Figure 6

Overexpression of Egr-1 reduces PDK1 promoter activity. A, H1299 cells (1x10⁵ cells) were transfected with control and Egr-1 expression reporter constructs, and together with a wild type PDK1 promoter construct and an internal control Renilla Luciferase Reporter Vector as described in Material and Methods section for 24 h, then treated with ciglitazone (20 μM) for an additional 24 h. The insert in upper panel represents Western blot results for Egr-1 protein. B, H1299 cells were lysed after exposure of ciglitazone (20 μM) for 24 h, and nuclei were isolated and then sonicated. Chromatin from H1650 cells was immunoprecipitated using antibodies against Egr-1 protein or preimmune serum (pre-immune). PCR analysis using primers surrounding the Egr-1 site shows that this DNA sequence (−4392 to −4402 bp) is specifically immunoprecipitated indicating that Egr-1 binds to endogenous DNA sites in the PDK1 promoter. A non- Egr-1 sequence was used as control. Aliquots of the chromatin were also analyzed before immunoprecipitation (input). C, Diagram demonstrates that ciglitazone inhibits PDK1 expression through AMPKα-mediated induction of Egr-1 protein expression and Egr-1 protein binding to the DNA sequence in the PDK1 gene promoter independent of PPARγ. Activation of AMPKα enhances the effect of ciglitazone on Egr-1 and PDK1 protein expression. In turn, this results in inhibition of NSCLC cell proliferation.