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Molecular and Cellular Biology, April 2008, p. 2213-2220, Vol. 28, No. 7
0270-7306/08/$08.00+0     doi:10.1128/MCB.01608-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Bifunctional Role of Rev-erb in Adipocyte Differentiation

Jing Wang and Mitchell A. Lazar^*

Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine and Department of Genetics, and Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Received 31 August 2007/ Returned for modification 9 October 2007/ Accepted 20 January 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The nuclear receptor Rev-erb is a potent transcriptional repressor that regulates circadian rhythm and metabolism. Here we demonstrate a dissociation between Rev-erb mRNA and protein levels that profoundly influences adipocyte differentiation. During adipogenesis, Rev-erb gene expression initially declines and subsequently increases. Remarkably, Rev-erb protein levels are nearly the opposite, increasing early in adipogenesis and then markedly decreasing in adipocytes. The Rev-erb protein is necessary for the early mitotic events that are required for adipogenesis. The subsequent reduction in Rev-erb protein, due to increased degradation via the 26S proteasome, is also required for adipocyte differentiation because Rev-erb represses the expression of PPAR2, the master transcriptional regulator of adipogenesis. Thus, opposite to what might be predicted from Rev-erb gene expression, Rev-erb protein levels must rise and then fall for adipocyte differentiation to occur.

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The conversion of fibroblastic preadipocytes to mature adipocytes involves the temporal and hierarchical coordination of intracellular signaling molecules and transcription factors (11, 34). Differentiation of the widely used 3T3-L1 preadipocyte cell line requires extracellular signals, including cell contact, glucocorticoids, and serum-derived factors, as well as intracellular accumulation of cyclic AMP (39). Together, these initiators lead confluent preadipocytes to undergo two rounds of cell division that are required for adipogenesis (41). They also induce early transcription factors, notably, CCAAT enhancer-binding proteins (C/EBP) β and , to instigate a cascade of transcriptional events that ultimately results in withdrawal from the cell cycle in the process of differentiation into mature postmitotic adipocytes (8, 47, 49). C/EBPβ, in particular, induces nuclear receptor peroxisome proliferator-activated receptor (PPAR) (17, 35, 36, 47), the master transcriptional regulator of adipogenesis whose high-level expression in adipocytes is both necessary and sufficient for adipocyte-specific gene expression and the adipocyte phenotypes (6, 42, 43). PPAR and C/EBP induce one another to maintain the differentiated adipocyte phenotype (9, 33). Other transcription factors, such as ADD1/SREBP and KLF5, also play important roles in adipogenesis (23, 30).

Rev-erb is an orphan nuclear receptor that has also been implicated in adipocyte differentiation (5, 12, 24). It is highly expressed in adipose tissue, and its gene expression is specifically induced in differentiating adipocytes (5, 14). Rev-erb mRNA is transcribed from the opposite strand of the thyroid hormone receptor (TR) gene and is antisense to the TR2 splice product which encodes a non-thyroid hormone-binding TR variant (26, 29). The Rev-erb protein lacks the classical nuclear receptor C-terminal activation domain but binds to nuclear receptor corepressor (N-CoR) and hence functions as a potent transcriptional repressor (18). Recently, Rev-erb has been shown to function as a core negative component of the circadian clock, driving antiphasic expression of the positive feedback loop involving Bmal1 and Clock (3, 31). Rev-erb protein stability is subject to regulated, ubiquitin-targeted proteasomal degradation that is required for synchronization of cellular clocks (51). Rev-erb expression is circadian in adipose tissue (52), and Rev-erb directly modulates the rhythmic expression of plasminogen activator inhibitor 1, which is an adipokine (46). However, the function of the Rev-erb protein in adipocyte differentiation remains obscure.

Here we report that, surprisingly, levels of Rev-erb mRNA and protein are dissociated during adipogenesis, with the protein increasing early and decreasing late in the process. Rev-erb is required for adipogenesis, where it is critical for the early mitotic events. However, constitutive expression of Rev-erb inhibits the adipogenic program by repressing the expression of the gene for PPAR2. Proteasomal degradation is responsible for the decrease in endogenous Rev-erb protein levels that is normally permissive for adipogenesis. Thus, the dynamic expression of Rev-erb is an important determinant of adipocyte differentiation.

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Plasmids and reagents. The murine PPAR2-luciferase reporter construct was generated by PCR amplifying 700 bp of the proximal PPAR2 promoter, ending at the ATG start codon in exon 1, from mouse genomic DNA with the following primers: forward, 5'-TTCCTTTTTATAGAATTTGGATAGCA-3'; reverse, 5'-CCCAGAGTTTCACCCATAACA-3'. The PCR product was digested with KpnI and SacI and subcloned into a short-half-life pGL4.15 luc2P/Hygro vector (Promega, Madison, WI). The expression vectors encoding human Rev-erb have been described previously (51). Components of the adipocyte differentiation cocktail, including dexamethasone, insulin, and 3-isobutyl-1-methylxanthine (IBMX), as well as the protein synthesis inhibitor cycloheximide (CHX), were purchased from Sigma (St. Louis, MO).

Mammalian cell culture and transfection. 3T3-L1 cells were obtained from the American Type Culture Collection (Manassas, VA). 293T-derived BOSC viral packaging cells were a gift from M. Birnbaum (University of Pennsylvania). All cells were maintained in high-glucose, antibiotic-free Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum. Cells were grown at 37°C in 5% CO₂. Stable Tet-Off 3T3-L1 cell lines expressing ectopic wild-type (WT) or 55/59SD mutant Rev-erb were constructed by transient transfection with pTet-Off and pTRE-Rev-erb vectors (51) and subsequent selection in G418 and hygromycin. For the repression assay, cells were transfected in 12-well plates in triplicate with FuGENE (Roche) according to the manufacturer's instructions. Luciferase activities were assayed 48 h later with a Reporter Assay kit (Promega, Madison, WI).

Retroviral infection. Ecotropic BOSC packaging cells were a gift from M. Birnbaum (University of Pennsylvania) and were grown in six-well plates and transfected with FuGENE with the pMSCV backbone vector or the pMSCV-mPPAR2 expression vector (15). At 48 h posttransfection, filtered viral supernatants were used to infect target 3T3-L1 preadipocytes. Infection was performed twice over 48 h, and target cells were harvested for protein analysis or plated for differentiation.

Adipocyte differentiation. For in vitro adipogenic induction, 3T3-L1 preadipocytes were grown to confluence in growth medium consisting of DMEM supplemented with 10% differentiation grade fetal bovine serum (U.S. Biotechnologies) at 37°C. Differentiation was induced on day 0 (cells at 2 days postconfluence) by addition of 10 µg/ml insulin, 0.5 mM IBMX, and 0.25 µM dexamethasone. After 48 h, the medium was replaced with DMEM containing 10% fetal bovine serum and 10 µg/ml insulin only. Medium was changed every 48 h until cells had differentiated into mature adipocytes, at 6 to 7 days. Differentiation outcome was assessed by morphological examination by phase-contrast microscopy and Oil Red O staining for lipid accumulation and by protein and RNA analysis for the adipocyte marker gene aP2.

Oil Red O staining. Dishes were washed twice with phosphate-buffered saline and fixed by 100% methanol for 1 min at room temperature. After fixation, cells were stained with Oil Red O solution (PolyScientific Reagents) for 1 h at room temperature. Cells were rinsed with deionized water and differentiated by 85% propylene glycol for 1 min. Cells were rinsed again in deionized water and counterstained with hematoxylin for 30 s, followed by a final rinse in water, and then imaged.

Quantitative reverse transcription (RT)-PCR. Total mRNA was prepared with the RNeasy kit (Qiagen, Valencia, CA). RT was performed with 3 µg total RNA and the ImpromII RT kit (Promega, Madison, WI) according to the manufacturer's instructions. The cDNA was subjected to quantitative RT-PCR with an ABI Prism 7900 HT detection system (Applied Biosystems, Foster City, CA). All primers and probes were purchased from Applied Biosystems. Target gene expression was normalized to the housekeeping gene 36B4. The average cycle threshold value from each triplicate was used to calculate the relative induction of the gene, with the control group normalized to 1.

RNA silencing. Vectors expressing small hairpin interfering RNAs (shRNAs) under the control of the human U6 promoter were previously described (20, 51). The target sequences were as follows: β-galactosidase (β-gal), 5'-GTGCACCTGGTAAATCTTAT-3'; Rev-erb, 5'-GCCGGAGCATCCAACAGAATA-3'. Plasmids were electroporated into target cells with an Amaxa Nucleofection apparatus according to the manufacturer's instructions. Cells were allowed to recover for 24 to 48 h before use for protein and RNA analysis.

Immunoprecipitation (IP) and Western blotting. Cells were lysed in whole-cell lysis buffer (150 mM NaCl, 10 mM Tris [pH 7.6], 0.1% sodium dodecyl sulfate, 5 mM EDTA) with protease inhibitor, homogenized by vortexing, and centrifuged for 10 min at 4°C at 14,000 x g. The protein concentration of the supernatant was determined by a NanoDrop spectrophotometer, and 500 µg of whole-cell extract per sample was incubated with anti-Flag M2 agarose beads (Sigma) overnight at 4°C in Flag IP buffer (150 mM NaCl, 50 mM Tris [pH 8.0], 0.1% NP-40, 2 mM EDTA). Beads were subsequently pelleted, washed four times in IP buffer, and eluted by boiling in 2x sodium dodecyl sulfate buffer at 95°C for 10 min. Western blots were probed with the following antibodies: rabbit anti-Rev-erb (Cell Signaling), mouse anti-PPAR E8 antibody and anti-β-actin antibody (Santa Cruz), anti-glyceraldehyde-3-phosphate dehydrogenase-horseradish peroxidase (Abcam, Cambridge, MA), and rabbit anti-aP2 (gift from D. Bernlohr, University of Minnesota, Minneapolis). Quantitation of Western blot bands was performed with Photoshop CS2 software by selecting each band area and integrating the mean intensity and pixel value and then dividing the product by that of the standard band, which was either β-actin or glyceraldehyde-3-phosphate dehydrogenase. Relative intensity was then normalized to the control treatment or the initial time point, which was assigned a value of 1.

Bromodeoxyuridine (BrdU) assay. BrdU incorporation was assessed by a BrdU Cell Proliferation Assay kit (Calbiochem). 3T3-L1 preadipocytes first received shRNA against β-gal or endogenous Rev-erb and were plated in 96-well plates. At 48 h later, cells were treated with differentiation medium and labeled with BrdU for 12 h, after which cells were fixed and stained with anti-BrdU antibody and visualized in a colorimetric immunoassay. Spectrum absorbance was measured on a Bio-Tek Synergy HT plate reader.

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Rev-erb mRNA and protein levels are uncoupled during adipogenesis. We confirmed that Rev-erb mRNA decreases in the first 24 h and then is markedly induced during adipocyte differentiation (5, 14) (Fig. 1A). Surprisingly, we found that Rev-erb protein levels increase during the initial 24 h but then decrease (Fig. 1B), the opposite of the mRNA expression pattern. Given that Rev-erb is regulated posttranscriptionally and represses its own gene expression (1, 51), we hypothesized that the discrepancy between Rev-erb mRNA levels in the mature adipocyte is due to enhanced proteasomal degradation of the protein. Indeed, an acute 4-h treatment with the proteasome inhibitor MG132 at 20 µM stabilized Rev-erb protein in differentiating adipocytes, an effect that was much more pronounced in cells after day 4 than in day 0 preadipocytes (Fig. 1C). Thus, Rev-erb protein appears to be regulated by increasing proteasomal degradation during late adipogenesis.

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FIG. 1. Uncoupling of Rev-erb mRNA and protein expressions during adipogenesis. (A) Quantitative RT-PCR showing initial repression and subsequent induction of Rev-erb mRNA during normal 3T3-L1 adipocyte differentiation. (B) Western blot assay showing the initial decline and the subsequent decrease in Rev-erb protein during adipogenesis. β-Actin served as a loading control, and aP2 was a positive control for differentiation. (C) Rev-erb 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.

Endogenous Rev-erb is required for adipocyte differentiation. To determine the role of Rev-erb expression in adipocyte differentiation, we used an shRNA against murine Rev-erb to inhibit the expression of Rev-erb in 3T3-L1 preadipocytes prior to subjecting the cells to differentiation induction (Fig. 2A). Compared to control cells treated with an irrelevant shRNA, knockdown of Rev-erb dramatically reduced the differentiation capacity of the cells, as assessed by morphological examination and Oil Red O staining (Fig. 2B).

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FIG. 2. Knockdown of endogenous Rev-erb blocks adipocyte differentiation. (A) Quantitative PCR and Western blot assay showing knockdown of Rev-erb in 3T3-L1 preadipocytes with a control β-gal- or Rev-erb-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-erb shRNA (lower panels). (C) Preadipocytes lacking Rev-erb 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-erb 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.

Since mitotic clonal expansion is required for adipocyte differentiation (41), we examined whether Rev-erb knockdown had altered the proliferative capacity of the cells. Indeed, cells depleted of Rev-erb had a significantly decreased mitotic rate, as determined by BrdU uptake upon exposure to differentiation medium (Fig. 2C). Thus, the early increase in Rev-erb protein is required for the mitotic events that are an obligatory step in adipocyte differentiation.

Proteasomal degradation of Rev-erb is required for adipogenesis. Since Rev-erb gene expression increases during adipocyte differentiation, we and others have suggested that Rev-erb would enhance adipogenesis (5, 12), an idea that is consistent with our observation that Rev-erb knockdown prevents adipogenesis. However, having noted that Rev-erb protein levels decrease as adipogenesis progresses, due to proteasomal degradation, we hypothesized that the loss of Rev-erb protein is also critical for late stages of adipocyte differentiation. To test this, we used a Tet-off system to conditionally express Flag-tagged WT Rev-erb or the S55D/S59D Rev-erb mutant (SD) that is resistant to proteasomal degradation (51). In the absence of doxycycline, these 3T3-L1-derived cell lines stably expressed the ectopic Rev-erb mRNA and protein, while the addition of 2 µg/ml doxycycline led to a marked decrease in the transgenes (Fig. 3A and B). Consistent with our expectations, steady-state levels of the SD Rev-erb protein were higher than WT Rev-erb levels despite similar mRNA expression levels, reflecting the increased stability of the SD protein. Analysis of protein levels following CHX treatment confirmed that the half-life of the SD Rev-erb protein was markedly longer than that of WT Rev-erb in 3T3-L1 cells (Fig. 3C).

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FIG. 3. 3T3-L1 cells conditionally expressing WT and degradation-resistant Rev-erb. (A) Expression of Flag-tagged WT or degradation-resistant S55D/S59D (SD) Rev-erb 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-erb proteins are also sensitive to doxycycline inhibition. (C) Western blot assay of WT and SD Rev-erb proteins in Tet-off stable 3T3-L1 preadipocytes at various times after treatment with 20 µM CHX. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

We next tested the functional outcome of Rev-erb protein stabilization in adipogenesis. In the presence of doxycycline, both the WT and SD cells displayed normal adipogenesis (Fig. 4A, upper panels), suggesting that the genetic manipulation of the cells had not nonspecifically altered their function as preadipocytes. In the absence of doxycycline, cells that expressed ectopic WT Rev-erb also differentiated normally (Fig. 4A, lower left panel). In contrast, expression of degradation-resistant SD Rev-erb markedly impaired adipocyte differentiation (Fig. 4A, lower right panel). Differentiation status was also monitored by induction of the adipogenic marker aP2, which was greatly diminished in SD Rev-erb-expressing cells, at both the mRNA (Fig. 4B) and protein (Fig. 4C) levels. Note that the ectopic WT and SD Rev-erb proteins followed different patterns of expression during adipogenesis. In the absence of doxycycline, the WT protein was initially expressed but became destabilized after day 4 (Fig. 4D), much like the endogenous protein (cf. Fig. 1A). By contrast, the SD protein was constitutively expressed at a higher level throughout adipogenesis (Fig. 4D), likely explaining its more marked effect on adipogenesis.

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FIG. 4. Ectopic expression of degradation-resistant Rev-erb blocks adipocyte differentiation. (A) Preadipocytes expressing WT or SD Rev-erb 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-erb. (C) aP2 protein levels in cells expressing ectopic WT and SD Rev-erb. β-Actin served as a loading control. (D) Expression of the ectopic SD and WT Rev-erb proteins during adipogenesis. β-Actin served as a loading control.

Stable expression of Rev-erb protein prevents induction of PPAR. To determine the mechanism by which constitutive Rev-erb protein expression inhibited adipogenesis, we examined the expression of PPAR2, the "master" transcriptional regulator of adipogenesis. As expected, PPAR2 expression robustly increased during adipogenesis of control preadipocytes, as well as in cells expressing ectopic WT Rev-erb (Fig. 5A). By contrast, the induction of PPAR2 was dramatically blunted in cells expressing degradation-resistant SD Rev-erb. To test whether the failure to induce PPAR2 was responsible for the inability of these cells to differentiate, we used retroviral vectors to force the expression of PPAR2 (Fig. 5B). Indeed, ectopic expression of PPAR2 rescued the adipogenic phenotype (Fig. 5C), indicating that the differentiation block resulted from repression of PPAR2 expression by the SD Rev-erb protein.

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FIG. 5. Rev-erb expression represses PPAR2. (A) Ectopic expression of degradation-resistant (SD), but not WT, Rev-erb blocks PPAR2 induction. (B) Retroviral expression of ectopic PPAR2 in 3T3-L1 preadipocytes. (C) Ectopic expression of PPAR2 rescues adipogenesis in 3T3-L1 cells ectopically expressing degradation-resistant SD Rev-erb, as assessed by Oil Red O staining on day 7. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Rev-erb represses PPAR2 gene expression. Because Rev-erb is a potent transcriptional repressor, we hypothesized that it might directly repress PPAR expression. Indeed, overexpression of WT Rev-erb repressed the luciferase activity of a murine PPAR2 reporter in 3T3-L1 cells (Fig. 6A). Expression of SD Rev-erb led to even greater repression of the mPPAR2 promoter, indicating that the SD Rev-erb mutant was not functionally defective and was, in fact, a more potent repressor, likely due to its expression at higher levels. Conversely, knockdown of endogenous Rev-erb led to increased mPPAR2 promoter activity, indicating that, at its endogenous level, Rev-erb suppresses PPAR2 gene expression (Fig. 6A). Ectopic expression of SD Rev-erb by removal of doxycycline significantly repressed endogenous PPAR2 and Bmal1 expression in mature adipocytes (Fig. 6B). Consistent with this, Rev-erb knockdown increased native PPAR2 mRNA, as well as the expression of Bmal1, a known Rev-erb target gene (Fig. 6C). A similar result was obtained with a second, nonoverlapping shRNA targeting Rev-erb (not shown), but not an off-target effect because it was not seen with a control shRNA directed at β-gal (Fig. 6C). Knockdown of Rev-erb also induced endogenous PPAR2 protein in preadipocytes (Fig. 6D). Thus, Rev-erb appears to be a regulator of PPAR2 in 3T3-L1 cells and constitutive expression of Rev-erb protein prevents adipogenesis by inhibiting PPAR2 induction.

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FIG. 6. Rev-erb directly represses PPAR2 promoter activity and expression. (A) Expression of PPAR2-luciferase reporter transfected into 3T3-L1 preadipocytes along with 1 µg of either a WT or SD Rev-erb expression plasmid or shRNA knockdown of endogenous Rev-erb. Data shown are the averages of three independent experiments. Error bars represent standard deviations. (B) Ectopic expression of SD Rev-erb in mature 3T3-L1 adipocytes reduces PPAR2 and Bmal1 gene expression. *, P < 0.05 (n = 3). (C) shRNA knockdown of endogenous Rev-erb increases the expression of the native PPAR2 mRNA in preadipocytes, as well as a known Rev-erb target gene, that for Bmal1. *, P < 0.05 (n = 3). (D) shRNA knockdown of endogenous Rev-erb increases the expression of endogenous PPAR2 protein in preadipocytes. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Dox, doxycycline.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Since expression of the gene for Rev-erb is induced during adipogenesis, it has been suggested that Rev-erb may be proadipogenic. Here we show that Rev-erb actually has a bipartite function, reflected by the dissociation between its mRNA and protein expressions. Rev-erb is indeed required for adipocyte differentiation; this requirement is early, during the period of greatest Rev-erb protein expression, and due to a permissive role for Rev-erb during the cell proliferation stage that is crucial for adipogenesis of 3T3-L1 cells. Remarkably, this period is when Rev-erb mRNA levels are lowest. Later in adipogenesis, when Rev-erb gene expression is highest, Rev-erb protein levels are actually low and forced expression of Rev-erb prevents adipogenesis by repressing expression of the master adipogenic transcription factor PPAR.

The lack of correlation between Rev-erb mRNA and protein levels is interesting, and similar observations have been reported for a number of other genes (21). Of note, while this work was in progress, the Nuclear Receptor Signaling Alliance confirmed our findings for Rev-erb protein, as well as mRNA, during adipogenesis (48). The patterns of Rev-erb protein and mRNA expression are nearly antiphasic, most likely because Rev-erb potently represses its own gene expression (1, 51) but is independently and posttranslationally regulated by proteasomal degradation. Increased proteasomal degradation of Rev-erb in late adipogenesis reduces the steady-state protein level, which depresses Rev-erb gene expression. Consistent with this, we have observed that ectopic expression of degradation-resistant SD Rev-erb markedly suppresses the endogenous Rev-erb mRNA level in 3T3-L1 cells (data not shown).

The coupling of Rev-erb protein stabilization to early clonal expansion in adipogenesis suggests a potential linkage between circadian cycle and cell cycle regulators. Consistent with this, other core circadian cycle proteins, such as PER and TIM, have been shown to interact with components of the cell cycle machinery (16, 44). Furthermore, the expression of cell cycle genes such as those for Wee1, cyclins, and c-Myc is under circadian regulation (13, 28). Given the role of Rev-erb as a major feedback circadian cycle regulator, Rev-erb may participate in the circadian control of cell cycle gene expression. It should be noted that while mitotic clonal expansion is a requirement for adipocyte differentiation of 3T3-L1 cells, this may be less critical in other models of adipogenesis (7, 10).

Rev-erb protein loss in late adipogenesis is due to reduced stability that is mediated by proteasomal degradation, which we have demonstrated by assessing the effect of the 26S proteasomal inhibitor MG132 on successive days in adipocyte differentiation. Rev-erb protein is stabilized by GSK3β-dependent phosphorylation at S55 and S59 (51), and hence, the reduced stability of Rev-erb is potentially explained by the decrease in GSK3β activity that has been shown to occur during 3T3-L1 adipocyte differentiation (4, 27). However, we and others have not consistently observed altered phosphorylation of GSK3β, which regulates its activity, during adipogenesis (40; data not shown). Thus, it is possible that other mechanisms play a role in destabilizing Rev-erb protein in adipogenesis.

Mutation of serines 55 and 59 to aspartate, which mimics phosphorylation, results in a protein (SD) that resists degradation and disrupts differentiation in adipocytes. Rev-erb protein stabilization leads to suppression of PPAR2 gene transcription, which is normally induced in adipogenesis by a hierarchical regulatory cascade that is initiated by C/EBPβ (36, 47) and perpetuated by C/EBP, as well as positive feedback from PPAR2 itself (11, 33, 37). Our data demonstrate that SD Rev-erb is capable of dominantly repressing PPAR2 induction during adipogenesis, and hence, the reduction in Rev-erb protein level seen in normal adipogenesis may play a permissive role in differentiation. Repression of PPAR2 promoter activity could be a direct effect of Rev-erb, although mutation of two putative Rev-erb-responsive elements in the PPAR2 promoter did not abrogate the effect of Rev-erb (data not shown). Thus, the effect of Rev-erb could be due to cryptic Rev-erb-responsive sequences or could be indirect, for example, by repression of the transcriptional activator Bmal1, which is encoded by a well-established Rev-erb target gene (50), which has been shown to promote adipogenesis (38).

The finding that Rev-erb protein decreases in adipogenesis is surprising given the increase in its mRNA, which raises the question of whether induction of Rev-erb mRNA during adipocyte differentiation has a function. Rev-erb has an important function in the circadian clock which may be important for mature adipocytes, whose circadian clock oscillations are robust and coordinated with the expression of many adipokines and metabolic enzymes (2, 52). Thus, it is possible that the relatively high level of Rev-erb gene expression in mature adipocytes reflects a circadian function. It is also intriguing that Rev-erb mRNA expression modulates the alternative splicing of the gene for TR, which governs the ratio of TR1 and TR2, two factors that facilitate or inhibit thyroid hormone action, respectively (19, 25). An accumulation of Rev-erb mRNA in mature adipocytes would therefore favor TR1 mRNA production and increase the cellular response to thyroid hormone, which is important for maintaining metabolic homeostasis (22, 32, 45).

In summary, we have uncovered a mechanism by which the rise and fall of Rev-erb protein may benefit adipocyte differentiation. The striking dissociation between Rev-erb mRNA and protein during adipogenesis indicates that Rev-erb may be regulated differently at the transcriptional and posttranslational levels. Indeed, stabilization of the Rev-erb protein, mediated by phosphorylation at serines 55 and 59, has a dominant effect in suppressing the adipogenic gene expression program. It will be interesting to explore, in future studies, the regulatory pathways that lead to altered Rev-erb protein expression in adipogenesis.

 
We thank Morris Birnbaum (University of Pennsylvania, Philadelphia) for the gift of BOSC retroviral packaging cells and David Bernlohr (University of Minnesota, Minneapolis) for anti-aP2 antiserum. We also thank Lei Yin for helpful discussions and other members of the Lazar laboratory for support.

This work was supported by NIH grant DK45586 (to M.A.L.).

 
* Corresponding author. Mailing address: University of Pennsylvania School of Medicine, 700 CRB, 415 Curie Blvd., Philadelphia, PA 19104-6149. Phone: (215) 898-0198. Fax: (215) 898-5408. E-mail: lazar{at}mail.med.upenn.edu

Published ahead of print on 28 January 2008.

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Molecular and Cellular Biology, April 2008, p. 2213-2220, Vol. 28, No. 7
0270-7306/08/$08.00+0     doi:10.1128/MCB.01608-07
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