Protein kinase C-δ and mitogen-activated protein/extracellular signal-regulated kinase-1 control GLI activation in hedgehog signaling

One third of all lethal cancers are associated with excessive activation of the Hedgehog (HH) pathway by mutations of its signaling components or by increased responsiveness of cells to the HH ligand. HH signaling through the GLI transcription factors leads to increased cell proliferation by up-regulation of the extracellular regulated kinase (ERK) pathway and by expression of S phase cyclins. In this study, we have tested the hypothesis that the HH pathway can integrate ERK signaling to modulate the activity of GLI. Using NIH 3T3 cells, we show that. phorbol esters, acting through protein kinase C-delta (PKC-delta) and mitogen-activated protein/extracellular signal-regulated kinase-1 (MEK-1), fully stimulate the transcriptional activity of endogenous and overexpressed GLI proteins, as assessed by GLI-luciferase reporter assays, and induce the expression of endogenous GLI1 and PTCH-1 target genes, as assessed by reverse transcription-PCR. Moreover, activation of GLI elicited by Sonic Hedgehog also requires PKC delta and MEK-1 function. Remarkably, coexpression of activated MEKA and GLI1 or GLI2 induced a 10-fold synergistic increase in GLI-luciferase activity that was totally blocked by PD98059. The NH2-terminal region of GLI1 (amino acids 1-130) is required for sensing the ERK pathway, as deletion of this domain produces active GLI1 protein with greatly reduced response to activation by MEK-1. Basic fibroblast growth factor activation of the ERK pathway also stimulated GLI1 activity through its NH2-terminal domain. Our results identify PKC delta and MEK-1 as essential, positive regulators of GL1-mediated HH signaling. Furthermore, our findings suggest that tumors with deregulated HH and ERK synergize to stimulate cell proliferation pathways.