Up-regulation of p21 Gene Expression by Peroxisome Proliferator-Activated Receptor γ in Human Lung Carcinoma Cells

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Abstract
Purpose: The peroxisome proliferator-activated receptor γ (PPARγ), a ligand-dependent transcription factor belonging to the family of nuclear receptors, has been implicated in the regulation of cell growth and differentiation although the exact mechanism(s) of this activity has not been elucidated. In this study, we explored the role of PPARγ signaling on the control of gene expression of the cycle-dependent kinase inhibitor p21 in human lung carcinoma cells. Experimental Design: Using several human lung carcinoma cell lines (small and non-small carcinoma cells), we assayed for cell growth inhibition and apoptosis induction. We also assayed for p21 mRNA and protein expression by reverse transcription-PCR, real-time reverse transcription-PCR, and Western blot analysis. Nuclear protein binding activities to three response elements located in the p21 promoter [nuclear factor (NF)-κB, Sp1, and NF-interleukin 6 (IL6) CAAT/enhancer binding protein (C/EBP)] were measured by gel mobility shift assays. We used transient transfection assays with p21 promoter reporter gene constructs to determine the transcriptional regulation by PPARγ ligands. Finally, by using p21 antisense oligonucleotides, we tested the link between PPARγ activation and p21 signaling in cell growth inhibition assays and by Western blot analysis. Results: We showed that the PPARγ ligands PGJ2 and ciglitazone inhibit the growth and induce the apoptosis of several human lung carcinoma cell lines, whereas the PPARα agonist WY14643 has little effect. Treatment of lung carcinoma cells with the PPARγ ligands PGJ2, ciglitazone, troglizaone, and GW1929 elevated p21 mRNA and protein levels and reduced cyclin D1 mRNA levels. These results were supported by transient transfection assays, which indicated that PPARγ ligands increased p21 gene promoter activity in human lung carcinoma cells. In addition, p21 antisense oligonucleotides inhibited PPARγ ligand-induced p21 protein expression and significantly blocked lung carcinoma cell growth inhibition induced by PPARγ ligands. Finally, electrophoresis mobility shift experiments demonstrated that PPARγ ligands increased the nuclear binding activities of Sp1 and NF-IL6 (C/EBP), two transcription factors with regulatory elements in the promoter region of the p21 gene. Conclusion: PPARγ ligands inhibit human lung carcinoma cell growth and induce apoptosis by stimulating the cyclin-dependent kinase inhibitor p21 and by reducing cyclin D1 gene expression. The induction of p21 gene expression by PPARγ ligands may be mediated through increased Sp1- and NF-IL6 (C/EBP)-dependent transcriptional activation. These observations unveil a mechanism for p21 gene regulation in lung carcinoma that represents a potential target for therapy.