Figure 5.

EGFR-mediated activation of JUN/AP-1 is essential for synergistic target gene activation and transformation of human keratinocytes by EGFR/GLI. A, top, gel shift assay demonstrating the activation of AP-1 DNA binding activity by EGF treatment of HaCaT keratinocytes. Inhibition of MEK/ERK function by U0126 treatment abolished EGF-induced activation of AP-1. As negative control, a mutated (− lanes) instead of wild-type (+ lanes) AP-1 binding oligonucleotide was used. Bottom, Western blot analysis showing that EGF treatment of HaCaT cells induces phosphorylation of JUN (pJun) via activation of EGFR/MEK/ERK signaling. EGFR was inhibited by gefitinib (Gef), MEK by UO126, AKT by wortmannin (Wort), and JNK by SP600125 treatment. B, siRNA knockdown of JUN expression selectively interferes with the synergistic activation of EGF-dependent GLI target genes, such as IL1R2, ARC, or EGR3. Synergistic activation of GLI/EGF targets was achieved by GLI1 expression and simultaneous EGF treatment (10 ng/mL; ref. 17). C, ChIP analysis showing JUN binding to AP-1 sites in the promoter region of EGF-dependent direct GLI target genes (IL1R2, JAG2, and S100A9). RPL30 and PTCH were used as negative and JUN bound to its own promoter as positive control (48). D, RNAi-mediated knockdown of JUN (shRNA-cJun) inhibits anchorage-independent growth induced by combined activation of EGFR and GLI1.