Volume 5 Supplement 3

3rd World Congress of the Global Arthritis Research Network (GARN): International Arthritis Summit

Open Access

TNF-alpha induced chondrocyte apoptosis in NF-κB suppression is augmented by inhibition of p38 mitogen-activated protein kinase or phosphatidylinositol 3-kinase

  • H Kim1
Arthritis Res Ther20035(Suppl 3):58

https://doi.org/10.1186/ar859

Published: 12 September 2003

Background

Therapeutics employing knowledge on various signaling pathway are being developed, with NF-κB one of the most promising targets. NF-κB has been suggested to play a role not only in the induction of inflammatory mediators, but also in the protection of apoptosis.

Objectives

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

Methods

NF-κB activation was specifically inhibited using an adenoviral vector encoding the transgene for IκB super-repressor (ad-IκB-SR). The human juvenile costal chondrocyte cell line (C28/I2) was used for the experiment. Infection with ad-IκB-SR was performed for 360 min using a multiple of infection of 1:200, after which the cells were incubated for 18 hours in medium containing 10% FCS. The cultures were then changed to serum-free medium, and treated with 10 ng/ml TNF-α. The proportion of cell death was analyzed by MTT assay. Activation of p38 mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase (PI3K) was inhibited by preincubation for 1 hour with SB202190 and Ly 294002, respectively. The expression of apoptosis-related protein was analyzed with Western blot assay. The activation of c-JUN N-terminal kinase was analyzed by gel kinase assay.

Results

Despite complete inhibition of NF-κB activation, treatment with TNF-α led to cell death in only about 23% of ad-IκB-SR-infected chondrocytes after 24 hours. Preincubation with SB202190 and Ly 294002 led to a significant increase in cell death, resulting in 53% and 30% cell death after 24 hours, respectively. The expression of Bcl-XL and XIAP significantly decreased, and activation of c-Jun N-terminal kinase was prolonged up to 4 hours in infected cells treated with TNF-α.

Conclusion

In our experimental system, specific inhibition of NF-κB activation rendered chondrocytes susceptible to cell death induced by TNF-α. However, completion of cell death required inhibition of another signaling pathway such as p38 MAP kinase and PI3K. The expression of Bcl-XL and XIAP, and activation of c-Jun N-terminal kinase was affected by ad-IκB-SR transduction, implying its role in the NF-κB-regulated cell survival signaling in human chondrocytes.

Authors’ Affiliations

(1)
Internal Medicine, Hallym University College of Medicine

Copyright

© The Author(s) 2003

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