AIM: To study the effects of I kappa B alpha and its mutants (I kappa B alpha M, I kappa B alpha 243N, I kappa B alpha M244C) on NF-kappa B, p53 and their downstream target genes. The relationship of NF-kappa B, p53, and I kappa B alpha was further discussed. METHODS: pECFP-I kappa B alpha, pECFP-I kappa B alpha M (amino acides 1-317, Ser32, 36A), pECFP-I kappa B alpha 243N (amino acides 1-243), pECFP-I kappa B alpha 244C (amino acides 244-317), pEYFP-p65 and pp53-DsRed were constructed and transfected to ASTC-alpha-1 cells. Cells were transfected with pECFP-C1 as a control. 30 h after the transfection, location patterns of NF-kappa B, p53 and I kappa B alpha (I kappa B alpha M, I kappa B alpha 243N, I kappa B alpha 244C) 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 I kappa B alpha and its mutants on the transprition level of NF-kappa B, NF-kappa B downstream target gene TNF-alpha, p53 and p53 downstream target gene Bax were observed by real time QT-PCR. In all experiments P-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-I kappa B alpha, CFP-I kappa B alpha M, and CFP-I kappa B alpha 243N respectively and revealed a predominant cytosolic localization. However, cells transfected of CFP-I kappa B alpha 244C revealed a predominant nuclear localization. The mRNA levels of p65, TNF-alpha, p53 and Bax in CFP-I kappa B alpha transfected cells did not change significantly, while in YFP-p65/CFP-I kappa B alpha co-transfected cells, I kappa Ba decreased the transcription of p65 downstream gene TNF-alpha (2.24 +/- 0.503) compared with the YFP-p65/ CFP-C1 co-transfected cells (5.08 +/- 0.891) (P < 0.05). Phosphorylation defective I kappa Ba (I kappa B alpha M) decreased the transcription levels of all the four genes compared with the control (P < 0.05). The N terminus Of I kappa B alpha (I kappa B alpha 243N) increased the transcription of NF-kappa B (1.84 +/- 0.176) and TNF-alpha (1.51 +/- 0.203) a little bit. However, the C terminus of IKBa (I kappa B alpha 244C) increased the transcription of NF-kappa B, TNF-alpha, 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 I kappa B alpha 244C and p53 synergistically mediate apoptosis. CONCLUSIONS: I kappa B alpha and its mutants (I kappa B alpha M, I kappa B alpha 243N, I kappa B alpha M244C) have different effects on NF-kappa B and p53 signaling pathways, according to their different structures. I kappa B alpha M bounds with NF-kappa B and p53 in cytoplasm steadily, and inhibits both of the two signaling pathways. p53 and I kappa B alpha 244C may be co-factor in inducing apoptosis. The C terminal Of I kappa B alpha enhanced cell death, which suggests that it may be a pro-apoptotic protein existed in cells. (C) 2006 The WJG Press. All rights reserved.
