Ruth C. Borghaei

INTERLEUKIN-4 SUPPRESSION OF INTERLEUKIN-1–INDUCED TRANSCRIPTION OF COLLAGENASE (MMP-1) AND STROMELYSIN 1 (MMP-3) IN HUMAN SYNOVIAL FIBROBLASTS

OBJECTIVE To determine the effects of interleukin-4 (IL-4) on IL-1 induction of collagenase (matrix metalloproteinase 1 [MMP-1]) and stromelysin-1 (MMP-3) in human synovial fibroblasts. METHODS Northern blot analysis was performed to determine the effects of IL-4 on IL-1 induction of MMP messenger RNA (mRNA). MMP protein levels were determined by enzyme-linked immunosorbent assay, and prostaglandin E2 (PGE2) levels were measured by enzyme immunoassay. Run-on transcription assays and transient transfection experiments were performed to determine whether the effects of IL-4 occur at the level of transcription. Activator protein 1 (AP-1) binding was assessed by electrophoretic mobility shift assay. RESULTS Northern blot analysis revealed that coincubation of synovial fibroblasts with IL-1 and IL-4 resulted in a significant decrease in both collagenase and stromelysin mRNA levels compared with IL-1 alone, with a concomitant decrease in MMP protein levels. This inhibition is dose dependent, with an IC50 (50% inhibition concentration) for both MMPs of approximately 0.3 ng of IL-4 per ml, and is at least somewhat selective, since IL-1 induction of c-fos mRNA is not affected. Nuclear run-on experiments and transient transfection studies demonstrated that the suppression of IL-1-induced collagenase and stromelysin expression by IL-4 occurs at least in part at the transcriptional level, and that binding of transcription factor AP-1 is not affected. Although IL-1-induced levels of PGE2 are reduced by IL-4, exogenous addition of PGE2 does not abrogate the inhibitory effects of IL-4 on MMP expression. CONCLUSION IL-4 inhibits IL-1 induction of both collagenase and stromelysin, as well as PGE2 production, in human synovial fibroblasts. The inhibition occurs at least in part at the level of transcription, does not affect binding of transcription factor AP-1, and appears to involve a mechanism that is independent of the ability of IL-4 to inhibit production of PGE2.

NF-κB AND ZBP-89 REGULATE MMP-3 EXPRESSION VIA A POLYMORPHIC SITE IN THE PROMOTER

A 5T/6T polymorphism in the human MMP-3 promoter affects gene expression and impacts the risk and/or severity of various pathological conditions. Chromatin immunoprecipitation (ChIP) in human fibroblasts homozygous for the 6T site demonstrate that it is bound by NF-kappaB and ZBP-89 transcription factors in its native chromatin. ChIP in COS-1 cells transfected with plasmids containing the 5T and 6T sites in the context of 2kb of the MMP-3 promoter showed that NF-kappaB p50 binds preferentially to the 6T site, while more ZBP-89 binding is detected to the 5T site. Over-expressed ZBP-89 increased transcription from the 5T promoter but not from the 6T, while NF-kappaB decreased transcription from both promoters, even in the presence of excess ZBP-89. A model is suggested in which the physiological impact of the polymorphism is dependent on the relative levels and activities of these competing factors in various cell types and conditions.

IL-4 inhibition of IL-1 induced Matrix Metalloproteinase-3 (MMP-3) expression in human fibroblasts involves decreased AP-1 activation via negative crosstalk involving of Jun N-terminal Kinase (JNK)

Matrix metalloproteinase-3 (MMP-3) over-expression is associated with tissue destruction in the context of chronic inflammation. Previous studies showed that IL-4 inhibits induction of MMP-3 by IL-1β, and suggested that AP-1 might be involved. Here we show that IL-1 induced binding of transcription factor AP-1 to the MMP-3 promoter consists primarily of c-Jun, JunB, and c-Fos and that binding of c-Jun and c-Fos is inhibited by the combination of cytokines while binding of Jun B is not. Mutation of the AP-1 site in the MMP-3 promoter decreased the ability of IL-4 to inhibit its transcription in transfected MG-63 cells. Western blotting showed that both cytokines activate Jun N-terminal kinase (JNK), but with somewhat different kinetics, and that activation of JNK by both cytokines individually is inhibited by the combination. These results indicate that IL-4 inhibition of MMP-3 expression is associated with reduction of IL-1 induced binding of active forms of the AP-1 dimer, while less active JunB-containing dimers remain, and suggest that these changes are associated with decreased activation of JNK.

Evidence that suppression of IL-1 induced collagenase mRNA expression by dihomo-γ-linolenic acid (DGLA) involves inhibition of NFκB binding

Joint destruction in rheumatoid arthritis is thought to be mediated by increased production of metalloproteinases (e.g. collagenase, stromelysin) by synovial fibroblasts. Evidence suggests that the cytokine interleukin-1 (IL-1) plays an important role in stimulating transcription of these genes. However, the mechanisms involved in the IL-1 induction of MMP have not yet been fully elucidated. Studies have suggested that diets supplemented with g-linolenic acid (GLA; 18:3q-6) or its botanical lipid precursors, may be a safe and effective anti-inflammatory treatment for rheumatoid arthritis, reducing symptoms such as joint pain and morning stiffness in humans [1, 2] and tissue injury in animal models [3, 4]. We have shown [5] that incorporation of dihomo-g-linolenic acid (DGLA, a precursor of GLA) into the membranes of synovial fibroblasts suppresses the IL-1 induction of collagenase mRNA, but has no effect on induction of stromelysin. Here we present evidence that this discoordinate regulation of MMP genes by DGLA may be mediated by inhibiting binding of transcription factor NF kB to the collagenase promoter.

Interleukin-4 inhibition of interleukin-1-induced expression of matrix metalloproteinase-3 (MMP-3) is independent of lipoxygenase and PPARγ activation in human gingival fibroblasts

BackgroundInterleukin 4 (IL-4) has been shown to suppress interleukin-1 (IL-1) induced expression of matrix metalloproteinase-3 (MMP-3) in human synovial and gingival fibroblasts, but the mechanism of suppression has not been determined. Activators of peroxisome proliferator-activated receptor-γ (PPARγ) have been shown to inhibit cytokine induced expression of MMPs in other cell types, and IL-4 has been shown to activate PPARγ by stimulating production of ligands through the lipoxygenase pathway. It has been suggested that PPARγ may inhibit expression of MMPs by competing with transcription factor AP-1 for binding to a putative composite binding element in the promoters. The objective of this study was to determine whether the suppressive effects of IL-4 on the IL-1 induced expression of MMP-3 involve activation of lipoxygenase and/or PPARγ.ResultsWestern blotting revealed the presence of PPARγ in nuclear extract of HGF. IL-1 induced binding of nuclear extract to the putative composite PPRE/AP-1 site was diminished in the presence of pioglitazone, but there was no evidence of any change in the composition of the retarded complexes, and no evidence of PPARγ binding to this site. Nordihydroguaiaretic acid (NDGA), a non-selective lipoxygenase inhibitor, and MK886, a specific inhibitor of 5-lipoxygenase, induced MMP-3 expression synergistically with IL-1. However IL-4 was still able to inhibit MMP-3 expression in the presence of NDGA or MK886 and IL-1. Activation of PPARγ with pioglitazone not only failed to inhibit IL-1 induced expression of MMP-3 mRNA, but rather super-induced MMP-3 in the presence of IL-1. PPARγ antagonist GW9662 failed to abolish the suppressive effects of IL-4. Another PPARγ activator, 15-deoxy-Delta12,14prostaglandin J2 (15dPGJ2), also super-induced MMP-3 mRNA, and this was due at least in part to increased transcription.ConclusionIL-4 suppression of IL-1-induced MMP-3 expression in HGF is independent of lipoxygenase activity and activation of PPARγ. Super-induction of MMP-3 by pioglitazone may have important implications for patients using pioglitazone to treat type II diabetes in the presence of chronic inflammation.

Expression of transcription factor zinc-binding protein-89 (ZBP-89) is inhibited by inflammatory cytokines.

Zinc-binding protein-89 (ZBP-89; ZNF148, BERF-1, BFCOL-1) is a zinc-finger transcription factor of the Kruppel family. It has been shown to regulate the expression of a number of genes, acting as either an activator or repressor of gene expression, depending on the context. It is over-expressed in several cancers, but has been shown to be involved in apoptosis and to have a negative influence on cell growth in part by interactions with p53. Previously, ZBP-89 was shown to activate transcription of the matrix metalloproteinase-3 (MMP-3) gene by binding to a polymorphic promoter element in competition with nuclear factor kappaB (NF-kappaB). NF-kappaB is known to be a key regulator of the inflammatory response, but relatively little is known about regulation of ZBP-89. In order to ascertain whether ZBP-89 is regulated during inflammation, we designed experiments to determine whether and to what extent ZBP-89 levels are affected by inflammatory cytokines. Here we show that ZBP-89 mRNA and protein expression are significantly inhibited in human fibroblasts by the inflammatory cytokine interleukin-1beta. Since any change in the levels of ZBP-89 would presumably impact the regulation of MMP-3 and other ZBP-89 target genes, these results provide important insight into mechanisms involved in fine-tuning the immune response.

Zinc-binding protein-89 (ZBP-89) cooperates with NF-κB to regulate expression of matrix metalloproteinases (MMPs) in response to inflammatory cytokines

Matrix metalloproteinases (MMPs) have both protective and pathological roles in inflammation, and transcriptional mechanisms are important in regulating physiological levels to maintain health. Zinc-binding protein-89 (ZBP-89) is a transcription factor with roles in regulating vital cellular processes, acting through complex interactions with other proteins to ensure appropriate expression of tightly regulated genes. ZBP-89 binds the MMP-3 promoter at a polymorphic (5A/6A) site along with NF-κB. This polymorphism affects MMP-3 protein levels in tissues. In disease association studies, both over- and under-expression has negative consequences to health, and this promoter element is important in maintaining balanced expression. There is evidence that effects of the polymorphism vary under different conditions, but the role of ZBP-89 in these differences is not known. ZBP-89 was stably knocked-down in MG-63 osteosarcoma cells in order to study its role in regulation of MMP-3 expression in response to cytokines, and evaluate the functionality of a putative binding site in the MMP-1 promoter. Results show ZBP-89 is needed for maximal induction of both genes by IL-1β and TNFα. Binding of both ZBP-89 and NF-κB to both promoters was decreased in the knock-down cells under basal and TNF-induced conditions, and protein interactions between ZBP-89 and NF-κB were suggested. These data provide the first evidence of a role for ZBP-89 in regulation of MMP-1 expression, and suggest the possibility of a larger role for ZBP-89 in inflammation through interactions with NF-κB.

DGLA discoordinately suppresses IL-1 induced metalloproteinase mRNA levels in human synovial fibroblasts

Joint destruction in rheumatoid arthritis (RA) is thought to be mediated by increased synovial fibroblast production of metalloproteinases (MMP e.g., collagenase, stromelysin) in response to cytokines such as interleukin-1 (IL-1). Studies [1, 2] in animal models and human trials have suggested that diets supplemented with omega-6 fatty acids ( glinolenic acid (GLA: 18:3), dihomogammalinolenic acid (DGLA: 20:3)) may be safe and effective anti-inflammatory treatments for rheumatoid arthritis (RA). In the present study, we examined whether DGLA affects the IL-1 induction of MMP mRNA expression in human synovial fibroblasts in culture.

Identification of IL-1 regulated genes in human synovial and gingival fibroblasts by differential display

Interleukin-1 (IL-1) is a pro-inflammatory, multifunctional cytokine, which affects nearly every cell type, often acting in concert with other cytokines or small mediator molecules. In rheumatoid arthritis, IL-1 and tumor necrosis factor-alpha (TNF-a) not only promote the inflammatory response but also induce cartilage degradation. It has also been shown that fibroblasts derived from inflammatory synovitis are one of the major sources of damaging mediators in this disorder [1]. Elevated levels of cytokines such as IL-1, IL-2, IL-6, TNF-a, TGFb have been detected in these cells as well as in the synovial fluid. Abnormal gene programs are activated, leading to enhanced inflammatory response and cell proliferation [1]. Differential display analysis of mRNA was utilized to identify genes that are regulated by IL-1 in human synovial fibroblasts.