The synergistic activities of WT Ras and TNF on CXCL8 up regulation are mediated by the following website NFB and AP 1 transcription factors Inhibitors,Modulators,Libraries Throughout this study, we found that CXCL8 up regulation took place at the mRNA level. Therefore, we asked Inhibitors,Modulators,Libraries which regulatory elements are inducing the transcription of CXCL8, thus leading to the ability of TNF WT Ras to eventually promote CXCL8 secretion. Here, we studied the roles of NFB and AP 1, two transcription Inhibitors,Modulators,Libraries factors known to up regulate CXCL8 in the immune context, although to different ex tents depending on cell type and stimulus. The activation of NFB comes into effect following down regulation of the I B inhibitor and Inhibitors,Modulators,Libraries phosphoryl ation of p65. Following TNF stimulation, the phosphorylation of p65 was increased and I B levels were reduced.

These general assays of NFB activation did not reveal coop erativity between TNF and WT Ras. However, more direct and sensitive analyses with dual luciferase assays using the NFB luciferase reporter, demonstrated that the stimulation Inhibitors,Modulators,Libraries of WT Ras expressing cells by TNF has increased the transcriptional activity of NFB. Also, siRNAs to p65 have down regulated p65 expression, and in cells stimu lated by TNF have led to almost complete shut off of the TNF WT Ras induced CXCL8 expression. These results provide evidence for direct roles of the NFB pathway in mediating the TNF WT Ras induced activation of CXCL8. In parallel, we found that TNF WT Ras induced co operative induction of c Jun phosphorylation, which is a major component of the AP 1 transcription factor.

The phosphorylation of c Jun indicates that there was a general process of AP 1 activation but it could not tell us whether the activation of AP 1 by TNF WT Ras has led directly to up regulation of CXCL8 expres sion. Looking for appropriate manners to determine the direct roles selleck kinase inhibitor of AP 1 in induction of CXCL8 upon TNF stimulation of WT Ras expressing cells, we wished to use siRNA shRNA to c Jun, however, we could not ob tain efficient enough down regulation of c Jun expres sion, being in line with the fact that c Jun is essential for cell proliferation. In the absence of a pharmaco logical inhibitor with high enough specificity, we used luciferase reporter assays in which the CXCL8 promoter expressed WT or mutated AP 1 binding sites. These tests have shown cooperativity between TNF and WT Ras in inducing luciferase activation, in addition, marked decrease was noted in luciferase levels when WT Ras cells were stimulated by TNF in the presence of AP 1 mutated promoter, compared to AP 1 WT promoter. Because the promoter was spe cifically the one of CXCL8, these results demonstrate that TNF cooperates with WT Ras in inducing AP 1 activation, together leading to an additive up regulation in the transcription of CXCL8.