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The transcription factor peroxisome proliferator-activated receptor γ (PPARγ) belongs to the family of nuclear hormone receptors and consists of two isotypes, PPARγ1 and PPARγ2. Our earlier studies have shown that troglitazone (TZD)-mediated activation of PPARγ2 in hepatocytes inhibits growth and attenuates cyclin D1 transcription via modulating CREB levels. Because this process of growth inhibition was also associated with an inhibition of β-catenin expression at a post-translational level, our aim was to elucidate the mechanism involved. β-Catenin is a multifunctional protein, which can regulate cell-cell adhesion by interacting with E-cadherin and other cellular processes via regulating target gene transcription in association with TCF/LEF transcription factors. Two adenomatous polyposis coli (APC)-dependent proteasomal degradation pathways, one involving glycogen synthase kinase 3β (GSK3β) and the other involving p53-Siah-1, degrade excess β-catenin in normal cells. Our immunofluorescence and Western blot studies indicated a TZD-dependent decrease in cytoplasmic and membrane-bound β-catenin, indicating no increase in its membrane translocation. This was associated with a reduction in E-cadherin expression. PPARγ2 activation inhibited GSK3β kinase activity, and pharmacological inhibition of GSK3β activity was unable to restore β-catenin expression following PPARγ2 activation. Additionally, this β-catenin degradation pathway was operative in cells, with inactivating mutations of both APC and p53. Inhibition of the proteasomal pathway inhibited PPARγ2-mediated degradation of β-catenin, and incubation with TZD increased ubiquitination of β-catenin. We conclude that PPARγ2-mediated suppression of β-catenin levels involves a novel APC/ GSK3β/p53-independent ubiquitination-mediated proteasomal degradation pathway. [ABSTRACT FROM AUTHOR]

Activation of peroxisome proliferator-activated receptor γ (PPARγ) following exposure to PPARγ-specific ligands resulted in growth inhibition in various carcinoma cell lines. Our aim was to elucidate the pathway of PPARγ2 activation-mediated modulation of cyclin D1 transcription in mouse hepatocytes. To address this we utilized stable control and PPARγ hepatocyte cell lines created via retroviral overexpression utilizing AML-12 hepatocytes. Addition of PPARγ ligand troglitazone (TZD) activated PPARγ2 in proliferating hepatocytes and resulted in growth arrest accompanied by a down. regulation of proliferating cell nuclear antigen, cyclin D1, and β-catenin expression. Furthermore activation of PPARγ2 attenuated cyclin D1 promoter activity indicating a transcriptional regulation of cyclin D1. Since β-catenin plays a pivotal role in regulating cyclin D1 transcription, we studied whether PPARγ2-mediated inhibition of cyclin D1 transcription involved β-catenin. Interestingly overexpression of either wild-type or S37A mutant β-catenin was unable to rescue PPARγ2-mediated suppression of cyclin D1 transcription, whereas overexpression of cAMP-response element-binding protein (CREB) was capable of antagonizing this inhibitory effect of PPARγ2. Additionally pretreatment with okadaic acid antagonized PPARγ2-mediated inhibition of cyclin D1 transcription without any effect on β-catenin expression. These studies also showed a TZD-mediated inhibition of total and phospho-CREBser133 levels, CREB promoter activity, and cAMP-response element-mediated transcription in PPARγ hepatocytes. Pretreatment of PPARγ hepatocytes with okadaic acid, however, maintained higher total and phospho-CREBset133 levels in the presence of TZD. These results indicated that PPARγ2 activation inhibited cyclin D1 transcription in hepatocytes via CREB-dependent and β-catenin-independent pathways. Hepatocytes. [ABSTRACT FROM AUTHOR]

Describes a functional interaction between mixed lineage kinase 3 (MLK3) and AKT1/protein kinase B alpha. Regulation of the interaction of endogenous MLK3 and AKT1 with HepG2 cells by insulin; Attenuation of the endogenous kinase activities in HepG2 cells by insulin treatment; Inhibition of MLK3-mediated cell death in a manner dependent on serine 67 phosphorylation by AKT1.