Selectivity of fatty acid ligands for PPARalpha which correlates both with binding to cis-element and DNA binding-independent transactivity in Caco-2 cells.
It is thought that peroxisome proliferator-activated receptor alpha (PPARalpha) is a major regulator for fatty acid metabolism. Long-chain fatty acids have been shown to induce expression of the genes related to fatty acid metabolism through PPARalpha. However, it is unclear whether the intensity of PPARalpha activation is different among various fatty acids. In this study, we compared various fatty acids in the capability of PPARalpha activation by differential protease sensitivity assay (DPSA), electrophoretic mobility shift assay and GAL4-PPAR chimera reporter assay in intestinal cell line, Caco-2. DPSA revealed that polyunsaturated fatty acids of 18 to 20 carbon groups with 3-5 double bonds strongly induced a PPARalpha conformational change. The ligand-induced changes in the sensitivity to protease corresponded to the enhancement of the binding of PPARalpha-RXRalpha heterodimer to the PPAR-response element (PPRE). The GAL4-PPAR chimera reporter assay revealed that the DNA binding-independent transactivity of PPARalpha was induced by various fatty acids with a wide spectrum of intensity which correlated with the conformational change of PPARalpha. These results suggest that PPARalpha has greater selectivity to certain types of polyunsaturated fatty acids, and that the ligand-induced conformational change of PPARalpha leads to parallel increases in both DNA binding to the PPAR-response element and the DNA binding-independent transactivity.
Mochizuki
Kazuki
K
Graduate School of Nutritional and Environmental Sciences, COE Program in the 21st Century, The University of Shizuoka, Shizuoka 422-8526, Japan.
Suruga
Kazuhito
K
Fukami
Harukazu
H
Kiso
Yoshinobu
Y
Takase
Sachiko
S
Goda
Toshinao
T
D001667:Binding, Competitive; D018938:Caco-2 Cells; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D008024:Ligands; D050356:Lipid Metabolism / Q000187:drug effects / Q000235:genetics; D047493:PPAR alpha / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics; D047490:Retinoid X Receptor alpha / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects*
D004268:DNA-Binding Proteins; D005231:Fatty Acids, Unsaturated; D008024:Ligands; D047493:PPAR alpha; D047490:Retinoid X Receptor alpha