Abstract
Dehydroepiandrosterone (DHEA), a C19 human adrenal steroid, activates peroxisome proliferator-activated receptor α (PPARα) in vivo but does not ligand-activate PPARα in transient transfection experiments. We demonstrate that DHEA regulates PPARα action by altering both the levels and phosphorylation status of the receptor. Human hepatoma cells (HepG2) were transiently transfected with the expression plasmid encoding PPARα and a plasmid containing two copies of fatty acyl coenzyme oxidase (FACO) peroxisome-proliferator activated receptor responsive element consensus oligonucleotide in a luciferase reporter gene. Nafenopin treatment increased reporter gene activity in this system, whereas DHEA treatment did not. Okadaic acid significantly decreased nafenopin-induced reporter activity in a concentration-dependent manner. Okadaic acid treatment of primary rat hepatocytes decreased both DHEA- and nafenopin-induced FACO activity in primary rat hepatocytes. DHEA induced both PPARα mRNA and protein levels, as well as PP2A message in primary rat hepatocytes. Western blot analysis showed that the serines at positions 12 and 21 were rapidly dephosphorylated upon treatment with DHEA and nafenopin. Results using specific protein phosphatase inhibitors suggested that protein phosphatase 2A (PP2A) is responsible for DHEA action, and protein phosphatase 1 might be involved in nafenopin induction. Mutation of serines at position 6, 12, and 21 to an uncharged alanine residue significantly increased transcriptional activity, whereas mutation to negative charged aspartate residues (mimicking receptor phosphorylation) decreased transcriptional activity. DHEA action involves induction of PPARα mRNA and protein levels as well as increased PPARα transcriptional activity through decreasing receptor phosphorylation at serines in the AF1 region.
Footnotes
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This work was supported in part by a Predoctoral Fellowship from the Ohio Valley American Heart Association (to K.K.M.), National Research Service Award Fellowship 1F32-ES05927-01 (to S.L.R.) and United States Public Health Service grant DK54774 (to R.A.P.) and the Center for Environmental Genomics and Integrative Biology (1P30-ES014443).
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ABBREVIATIONS: PPARα, peroxisome proliferator-activated receptor α; PP, peroxisome proliferator; DHEA, dehydroepiandrosterone; MAPK, mitogen-activated protein kinase; FACO, fatty acyl coenzyme oxidase; Wy-14643, [[4-chloro-6-[(2,3-dimethylamino]-2-pyrimidinyl]thio] acetic acid; PPRE, peroxisome proliferator-activated receptor responsive element; RT, reverse transcription; DMSO, dimethyl sulfoxide; PP2A, protein phosphatase 2A; RT, reverse transcription; PCR, polymerase chain reaction.
- Received April 4, 2007.
- Accepted December 7, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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