MMP13 is a primary catabolic factor involved in cartilage degradation through its ability to cleave type II collagen. Transcriptionally, MMP13 is regulated by 2 main elements; proximal promoter and distal enhancers. The aim of the study is to identify transcriptional elements that regulate the MMP13 gene in order to control the substantial rise in MMP13 expression observed in Osteoarthritis.
Possible enhancers have been determined by using the Encyclopaedia of DNA Elements (ENCODE), based on histone modifications (Limb H3K4ME1 and Limb H3k27AC), Evolutionarily conserved sequences, fibroblast and muscle peaks, ChIP peaks for RUNX2, and also based on published data on vitamin D elements. Each possible enhancer sequence has been ligated to the silent HSP68 proximal promoter in a vector that expresses the LacZ gene. Each of these constructs has been tested in transgenic embryos at E15.5 days.
We have identified several active enhancers in distal and intronic sequences. Expressions in skeletal elements were detected in the 5th Intron, proximal promoter, and the distal enhancers at -19.4, and -21.5kb). In addition, expression was also seen in other cell types such as developing skin, tendon, and fibroblasts in other tissues. However, the sequence overlapping the highest peak of RunX2 at -29kb did not show any significant expression.
Our results showed that the MMP13 is regulated at the level of transcription in at least three different regions, some of which coincide with RunX2 peak activity. The main distal enhancer is around -19 to22kb where expression goes beyond skeletal elements and includes other cell types. Intronic 5’ sequence reporter to respond to1l-1also show exclusively skeletal expression.