In osteoarthritis (OA) and rheumatoid arthritis (RA), patients experience erosion of articular cartilage, a process that involves degradation of type II collagen and proteoglycans (1, 2). Although the loss of proteoglycan reduces the shock-absorbing properties of articular cartilage, it is the collagen loss that correlates positively with the severity of arthritic lesions (3). This has led to the suggestion that degradation of type II collagen disrupts the architecture of articular cartilage and is, therefore, a committed step in the pathogenesis of OA (4) and RA (5).
The degradation of type II collagen in articular cartilage is facilitated by enzymes that are members of a family of neutral proteases named the matrix metalloproteinases (MMPs). The MMPs are zinc- and calcium-dependent enzymes responsible for degradation of the protein components of the extracellular matrix (6). Three members of this group of enzymes, the interstitial collagenases (MMP-1, MMP-8, and MMP-13), effectively digest type II collagen, and all 3 enzymes have been localized to arthritic lesions (4). Of these 3 enzymes, MMP-1 is the most ubiquitously expressed in connective tissue cells (7). Immunolocalization studies demonstrate that MMP-1 is the predominant collagenase produced by rheumatoid synovial fibroblasts and chondrocytes (8). While MMP-13 contributes primarily to the initial degradation of collagen in OA (9), inhibition of MMP-1, but not MMP-13, enhances new collagen synthesis in OA chondrocytes (10). Thus, MMP-1 plays a central role in the cartilage destruction observed in both RA and OA.
The nuclear factor κB (NF-κB) pathway plays a central role in inflammatory responses that are pertinent to arthritis (11), including activation of the MMP-1 gene (12–15). Heterodimers of NF-κB1/p50 and RelA/p65 are sequestered in the cytoplasm of resting cells by inhibitor of NF-κB (IκB). When cells are stimulated with inflammatory signals such as interleukin-1β (IL-1β), IκB becomes phosphorylated by a protein complex known as IκB kinase (IKK). IKK-dependent phosphorylation targets IκB for ubiquitination and proteosome-dependent degradation. With IκB degraded, the NF-κB1–RelA complex can translocate to the nucleus and bind to the promoters of responsive genes (16). We have previously demonstrated that the NF-κB pathway activates the MMP-1 promoter in IL-1β–stimulated synovial fibroblasts (SF) (14). Interestingly, we found that NF-κB1 bound to the MMP-1 IL-1β–responsive element, but that RelA was not detected in this complex. This result was unexpected because RelA contains a potent transactivation domain, but NF-κB1 does not (17).
NF-κB1 homodimers also bind to promoter sequences, and like heterodimers, nuclear translocation of these complexes is regulated in an IKK-dependent manner. The precursor form of NF-κB1, p105, associates with mature NF-κB1, and the p50–p105 complex is sequestered in the cytoplasm through the carboxy-terminal ankyrin repeats of p105 (18). In stimulated cells, IKK phosphorylates p105, and like IκB, this results in ubiquitination and degradation of p105 (19, 20). Degradation of p105 liberates p50, which forms homodimers that translocate to the nucleus. An IκB-related protein, Bcl-3, has recently been shown to complex with NF-κB1 dimers and assist with nuclear translocation and transcription activity (20–22). Since Bcl-3 contributes to the transcriptional competence of NF-κB1, we have examined the role of Bcl-3 in IL-1β–induced MMP-1 gene expression.
Here, we describe Bcl-3 as an IL-1β–responsive gene in chondrocytic cells, and we demonstrate that overexpression of Bcl-3 in these cells and in SF enhances basal and IL-1β–induced MMP-1 transcription. Furthermore, inhibition of Bcl-3 with antisense oligonucleotides in chondrocytes represses IL-1β–induced MMP-1 gene expression. This inhibition of MMP-1 in Bcl-3–repressed cells is not due to enhanced apoptosis. These studies demonstrate a novel role for Bcl-3 in inflammatory responses and implicate this gene in MMP-1–dependent cartilage degradation in arthritis.
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In this study, we showed that Bcl-3 is an IL-1β–inducible gene in chondrocytes and that this IκB-related factor can contribute to IL-1β–induced expression of MMP-1 in chondrocytes and synovial cells. Bcl-3 activates MMP-1 at the transcriptional level, in part, through cooperation with NF-κB1. These findings present a novel role for Bcl-3 as a mediator of IL-1β–dependent tissue remodeling, such as that occurring in OA and RA.
Studies from several laboratories, including our own, have shown that the NF-κB pathway is a critical component of IL-1β–induced MMP-1 gene expression in connective tissue cells (12–15). Specifically, we have identified an NF-κB1–binding site in the distal MMP-1 promoter (14). Others have shown that NF-κB1 homodimers physically interact with Bcl-3 and require Bcl-3 in order to be transcriptionally active (21, 22, 28, 29). Importantly, IL-1β induction of MMP-1 gene expression in stromal cells requires de novo gene expression (30). Thus, Bcl-3 may be an early IL-1β–responsive gene that is required for maximal MMP-1 transcription. This model is supported by our findings, that exogenous expression of Bcl-3 enhances MMP-1 transcription, and that inhibition of Bcl-3 expression reduces IL-1β–induced MMP-1 gene activation.
In contrast to reports from other laboratories (27, 31, 32), we did not find that inhibition of Bcl-3 expression caused increased programmed cell death in SW-1353 cells. This may be due to the fact that we were able to see substantial MMP-1 inhibition without complete ablation of Bcl-3 gene expression (Figure 3). However, our data do not preclude the possibility that apoptosis would occur in SW-1353 cells completely devoid of Bcl-3. Perhaps the concentration of Bcl-3 required to mediate IL-1–induced MMP-1 gene expression is higher than that required to maintain cell viability.
Our data demonstrate that Bcl-3 can enhance transactivation of the MMP-1 promoter by NF-κB1. The cooperative induction of transcription was somewhat modest, suggesting that additional factors are required to activate the promoter. Indeed, we have reported previously that NF-κB1 transactivation of MMP-1 requires an intact activator protein 1 (AP-1) site (14). Therefore, the interaction of Bcl-3 and NF-κB1 may only be fully functional in the context of other IL-1–induced transcription factors, such as AP-1 family members.
The interaction between Bcl-3 and NF-κB1 may explain how IL-1β stimulation of the NF-κB pathway can have specific effects in different cell types. While the NF-κB1/RelA heterodimer complex is essential for activation of cytokine gene expression in immune cells, NF-κB1 homodimers associated with Bcl-3 may be more critical for the activation of matrix remodeling genes in SF and chondrocytes. Indeed, investigators in our group have reported previously that IL-1β stimulation of SW-1353 cells results in a profound induction of NF-κB1 gene expression, while RelA was expressed at lower levels (26). Concurrent expression of Bcl-3 and NF-κB1 in connective tissue cells would elevate the levels of NF-κB1–Bcl-3 complexes, and this might skew transcription toward genes involved in matrix degradation, such as MMP-1. In future studies, it will be important to determine whether other genes known to contribute to OA and RA pathophysiology are targets of Bcl-3.