PPARα inhibits vascular smooth muscle cell proliferation underlying intimal hyperplasia by inducing the tumor suppressor p16^INK4a
J. Clin. Invest. Florence Gizard, et al. 115:3228 doi:10.1172/JCI22756 [Go to this article.]

Figure 2
PPARα activation increases p16 gene transcription. (A) Regulation of p16 mRNA levels in hSMCs and mSMCs. Quantitative RT-PCR analyses were performed on RNA from Ad-GFP or Ad-PPARα–infected hSMCs treated or not treated with GW7647 (600 nM, upper panel) and on PPARα^–/– and PPARα^+/+ Sv/129 mSMCs treated or not treated with Wy14,643 (6 μM, lower panel). Values are expressed relative to the controls (mRNA levels in PPARα^+/+ mSMCs or hSMCs at T0) set as 1. (B) Induction of human p16 promoter activity by ligand-activated PPARα. pGL2 luciferase reporter constructs driven by the indicated p16 promoter deletion fragments (0.1 μg) were cotransfected in HeLa cells with or without pSG5 PPARα expression plasmid (0.3 μg). Cells were subsequently treated with fenofibric acid (100 μM) for 10 hours. Results (mean ± SD) from 1 representative experiment (n = 3) out of 3 is shown. (C) In vitro binding of PPARα to the p16DR1. EMSAs were carried out with end-labeled consensus DR1 (DR1cons), wild-type p16DR1 (p16DR1wt), or mutated p16DR1 (p16DR1mt) probes in the presence of unprogrammed reticulocyte lysate, RXR, PPARα, or both RXR and PPARα as indicated. (D) In-cell occupation of the p16 gene promoter by PPARα. Soluble chromatin was prepared from hSMCs treated with or without fenofibric acid (250 μM). IP was performed with anti-Sp1 or anti-PPARα antibodies, and DNA was amplified using specific primer pairs. Bottom lane shows schematic diagram depicting the fragments of the p16 and β-actin genes that were amplified. The nucleotide positions are indicated relative to the ATG. In panels B and D, A, B, and C denote the 3 Sp1-binding sites identified by Myohanen et al. (26) (see Results), and the putative transcription start site of the p16 promoter is indicated by an arrow.