Grzegorz Gorski

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.

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.

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.

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.