217 MECHANISMS OF ACTION OF ESE1, A NOVEL TRANSCRIPTIONAL REGULATOR OF CARTILAGE REMODELING, IN MMP-13 REGULATION

Purpose: Matrix metalloproteinase (MMP)-13 plays a major role in cartilage degradation. MMP-13 expression and activity are up-regulated in osteoarthritis (OA) cartilage as compared to non-OA cartilage. We previously reported that ELF3/ESE1 levels are higher in OA cartilage and that ESE-1 transactivates the MMP13 promoter via a proximal highly conserved ETS binding site. The aim of this study was to better define the mechanism/s and signalling pathways that modulate the ESE-1-driven MMP13 promoter activation in chondrocytes. Methods: We investigated the contribution of ESE-1 to MMP-13 expression by siRNA knock down (KD) transfection experiments in human primary chondrocytes and real time PCR analysis of MMP-13 expression induced by IL-1β. The response of the MMP13 promoter to ESE-1 was analyzed in luciferase reporter assays, after co-transfection of immortalized chondrocytes with human MMP13 promoter constructs and expression vectors encoding ESE-1, p38, JNK, ERK-1, MKK-6, MKK-7, MEK-1 and MKP-1. ESE-1 binding to the MMP13 promoter was analyzed by chromatin immunoprecipitation (ChIP) assays. IL-1β-induced ESE-1 nuclear translocation was addressed by Western blotting analysis of cytoplasmic and nuclear fractions of human immortalized chondrocytes. Results: Real time PCR analysis revealed a significant reduction of IL1β-induced MMP-13 mRNA levels associated with ESE-1 KD in human primary chondrocytes. Luciferase reporter assays showed that MEK1/ERK1 overexpression enhanced ESE1-driven activities of the -1528/+27 and 267/+27 MMP13 promoter constructs, whereas MKK6/p38 or MKK7/JNK overexpression did not affect ESE-1 transactivation of MMP-13. Overexpression of MKP-1 reduced both the ESE-1 activation of MMP13 and also the MEK1/ERK1 enhancement of ESE-1-driven MMP13 activation. Accordingly, pre-treatment with the MEK1/2 inhibitor, U0126, decreased the MMP13 promoter activity induced by ESE-1 overexpression. Finally, Western blotting analysis revealed that IL-1β stimulation induced ESE-1 nuclear translocation, which correlated with increased ESE-1 binding to the endogenous MMP13 promoter, as addressed by ChIP assays. Conclusions: The role of MMP-13 as a central factor for OA progression has been highlighted by recent studies in Mmp13 knockout mice. We previously reported that ESE-1 is a key factor in controlling MMP13 transcription. ESE-1 belongs to the ETS family of transcription factors, which are classic MAPK effectors in different tissues and cell types. Here, we show that ESE-1 participates in the IL-1β-induced MMP-13 expression and that IL-1β induces ESE-1 nuclear translocation and binding to the MMP13 promoter. In addition, we show that the ESE1-driven transactivation of MMP13 is enhanced by MEK1/ERK1. In chondrocytes, ERK1/2 phosphorylation is increased in vitro in response to IL-1β and in vivo in OA cartilage, and MEK/ERK activation has been reported to participate in the cytokine-induced MMP-13 expression. Therefore, ESE-1 could be one of the effectors of the MEK/ERK pathway in controlling MMP-13 expression in chondrocytes, and the relative contributions of the MEK/ERK/ESE1 axis in OA with regard to MMP-13 expression and activity will merit further investigation.