Background: We previously classified human OA chondrocytes into L (Low)- and H (High)-OA according to MMP-13 basal levels and IL-1β inducibility. In H-OA chondrocytes, the regulatory proteins p130cas and NMP4 acting on the MMP-13 promoter were identified.
Objective: To identify regulators of MMP-13 expression/production in human L-OA chondrocytes, determine their effect on the expression of other MMPs and the effect of IL-1β on these molecules.
Methods: The identification of the L-OA chondrocyte proteins interacting specifically with the MMP-13 promoter AGRE-site was performed by mass spectrometry. Hsp90β, p130cas and NMP4 siRNAs were transfected into L-OA chondrocytes and incubated with or without IL-1β. Gene expression was determined by real-time PCR, MMP-1 and -13 production by ELISA, and signaling pathway activation by Western blotting and ELISA.
Results: Hsp90β was identified as a protein of the L-OA/AGRE specific complex. Silencing p130cas and Hsp90β significantly increased the expression (about 4 and 2 fold, respectively) and production of MMP-13. sip130cas affected to a lesser extent MMP-1 expression (2 fold) and production. siNMP4 showed no effect. MMP-2, -3, -9 and -14 expressions were unaffected. Silencing both Hsp90β and p130cas had a significant additive effect on MMP-13, but not MMP-1 expression, the level of which was similar to sip130cas alone. IL-1β decreased p130cas and Hsp90β expression/production, indicating another pathway by which this cytokine up-regulates MMP expression. The IL-1β-triggered signaling pathways responsible for MMP up-regulation were unaffected in the silenced cells.
Conclusion: This study illustrates the complex regulation of MMP-13 by showing the inhibitory effect of the two cytoplasmic molecules, p130cas and Hsp90β, in L-OA chondrocytes.