title: Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) inhibition by 4-hydroxynonenal leads to increased Akt activation in hepatocytes.
abstract: The production of reactive aldehydes such as 4-hydroxynonenal (4-HNE) is proposed to be an important factor in the etiology of alcoholic liver disease. To understand the effects of 4-HNE on homeostatic signaling pathways in hepatocytes, cellular models consisting of the human hepatocellular carcinoma cell line (HepG2) and primary rat hepatocytes were evaluated. Treatment of both HepG2 cells and primary hepatocytes with subcytotoxic concentrations of 4-HNE resulted in the activation of Akt within 30 min as demonstrated by increased phosphorylation of residues Ser473 and Thr308. Quantification and subsequent immunocytochemistry of phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P(3)[rsqb] resulted in a 6-fold increase in total PtdIns(3,4,5)P(3) and increased immunostaining at the plasma membrane after 4-HNE treatment. Cotreatment of HepG2 cells with 4-HNE and the phosphatidylinositol 3-kinase (PI3K) inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (Ly294002) or the protein phosphatase 2A (PP2A) inhibitor okadaic acid revealed that the mechanism of activation of Akt is PI3K-dependent and PP2A-independent. Using biotin hydrazide detection, it was established that the incubation of HepG2 cells with 4-HNE resulted in increased carbonylation of the lipid phosphatase known as "phosphatase and tensin homolog deleted on chromosome 10" (PTEN), a key regulator of Akt activation. Activity assays both in HepG2 cells and recombinant PTEN revealed a decrease in PTEN lipid phosphatase activity after 4-HNE application. Mass spectral analysis of 4-HNE-treated recombinant PTEN detected a single 4-HNE adduct. Subsequent analysis of Akt dependent physiological consequences of 4-HNE in HepG2 cells revealed significant increases in the accumulation of neutral lipids. These results provide a potential mechanism of Akt activation and cellular consequences of 4-HNE in hepatocytes.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Denver, Aurora, Colorado, USA.
authors.forename: Colin T
authors.initials: CT
authors.lastname: Shearn
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Smathers
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Stewart
authors.forename: Kristofer S
authors.initials: KS
authors.lastname: Fritz
authors.forename: James J
authors.initials: JJ
authors.lastname: Galligan
authors.forename: Numsen
authors.initials: N
authors.lastname: Hail
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Petersen
mesh_terms: D000447:Aldehydes / Q000494:pharmacology*; D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D017931:DNA Primers; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D018613:Microscopy, Confocal; D051059:PTEN Phosphohydrolase / Q000037:antagonists & inhibitors*; D016133:Polymerase Chain Reaction; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors
chemical_list: D000447:Aldehydes; D017931:DNA Primers; D011994:Recombinant Proteins; D051057:Proto-Oncogene Proteins c-akt; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human; C487216:Pten protein, rat; C027576:4-hydroxy-2-nonenal