Figure 6.

TonEBP controls the activity of the CXCL1 promoter via a highly conserved NF-κB-binding site. A, diagram showing predicted TonEBP- and NF-κB-binding sites in the rat CXCL1 proximal promoter spanning −1.5 kb upstream of the transcription start site. Predicted TonE is depicted by gray circle and NF-κB-binding sites by open squares. NF-κB-mutant reporter contains mutation in binding site 2 (star), which has high species conservation and activity in other cell types. B, under basal conditions, TonEBP increased activities of WT and TonE-mutant promoters, whereas NF-κB-mutant was unaffected. C, WT, TonE-, and NF-κB-mutant reporters were all induced by TNF-α treatment. During TNF-α treatment, addition of TonEBP further induced activity of WT and TonE-mutant reporters only. D, WT and TonE-mutant reporters, but not NF-κB-mutant, were induced by IL-1β treatment. During IL-1β stimulation, addition of TonEBP did not affect inducibility of any of the reporters, whereas DN-TonEBP blocked induction of WT and TonE-mutant reporters. E, none of the reporters responded to LPS treatment alone. Addition of TonEBP during LPS treatment induced activity in WT and TonE-mutant reporters only. F, addition of small amount (15 ng) of p65 plasmid resulted in a trend of increased activity in all reporters, which was further enhanced by addition of TonEBP. G, expression of TonEBP further increased TNF-α-dependent induction of NF-κB reporter activity. H, treatment with TNF-α, but not NaCl, induced mRNA expression of CXCL1. TNF-α-mediated induction was unaffected by co-treatment with NaCl. Quantitative measurements represent mean ± S.E. of ≥3 biological replicates and, for transfection experiments, 3 technical replicates per biological replicate. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. ns, not statistically significant.