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Map4k4 negatively regulates peroxisome proliferator-activated receptor (PPAR) gamma protein translation by suppressing the mammalian target of rapamycin (mTOR) signaling pathway in cultured adipocytes.

ABSTRACT: The receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is considered a master regulator of adipocyte differentiation and promotes glucose and lipid metabolism in mature adipocytes. We recently identified the yeast Sterile 20 (Ste20) protein kinase ortholog, Map4k4, in an RNA interference-based screen as an inhibitor of PPARgamma expression in cultured adipocytes. Here, we show that RNA interference-mediated silencing of Map4k4 elevates the levels of both PPARgamma1 and PPARgamma2 proteins in 3T3-L1 adipocytes without affecting PPARgamma mRNA levels, suggesting that Map4k4 regulates PPARgamma at a post-transcriptional step. PPARgamma degradation rates are remarkably rapid as measured in the presence of cycloheximide (t(1/2) = 2 h), but silencing Map4k4 had no effect on PPARgamma degradation. However, depletion of Map4k4 significantly enhances [(35)S]methionine/cysteine incorporation into proteins, suggesting that Map4k4 signaling decreases protein translation. We show a function of Map4k4 is to inhibit rapamycin-sensitive mammalian target of rapamycin (mTOR) activity, decreasing 4E-BP1 phosphorylation. In addition, our results show mTOR and 4E-BP1 are required for the increased PPARgamma protein expression upon Map4k4 knockdown. Consistent with this concept, adenovirus-mediated expression of Map4k4 decreased PPARgamma protein levels and mTOR phosphorylation. These data show that Map4k4 negatively regulates PPARgamma post-transcriptionally, by attenuating mTOR signaling and a 4E-BP1-dependent mechanism.

SUBMITTER: Guntur KV

PROVIDER: S-EPMC2825455 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Map4k4 negatively regulates peroxisome proliferator-activated receptor (PPAR) gamma protein translation by suppressing the mammalian target of rapamycin (mTOR) signaling pathway in cultured adipocytes.

Guntur Kalyani V P KV Guilherme Adilson A Xue Liting L Chawla Anil A Czech Michael P MP

The Journal of biological chemistry 20091228 9

The receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is considered a master regulator of adipocyte differentiation and promotes glucose and lipid metabolism in mature adipocytes. We recently identified the yeast Sterile 20 (Ste20) protein kinase ortholog, Map4k4, in an RNA interference-based screen as an inhibitor of PPARgamma expression in cultured adipocytes. Here, we show that RNA interference-mediated silencing of Map4k4 elevates the levels of both PPARgamma1 and PPARgam ...[more]

PMID: 20038583

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Project description:PPAR (peroxisome-proliferator-activated receptor) gamma is expressed in brown and white adipose tissues and is involved in the control of differentiation and proliferation. Noradrenaline stimulates brown pre-adipocyte proliferation and brown adipocyte differentiation. The aim of the present study was thus to investigate the influence of noradrenaline on PPARgamma gene expression in brown adipocytes. In primary cultures of brown adipocytes, PPARgamma2 mRNA levels were 20-fold higher than PPARgamma1 mRNA levels. PPARgamma expression occurred during both the proliferation and the differentiation phases, with the highest mRNA levels being found at the time of transition between the phases. PPARgamma2 mRNA levels were downregulated by noradrenaline treatment (EC50, 0.1 microM) in both proliferative and differentiating cells, with a lagtime of 1 h and lasting up to 4 h, after which expression gradually recovered. The down-regulation was beta-adrenoceptor-induced and intracellularly mediated via cAMP and protein kinase A; the signalling pathway did not involve phosphoinositide 3-kinase, Src, p38 mitogen-activated protein kinase or extracellular-signal-regulated kinases 1 and 2. Treatment of the cells with the protein synthesis inhibitor cycloheximide not only abolished the noradrenaline-induced down-regulation of PPARgamma2 mRNA, but also in itself induced PPARgamma2 hyperexpression. The down-regulation was probably the result of suppression of transcription. The down-regulation of PPARgamma2 mRNA resulted in similar down-regulation of PPARgamma2 and phosphoPPARgamma2 protein levels. Remarkably, the level of PPARgamma1 protein was similar to that of PPARgamma2 (despite almost no PPARgamma1 mRNA), and the down-regulation by noradrenaline demonstrated similar kinetics to that of PPARgamma2; thus PPARgamma1 was apparently translated from the PPARgamma2 template. It is suggested that beta-adrenergic stimulation via cAMP and protein kinase A represses PPARgamma gene expression, leading to reduction of PPARgamma2 mRNA levels, which is then reflected in down-regulated levels of PPARgamma2, phosphoPPARgamma2 and PPARgamma1.

Project description:Although MK886 was originally identified as an inhibitor of 5-lipoxygenase activating protein (FLAP), recent data demonstrate that this activity does not underlie its ability to induce apoptosis [Datta, Biswal and Kehrer (1999) Biochem. J. 340, 371--375]. Since FLAP is a fatty-acid binding protein, it is conceivable that MK886 may affect other such proteins. A family of nuclear receptors that are activated by fatty acids and their metabolites, the peroxisome-proliferator-activated receptors (PPARs), have been implicated in apoptosis and may represent a target for MK886. The ability of MK886 to inhibit PPAR-alpha, -beta and -gamma activity was assessed using reporter assay systems (peroxisome-proliferator response element--luciferase). Using a transient transfection system in monkey kidney fibroblast CV-1 cells, mouse keratinocyte 308 cells and human lung adenocarcinoma A549 cells, 10--20 microM MK886 inhibited Wy14,643 activation of PPAR alpha by approximately 80%. Similar inhibition of PPAR alpha by MK886 was observed with a stable transfection reporter system in CV-1 cells. Only minimal inhibitory effects were seen on PPAR beta and PPAR gamma. MK886 inhibited PPAR alpha by a non-competitive mechanism as shown by its effects on the binding of arachidonic acid to PPAR alpha protein, and a dose-response study using a transient transfection reporter assay in COS-1 cells. An assay assessing PPAR ligand-receptor interactions showed that MK886 prevents the conformational change necessary for active-complex formation. The expression of keratin-1, a protein encoded by a PPAR alpha-responsive gene, was reduced by MK886 in a culture of mouse primary keratinocytes, suggesting that PPAR inhibition has functional consequences in normal cells. Although Jurkat cells express all PPAR isoforms, various PPAR alpha and PPAR gamma agonists were unable to prevent MK886-induced apoptosis. This is consistent with MK886 functioning as a non-competitive inhibitor of PPAR alpha, but may also indicate that PPAR alpha is not directly involved in MK886-induced apoptosis. Although numerous PPAR activators have been identified, the results show that MK886 can inhibit PPAR alpha, making it the first compound identified to have such an effect.

Dataset Information

Map4k4 negatively regulates peroxisome proliferator-activated receptor (PPAR) gamma protein translation by suppressing the mammalian target of rapamycin (mTOR) signaling pathway in cultured adipocytes.

Publications

Map4k4 negatively regulates peroxisome proliferator-activated receptor (PPAR) gamma protein translation by suppressing the mammalian target of rapamycin (mTOR) signaling pathway in cultured adipocytes.

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