Inhibition of NF-kappaB renders human juvenile costal chondrocyte cell lines sensitive to TNF-alpha-mediated cell death.
BACKGROUND
Recently, therapeutics employing knowledge on various signaling pathways are being developed, with NF-kappaB being one of the most promising targets. NF-kappaB has been suggested to play a role not only in the induction of inflammatory mediators, but also in the protection from cell death.


OBJECTIVE
This study pursued the role of the NF-kappaB pathway in the regulation of chondrocyte death induced by tumor necrosis factor alpha (TNF-alpha) and of the pertinent target molecules involved.


METHODS
The human chondrocyte cell line C28/I2 was used for the experiment. Chondrocytes were transduced with adenovirus-encoding IkappaB (IkappaB) superrepressor which inhibits NF-kappaB activation, and treated with TNF-alpha. The proportion of cell death was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazdium bromide (MTT) assay. Activation of p38 mitogen activated protein (MAP) kinase and phosphatidylinositol 3-kinase (PI3K) by TNF-alpha was inhibited with SB202190 and Ly 294002 respectively. The expression of apoptosis related protein was analyzed with western blot assay, and the activation of c-Jun N-terminal kinase (JNK) by solid-phase kinase assay.


RESULTS
Treatment with TNF-alpha led to cell death in 23% and 50% of ad-IkappaB-SR infected chondrocytes after 24 and 72 h respectively. The expression of Bcl-XL, Bcl-2, and XIAP significantly decreased, and activation of JNK was prolonged for up to 6 h in infected cells treated with TNF-alpha. Preincubation with p38 inhibitor or PI3K inhibitor before TNF-alpha led to a significant increase in cell death in ad-IkappaB-SR transduced chondrocytes, resulting in 53% and 30% cell death after 24 h for p38 inhibitor and PI3K inhibitor respectively.


CONCLUSIONS
In our experimental system, specific inhibition of NF-kappaB activation rendered chondrocytes susceptible to cell death induced by TNF-alpha. The cell death was enhanced by inhibition of another signaling pathway such as p38 MAP kinase or PI3K. The expression of Bcl-XL, Bcl-2 and XIAP and activation of JNK were affected by ad-IkappaB-SR transduction, implying a role in the NF-kappaB regulated cell survival signaling in human chondrocytes.
Yoon
Ho Sung
HS
Department of Internal Medicine, Hallym University Kangdong Sacred Heart Hospital, 445 Gil-dong, Kangdong-gu, Seoul, 134-701, South Korea.
Kim
Hyun Ah
HA
Song
Yeong Wook
YW
D000256:Adenoviridae; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism / Q000821:virology; D002867:Chromones / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011725:Pyridines / Q000494:pharmacology; D014161:Transduction, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D002867:Chromones; D004791:Enzyme Inhibitors; D007093:Imidazoles; D048031:JNK Mitogen-Activated Protein Kinases; D009025:Morpholines; D016328:NF-kappa B; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011725:Pyridines; D014409:Tumor Necrosis Factor-alpha; D048051:p38 Mitogen-Activated Protein Kinases; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C090942:4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole