Cyclin-dependent kinase 9 inhibition protects cartilage from the catabolic effects of proinflammatory cytokines.
OBJECTIVE
Cyclin-dependent kinase 9 (CDK-9) controls the activation of primary inflammatory response genes. The purpose of this study was to determine whether CDK-9 inhibition protects cartilage from the catabolic effects of proinflammatory cytokines.


METHODS
Human chondrocytes were challenged with different proinflammatory stimuli (interleukin-1β [IL-1β], lipopolysaccharides, and tumor necrosis factor α) in the presence or absence of either the CDK-9 inhibitor flavopiridol or small interfering RNA (siRNA). The expression of messenger RNA (mRNA) for inflammatory mediator genes, catabolic genes, and anabolic genes were determined by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. Cartilage explants were incubated for 6 days with IL-1β in the presence or absence of flavopiridol. Cartilage matrix degradation was assessed by the release of glycosaminoglycan (GAG) and cleaved type II collagen (COL2A) peptides.


RESULTS
CDK-9 inhibition by flavopiridol or knockdown by siRNA effectively suppressed the induction of mRNA for inducible nitric oxide synthase by all 3 proinflammatory stimuli. Results from NF-κB-targeted PCR array analysis showed that flavopiridol suppressed IL-1β induction of a broad range of inflammatory mediator genes (59 of 67 tested). CDK-9 inhibition also suppressed the induction of catabolic genes (matrix metalloproteinase 1 [MMP-1], MMP-3, MMP-9, MMP-13, ADAMTS-4, and ADAMTS-5), but did not affect the basal expression of anabolic genes (COL2A, aggrecan, and cartilage oligomeric matrix protein) and housekeeping genes. Flavopiridol had no apparent short-term cytotoxicity, as assessed by G6PDH activity. Finally, in IL-1β-treated cartilage explants, flavopiridol reduced the release of the matrix degradation product GAG and cleaved COL2A peptides, but did not affect long-term chondrocyte viability.


CONCLUSIONS
CDK-9 activity is required for the primary inflammatory response in chondrocytes. Flavopiridol suppresses the induction of inflammatory mediator genes and catabolic genes to protect cartilage from the deleterious effects of proinflammatory cytokines, without affecting cell viability and functions.
University of California Davis, Sacramento, California.
Jasper H N
JH
Yik
Zi'ang
Z
Hu
Ratna
R
Kumari
Blaine A
BA
Christiansen
Dominik R
DR
Haudenschild
D051722:ADAM Proteins / Q000378:metabolism; D000071121:ADAMTS4 Protein; D000071116:ADAMTS5 Protein; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002358:Cartilage, Articular / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D024043:Collagen Type II / Q000378:metabolism; D042863:Cyclin-Dependent Kinase 9 / Q000037:antagonists & inhibitors* / Q000187:drug effects; D016207:Cytokines / Q000494:pharmacology*; D005419:Flavonoids / Q000494:pharmacology*; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007249:Inflammation / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D053583:Interleukin-1beta / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D020782:Matrix Metalloproteinases / Q000378:metabolism; D008875:Middle Aged; D010880:Piperidines / Q000494:pharmacology*; D011348:Procollagen N-Endopeptidase / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
C499177:COL2A1 protein, human; D024043:Collagen Type II; D016207:Cytokines; D005419:Flavonoids; D006025:Glycosaminoglycans; D053583:Interleukin-1beta; D008070:Lipopolysaccharides; D010880:Piperidines; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; C077990:alvocidib; C467969:CDK9 protein, human; D042863:Cyclin-Dependent Kinase 9; D051722:ADAM Proteins; D000071116:ADAMTS5 Protein; C121532:ADAMTS5 protein, human; D020782:Matrix Metalloproteinases; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein; C000603918:ADAMTS4 protein, human