Effect Of PPARβ/δ and PPARγ on Cell Proliferation and Gene Expression in the A431 Human Skin Cancer Cell Line

Open Access
Lee, Christina
Area of Honors:
Biochemistry and Molecular Biology
Bachelor of Science
Document Type:
Thesis Supervisors:
  • Jeffrey Maurice Peters, Thesis Supervisor
  • Joseph C. Reese, Honors Advisor
  • Wendy Hanna Rose, Faculty Reader
  • PPARβ/δ
  • PPARγ
  • Skin Cancer
  • A431
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear hormone receptors that play distinct roles in the β-oxidation of fatty acids. These receptors regulate gene expression by heterodimerization with retinoid X receptor (RXR), binding to PPAR response elements (PPREs), and recruitment of coactivators. Of the three PPAR isoforms, PPARα, PPARβ/δ, and PPARγ, PPARβ/δ is the most prevalent in the skin, but PPARγ is also present. While much controversy exists as to whether PPARs, especially PPARβ/δ, either promote or inhibit cell proliferation, increasing evidence suggests that PPARβ/δ promotes differentiation and inhibits proliferation. The goal of this thesis was to characterize the roles of PPARβ/δ and PPARγ in the A431 human skin cancer cell line by stably overexpressing the receptors. A431 cells were stably infected with either empty Migr1 retroviral vector, Migr1-hPPARβ/δ, or Migr1-hPPARγ. The Migr1 vector allows for expression of a protein such as PPARβ/δ or PPARγ and enhanced green fluorescent protein (eGFP). After stable infection, cells were sorted using flow cytometry and then the presence of elevated levels of PPARβ/δ and PPARγ was confirmed through western blot analysis. To confirm that the protein being expressed was functional, qPCR was performed to examine the expression of a PPAR target gene upon receptor-specific ligand treatments. Two synthetic ligands were used: GW0742, a PPARβ/δ-specific agonist, and rosiglitazone, a PPARγ-specific agonist. Parent and Migr1 cell lines were treated with these ligands over a 1000-fold dose response range. Receptor function was determined by measuring induction of the PPAR target gene, angiopoietin-like protein 4 (ANGPTL4), which responds to either PPARβ/δ or PPARγ. The overexpressed PPARβ/δ and PPARγ proteins were found to be functional as indicated by a significant increase in the induction of ANGPTL4 in the cells overexpressing PPARβ/δ or PPARγ. The effects of PPARβ/δ and PPARγ overexpression on cell proliferation were then examined using direct cell counting. Cells were treated with 0.01, 0.1, 1.0, or 10 µM GW0742 or rosiglitazone and cell number quantified with a Coulter counter after 24, 48, and 72 hours of treatment. Ligand activation of PPARβ/δ modestly inhibited cell proliferation of A431 cells as compared to control. Overexpression of PPARβ/δ in the A431-Migr1-hPPARβ/δ cells only marginally influenced this effect. Ligand activation of PPARγ more strongly inhibited cell proliferation of A431 cells as compared to control, and overexpression of PPARγ in A431-Migr1-hPPARγ cells enhanced this effect. While the results provide further evidence for PPARβ/δ and PPARγ as inhibitors of cell proliferation, future experiments must be performed to determine the mechanisms underlying this effect. Additionally, the experiments described in this thesis do not give any indication of how PPAR activation affects cell differentiation, which provides another area where further experiments are needed. Overall, the results of this experiment suggest that ligand activation of PPARβ/δ and PPARγ have some role in decreasing cell proliferation in human A431 skin cancer cells.