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.

OBJECTIVES

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.

CONCLUSION

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.