In periodontitis, as well as rheumatoid arthritis and several other chronic inflammatory conditions, levels of inflammatory cytokine IL-1 are high and correlate with disease severity, while levels of anti-inflammatory IL-4 are low or undetectable. Furthermore, decreasing levels of IL-4 are correlated with increasing severity. IL-1 contributes to loss of attachment and alveolar bone destruction by increasing expression of matrix metalloproteinases. MMP-3 has broad substrate specificity and can activate other pro-MMP. It is found in increased levels in diseased sites, correlated with severity and progression. IL-4 suppresses the IL-1 induced expression of MMP-3 in human gingival fibroblasts isolated from patients with periodontitis. The SIRE site (stromelysin IL-1 responsive element) was identified as a repressor element involved in suppressing the IL-1 induction of MMP-3. It is also the site of a common 5T/6T polymorphism that affects transcription, with the 5T allele associated with higher levels of expression. Genotype at this site has also been linked to tissue levels of MMP-3 and to susceptibility or severity of a number of diseases, including periodontitis. In cardiovascular disease (a condition associated with periodontitis), homozygosity for the higher- expressing 5T allele is associated with myocardial infarction and aneurysm, while the lower- expressing 6T allele is associated with atherosclerosis. It is therefore clear that regulation of this gene must be tightly controlled to maintain correct tissue homeostasis, and that understanding these control mechanisms is important for a variety of pathologies. Transcription factors ZBP-89 and NFkB bind to the SIRE site, and evidence suggests that ZBP-89 activates transcription, especially from the 5T site, while NF-?B represses from both 5T and 6T The specific aims of this proposal are to: 1) Study the roles of transcription factors interacting with the MMP-3 promoter, focusing in particular on the polymorphic SIRE site. The chromatin immunoprecipitation (ChIP) assay will be used to determine which transcription factor(s) bind in vivo at the 5T and 6T sites in several cell types and conditions, and interactions with co-factors will also be compared. The effects of knock-down of each of the factors by small-interfering RNA (siRNA) and/or by using chemical inhibitors will also be tested. 2) Determine the mechanism of suppression of MMP-3 expression by IL-4, concentrating in particular on the roles of STAT6, p300/CBP and/or protein phosphatase 2A (PP2A). These studies will utilize transient transfection, siRNA and real-time PCR. In doing so, we hope to gain information about complex gene regulatory mechanisms involved in MMP-3 regulation, but also continue to contribute to the research environment at PCOM and to the training of its students in research in general, and molecular biology in particular. Periodontitis is the most common cause of adult tooth loss in the U.S., and contributes to the development of several other diseases. MMP-3 expression is associated with destruction of support structures in periodontitis, and also plays a role in other diseases with chronic inflammation. A common promoter polymorphism in the MMP-3 promoter has been shown to affect transcription and to have a number of disease associations (including with periodontitis), suggesting that either too much or too little MMP-3 can have pathological consequences and that expression of this gene must be tightly controlled in order to maintain proper tissue homeostasis. Our increased understanding of these transcriptional control mechanisms, and how they sometimes fail, will be important to improve our understanding of a variety of pathologies. ? ? ?
