Effects of IκBα and its mutants on NF-κB and p53 signaling pathways

AIM： To study the effects of IκBα and its mutants （IκBαM, IκBα3N, IκBαM44C） 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 24±317）, pEYFP-p65 and pp53-DsRed were constructed and transfected to ASTC-α-1 cells. Cells were transfected with pECFP-Cl as a control. 30 h after the transfection, location patterns of NF-κB, p53 and IκBα（IκBαM, IκBα243N, IκBα224C） 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 transprition 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 rnRNA 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- KB and p53 signaling pathways, according to their different structures. IκBαbounds with NF-KB 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 IκBαnhanced cell death, which suggests that it may be a pro-apoptotic protein existed in cells.