Basic Research
Copyright ©2006 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Nov 7, 2006; 12(41): 6658-6664
Published online Nov 7, 2006. doi: 10.3748/wjg.v12.i41.6658
Effects of IκBα and its mutants on NF-κB and p53 signaling pathways
Xian Li, Da Xing, Ju Wang, De-Bin Zhu, Lan Zhang, Xiao-Jia Chen, Fen-Yong Sun, An Hong
Xian Li, Da Xing, De-Bin Zhu, Lan Zhang, Institute of Laser Life Science, South China Normal University, Guangzhou 510631, Guangdong Province, China
Ju Wang, Xiao-Jia Chen, Fen-Yong Sun, An Hong, Institute of Biology Engineering, Jinan University, Guangzhou 510632, Guangdong Province, China
Supported by the National Natural Science Foundation of China No. 60378043 and 30470494), and the Natural Science Foundation of Guangdong Province No. 015012 and 04010394
Correspondence to: Professor Da Xing, Institute of Laser Life Science, South China Normal University, Guangzhou 510631, Guangdong Province, China. xingda@scnu.edu.cn
Telephone: +86-20-85210089 Fax: +86-20-85216052
Received: May 15, 2006
Revised: July 12, 2006
Accepted: August 19, 2006
Published online: November 7, 2006
Abstract

AIM: To study the effects of IκBα and its mutants (IκBαM, IκBα243N, IκBαM244C) on NF-κB, p53 and their downstream target genes. The relationship of NF-κB, p53, and IκBα was further discussed.

METHODS: pECFP-IκBα, pECFP-IκBαM (amino acides 1-317, Ser32, 36A), pECFP-IκBα243N (amino acides 1-243), pECFP-IκBα244C (amino acides 244-317), pEYFP-p65 and pp53-DsRed were constructed and transfected to ASTC-α-1 cells. Cells were transfected with pECFP-C1 as a control. 30 h after the transfection, location patterns of NF-κB, p53 and IκBα (IκBαM, IκBα243N, IκBα244C) were observed by a laser scanning microscope (LSM510/ConfoCor2, Zeiss). RNA extraction and reverse transcription were performed in cells transfected or co-transfected with different plasmids. Effects of IκBα and its mutants on the transpiration level of NF-κB, NF-κB downstream target gene TNF-α, p53 and p53 downstream target gene Bax were observed by real time QT-PCR. In all experiments β-actin was reference. Results are expressed as the target/reference ratio of the sample divided by the target/reference ratio of the control. Different transfected cells were incubated with CCK-8 for 2 h in the incubator. Then the absorbance at 450 nm was measured by using a microplate reader.

RESULTS: Cells that were transfected with p53-DsRed revealed a predominant nuclear localization. YFP-p65 mainly existed in the cytoplasm. Cells were transfected with CFP-IκBα, CFP-IκBαM, and CFP-IκBα243N respectively and revealed a predominant cytosolic localization. However, cells transfected of CFP-IκBα244C revealed a predominant nuclear localization. The mRNA levels of p65, TNF-α, p53 and Bax in CFP-IκBα transfected cells did not change significantly, while in YFP-p65/CFP-IκBα co-transfected cells, IκBα decreased the transcription of p65 downstream gene TNF-α (2.24 ± 0.503) compared with the YFP-p65/CFP-C1 co-transfected cells (5.08 ± 0.891) (P < 0.05). Phosphorylation defective IκBα (IκBαM) decreased the transcription levels of all the four genes compared with the control (P < 0.05). The N terminus of IκBα (IκBα243N) increased the transcription of NF-κB (1.84 ± 0.176) and TNF-α (1.51 ± 0.203) a little bit. However, the C terminus of IκBα (IκBα244C) increased the transcription of NF-κB, TNF-α, p53 and Bax significantly (8.29 ± 1.662, 14.16 ± 2.121, 10.2 ± 0.621, 3.72 ± 0.346) (P < 0.05). The CCK-8 experiment also showed that IκBα244C and p53 synergistically mediate apoptosis.

CONCLUSIONS: IκBα and its mutants (IκBαM, IκBα243N, IκBαM244C) have different effects on NF-κB and p53 signaling pathways, according to their different structures. IκBαM bounds with NF-κB and p53 in cytoplasm steadily, and inhibits both of the two signaling pathways. p53 and IκBα244C may be co-factor in inducing apoptosis. The C terminal of IκBα enhanced cell death, which suggests that it may be a pro-apoptotic protein existed in cells.

Keywords: Nuclear factor-κB; Inhibitor of NF-κB alpha; p53; Real-time QT-PCR