MAPKs are essential upstream signaling pathways in proteolytic cartilage degradation--divergence in pathways leading to aggrecanase and MMP-mediated articular cartilage degradation.
OBJECTIVE
Matrix metalloproteinases (MMPs) and aggrecanases are essential players in cartilage degradation. However, the signaling pathways that results in MMP and/or aggrecanase synthesis and activation are not well understood. We investigated the molecular events leading to MMP- and aggrecanase-mediated cartilage degradation.


METHODS
Cartilage degradation was induced in bovine articular cartilage explants by oncostatin M (OSM) and tumor necrosis factor (TNF), in the presence or absence of specific inhibitors of the mitogen-activated protein kinases (MAPKs) P38, P44/42 and Src family. Toxicity was followed by the AlamarBlue colorimetric assay. MMP-activity was assessed using a fluorescent substrate assay and MMP-9 and -2 activities by gelatinase zymography. MMP-mediated collagen type II degradation and MMP as well as aggrecanase-mediated aggrecan degradation was investigated with specific ELISA and hydroxyproline release by standard methods. The findings were verified by immunohistochemistry and histology.


RESULTS
Stimulation of cartilage degradation by OSM+TNF resulted in 100-fold induction of CTX-II release (P<0.01). This was dose-dependently inhibited by MAPK P38 inhibitors and by the MAPK P44/42 inhibitors. MMP-activity and expression was significantly decreased, as evaluated by cleavage of fluorescence MMP-substrate and zymography. Immunohistochemistry confirmed these findings. Interestingly, only the P44/42 inhibitors abrogated aggrecanase-mediated aggrecan degradation.


CONCLUSIONS
We found that inhibition of MAPK P38, P44/42 and Src family abrogated proteolytic cartilage degradation by blocking MMP synthesis and activity. However, only MAPK P44/42 was essential for aggrecanase-mediated aggrecan degradation. These data suggest that various aspects of cartilage degradation can be targeted independently by inhibiting specific upstream signaling pathway.
Sondergaard
B-C
BC
Nordic Bioscience A/S, Herlev, Denmark. bcs@nordicbioscience.com
Schultz
N
N
Madsen
S H
SH
Bay-Jensen
A-C
AC
Kassem
M
M
Karsdal
M A
MA
D000818:Animals; D002358:Cartilage, Articular / Q000378:metabolism* / Q000473:pathology; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D010450:Endopeptidases / Q000378:metabolism*; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D010003:Osteoarthritis / Q000378:metabolism* / Q000473:pathology; D015398:Signal Transduction / Q000187:drug effects*; D013223:Statistics as Topic
D010450:Endopeptidases; D020782:Matrix Metalloproteinases; C083641:aggrecanase