Induction of increased cAMP levels in articular chondrocytes blocks matrix metalloproteinase-mediated cartilage degradation, but not aggrecanase-mediated cartilage degradation.
OBJECTIVE
Calcitonin has been suggested to have chondroprotective effects. One signaling pathway of calcitonin is via the second messenger cAMP. We undertook this study to investigate whether increased cAMP levels in chondrocytes would be chondroprotective.


METHODS
Cartilage degradation was induced in bovine articular cartilage explants by 10 ng/ml oncostatin M (OSM) and 20 ng/ml tumor necrosis factor (TNF). In these cultures, cAMP levels were augmented by treatment with either forskolin (4, 16, or 64 microM) or 3-isobutyl-1-methyl xanthine (IBMX; 4, 16, or 64 microM). Cartilage degradation was assessed by 1) quantification of C-terminal crosslinking telopeptide of type II collagen fragments (CTX-II), 2) matrix metalloproteinase (MMP)-mediated aggrecan degradation by (342)FFGV- G2 assay, 3) aggrecanase-mediated degradation by (374)ARGS-G2 assay, 4) release of sulfated glycosaminoglycans (sGAG) into culture medium, 5) immunohistochemistry with a monoclonal antibody recognizing the CTX-II epitope, and 6) toluidine blue staining of proteoglycans. MMP expression and activity were assessed by gelatin zymography.


RESULTS
OSM and TNF induced an 8,000% increase in CTX-II compared with control (P < 0.001). Both forskolin and IBMX dose-dependently inhibited release of CTX-II (P < 0.001). OSM and TNF induced a 6-fold increase in (342)FFGV-G2, which was abrogated by forskolin and IBMX (by >80%). OSM and TNF stimulated MMP expression as visualized by zymography, and MMP expression was dose-dependently inhibited by forskolin and IBMX. The highest concentration of IBMX lowered cytokine-induced release of sGAG by 72%.


CONCLUSIONS
Levels of cAMP in chondrocytes play a key role in controlling catabolic activity. Increased cAMP levels in chondrocytes inhibited MMP expression and activity and consequently strongly inhibited cartilage degradation. Specific cAMP modulators in chondrocytes may be potential treatments for cartilage degenerative diseases.
Karsdal
Morten Asser
MA
Nordic Bioscience Diagnostics, Herlev, Denmark. mk@nordicbioscience.com
Sumer
Eren Ufuk
EU
Wulf
Helle
H
Madsen
Suzi H
SH
Christiansen
Claus
C
Fosang
Amanda J
AJ
Sondergaard
Bodil-Cecilie
BC
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D002116:Calcitonin / Q000502:physiology; D002358:Cartilage, Articular / Q000378:metabolism* / Q000473:pathology; D002417:Cattle; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006025:Glycosaminoglycans / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000235:genetics / Q000378:metabolism*; D053683:Oncostatin M / Q000494:pharmacology; D010003:Osteoarthritis / Q000378:metabolism* / Q000473:pathology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D006025:Glycosaminoglycans; D010726:Phosphodiesterase Inhibitors; D014409:Tumor Necrosis Factor-alpha; D053683:Oncostatin M; D005576:Colforsin; C013786:A73025; D002116:Calcitonin; D000242:Cyclic AMP; D010450:Endopeptidases; D020782:Matrix Metalloproteinases; C083641:aggrecanase; D015056:1-Methyl-3-isobutylxanthine