Selective inhibition of proinflammatory chemokines such as IL-8 is an important approach to combat inflammatory and infection diseases. Previous studies suggested that interaction of transcription factors NFκB repressing factor (NRF) and NFκB play a crucial role in activation of IL-8 gene expression. In a search for a specific inhibitor of IL-8 expression, we applied tandem affinity purification to investigate interaction of NRF and NFκB p65 in cells. We identified a synthetic peptide corresponding to aa 223–238 of NRF interfering with binding of endogenous p65 to NRF. Furthermore, nucleofection experiments were established to introduce this inhibitory peptide into the nucleus of IL-1 stimulated human cervical and Helicobacter pylori infected gastric epithelial cells. Our data clearly show that the specific peptide disturbing NRF/NFκB interaction is able to significantly decrease endogenous IL-8 gene transcription in response to IL-1 or Helicobacter pylori infection. Thus, our study provides novel insights into NRF and NFκB interaction in vivo and may facilitate the design of new anti-IL-8 drugs based on novel strategies.

NF-kappaB repressing factor (NRF) is a nuclear transcription factor that binds to a specific DNA sequence in NF-kappaB target promoters. Previous reports suggested that NRF interferes with the transcriptional activity of NF-kappaB binding sites through a direct interaction with NF-kappaB subunits. The aim of this study was to map specific NRF binding domains in the NF-kappaB proteins, p65 and p50. Our data demonstrate that NRF is able to interact with the p65 subunit and inhibit its transcription enhancing activity in reporter gene experiments. Using tandem affinity purifications (TAP), we show that NRF protein significantly binds to the endogenous p65, subunit but not to the p50 subunit. The selective binding activity of the NRF protein is consistently mediated by the N-terminal domain of NRF (aa 1-380). Moreover, the Rel homology domain (RHD) of p65 is sufficient for binding to the N-terminal domain of NRF. Using detailed peptide mapping studies, we finally identify three peptide motifs in p65 RHD showing distinctive binding specificities for the NRF protein. According to the predicted structure of p65, all three peptide motifs align within an exposed region of p65 and might hint at promising targets for inhibitors.

NF-κB-repressing factor (NKRF) inhibits transcription elongation by binding to specific sequences in target promoters. Stimuli such as IL-1 have been shown to overcome this inhibitory action and enable the resumption of transcription elongation machinery by an unknown mechanism. Using mass spectrometry and in vitro phosphorylation analyses, we demonstrate that NKRF is phosphorylated within 3 different domains in unstimulated HeLa cells. Phosphoamino acid mapping and mutation analysis of NKRF further suggest that only Ser phosphorylation within aa 421-429 is regulated by IL-1 stimulation. In copurification studies, aa 421-429 is required for interactions between NKRF, 5'→3' exoribonuclease 2 (XRN2) and the negative elongation factor (NELF)-E in HeLa cells. Chromatin immunoprecipitation experiments further show that IL-1 stimulation leads to decrease in NKRF aa 421-429 phosphorylation and dissociation of NELF-E and XRN2 by concomitant resumption of transcription elongation of a synthetic reporter or the endogenous NKRF target gene, IL-8. Together, NKRF phosphorylation modulates promoter-proximal transcription elongation of NF-κB/NKRF-regulated genes via direct interactions with elongation complex in response to specific stimuli.