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 1beta (IL1beta) inducibility. In H-OA chondrocytes, the regulatory proteins p130(cas) and nuclear matrix protein 4 (NMP4) acting on the MMP-13 promoter were identified.
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 IL1beta on these molecules.
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 90beta (Hsp90beta), p130(cas) and NMP4 small interfering RNAs (siRNAs) were transfected into L-OA chondrocytes and incubated with or without IL1beta. 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.
Hsp90beta was identified as a protein of the L-OA/AGRE-specific complex. Silencing p130(cas) and Hsp90beta significantly increased MMP-13 expression (about four- and twofold, respectively) and production. sip130(cas) affected to a lesser extent MMP-1 expression (twofold) and production. siNMP4 showed no effect. Expression of MMP-2, -3, -9 and -14 was unaffected. Silencing both Hsp90beta 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 sip130(cas) alone. IL1beta decreased p130(cas) and Hsp90beta expression/production, indicating another pathway by which this cytokine upregulates MMP expression. The IL1beta-triggered signalling pathways responsible for MMP upregulation were unaffected in the silenced cells.
This study illustrates the complex regulation of MMP-13 by showing the inhibitory effect of the two cytoplasmic molecules, p130(cas) and Hsp90beta, in L-OA chondrocytes.