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.
Previously we showed that IL‐4 inhibits the IL‐1 induction of MMP‐3 in human fibroblasts. Since STAT6 is a transcription factor prominently involved in IL‐4 transcriptional responses, experiments were designed to determine its role in IL‐4 inhibition of MMP‐3 expression. Chemical activation of STAT6 using chromeceptin caused a super‐induction of MMP‐3 mRNA in IL‐1 stimulated fibroblasts, but IL‐4 was still able to suppress MMP‐3 expression in its presence. Chromeceptin also increased MMP‐3 transcription in transfected COS‐1 cells. Chemical inhibition of STAT6 using pifithrin‐á caused a dose‐dependent inhibition of IL‐1 induced MMP‐3 expression, which was not due to inhibition of p53, since nutlin, an activator of p53, had no effect. Interestingly, overexpression of STAT6 in transiently transfected COS‐1 cells was able to increase transcription from the MMP‐3 promoter, but only when it contained the 6T version of the 5T/6T polymorphism at −1610. Experiments are currently underway to determine whether the effects of STAT6 on MMP‐3 expression are mediated through interaction with NF‐êB binding to the polymorphic SIRE site. This work is supported by grant R15DE16277 from the NIDCR to R.C. Borghaei.
Periodontitis is the most common cause of adult tooth loss in the U.S., with an estimated 1 in 3 adults suffering from some form and 10–15% of adults developing severe forms. In addition to its direct impact, periodontitis also contributes to the development of several other diseases, including cardiovascular disease, pre‐term low birth weight, and diabetes. Although the primary function of HO‐1 is the breakdown of heme to carbon monoxide, iron and bilirubin, it has also been shown to play an important role in wound repair and the resolution of inflammation by mechanisms involving homeostatic regulation of the redox state of cells. A series of experiments has been designed to determine whether and to what extent the levels of HO‐1 mRNA and protein are regulated by inflammatory cytokines in HGF isolated from individuals with or without periodontitis. Preliminary results show that HO‐1 mRNA is expressed in HGF cultures derived from patients with periodontitis and that mRNA levels are inhibited over 60% by Interleukin‐1 at 6 hours (10 ng/ml, p < 0.001). Interestingly however, HO‐1 protein levels as measured by ELISA are not decreased by IL‐1. Experiments are currently underway to address this apparent paradox, as well as the potential role of HO‐1 in regulation of inflammatory mediators in HGF.
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