Regulation of Matrix Metalloproteinases and Their Inhibitors by DNA Methylation in Human Gestational Tissues

Abstract

An appropriate transcriptional profile in the placenta and fetal membranes is a necessary requirement for the successful completion of pregnancy since any aberrant variations may lead to the inappropriate timing of birth. Epigenetic regulation through reversible modification of chromatin has emerged as a fundamental mechanism for the control of gene expression in a wide range of biological systems and can be modified by pharmacological intervention, thus providing novel therapeutic avenues. Matrix metalloproteinases (MMPs) and specific endogenous tissue inhibitors of metalloproteinases (TIMPs) are key mediators of fetal membrane rupture during physiological and pathological labour. Membrane type MMPs (MT-MMPs) activate secreted MMPs in addition to degrading the extracellular matrix (ECM); however, their role in fetal membrane rupture is unclear. We have characterised MTMMPs by quantitative real-time PCR (qRT-PCR) and immunohistochemistry in term placenta and fetal membranes and discovered tissue-specific expression patterns in relation to the process of labour. To determine if MMPs and TIMPs are regulated by DNA methylation in gestational tissues we employed an in vitro model in which gestational tissue explants were treated with the demethylating agent 5-aza-2'-deoxycytidine (AZA) and/or lipopolysaccharide (LPS). qRT-PCR and western blotting revealed synergistic up-regulation of MT1-MMP and TIMP-1, by combined AZA+LPS treatments. Secreted MMP-2 and -9 activities were evaluated by gelatin zymography and found to be increased in placenta and amnion at specific time points and treatments, correlating with mRNA expression levels. Interrogation of the TIMP-1 and MT1-MMP promoters using Sequenom™ EpiTyper® MassARRAY revealed general hypomethylation of the two genes, with sex-specific differential methylation observed in the TIMP-1 promoter, in part explained by x-linked methylation. These studies reveal that although selected MMPs and TIMPs may not be directly regulated by DNA methylation in the placenta and fetal membranes, the synergistic response to AZA+LPS suggest that DNA methylation may be involved in the regulation of these genes within an infectious setting, through alterations in chromatin structure, direct interactions with upstream mediators, or through the regulation of specific targets of LPS signalling. Collectively, these observations support a mechanistic link between inflammation and epigenetic alterations in the placenta and fetal membranes.