in all SF, it is the secretion of annexin-1, in response to high local concentrations of cytokines, that may distinguish RA SF. In contrast, neither TNF-{alpha} nor IL-1β stimulated annexin-2 secretion, suggesting that annexin-1 secretion might play a unique role. Annexin-1 secretion was biphasic: an initial, early secretion event was protein synthesis-independent, and accompanied by depletion of intracellular annexin-1 stores, whereas a later wave of annexin-1 secretion was protein synthesis-dependent and accompanied by re-accumulation of intracellular annexin-1. Interestingly, different cytokines variably affected the early and late phases of secretion, with TNF-{alpha} most potently stimulating both phases. Annexin-1 secretion is therefore tightly and specifically regulated. The kinetics of early-phase annexin-1 secretion in particular suggested a possible role in regulating RA SF MMP-1 secretion in response to TNF-{alpha} and other cytokines.

Full-length annexin-1 is secreted from cells and may be cleaved into fragments via proteases synthesized by resident or invasive inflammatory cells; proteinase 3 and human neutrophil elastase have been implicated (44, 45). The physiologic effects of extracellular annexin-1 are duplicated by the proteolytically cleaved N-terminus of the intact molecule (46). Because annexin-1 has inhibitory effects on various cell types including polymorphonuclear leukocytes (16), we hypothesized that annexin-1 and its peptide derivatives would inhibit MMP secretion in RA SF. Contrary to that expectation, both exogenously-applied annexin-1, and its N-terminal peptide Ac2-26, stimulated MMP-1 secretion in a dose-dependent manner. These effects were observed both in isolation and in the presence of TNF-{alpha} or IL-lβ, which are found in high concentrations in the rheumatoid joint. Thus, the annexin-1 effect on MMP-1 secretion is likely to occur in the actual RA environment and in the presence of cytokines that themselves stimulate annexin-1 secretion. The ability of an annexin-1 siRNA, but not a control siRNA, to inhibit both early annexin-1, and later MMP-1 secretion, confirmed that secreted annexin-1 plays a necessary role in regulating RA SF MMP-1 secretion in response to TNF-{alpha}. We also observed increases in MMP-3 but not MMP-13 secretion in response to Ac2-26, alone or in combination with TNF-{alpha} (data not shown), suggesting that secreted annexin-1 may specifically regulate the secretion of multiple proteolytic enzymes.

Erk, Jnk, and NF-{kappa}B mediate MMP-1 secretion from RA SF, but their places in the regulatory circuits for MMP-1 secretion are not fully established. In our present studies, inhibiting Erk, Jnk, or NF-{kappa}B failed to inhibit TNF-{alpha}-stimulated annexin-1 secretion. Thus, regulation of annexin-1 secretion is independent of MAPK and NF-{kappa}B, and the signaling pathway(s) promoting annexin-1 secretion remains to be determined. In contrast, Ac2-26 stimulated the activation of Erk and Jnk, as well as NF-{kappa}B, and inhibitors of each of these molecules inhibited MMP-1 secretion in response to Ac2-26 as well as TNF-{alpha}. These results place annexin-1 secretion upstream of Erk, Jnk, and NF-{kappa}B activation in the regulation of MMP-1 secretion, although they do not formally exclude the possibility that Erk, Jnk, and/or NF-{kappa}B may also be activated via annexin-1-independent pathways in response to cytokine signals. In contrast to Erk and Jnk, prior reports suggest that the alternate MAPK p38 does not regulate MMP-1 secretion from RA SF (4). Consistent with these reports, we observed that Ac2-26 did not stimulate p38 activation, and that a p38 inhibitor had no effect on Ac2-26-stimulated MMP-1 secretion.

Although our studies represent the first demonstration of annexin-1-induced MMP secretion from RA SF, annexin-1 has previously been demonstrated to stimulate other secretory activities, in particular insulin secretion from rat pancreatic islet cells. Insulin secretion in that model was Ca2+-independent and, as in the case of RA SF, was an autacoid event preceded by annexin-1 secretion (23). Other studies have demonstrated additional stimulatory effects of annexin-1, in particular its ability to increase the invasiveness of epithelial cells (cell line SKCO-15) into Matrigel, a process likely to depend upon MMP secretion (47). Thus the proinflammatory and/or prosecretory effects of annexin-1 appear not to be limited to RA SF. These effects of annexin-1 are distinct from those classically described in leukocytes; extracellular annexin-1 exerts a net inhibitory effect on monocytes, macrophages and polymorphonuclear leukocytes, decreasing their response to chemotactic stimuli as well as their adhesiveness, and reducing the accumulation of these cells at sites of inflammation (21).

Previous studies demonstrate that the effects of extracellular annexin-1 are receptor-mediated, and the primary candidates for annexin-1 receptors are members of the FPR family of seven transmembrane, Gi-protein coupled receptors. The human FPR family includes three related receptors, FPR-1, FPRL-1, and FPRL-2, and Ac2-26 has been reported to activate all three (25, 48). Our studies using a FPR receptor inhibitor (boc peptide) and pertussis toxin confirmed that annexin-1 stimulation of MMP-1 secretion is FPR- and Gi-dependent, but did not identify which particular receptors were responsible for the annexin-1 effects. Because FPRL-1 is expressed on RA SF (where its engagement by serum amyloid A stimulates both SF hyperplasia and MMP-1 secretion) (26, 49), it may represent the primary candidate molecule for the annexin-1 receptor. However, our data do not formally exclude the possibility that annexin-1 may also signal through FPRL-2; indeed, microarray analysis of both normal and RA synovium confirmed the presence of message for both FPRL-1 and FPRL-2. In contrast, FPR-1 is unlikely to play a role in annexin-1 RA SF signaling, because in our experiments, selective ligation of FPR-1 by fMLP failed to stimulate MMP-1 secretion.

In summary, our data indicate that annexin-1, secreted by RA SF in response to cytokine stimulation, acts in an autacoid manner to engage FPR, stimulate Erk, Jnk, and NF-{kappa}B, and induce MMP-1 synthesis and secretion (Fig. 7). Further study will be needed to evaluate the importance of annexin-1 secretion to RA joint destruction, and its possible value as a therapeutic target.


Figure 7
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  		FIGURE 7. Annexin-1 secretion regulates MMP-1 secretion stimulated by TNF-{alpha}. Our data suggest that TNF-{alpha} rapidly stimulates annexin-1 secretion, and that annexin-1 acts in an autacoid manner to engage a FPR, most likely FPRL-1, leading to Erk, Jnk, and NF-{kappa}B activation, all of which contribute to the secretion of MMP-1.

 

   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by a research grant from the Arthritis Foundation, New York Chapter. Back

2 C.E.T. and N.M. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Clement E. Tagoe, Division of Rheumatology, Montefiore Medical Center, 111 East 210th Street, Bronx, NY 10467. E-mail address: ctagoe{at}montefiore.org Back

4 Abbreviations used in this paper: RA, rheumatoid arthritis; SF, synovial fibroblasts; MMPs, matrix metalloproteinases; FPR, formyl peptide receptor; FPRL-1, FPR-like 1 receptors; PDTC, pyrollidine dithiocarbamate; OA, osteoarthritis; siRNA, small interfering RNA. Back

Received for publication December 20, 2007. Accepted for publication June 6, 2008.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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