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.

FIGURE 1.

Gene silencing of Map4k4 enhances PPARγ protein but not PPARγ mRNA in 3T3-L1 adipocytes. 3T3-L1 adipocytes 4 days post-differentiation induction were transfected with 7.5 nmol of either scrambled (Scr) or Map4k4 siRNA. 72 h later, cell lysates were examined by Western blot (A) and densitometry analysis for PPARγ1 and PPARγ2 (B). Total RNA was harvested and analyzed by quantitative real time PCR for PPARγ mRNA expression (C) and Map4k4 mRNA expression (E). D, Map4k4 protein expression. *, p < 0.01 when compared with scrambled siRNA-transfected samples by ANOVA (n = 4).

FIGURE 2.

Depletion of Map4k4 does not prolong the half-life of PPARγ protein in 3T3-L1 adipocytes. 3T3-L1 adipocytes 4 days post-differentiation induction were transfected with 7.5 nmol of either scrambled (Scr) or Map4k4 siRNA. 72 h later, the cells were treated with 5 μg/ml cycloheximide for the indicated times. Cell lysates were examined by Western blot (A) and densitometry analysis (B) for PPARγ1 and PPARγ2. Densitometry is representative of three independent experiments.

FIGURE 3.

Map4k4 gene silencing enhances protein synthesis and mTOR phosphorylation. 3T3-L1 adipocytes were transfected with 7.5 nmol of either scrambled (Scr) or Map4k4 siRNA 4 days post-differentiation induction. 72 h later, [³⁵S]Met/Cys protein labeling mix was added to cells for 1, 3, and 6 h. A, graphical representation of ³⁵S incorporation as detected by a scintillation counter (n = 3). The counts/min from each knockdown condition were normalized to total DNA content of that sample. Cell lysates were examined for phospho-mTOR (Ser-2448) by Western blot (B) and densitometry analysis (C). D, model depicting possible mechanism of PPARγ translational regulation through mTOR. Densitometry and reverse transcription-PCR are representative of three independent experiments. **, p < 0.01, *, p < 0.05 when compared with scrambled siRNA-transfected samples by ANOVA.

FIGURE 4.

Map4k4 gene silencing in 3T3-L1 adipocytes enhances 4E-BP1 phosphorylation. 4 days post-differentiation induction, 3T3-L1 adipocytes were transfected with 7.5 nmol of either scrambled (Scr) or Map4k4 siRNA. 72 h later, cell lysates were examined for phospho-4E-BP1 (Thr-36/Thr-45) by Western blot (A) and densitometry analysis (B). *, p < 0.05 when compared with scrambled siRNA-transfected samples by ANOVA. Densitometry is representative of three independent experiments.

FIGURE 5.

4E-BP1 and 4e-BP2 silencing does not enhance PPARγ protein level. 3T3-L1 adipocytes were transfected with 7.5 nmol of scrambled (Scr) or Map4k4 and 1.87 nmol of (each) 4E-BP1 and 4e-BP2 siRNA. 72 h later, cell lysates were examined for PPARγ protein levels as well as for efficiency of 4E-BP1 and 4e-BP2 gene silencing by SDS-PAGE followed by Western blot (A) and densitometry analysis (B). *, p < 0.05 when compared with scrambled siRNA transfected samples by ANOVA. Densitometry is representative of three independent experiments.

FIGURE 6.

4E-BP1 and 4e-BP2 are required for increased PPARγ protein levels upon Map4k4 gene silencing. 3T3-L1 adipocytes were transfected with 7.5 nmol of either scrambled (Scr) or Map4k4 and 1.87 nmol of 4E-BP1 and 4e-BP2 siRNA. 72 h later cell lysates were examined for PPARγ protein levels by Western blot (A) and densitometry analysis (B). *, p < 0.01 when compared with scrambled siRNA transfected samples by ANOVA. Densitometry is representative of three independent experiments.

FIGURE 7.

Rapamycin inhibits Map4k4 regulation of 4E-BP1 phosphorylation and PPARγ expression. 3T3-L1 adipocytes 4 days post-differentiation induction were transfected with 7.5 nmol of either scrambled (Scr) or Map4k4 siRNA. 72 h later, cells were treated with 20 nm of rapamycin for 0.5, 1, or 2 h. Cell lysates were examined for the appearance of the hypophosphorylated form (4E-BP1α), and changes in the phosphorylation levels of 4E-BP1 at Ser-64, Thr-69, and Thr-36/45 were analyzed using appropriate antibodies by Western blot (A) (representative blots) and densitometry (B) analysis (n = 5). *, p < 0.05 when compared with scrambled siRNA transfected samples by ANOVA.

FIGURE 8.

Adenovirus-mediated overexpression of Map4k4 decreases PPARγ protein level as well as mTOR (Ser-2448) phosphorylation in 3T3-L1 adipocytes. 4 days post-differentiation induction, 3T3-L1 adipocytes were infected with 40 μl (1.4 × 10¹² particles/ml) of either HA control (Ad-control) or 3HA-Map4k4-HA (Ad-Map4k4) adenovirus. 72 h post-infection, cell lysates were examined for PPARγ protein levels as well as Map4k4, HA, phospho-mTOR (Ser-2448), and total mTOR by Western blot (A) (representative blots) and densitometry (B) analysis (n = 4). *, p = 0.05 when compared with control adenovirus infected samples by ANOVA.

FIGURE 9.

Model for Map4k4-mediated PPARγ protein regulation. Our data support the following hypothesis. Silencing Map4k4 in 3T3-L1 adipocytes using siRNA enhances mTOR activity, as indicated by an increase in phosphorylation at Ser-2448. This in turn inhibits 4E-BP1 activity by enhancing phosphorylation at Thr-36, Thr-45, Ser-64, and Thr-69. Phosphorylation of 4E-BP1 renders it inactive leading to its dissociation from the translational initiation factor eIF4E. Thus eIF4E is activated, and cap-dependent translational initiation, responsible for the increase in PPARγ protein levels, is enhanced. However, 4E-BP1 silencing alone does not mimic Map4k4 depletion but blocks the Map4k4 effect, indicating it plays an additional role in regulation of PPARγ protein expression.