Regulation of Sox9 and NFB activity by TNF are independent of MEKERK signalling We next wanted to determine the possible molecular basis for TNF modulated, U0126 sensitive gene expression. First, we investigated whether U0126 affected the ability of TNF to regulate the activity of the transcription factors Sox9 and NFB, which are known to be regulated by TNF in chondro cytes. As expected, TNF significantly reduced the level of Sox9 activity and increased the level of NFB activity in chondrocytes. There was no signif icant effect, however, on the level of inhibition or the induction of Sox9 and NFB activity, respectively, by either U0124 or U0126. Furthermore, we found that TNF induced DNA binding of NFB was reduced by pre treatment with DMSO and was not further reduced by pretreatment with U0124, U0126 or the selective epidermal growth factor receptor inhibitor, PD153035.
These results indicate that transcrip tion factors other than Sox9 and NFB are targets of TNF induced MEKERK signalling. Egr 1 DNA binding is increased in a TNF induced MEK ERK dependent manner read this post here To determine additional, candidate transcription factors that may regulated by MEKERK, we considered that Egr 1 is a known early target of MEKERK signalling and that IL 1 induc tion of Egr 1 inhibits the activity of the human type II collagen proximal promoter. We therefore focused the remainder of our study on Egr 1 and its possible role in regulating U0126 sensitive TNF induced genes. We identified multiple putative Egr 1 binding sites in the pro moter regions of the rat Col2a1 and Agc1 genes that were proximal to the transcription initiation site and overlapped with putative Sp1 binding sites.
TNF treatment of chondrocytes selelck kinase inhibitor over 24 hours did not alter the Egr 1 protein lev els, and neither did treatment for 90 minutes alter the nuclear localization of Egr 1. We then used electrophoretic mobility shift assays to investi gate whether the binding of Egr 1 to DNA was dependent on TNF induced MEKERK signalling. Nuclear extracts from chondrocytes treated with TNF for 90 minutes increased the DNA binding of two complexes containing Egr 1 to an Egr consensus DNA binding site. Both complexes were reduced when extracts were preincubated with a 100 fold molar excess of double stranded cold Egr con sensus ODNs, but not with cold mutant Egr ODNs or NFB consensus ODNs. Compared with pre incubation of extracts with the anti NFB p65 antibody, prein cubation of extracts with the anti Egr 1 antibody specifically reduced the DNA protein complexes attributed by the Egr consensus ODN competition studies to be a result of Egr DNA binding. Pretreatment of cels with U0126 attenuated the increase in complex formation of both identified complexes. l