Hsp90β and p130cas: novel regulatory factors of MMP-13 expression in human osteoarthritic chondrocytes

Background: Human osteoarthritic (OA) chondrocytes were previously classified into L (low)- and H (high)-OA according to matrix metalloproteinase-13 (MMP-13) basal levels and interleukin 1 beta (IL1 beta) inducibility. In H-OA chondrocytes, the regulatory proteins p130(cas) and nuclear matrix protein 4 (NMP4) acting on the MMP-13 promoter were identified. Objective: To identify regulators of MMP-13 expression/production in human L-OA chondrocytes, to determine their effect on the expression of other MMPs and the effect of IL1 beta on these molecules. Methods: The identification of the L-OA chondrocyte proteins interacting specifically with the AGRE site of the MMP-13 promoter was performed by mass spectrometry. Heat shock protein 90 beta (Hsp90 beta), p130(cas) and NMP4 small interfering RNAs (siRNAs) were transfected into LOA chondrocytes and incubated with or without IL1 beta. Gene expression was determined by real-time PCR, MMP-1 and MMP-13 production by ELISA, and signalling pathway activation by western blotting and ELISA. Results: Hsp90 beta was identified as a protein of the L-OA/AGRE-specific complex. Silencing p130(cas) and Hsp90 beta significantly increased MMP-13 expression (about four- and twofold, respectively) and production. sip130(cas) affected to a lesser extent MMP-13 expression (twofold) and production. siNMP4 showed no effect. Expression of MMP-2, -3, -9 and -14 was unaffected. Silencing both Hsp90 beta and p130(cas) had a significant additive effect on MMP-13, but not on MMP-1 expression, the level of which was similar to that with sip130cas alone. IL1 beta decreased p130(cas) and Hsp90 beta expression/production, indicating another pathway by which this cytokine upregulates MMP expression. The IL1 beta-triggered signalling pathways responsible for MMP upregulation 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, p130(cas) and Hsp90 beta, in L-OA chondrocytes.