Figure 6. The role of Sp1 in mediating the effect of nicotine on PPARβ/δ expression.

A, Protein from H1838 cells treated with mithramycin A (100 nM) for 2 h before exposure to nicotine for 24 h was analyzed by Western Blot for PPARβ/δ and AP-2α (upper panel). H1838 cells were transfected with wild type PPARβ/δ promoter construct (PPARβ/δ -1880 bp luc) and treated with or without mithramycin A (100 nM) for 24 h, followed by nicotine exposure for 24 h. Firefly/renilla luciferase activity was quantified. Bars represent the mean ± SD of at least four independent experiments (lower panel). B, Upper panel, H1838 cells transfected with control or Sp1 siRNA (100 nM) for 30 h were analyzed by Western blot for Sp1 protein. Lower panel, H1838 cells transfected with control or Sp1 siRNA for 30 h were re-transfected with the wild type PPARβ/δ promoter construct (PPARβ/δ -1880 bp luc) along with an internal control Renilla Vector and treated with or without nicotine for an additional 24 h. The ratio of firefly/renilla luciferase activity was quantified; bars represent mean ± SD of at least four independent experiments. * indicates significant increase of activity as compared to controls. ** indicates significance of combination treatment as compared to the nicotine alone. Con, untreated control cells. C, Upper panel, AP-2 oligonucleotides were endlabeled with [γ-³²P]-ATP and incubated with nuclear extracts (5 μg) from H1838 cells treated with nicotine for 24 h in the absence or presence of Sp1 antibody (2 μg/μl each). Lower panel, Sp1 oligonucleotides were end-labeled with [γ-³²P]-ATP and incubated with nuclear extracts (5 μg) from H1838 cells treated with 0.1 μM nicotine for 24 h. For competition assays, a molar excess (x100) of Sp1 (Cold Sp1) oligonucleotide was added to the binding reaction. Mutated Sp1 (Mut Sp1) oligonucleotides end-labeled with γ³²PATP were used to confirm binding specificity. Con, indicates untreated control cells. D, Diagram demonstrates that nicotine increases PPARβ/δ expression through α7 nAChR-mediated activation of PI3-K and mTOR pathways and inhibition of AP-2α expression and DNA binding activity in the PPARβ/δ gene promoter. Sp1 modulates these processes. Nicotine also enhances the formation of α7 nAChR and PPARβ/δ protein complex. In turn, this may further stimulate NSCLC cell proliferation.