Hsp90{beta} and p130(cas): 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 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.


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 IL1beta 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 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.


RESULTS
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.


CONCLUSIONS
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.
Fan
Z
Z
University of Montreal Hospital Centre, Notre-Dame Hospital, Quebec, Canada.
Tardif
G
G
Hum
D
D
Duval
N
N
Pelletier
J-P
JP
Martel-Pelletier
J
J
D000368:Aged; D001483:Base Sequence; D002358:Cartilage, Articular / Q000378:metabolism*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism*; D050719:Crk-Associated Substrate Protein / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005786:Gene Expression Regulation; D018841:HSP90 Heat-Shock Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D053583:Interleukin-1beta / Q000494:pharmacology; D020781:Matrix Metalloproteinase 1 / Q000235:genetics / Q000378:metabolism; D053509:Matrix Metalloproteinase 13 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008875:Middle Aged; D008969:Molecular Sequence Data; D020370:Osteoarthritis, Knee / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D018709:Statistics, Nonparametric; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods
C493841:BCAR1 protein, human; D050719:Crk-Associated Substrate Protein; D017931:DNA Primers; D018841:HSP90 Heat-Shock Proteins; C493165:HSP90AB1 protein, human; C508585:IL1B protein, human; D053583:Interleukin-1beta; D034741:RNA, Small Interfering; D015534:Trans-Activators; C498621:ZNF384 protein, human; D053509:Matrix Metalloproteinase 13; D020781:Matrix Metalloproteinase 1