Inhibition Of NF-κB Stabilizes Gadd45α MRNA

The nuclear factor-κB (NF-κB) is known as a key transcription factor that regulates the gene expression of apoptosis, cell cycle, immune, and tumorgenesis. The growth arrest and DNA damage-inducible (Gadd) gene Gadd45αis an important regulator for cell cycle, genomic stability, and cell apoptosis. Recent results show that NF-κB is an inhibitory signal for the expression of gadd45α. However, the regulation mechanism remains unknown. In this report, we focused on the molecular mechanism for the induction of gadd45αby the inhibition of NF-κB. Ikkβis identified to be responsible for the activation of NF-κB. Inhibition of Ikkβleads to the loss of activity of NF-κB. Using embryo fibroblast cells derived from wild type (Ikkβ+/+) or Ikkβgene knockout (Ikkβ-/-) mice, we found enhanced expression of gadd45αmRNA in Ikkβ-/-cells compared with Ikkβ+/+ cells. Further experiments demonstrated that the induction of gadd45αmRNA was not due to the enhanced transcription. First, Ikkβdeficiency had no effect on the basal expression or activity of Akt, FoxO3a, p53, and c-myc that regulate the transcription of gadd45αgene positively or negatively. Second, there was no difference in the activity of promoter of gadd45αgene between Ikkβ-/-and Ikkβ+/+ cells. Thus, NF-κB may regulate the expression of gadd45αmRNA at post-transcriptional level. Analysis of gadd45αmRNA stability showed a ROS (reactive oxygen species)-dependent increase in the half-life of gadd45αmRNA in Ikkβ-/-cells. Increased accumulation of ROS was also found in Ikkβ-/-cells. Immunoprecipitation experiments indicated an increased binding of a RNA stabilizing protein, nucleolin, to gadd45αmRNA in Ikkβ-/-cells. The binding of nucleolin to gadd45αmRNA could be prevented by the antioxidant, N-acetyl-cysteine (NAC). In summary, inhibition of Ikkβ-NF-κB signaling up-regulates the expression of gadd45αmRNA through a post-transcription mechanism, which implicates an important role of NF-κB-gadd45αregulatory pathway in cell cycle control, apoptosis, and tumorgenesis.