Bifunctional role of Rev-erbalpha in adipocyte differentiation

Abstract

The nuclear receptor Rev-erbalpha is a potent transcriptional repressor that regulates circadian rhythm and metabolism. Here we demonstrate a dissociation between Rev-erbalpha mRNA and protein levels that profoundly influences adipocyte differentiation. During adipogenesis, Rev-erbalpha gene expression initially declines and subsequently increases. Remarkably, Rev-erbalpha protein levels are nearly the opposite, increasing early in adipogenesis and then markedly decreasing in adipocytes. The Rev-erbalpha protein is necessary for the early mitotic events that are required for adipogenesis. The subsequent reduction in Rev-erbalpha protein, due to increased degradation via the 26S proteasome, is also required for adipocyte differentiation because Rev-erbalpha represses the expression of PPARgamma2, the master transcriptional regulator of adipogenesis. Thus, opposite to what might be predicted from Rev-erbalpha gene expression, Rev-erbalpha protein levels must rise and then fall for adipocyte differentiation to occur.

FIG. 1.

Uncoupling of Rev-erbα mRNA and protein expressions during adipogenesis. (A) Quantitative RT-PCR showing initial repression and subsequent induction of Rev-erbα mRNA during normal 3T3-L1 adipocyte differentiation. (B) Western blot assay showing the initial decline and the subsequent decrease in Rev-erbα protein during adipogenesis. β-Actin served as a loading control, and aP2 was a positive control for differentiation. (C) Rev-erbα protein levels in adipocytes are increased by a 4-h treatment with the proteasome inhibitor MG132 at 20 μM. Ethanol (EtOH) was the solvent and served as a vehicle control.

FIG. 2.

Knockdown of endogenous Rev-erbα blocks adipocyte differentiation. (A) Quantitative PCR and Western blot assay showing knockdown of Rev-erbα in 3T3-L1 preadipocytes with a control β-gal- or Rev-erbα-specific shRNA. *, P < 0.05 (n = 3). (B) Phase-contrast microscopy and Oil Red O staining of day 7 3T3-L1 cells treated with control β-gal shRNA (upper panels) or Rev-erbα shRNA (lower panels). (C) Preadipocytes lacking Rev-erbα do not undergo the cell division normally required for adipogenesis. BrdU incorporation at day 2 in cells treated with no shRNA, β-gal control shRNA, or specific Rev-erbα shRNA is shown. Black bars indicate growth medium (GM), which was the negative control; gray bars indicate treatment with differentiation medium (DM). *, P < 0.05 (n = 3) compared with other shRNA treatments in differentiation medium. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIG. 3.

3T3-L1 cells conditionally expressing WT and degradation-resistant Rev-erbα. (A) Expression of Flag-tagged WT or degradation-resistant S55D/S59D (SD) Rev-erbα mRNA in Tet-off 3T3-L1 cells. Transgene expression is sensitive to inhibition by 2 μg/ml doxycycline (Dox). (B) Flag IP, followed by Western blotting, showing that the Tet-off WT and SD Rev-erbα proteins are also sensitive to doxycycline inhibition. (C) Western blot assay of WT and SD Rev-erbα proteins in Tet-off stable 3T3-L1 preadipocytes at various times after treatment with 20 μM CHX. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIG. 4.

Ectopic expression of degradation-resistant Rev-erbα blocks adipocyte differentiation. (A) Preadipocytes expressing WT or SD Rev-erbα expression vectors were differentiated for 9 days with or without 2 μg/ml doxycycline (Dox) and stained with Oil Red O. (B) aP2 mRNA levels in cells expressing ectopic WT and SD Rev-erbα. (C) aP2 protein levels in cells expressing ectopic WT and SD Rev-erbα. β-Actin served as a loading control. (D) Expression of the ectopic SD and WT Rev-erbα proteins during adipogenesis. β-Actin served as a loading control.

FIG. 5.

Rev-erbα expression represses PPARγ2. (A) Ectopic expression of degradation-resistant (SD), but not WT, Rev-erbα blocks PPARγ2 induction. (B) Retroviral expression of ectopic PPARγ2 in 3T3-L1 preadipocytes. (C) Ectopic expression of PPARγ2 rescues adipogenesis in 3T3-L1 cells ectopically expressing degradation-resistant SD Rev-erbα, as assessed by Oil Red O staining on day 7. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIG. 6.

Rev-erbα directly represses PPARγ2 promoter activity and expression. (A) Expression of PPARγ2-luciferase reporter transfected into 3T3-L1 preadipocytes along with 1 μg of either a WT or SD Rev-erbα expression plasmid or shRNA knockdown of endogenous Rev-erbα. Data shown are the averages of three independent experiments. Error bars represent standard deviations. (B) Ectopic expression of SD Rev-erbα in mature 3T3-L1 adipocytes reduces PPARγ2 and Bmal1 gene expression. *, P < 0.05 (n = 3). (C) shRNA knockdown of endogenous Rev-erbα increases the expression of the native PPARγ2 mRNA in preadipocytes, as well as a known Rev-erbα target gene, that for Bmal1. *, P < 0.05 (n = 3). (D) shRNA knockdown of endogenous Rev-erbα increases the expression of endogenous PPARγ2 protein in preadipocytes. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Dox, doxycycline.