The Function Of Importin 8 In The Regulation Of NF-κB Signaling Pathway

Nuclear factor-κB (NF-κB) is a ubiquitous transcription factor which regulates gene expression and plays an important role in immune and inflammatory responses and the regulation of cell apoptosis. In unstimulated cells, NF-κB exists in the cytosol with an inhibitor protein termed IκB. Various stimulation lead to the proteolytic degradation of IκB and then NF-κB is released. The free NF-κB is transported into nucleus and regulates its downstream gene expression. Therefore, the nuclear translocation of NF-κB plays an important role in maintaining cell normal physiological function.The mechanism for activation of NF-κB signaling pathway has now been illustrated clearly. However, the molecular mechanism of NF-κB nuclear translocation remains incompletely understood. Many studies have demonstrated that NF-κB contains classical nuclear location signal (cNLS) and can be transported into the nucleus via importinα/β1 pathway. Members of importinαfamily that recognize cNLS of NF-κB have been validated, but only one member of importinβfamily (imprortinβ1) was reported for NF-kB translocation. So we wonder whether there are other importinβs involved in NF-κB translocation.Importin 7 and importin 8 are two members of importinβfamily, importin 7 shares over 60% identity in amino acid sequence with importin 8. In our study, we depleted importin 7 and importin 8 by RNA interference and evaluated the functional consequences on NF-κB signal pathway. Firstly, we designed siRNA targeting for importin 7 or importin 8 respectively. The silent effect of these siRNA was tested by RT-PCR and Western Blotting analysis. After selective depletion of expression of importin 7 or importin 8, the activity of NF-κB-dependent reporter gene was detected using luciferase reporter assay. Then we observed subcellular location of NF-κB subunit p65 with Immunofluorescence. Finally, we extracted the fractions of cytoplasmic and nuclear and then these proteins were separated on SDS-PAGE to detect p65 expression. Our results showed that the activity of NF-κB was reduced significantly and TNF-α-induced p65 nuclear accumulation was inhibited obviously by siRNA targeting importin 8. These studies implied that importin 8 regulated NF-κB signal pathway at the level of nucleocytoplasmic transport. And this finding provided foundation for further understanding the mechanisms of NF-κB nuclear translocation.