Allele-Specific Regulation of Matrix Metalloproteinase-3 Gene by Transcription Factor NFκB

Veronika Souslova,Anna Motterle,Chris M Van Der Loos,Paul A Townsend,Derek A Mann,Jelena Mann,Shu Ye,Fulvio D'Acquisto

doi:10.1371/journal.pone.0009902

Abstract

BackgroundMatrix metalloproteinase-3 (MMP3) is implicated in the pathogenesis and progression of atherosclerotic lesions. Previous studies suggested that MMP3 expression is influenced by a polymorphism (known as the 5A/6A polymorphism) in the promoter of the MMP3 gene and that this polymorphism is located within a cis-element that interacts with the transcription factor NFκB. In the present study, we sought to investigate whether MMP3 and NFκB were co-localized in atherosclerotic lesions and whether NFκB had differential effects on the two alleles of the MMP3 5A/6A polymorphism.Methodology/Principal FindingsImmunohistochemical examination showed that MMP3 and both the NFκB p50 and p65 subunits were expressed abundantly in macrophages in atherosclerotic lesions and that MMP3 expression was co-localized with p50 and p65. Chromatin immunoprecipitation experiments showed interaction of p50 and p65 with the MMP3 promoter in macrophages, with greater binding to the 5A allele than to the 6A allele. Reporter gene assays in transiently transfected macrophages showed that the 5A allele had greater transcriptional activity than the 6A allele, and that this allele-specific effect was augmented when the cells were treated with the NFκB activator lipopolysaccharides or co-transfected with p50 and/or p65 expressing plasmids, but was reduced when the cells were treated with the NFκB inhibitor 6-Amino-4-(4-phenoxyphenylethylamino)-quinazoline or transfected with a dominant negative mutant of IkB kinase-β.ConclusionThese results corroborate an effect of the 5A/6A polymorphism on MMP3 transcription and indicate that NFκB has differential effects on the 5A and 6A alleles.

Highlights

Atherosclerotic plaque rupture is the most common cause of acute clinical ischemic events associated with coronary artery disease [1]
We carried out an immunohistochemical analysis to determine whether Matrix metalloproteinase-3 (MMP3) and NFkB were co-expressed in atherosclerotic lesions
The analysis showed that MMP3 and both the NFkB p50 and p65 subunits were expressed abundantly in macrophages in atherosclerotic lesions and were present in smooth muscle cells in these tissues (Figure 1)

Summary

Introduction

Atherosclerotic plaque rupture is the most common cause of acute clinical ischemic events associated with coronary artery disease [1]. There is evidence indicating that increased expression of matrix metalloproteinase-3 (MMP3) in the atherosclerotic plaque promotes plaque rupture [3,4,5]. MMP3 is abundantly expressed by macrophages in atherosclerotic plaques, in the lateral regions of the fibrous cap where fissuring is most likely to occur [3,4]. This protease is capable of degrading several major structural matrix proteins present in atherosclerotic plaques [6,7]. A study of a mouse model of atherosclerosis has shown that knocking out MMP3 results in increased amounts of matrix proteins in atherosclerotic lesions, a feature of more stable plaques [5]. We sought to investigate whether MMP3 and NFkB were colocalized in atherosclerotic lesions and whether NFkB had differential effects on the two alleles of the MMP3 5A/6A polymorphism

Methods

Results

Discussion

Conclusion