Development of treatment for Osteoarthritis by blocking metalloproteinases

We aim to understand how MMP13 is transcriptionally regulated and whether a newly identified enhancer is involved in normal and osteoarthritic cartilage. This is important because in osteoarthritis, collagen degradation is mediated almost exclusively by matrix metalloproteinases (MMPs) and their collagenolytic activity are considered the rate-limiting in irreversible cartilage destruction. MMP13 (collagenase 3) plays a vital role in the pathogenesis of osteoarthritis (OA) because MMP13 preferentially cleaves type II collagen, the main component of cartilage, and both MMP13 levels and enzymatic activity are enhanced in OA.

Transcriptional regulation of a gene is normally governed by two elements in proximal promoter where initiation of transcription occurs, and by distal enhancers that potentiate or direct specificity of a gene. Currently, very little is known about the expression of the MMP13 proximal promoter up to 3kb away from transcription start site. Furthermore, we have recently identified a novel enhancer located upstream in the MMP13 gene, which is only expressed in chondrocytes. By unraveling the pathway that drives MMP13 expression in human and mouse, we hope to limit the increase of MMP13 expression and possibly proteolytic activity in a cell specific manner to prevent the critical connective tissue destruction seen in OA.

The regulation of a gene such as MMP13 is governed by two types of transcriptional regulatory DNA elements: promoters (core and proximal), and distal regulatory elements such as enhancers. Thus far, studies into the transcriptional regulation in the MMP13 gene have concentrated on the proximal promoter, up to 3kb away from transcription start site, where the MMP13 promoter contains multiple factor binding sites, such as a TATA box, and those for AP-1, PEA-3, and OSE-2 21 . The AP-1 members such as c-Jun/c-Fos have been implicated in the activation of MMP13 in articular chondrocyte and a Runx-2 binding site in the MMP13 proximal promoter cooperates with the AP-1 site to mediate MMP13 transcription.

Enhancers are densely clustered aggregations of transcription factor binding motifs, and activate transcription independent of location, distance or orientation. The regulation of spatial and temporal patterns of gene transcription during development is primarily accomplished by enhancers, although those located close to core promoters are often referred to as proximal promoters . A single gene can be regulated by multiple different enhancers, each responsible for a particular aspect of expression. Enhancer regions are frequently marked by cell-type specific histone modification patterns, particularly enrichment of mono-methylation of histone H3 lysine 4 (H3K4me1) and acetylation of histone H3 lysine 27 (H3K27Ac). In addition, the majority of enhancers contain evolutionarily conserved sequences, although the depth of conservation can vary. Enhancers can be found in intronic sequences, 5’ or 3’ to the core promoter, and although they have been found over 1MB from the gene they control, recent large screens suggest that the vast majority of enhancers are located within 100kb of a core promoter, with most located within 10kb of the core promoter.

Our hypothesis is that by identifying the mechanisms of transcriptional control of MMP13 in chondrocytes we will identify specific pathways and methods that can be used to block MMP13 expression in a cartilage specific manner.

Our specific aims are:
i. To define the enhancer expression in adult mice and in response to induction of OA.
ii. To characterise the transcription factors and the pathway that leads to expression of MMP13.
iii. To establish whether exogenous cytokine mediated MMP13 expression depends on this novel

The student will learn a number of techniques during the course of this PhD ranging from cell culture to transfection and molecular biology to generate vectors to express specifically in chondrocytes. The major task will be identifying transcription factors by EMSA ChIP seq etc. The student will have a chance to engage in in vivo models of osteoarthritis and test the effect of blocking MMPs in such a model.

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