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. Author manuscript; available in PMC: 2007 May 4.
Published in final edited form as: Osteoarthritis Cartilage. 2006 Nov 13;15(4):431–441. doi: 10.1016/j.joca.2006.09.011

RelA is Required for IL-1β Stimulation of Matrix Metalloproteinase-1 Expression In Chondrocytes

Lauren Raymond 1,2, Sarah Eck 2, Ezra Hays 2, Ivan Tomek 3, Stephen Kantor 3, Matthew Vincenti 1,2
PMCID: PMC1865522  NIHMSID: NIHMS20653  PMID: 17097317

Abstract

Objective

IL-1β stimulates collagenase-1 (MMP-1) expression in articular chondrocytes, leading to cleavage of type II collagen and irreversible cartilage degradation. The nuclear factor kappa B (NF-κB) pathway is potently activated in IL-1β-stimulated cells and has been implicated as an intermediate in MMP-1 gene expression. However, the roles of individual NF-κB family members during IL-1β-induced MMP-1 gene expression have not been defined.

Results

To address the relationship between the NF-κB pathway and MMP-1 gene activation in chondrocytes, primary human articular chondrocyte cultures (HAC) and SW-1353 cells were stimulated with IL-1β over a 24-hour time course and MMP-1, NF-κB1, NF-κB2 and RelA gene expression was assayed. IL-1β-induced MMP-1 expression was comparable in HAC and SW-1353 cells both temporally and quantitatively. MMP-1 gene expression was mirrored by increases in NF-κB gene expresssion, and inhibition of NF-κB nuclear translocation with dominant negative IκBα reduced IL-1β-dependent MMP-1 gene expression. IL-1β activated the NF-κB pathway in chondrocytes, both through phosphorylation and transient degradation of IκBα, as well as through sustained phosphorylation of RelA. Small inhibitory RNAs (siRNA) specific for RelA resulted in significant reduction of MMP-1 mRNA, whereas siRNA for NF-κB1 and NF-κB2 augmented IL-1β-induced MMP-1 expression.

Conclusions

Our data demonstrate that IL-1β activation of the NF-κB pathway is required for IL-1β induction of MMP-1 in chondrocytes and that RelA can work independently of NF-κB1 or NF-κB2 to activate this gene expression program.

Keywords: Matrix Metalloproteinase-1, Nuclear Factor-Kappa B, Chondrocytes, Interleukin-1, Arthritis

Introduction

Rheumatoid arthritis (RA) and osteoarthritis (OA) have distinct etiologies, yet both diseases result in the permanent destruction of articular cartilage. Articular cartilage consists primarily of the matrix proteins type II collagen and aggrecan. In RA and OA, the Matrix Metalloproteinases (MMP) degrade the collagen present in the cartilage, while members of the A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS) family target aggrecan. Although digestion of aggrecan can be transient, cleavage of type II collagen is believed to be the committed step for irreversible cartilage degradation 1. There are four MMP family members that are interstitial collagenases, including collagenase-1 (MMP-1), collagenase-2 (MMP-8), collagenase-3 (MMP-13) and membrane-type-1 MMP (MMP-14) 2, 3. MMP-14 is expressed in RA synovium, while MMP-1, MMP-8 and MMP-13 are expressed in both synovium and cartilage of RA and OA patients 4–9. The inflammatory cytokines IL-1β and TNFα promote MMP-1 and MMP-13 expression in articular chondrocytes 7, 8 and this correlates with cartilage loss in arthritic lesions 7, 10, 11. MMP-1 and MMP-13 are both expressed in cartilage lesions that form before the onset of clinical disease 10, suggesting that both enzymes are important for disease initiation. While MMP-13 degrades resident collagen fibrils in the cartilage, MMP-1 may be more important for remodeling of newly synthesized collagen 12. Thus, elucidating how cytokines like IL-1β activate MMP-1 gene expression in chondrocytes is critical for understanding the pathogenesis of arthritis.

The Nuclear Factor Kappa B (NF-κB) pathway is an important mediator of cyotkine-induced gene expression in inflammation and immunity. The NF-κB family of transcription factors consists of NF-κB1, NF-κB2, RelA, c-Rel and RelB. NF-κB1 and NF-κB2 are synthesized as propeptides that contain a Rel homology domain involved in DNA binding activity and nuclear localization, and an ankyrin-rich cytoplasmic domain that sequesters these proteins in the cytoplasm. During translation (NF-κB1) or in response to signal-dependent phosphorylation (NF-κB2), the cytoplasmic domains are proteolytically removed and these proteins translocate to the nucleus 13. Both NF-κB1 and NF-κB2 lack transactivation domains and must dimerize with Rel family members to create transcriptionally competent complexes. RelA forms heterodimers with processed NF-κB1 or NF-κB2, and these are retained in the cytoplasm by the inhibitor of kappa B (IκB) 14. Following cellular stimulation with cytokines, IκB is phosphorylated by the IκB kinases (IKK) and degraded by the proteosome, resulting in nuclear translocation of RelA complexes. RelA can also be released from IκB repression by direct phosphorylation on serine 536, and in this phosphorylated state, RelA does not associate with NF-κB1 15. Thus, cytokine stimulation results in a complex NF-κB activation program that can impact MMP-1 gene expression.

IL-1β activation of the MMP-1 gene requires the integration of several signaling pathways and activated transcription factors 16. Recent work in our laboratory has demonstrated that IL-1β stimulates MMP-1 transcription in chondrocytes through extracellular-regulated kinase (ERK)-dependent activation of CCAAT enhancer binding protein-beta (C/EBPβ) 17. In addition to the ERK pathway, IL-1β activation of the NF-κB pathway contributes to increased MMP-1 gene expression in synovial fibroblasts and vascular smooth muscle cells 18, 19. However, there have been conflicting reports regarding the importance of NF-κB during MMP-1 expression in chondrocytic cells. Bondeson et al. 20 reported that over expression of wild type IκB-α reduced MMP-1 gene expression, while Mengshol et al. 21 found that a dominant negative allele of this gene had no effect on MMP-1. Work in our laboratory has demonstrated that IL-1β elevated NF-κB and MMP-1 gene expression in SW-1353 chondrosarcoma cells 22, 23. Gebauer et al. recently confirmed this correlation between NF-κB and MMP-1 gene expression24, and these authors concluded that the SW-1353 cell line is an appropriate model of IL-1β induced MMP-1 and NF-κB gene expression in human articular chondrocytes.

In the present study, we set out to determine if there is a mechanistic link between IL-1β induction of the NF-κB pathway, and IL-1β induction of MMP-1 gene expression in chondrocytes. We found a temporal correlation between IL-1β-stimulated NF-κB and MMP-1 gene activation and that dominant negative IκBα repressed IL-1β-dependent MMP-1 expression. While IL-1β stimulates a transient phosphorylation and degradation of IκBα in chondrocytes, RelA phosphorylation on serine 536 is more sustained. Moreover, of the NF-κB genes assayed, only RelA was found to be required for IL-1β induced MMP-1 gene expression. These data provide a new model of NF-κB-dependent MMP-1 gene expression in chondrocytes, which involves heterodimer-independent mechanisms.

Methods and Materials

Cell Culture and Reagents

SW-1353 cells were purchased from ATCC and cultured in DMEM supplemented with 10% FBS (Hyclone, Logan, UT), penicillin/streptomycin, and glutamine (Cellgro, Mediatech, Herndon, VA). Human articular chondrocyte cultures (HAC) were prepared as previously described 25. For assays of gene expression, cells were washed with HBSS and placed in DMEM supplemented with 0.2 % lactalbumin hydrolysate (Invitrogen, Carlsbad, CA). Recombinant human IL-1β was purchased from Promega (Madison, WI).

Analysis of Steady State mRNA

Analysis of gene expression was performed by quantitative reverse transcriptase polymerase chain reaction as described previously 22, 25. The following primers were used to amplify reverse-transcribed mRNA.

RelA: 5′-CTGCCGGGATGGCTTCTAT-3′

   5′-CCGCTTCTTCACACACTGGAT-3′

NF-κB1: 5′-TGGAGTCTGGGAAGGATTT-3′

   5′-CGAAGCTGGACAAACACAG-3′

NF-κB2: 5′-GCGCAGGACGAGAACGGAGAC-3′

   5′-TGGGCGTGGTGGATGACATA-3′

MMP-1: 5′-AGCTAGCTCAGGATGACATTGATG-3′

   5′-GCCGATGGGCTGGACAG-3′

GAPDH: 5′-CGACAGTCAGCCGCATCTT-3′

   5′-CCCCATGGTGTCTGAGCG-3′

Transfection and creation of stable cell lines

SW-1353 cells were transfected with an empty vector or a plasmid expressing a HA-tagged dominant negative IκBα (Biomyx Technology, San Diego, CA) using the Nucleofector transfection system (Amaxa Inc. Gaithersburg, MD) according to the manufacturer’s protocol. Transfectants were placed under selection with Geneticin (Invitrogen Carlsbad, CA) and pooled stable lines were established. IKBDN transgene expression was confirmed by Western blot using an HA antibody (data not shown). In order to confirm the functionality of the IKBDN transgene, the stable line was transiently transfected in triplicate with a kappa B luciferase reporter gene (Stratagene, La Jolla, CA) and assayed using Luciferase Assay Reagent (Promega Corporation, Madison, WI). Triplicate cultures of this stable line were also assayed for MMP-1 and GAPDH gene expression by QRT-PCR as described above.

In separate experiments, SW-1353 were transfected with a 3292 nucleotide human MMP-1 promoter/luciferase reporter plasmid 26 along with a plasmid containing the neomycin gene. Transfectants were placed under selection with Geneticin (Invitrogen Carlsbad, CA) and pooled stable lines were established. This stable line was plated in triplicate and assayed using Luciferase Assay Reagent (Promega Corporation, Madison, WI).

Western blot

Confluent monolayers of HAC were stimulated as indicated, washed briefly in phosphate buffered saline and then scraped into 2X laemmli buffer. The samples were heated to 100ºC for 5 minutes and then run on 10% reducing SDS-PAGE gels. The proteins were transferred to PVDF membranes (Millipore, Bedford, MA) and probed using commercially available antibodies for NF-κB1 (Santa Cruz Biotechnology, Santa Cruz, CA, SC-114), NF-κB2 (Upstate Cell Signaling Solutions, Charlottesville, VA, 06–413), RelA (Santa Cruz Biotechnology, SC-109), RelA phospho-serine 536 (Cell Signaling Technology, Danvers, MA, #3031), IκBα (Cell Signaling Technology, #9242), IκBα phospho-serine 32 (Cell Signaling Technology #9241) and Pan Actin (Cell Signaling Technology #4968). Blots were visualized using the Pierce Biotechnology (Rockford, IL) Supersignal West Femto detection kit.

Gene silencing

The following double-stranded siRNAs were designed using Oligoengine software and purchased from Dharmacon Research, Inc. (Lafayette, CO).

Rel A: 5′-UCCAGUGUGUGAAGAAGCGdTdT-3′

3′-dTdTAGGUCACACACUUCUUCGC-5′

NF-κB1: 5′-CACUGGAAGCACGAAUGACdTdT-3′

3′-dTdTGUGACCUUCGUGCUUACUG-5′

NF-κB2: 5′-GAUUUCUCGAAUGGACAAGdTdT-3′

3′-dTdTCUAAAGAFCUUACCUGUUC-5′

Control: 5′-AGAGAUGCAUGCACGCACAdTdT-3′

3′-dTdTUCUCUACGUACGUGCGUGU-5′

For siRNA experiments, SW-1353 cells were transfected with siRNA using Lipofectamine 2000 according to the manufacturer’s protocol. HAC were transfected using the Nucleofector transfection system (Amaxa Inc. Gaithersburg, MD) according to the manufacturer’s protocol. After 48 hours of culture, cells were washed, placed in DMEM supplemented with LH, with or without IL-1β (10 ng/ml). After 24 hours of culture, RNA was harvested with TRIzol reagent (Invitrogen, Carlsbad, CA) for gene expression analysis.

Statistical Analysis

Data are presented as means of triplicate cultures with standard deviations. Statistical significance was assessed by two-tailed Student’s T-test. Unless otherwise indicated, *indicates P≤0.05.

Results

IL-1β activates MMP-1 and NF-κB gene expression in chondrocytes

Gene-profiling studies have indicated that the SW-1353 chondrosarcoma cell line is an appropriate model of IL-1β-induced protease and NF-κB gene expression in human chondrocytes 23, 24. To extend this work, we examined basal and IL-1β-stimulated MMP-1 gene expression in the SW-1353 cell line, and in cultured human articular chondrocytes (HAC) over a twenty-four hour period. IL-1β treatment significantly increased MMP-1 mRNA levels in SW-1353 cells and HAC within two hours and with a similar magnitude and time course over twenty-four hours (figure 1). Western blot analysis of MMP-1 protein secreted into the culture media confirmed that IL-1β dramatically increased MMP-1 gene expression after 24 hours in both SW-1353 and HAC. Furthermore, both latent and active forms of MMP-1 were present in the media of these cells. These results confirm earlier findings 24 demonstrating that SW-1353 cells accurately represent IL-1β-induced MMP-1 gene expression in human chondrocytes.

Figure 1.

Figure 1

Figure 1

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IL-1 stimulation of MMP-1 mRNA and protein levels in chondrocytes. Triplicate confluent cultures of HAC (A) and SW-1353 cells (B) were placed in serum-free media, both with and without IL-1β (10 ng/ml). Culture medium was collected after 24 hours and assayed for MMP-1 protein by Western blot. Total RNA was harvested at the indicated time periods and steady state levels of MMP-1 and GAPDH mRNA were assayed by QRT-PCR. MMP-1 mRNA values were normalized to GAPDH and then plotted as fold of control. The data presented are representative of two separate experiments. *P≤0.05.

We next examined NF-κB gene expression in IL-1β treated chondrocytes to determine how this compared with MMP-1 gene expression. HAC stimulated with IL-1β contained increasing mRNA levels for all three genes over the time period assayed (figure 2). This increase in mRNA was followed closely by increases in cellular protein. NF-κB1 consisted of the 105 kD proform and the 50 kDA processed form in both untreated and IL-1β treated cells (figure 2A). Dramatic increases of both p105 and p50 were observed in IL-1β stimulated chondrocytes by 8 hours of culture. NF-κB2 consisted of the 100 kDa proform and the 52 kDa processed form in both untreated and IL-1β treated cells (figure 2B). An increase in the p100 proform was observed in IL-1β treated cells by 8 hours of culture, without a parallel increase in the processed 52 kDa form. Consistent with NF-κB1 and NF-κB2, RelA protein levels increased in IL-1β stimulated chondrocytes between 8 and 24 hours of culture (figure 2C). These analyses demonstrate that IL-1β induces individual NF-κB proteins in chondrocytes with a time course similar to that of MMP-1, and suggests that these proteins may contribute to IL-1β-induced MMP-1 gene expression in these cells.

Figure 2.

Figure 2

Figure 2

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IL-1β stimulation of NF-κB gene expression in chondrocytes. Confluent cultures of HAC were placed in serum-free media, both with and without IL-1β (10 ng/ml). Total cellular RNA and protein were harvested at the indicated time points and assayed by QRT-PCR and Western blot. Steady state mRNA and protein levels for (A) NF-κB1, (B) NF-κB2 and (C) RelA are presented. All mRNA levels were normalized to GAPDH and presented as fold of control. The data presented are representative of two separate experiments. *P≤0.05.

IL-1 activates the NF-κB pathway at two different levels in chondrocytes

Conflicting reports about the requirement of NF-κB for IL-1β-induced MMP-1 gene expression in chondrocytes 20, 21 prompted us to address this question in SW-1353 cells. For these studies, SW-1353 cells were stably transfected with an expression plasmid for dominant negative IκBα (IKBDN), which contains alanine substitutions at serines 32 and 36. These substitutions render IKBDN resistant to phosphorylation and degradation so that it acts as a constitutive repressor of NF-κB nuclear translocation 27–29. IL-1β induction of a transiently transfected kappa B luciferase reporter plasmid was significantly reduced, confirming the functionality of IKBDN (figure 3A). Similarly, expression of IKBDN in SW-1353 cells significantly reduced IL-1β-induced endogenous MMP-1 mRNA levels (figure 3B). Transfection of SW-1353 cells enhanced IL-1β-induced MMP-1 expression (compare figures 1B and 3B) and this may reflect cellular responses to the expression of exogenous DNA. Nonetheless, our results agree with those of Bondeson et al. 20, which demonstrate that nuclear translocation of RelA-containing NF-κB complexes is required for optimal IL-1β-induced MMP-1 expression.

Figure 3.

Figure 3

Figure 3

Figure 3

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IL-1β stimulation of the NF-κB pathway in chondrocytes. SW-1353 were stably transfected with an empty expression plasmid (CMV) or a plasmid expressing dominant negative IκBβ (IKBDN). (A) Stably transfected cells were then transiently transfected with a kappa B luciferase reporter plasmid, placed in serum-free media both with and without IL-1β for 24 hours, and luciferase activity was assayed. Data are from triplicate cultures and are presented as fold of CMV-transfected, untreated cultures. * P≤0.05. (B) Triplicate cultures of the IKBDN line were cultured for 24 hours in serum-free media, both with and without IL-1β. Total RNA was harvested and assayed for MMP-1 and GAPDH by QRT-PCR. Data are presented as fold of CMV-transfected, untreated cultures * P≤0.05. (C and D) Confluent cultures of HAC were placed in serum-free media, both with and without IL-1β (10 ng/ml). Total cellular protein was harvested at early (C) and late (D) time points and assayed by Western blot for IκBα-phospho-serine 32, total IκBα, RelA-phospho-serine 536, total RelA and Actin. The data presented are representative of two separate experiments.

We next assayed the ability of IL-1β to activate the NF-κB pathway over this period. Phosphorylation of IκBα on serine 32 results in ubiquitination and proteosome-mediated degradation of this protein. IL-1β treatment caused phosphorylation of IκBα within five minutes, resulting in degradation of IκBα protein levels between five and thirty minutes (figure 3C). The fact that phospho-IκBα is readily detectable at five minutes, despite very low levels of total IκBα at this time point, likely reflects the higher sensitivity of the phospho-specific antibody. Within sixty minutes, IκBα re-synthesis was evident in IL-1β-treated cells (figure 3C) and protein levels returned to control levels by four hours, despite low levels of IκBα phosphorylation (figure 3D). Recent work by other laboratories has demonstrated that IL-1β stimulation can promote phosphorylation of RelA on serine 536, which causes RelA to dissociate from IκBα and NF-κB1 15, 30. To determine if this pathway is activated in chondrocytes, we treated HAC with IL-1β and assayed RelA phospho-serine 536 at various time points. RelA phospho-serine 536 increased dramatically in IL-1β-treated cells within five minutes despite no increase in total RelA (figure 3C), and remained above levels in untreated cells at each time point assayed over twenty-four hours (figure 3D). Together, these data demonstrate that in addition to inducing NF-κB gene expression, IL-1β activates the NF-κB pathway through a transient degradation of IκBα and a sustained phosphorylation of RelA.

The effect of NF-κB gene silencing on MMP-1 expression

Our data suggest that NF-κB activation, and specifically RelA nuclear localization, is required for IL-1β induction of MMP-1 in chondrocytes. To assess the roles of individual NF-κB family members during IL-1β-induced MMP-1 gene expression, we transiently transfected SW-1353 cells with double-stranded small interfering RNA molecules (siRNA) and then assayed MMP-1 mRNA levels in untreated and IL-1β-treated cells. Transfection of specific siRNA effectively silenced NF-κB1 (figure 4A), NF-κB2 (figure 5A) and RelA (figure 6A) mRNA and protein in SW-1353 cells treated with IL-1β for 24 hours. Surprisingly, silencing of NF-κB1 (figure 4B) or NF-κB2 (figure 5B) in these cells actually increased IL-1β-induced MMP-1 mRNA levels. In contrast, silencing of RelA (figure 6B) significantly reduced basal and IL-1β-induced MMP-1 mRNA levels in SW-1353 cells. To ensure that the requirement for RelA is not specific to the SW-1353 cell line, we transfected HAC with the same siRNA and assayed MMP-1 mRNA expression. We found that while NF-κB1 siRNA had no effect, NF-κB2 siRNA significantly increased and RelA siRNA significantly reduced, IL-1-induced MMP-1 mRNA (figure 7). These results reemphasize that SW-1353 cells are an appropriate model for primary chondrocytes, and confirm that RelA alone is required for IL-1-induced MMP-1 gene expression.

Figure 4.

Figure 4

Figure 4

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Gene expression in NF-κB1-silenced SW-1353 cells. Triplicate cultures of SW-1353 cells were transfected with NF-κB1-specific siRNA. After 48 hours, the cells were placed in serum-free media, both with and without IL-1β (10 ng/ml) for an additional 24 hours. Total cellular protein and RNA was then harvested and assayed for (A) NF-κB1 mRNA and protein and (B) MMP-1 mRNA. NF-κB1 and MMP-1 mRNA levels, as assayed by QRT-PCR, were normalized to GAPDH mRNA levels in each sample and presented as fold of control.

Figure 5.

Figure 5

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Gene expression in NF-κB2-silenced SW-1353 cells. Triplicate cultures of SW-1353 cells were transfected with NF-κB2-specific siRNA. After 48 hours, the cells were placed in serum-free media, both with and without IL-1β (10 ng/ml), for an additional 24 hours. Total cellular protein and RNA was then harvested and assayed for (A) NF-κB2 mRNA and protein and (B) MMP-1 mRNA. NF-κB2 and MMP-1 mRNA levels, as assayed by QRT-PCR, were normalized to GAPDH mRNA levels in each sample and presented as fold of control.

Figure 6.

Figure 6

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Gene expression in RelA-silenced SW-1353 cells. Triplicate cultures of SW-1353 cells were transfected with RelA-specific siRNA. After 48 hours, the cells were placed in serum-free media, both with and without IL-1β (10 ng/ml), for an additional 24 hours. Total cellular protein and RNA was then harvested and assayed for (A) RelA mRNA and protein and (B) MMP-1 mRNA. RelA and MMP-1 mRNA levels, as assayed by QRT-PCR, were normalized to GAPDH mRNA levels in each sample and presented as fold of control.

Figure 7.

Figure 7

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MMP-1 gene expression in NF-κB1-, NF-κB2- and RelA-silenced HAC. Triplicate cultures of HAC were transfected with NF-κB1, NF-κB2 and RelA-specific siRNA. After 48 hours, the cells were placed in serum-free media, both with and without IL-1β (10 ng/ml), for an additional 24 hours. Total RNA was then harvested and assayed for MMP-1 mRNA by QRT-PCR. MMP-1 mRNA levels were normalized to GAPDH mRNA levels in each sample and presented as fold of control. * P≤0.05.

In order to determine if the role of RelA in MMP-1 gene expression is transcriptional, we created a stable line of SW-1353 cells expressing the luciferase gene under the transcriptional control of a 3292 nucleotide MMP-1 promoter fragment. We have recently demonstrated that this promoter construct is IL-1β-responsive in chondrocytes 17. We next transfected this stable line with control or RelA-specific siRNA and then assayed luciferase gene expression in untreated and IL-1β treated cells (figure 8). IL-1β induced luciferase activity in control siRNA-transfected cells, but IL-1β induction was significantly reduced in RelA siRNA transfected cells. These data demonstrate that silencing of RelA in chondrocytes reduces IL-1β stimulation of the MMP-1 promoter. Together, our findings indicate that IL-1β activates the NF-κB pathway in chondrocytes through increased NF-κB gene expression, IκBα degradation and RelA phosphorylation. Furthermore, among the NF-κB proteins that can contribute to MMP-1 gene expression, only RelA is required.

Figure 8.

Figure 8

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RelA siRNA targets the MMP-1 promoter. SW1353 cells were stably transfected with a 3292-nucleotide MMP-1 promoter/luciferase reporter construct. Triplicate cultures were then transfected with RelA siRNA, allowed to recover for 48 hours and then cultured with and without IL-1β for 24 hours under serum-free conditions. Cultures were harvested in lysis buffer and assayed for luciferase activity. * P≤0.05.

Discussion

The NF-κB pathway controls a variety of cellular responses through the activation of genes controlling inflammation, apoptosis and matrix remodeling. Recent studies suggest that NF-κB plays a particularly complex role in the biology of articular chondrocytes. Activation of this pathway promotes chondrocyte differentiation through stimulation of Sox9 gene expression 31, and IL-1β induction of NF-κB protects chondrocytes from apoptotic signals 32. We showed previously that Bcl-3, which is structurally related to IκBα, contributes to IL-1β-induced MMP-1 gene expression in chondrocytes 22, 25. Here we show that concurrent with increasing MMP-1 mRNA levels, IL-1β stimulation of chondrocytes activates NF-κB gene expression. Nuclear translocation of RelA-containing complexes is required for maximal IL-1β-induced MMP-1 expression, and this is mediated through a transient degradation of IκBα and a sustained phosphorylation of RelA. Moreover, we demonstrate that RelA is absolutely required for maximal IL-1β induction of MMP-1 mRNA through activation of the MMP-1 promoter. This work defines RelA as a central mediator of IL-1β-induced MMP-1 gene expression in chondrocytes and establishes this transcription factor as a potential target for modulating collagen degradation in arthritis.

Expression of a dominant-negative IκBα, and silencing of RelA expression with siRNA, did not completely inhibit IL-1β-induced MMP-1 gene expression. From these results, one must conclude that IL-1β targets signaling pathways in addition to NF-κB to activate the MMP-1 promoter. In support of this, we have recently reported that IL-1β activates the extracellular signal-regulated kinase (ERK) pathway in chondrocytes, leading to MMP-1 transcriptional activation by CCAAT enhancer binding protein beta17. Interestingly, gene silencing NF-κB1 and NF-κB2 in SW-1353 cells, and NF-κB2 in HAC, actually augmented IL-1β-induced MMP-1 gene expression. These effects were not due to a compensatory increase in RelA expression (data not shown). Both NF-κB1 and NF-κB2 can act as IκB molecules, because their unprocessed forms possess ankyrin repeats that can sequester RelA proteins in the cytoplasm 13. In addition, homodimers of processed NF-κB1 can bind to some kappa B sites and act as transcriptional repressors 33. Thus, the dramatic increase of unprocessed NF-κB1 and NF-κB2 proteins present after 8 and 24 hours of IL-1β treatment may function to dampen IL-1β-induced MMP-1 gene expression. Bcl-3, which is an IL-1β inducible gene in chondrocytes that activates MMP-1 expression 22, can bind to and remove repressive NF-κB1 homodimers from the nucleus 33, 34. Taken together, these findings may explain our observations that Bcl-3 activates 22, while NF-κB1 inhibits, IL-1β-induced MMP-1 gene expression in SW-1353 cells.

Our finding that RelA activates MMP-1 independently of NF-κB1 is supported by the fact that RelA phosphorylation on serine 536 is elevated in IL-1β stimulated chondrocytes over a 24-hour period (figure 3). Sasaki et al. 15 recently reported that this phosphorylation creates a pool of RelA that translocates to the nucleus because it does not associate with NF-κB1 or IκBα Thus, RelA could promote MMP-1 gene expression even as IκBα levels return to pre-stimulation levels. Once in the nucleus, RelA phospho-serine 536 is a more potent transactivating protein because it associates more readily with p300 35.

RelA could activate the MMP-1 promoter in IL-1β stimulated chondrocytes through multiple mechanisms. We recently reported that C/EBPβ activates the MMP-1 promoter in IL-1β stimulated chondrocytes 17 and RelA cooperates with C/EBPβ at several cytokine-responsive promoters. Using electrophoresis mobility shift assays, we were unable to detect RelA binding activity at the C/EBPβ site in the MMP-1 promoter. However, RelA could be cooperating with C/EBPβ through a yet undefined binding site in the promoter. RelA has also been reported to antagonize other transcription factors that are established repressors of MMP-1 transcription. The tumor suppressor p53 represses MMP-1 transcription by disrupting AP-1 interactions with CBP/p300 36, 37. When RelA is phosphorylated on serine 536 (as is the case in IL-1β treated chondrocytes), it inhibits p53 function 38 and has the potential to reverse the repressive effects of p53. Similarly, transforming growth factor beta (TGF-β) represses IL-1β stimulated MMP-1 transcription in dermal fibroblasts through Smad3 and Smad4 39. Since RelA interferes with the DNA binding activity of Smad3 and Smad4 40, RelA could overcome MMP-1 repression in TGF-β treated cells. Finally, RelA could contribute to MMP-1 gene expression by promoting the expression of other essential factors. Bcl-3 activates MMP-1 in IL-1β stimulated chondrocytes 22 and expression of constitutively active RelA is sufficient to induce Bcl-3 expression 34. These few examples demonstrate that RelA has the potential to activate MMP-1 gene expression at multiple levels, through direct interactions and through the expression of other transcription factors.

IL-1β stimulation of MMP-1 gene expression in articular chondrocytes is a pathway that can contribute directly to cartilage loss. This gene expression program is extremely complex and requires a well-orchestrated interplay of multiple protein kinases and transcription factors. The present study, which defines the roles of individual components of the NF-κB pathway, should provide a clearer perspective toward therapeutic intervention in arthritis.

Acknowledgments

The authors would like to thank Dr. Brooks Robey for critical reading of this manuscript. This work was supported by a VA Merit Review Grant and a grant from the National Institutes of Arthritis and Musculoskeletal and Skin Diseases (R01 AR46977), both awarded to Matthew Vincenti.

Footnotes

Support: VA Merit Review and NIH/NIAMS R01 AR46977

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References

  • 1.Burrage PS, Mix KS, Brinckerhoff CE. Matrix metalloproteinases: role in arthritis. Front Biosci. 2006;11:529–43. doi: 10.2741/1817. [DOI] [PubMed] [Google Scholar]
  • 2.Holmbeck K, Bianco P, Yamada S, Birkedal-Hansen H. MT1-MMP: a tethered collagenase. J Cell Physiol. 2004;200:11–9. doi: 10.1002/jcp.20065. [DOI] [PubMed] [Google Scholar]
  • 3.Murphy G, Knauper V, Atkinson S, Butler G, English W, Hutton M, et al. Matrix metalloproteinases in arthritic disease. Arthritis Res. 2002;4(Suppl 3):S39–49. doi: 10.1186/ar572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Firestein GS, Paine MM, Littman BH. Gene expression (collagenase, tissue inhibitor of metalloproteinases, complement, and HLA-DR) in rheumatoid arthritis and osteoarthritis synovium. Quantitative analysis and effect of intraarticular corticosteroids. Arthritis Rheum. 1991;34:1094–105. doi: 10.1002/art.1780340905. [DOI] [PubMed] [Google Scholar]
  • 5.Kevorkian L, Young DA, Darrah C, Donell ST, Shepstone L, Porter S, et al. Expression profiling of metalloproteinases and their inhibitors in cartilage. Arthritis Rheum. 2004;50:131–41. doi: 10.1002/art.11433. [DOI] [PubMed] [Google Scholar]
  • 6.Petrow PK, Wernicke D, Schulze Westhoff C, Hummel KM, Brauer R, Kriegsmann J, et al. Characterisation of the cell type-specificity of collagenase 3 mRNA expression in comparison with membrane type 1 matrix metalloproteinase and gelatinase A in the synovial membrane in rheumatoid arthritis. Ann Rheum Dis. 2002;61:391–7. doi: 10.1136/ard.61.5.391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Shlopov BV, Lie WR, Mainardi CL, Cole AA, Chubinskaya S, Hasty KA. Osteoarthritic lesions: involvement of three different collagenases. Arthritis Rheum. 1997;40:2065–74. doi: 10.1002/art.1780401120. [DOI] [PubMed] [Google Scholar]
  • 8.Tetlow LC, Adlam DJ, Woolley DE. Matrix metalloproteinase and proinflammatory cytokine production by chondrocytes of human osteoarthritic cartilage: associations with degenerative changes. Arthritis Rheum. 2001;44:585–94. doi: 10.1002/1529-0131(200103)44:3<585::AID-ANR107>3.0.CO;2-C. [DOI] [PubMed] [Google Scholar]
  • 9.Tetlow LC, Woolley DE. Comparative immunolocalization studies of collagenase 1 and collagenase 3 production in the rheumatoid lesion, and by human chondrocytes and synoviocytes in vitro. Br J Rheumatol. 1998;37:64–70. doi: 10.1093/rheumatology/37.1.64. [DOI] [PubMed] [Google Scholar]
  • 10.Tchetina EV, Squires G, Poole AR. Increased type II collagen degradation and very early focal cartilage degeneration is associated with upregulation of chondrocyte differentiation related genes in early human articular cartilage lesions. J Rheumatol. 2005;32:876–86. [PubMed] [Google Scholar]
  • 11.Wu W, Billinghurst RC, Pidoux I, Antoniou J, Zukor D, Tanzer M, et al. Sites of collagenase cleavage and denaturation of type II collagen in aging and osteoarthritic articular cartilage and their relationship to the distribution of matrix metalloproteinase 1 and matrix metalloproteinase 13. Arthritis Rheum. 2002;46:2087–94. doi: 10.1002/art.10428. [DOI] [PubMed] [Google Scholar]
  • 12.Poole AR, Kobayashi M, Yasuda T, Laverty S, Mwale F, Kojima T, et al. Type II collagen degradation and its regulation in articular cartilage in osteoarthritis. Ann Rheum Dis. 2002;61(Suppl 2):ii78–81. doi: 10.1136/ard.61.suppl_2.ii78. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Karin M, Yamamoto Y, Wang QM. The IKK NF-kappa B system: a treasure trove for drug development. Nat Rev Drug Discov. 2004;3:17–26. doi: 10.1038/nrd1279. [DOI] [PubMed] [Google Scholar]
  • 14.Baeuerle PA, Baltimore D. A 65-kappaD subunit of active NF-kappaB is required for inhibition of NF-kappaB by I kappaB. Genes Dev. 1989;3:1689–98. doi: 10.1101/gad.3.11.1689. [DOI] [PubMed] [Google Scholar]
  • 15.Sasaki CY, Barberi TJ, Ghosh P, Longo DL. Phosphorylation of RelA/p65 on serine 536 defines an I{kappa}B{alpha}-independent NF-{kappa}B pathway. J Biol Chem. 2005;280:34538–47. doi: 10.1074/jbc.M504943200. [DOI] [PubMed] [Google Scholar]
  • 16.Vincenti MP, Brinckerhoff CE. Transcriptional regulation of collagenase (MMP-1, MMP-13) genes in arthritis: integration of complex signaling pathways for the recruitment of gene-specific transcription factors. Arthritis Res. 2002;4:157–64. doi: 10.1186/ar401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Raymond L, Eck S, Mollmark J, Hays E, Tomek I, Kantor S, et al. Interleukin-1 beta induction of matrix metalloproteinase-1 transcription in chondrocytes requires ERK-dependent activation of CCAAT enhancer-binding protein-beta. J Cell Physiol. 2006;207:683–8. doi: 10.1002/jcp.20608. [DOI] [PubMed] [Google Scholar]
  • 18.Barchowsky A, Frleta D, Vincenti MP. Integration of the NF-kappaB and mitogen-activated protein kinase/AP-1 pathways at the collagenase-1 promoter: divergence of IL-1 and TNF-dependent signal transduction in rabbit primary synovial fibroblasts. Cytokine. 2000;12:1469–79. doi: 10.1006/cyto.2000.0743. [DOI] [PubMed] [Google Scholar]
  • 19.Bond M, Chase AJ, Baker AH, Newby AC. Inhibition of transcription factor NF-kappaB reduces matrix metalloproteinase-1, -3 and -9 production by vascular smooth muscle cells. Cardiovasc Res. 2001;50:556–65. doi: 10.1016/s0008-6363(01)00220-6. [DOI] [PubMed] [Google Scholar]
  • 20.Bondeson J, Brennan F, Foxwell B, Feldmann M. Effective adenoviral transfer of IkappaBalpha into human fibroblasts and chondrosarcoma cells reveals that the induction of matrix metalloproteinases and proinflammatory cytokines is nuclear factor-kappaB dependent. J Rheumatol. 2000;27:2078–89. [PubMed] [Google Scholar]
  • 21.Mengshol JA, Vincenti MP, Coon CI, Barchowsky A, Brinckerhoff CE. Interleukin-1 induction of collagenase 3 (matrix metalloproteinase 13) gene expression in chondrocytes requires p38, c-Jun N-terminal kinase, and nuclear factor kappaB: differential regulation of collagenase 1 and collagenase 3. Arthritis Rheum. 2000;43:801–11. doi: 10.1002/1529-0131(200004)43:4<801::AID-ANR10>3.0.CO;2-4. [DOI] [PubMed] [Google Scholar]
  • 22.Elliott SF, Coon CI, Hays E, Stadheim TA, Vincenti MP. Bcl-3 is an interleukin-1-responsive gene in chondrocytes and synovial fibroblasts that activates transcription of the matrix metalloproteinase 1 gene. Arthritis Rheum. 2002;46:3230–9. doi: 10.1002/art.10675. [DOI] [PubMed] [Google Scholar]
  • 23.Vincenti MP, Brinckerhoff CE. Early response genes induced in chondrocytes stimulated with the inflammatory cytokine interleukin-1beta. Arthritis Res. 2001;3:381–8. doi: 10.1186/ar331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Gebauer M, Saas J, Sohler F, Haag J, Soder S, Pieper M, et al. Comparison of the chondrosarcoma cell line SW1353 with primary human adult articular chondrocytes with regard to their gene expression profile and reactivity to IL-1beta. Osteoarthritis Cartilage. 2005;13:697–708. doi: 10.1016/j.joca.2005.04.004. [DOI] [PubMed] [Google Scholar]
  • 25.Elliott S, Hays E, Mayor M, Sporn M, Vincenti M. The triterpenoid CDDO inhibits expression of matrix metalloproteinase-1, matrix metalloproteinase-13 and Bcl-3 in primary human chondrocytes. Arthritis Res Ther. 2003;5:R285–91. doi: 10.1186/ar792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Rutter JL, Benbow U, Coon CI, Brinckerhoff CE. Cell-type specific regulation of human interstitial collagenase-1 gene expression by interleukin-1 beta (IL-1 beta) in human fibroblasts and BC-8701 breast cancer cells. J Cell Biochem. 1997;66:322–36. [PubMed] [Google Scholar]
  • 27.Brown K, Gerstberger S, Carlson L, Franzoso G, Siebenlist U. Control of I kappa B-alpha proteolysis by site-specific, signal-induced phosphorylation. Science. 1995;267:1485–8. doi: 10.1126/science.7878466. [DOI] [PubMed] [Google Scholar]
  • 28.Chen Z, Hagler J, Palombella VJ, Melandri F, Scherer D, Ballard D, et al. Signal-induced site-specific phosphorylation targets I kappa B alpha to the ubiquitin-proteasome pathway. Genes Dev. 1995;9:1586–97. doi: 10.1101/gad.9.13.1586. [DOI] [PubMed] [Google Scholar]
  • 29.Traenckner EB, Pahl HL, Henkel T, Schmidt KN, Wilk S, Baeuerle PA. Phosphorylation of human I kappa B-alpha on serines 32 and 36 controls I kappa B-alpha proteolysis and NF-kappa B activation in response to diverse stimuli. Embo J. 1995;14:2876–83. doi: 10.1002/j.1460-2075.1995.tb07287.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Egan LJ, Mays DC, Huntoon CJ, Bell MP, Pike MG, Sandborn WJ, et al. Inhibition of interleukin-1-stimulated NF-kappaB RelA/p65 phosphorylation by mesalamine is accompanied by decreased transcriptional activity. J Biol Chem. 1999;274:26448–53. doi: 10.1074/jbc.274.37.26448. [DOI] [PubMed] [Google Scholar]
  • 31.Sitcheran R, Cogswell PC, Baldwin AS., Jr NF-kappaB mediates inhibition of mesenchymal cell differentiation through a posttranscriptional gene silencing mechanism. Genes Dev. 2003;17:2368–73. doi: 10.1101/gad.1114503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Kuhn K, Hashimoto S, Lotz M. IL-1 beta protects human chondrocytes from CD95-induced apoptosis. J Immunol. 2000;164:2233–9. doi: 10.4049/jimmunol.164.4.2233. [DOI] [PubMed] [Google Scholar]
  • 33.Franzoso G, Bours V, Azarenko V, Park S, Tomita-Yamaguchi M, Kanno T, et al. The oncoprotein Bcl-3 can facilitate NF-kappa B-mediated transactivation by removing inhibiting p50 homodimers from select kappa B sites. Embo J. 1993;12:3893–901. doi: 10.1002/j.1460-2075.1993.tb06067.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Brasier AR, Lu M, Hai T, Lu Y, Boldogh I. NF-kappa B-inducible BCL-3 expression is an autoregulatory loop controlling nuclear p50/NF-kappa B1 residence. J Biol Chem. 2001;276:32080–93. doi: 10.1074/jbc.M102949200. [DOI] [PubMed] [Google Scholar]
  • 35.Chen LF, Williams SA, Mu Y, Nakano H, Duerr JM, Buckbinder L, et al. NF-kappaB RelA phosphorylation regulates RelA acetylation. Mol Cell Biol. 2005;25:7966–75. doi: 10.1128/MCB.25.18.7966-7975.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Sun Y, Sun Y, Wenger L, Rutter JL, Brinckerhoff CE, Cheung HS. Human metalloproteinase-1 (collagenase-1) is a tumor suppressor protein p53 target gene. Ann N Y Acad Sci. 1999;878:638–41. doi: 10.1111/j.1749-6632.1999.tb07749.x. [DOI] [PubMed] [Google Scholar]
  • 37.Sun Y, Zeng XR, Wenger L, Firestein GS, Cheung HS. P53 down-regulates matrix metalloproteinase-1 by targeting the communications between AP-1 and the basal transcription complex. J Cell Biochem. 2004;92:258–69. doi: 10.1002/jcb.20044. [DOI] [PubMed] [Google Scholar]
  • 38.Jeong SJ, Pise-Masison CA, Radonovich MF, Park HU, Brady JN. A novel NF-kappaB pathway involving IKKbeta and p65/RelA Ser-536 phosphorylation results in p53 Inhibition in the absence of NF-kappaB transcriptional activity. J Biol Chem. 2005;280:10326–32. doi: 10.1074/jbc.M412643200. [DOI] [PubMed] [Google Scholar]
  • 39.Yuan W, Varga J. Transforming growth factor-beta repression of matrix metalloproteinase-1 in dermal fibroblasts involves Smad3. J Biol Chem. 2001;276:38502–10. doi: 10.1074/jbc.M107081200. [DOI] [PubMed] [Google Scholar]
  • 40.Bitzer M, von Gersdorff G, Liang D, Dominguez-Rosales A, Beg AA, Rojkind M, et al. A mechanism of suppression of TGF-beta/SMAD signaling by NF-kappa B/RelA. Genes Dev. 2000;14:187–97. [PMC free article] [PubMed] [Google Scholar]

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