Inhibition of Adipocyte Differentiation by Nur77, Nurr1, and Nor1

Abstract

Members of the nuclear receptor 4A (NR4A) subgroup of nuclear receptors have been implicated in the regulation of glucose and lipid metabolism in insulin-sensitive tissues such as liver and skeletal muscle. However, their function in adipocytes is not well defined. Previous studies have reported that these receptors are rapidly up-regulated after treatment of 3T3-L1 preadipocytes with an adipogenic cocktail. We show here that although Nur77 expression is acutely induced by cAMP agonists in 3T3-L1 cells, it is not induced by other adipogenic stimuli, such as peroxisome proliferator-activated receptor-γ ligands, nor is it induced during the differentiation of 3T3-F442A preadipocytes, suggesting that Nur77 induction is not an obligatory feature of preadipocyte differentiation. We further demonstrate that inflammatory signals that antagonize differentiation, such as TNFα and lipopolysaccharide, acutely induce Nur77 expression both in vitro and in vivo. We also show that NR4A expression in adipose tissue is responsive to fasting/refeeding. Retroviral transduction of each of the NR4A receptors (Nur77, Nurr1, and NOR1) into either 3T3-L1 or 3T3-F442A preadipocytes potently inhibits adipogenesis. Interestingly, NR4A-mediated inhibition of adipogenesis cannot be rescued by peroxisome proliferator-activated receptor-γ overexpression or activation. Transcriptional profiling of Nur77-expressing preadipocytes led to the identification of gap-junction protein α1 (Gja1) and tolloid-like 1 (Tll1) as Nur77-responsive genes. Remarkably, retroviral expression of either Gja1 or Tll1 in 3T3-L1 preadipocytes also inhibited adipocyte differentiation, implicating these genes as potential mediators of Nur77’s effects on adipogenesis. Finally, we show that Nur77 expression inhibits mitotic clonal expansion of preadipocytes, providing an additional mechanism by which Nur77 may inhibit adipogenesis.

STUDIES UTILIZING various in vitro preadipocyte cell lines, mouse embryonic fibroblasts, and in vivo murine models have revealed adipogenesis to be a well-orchestrated process marked by changes in cell proliferation/differentiation and the sequential induction of key transcription factors. In the 3T3-L1 preadipocyte cell line originally isolated from nonclonal Swiss 3T3 cells (1–3), proliferation of preadipocytes ceases upon confluence. The addition of commonly used differentiation cocktail, consisting of dexamethasone, phosphodiesterase inhibitor, and insulin, triggers mitotic clonal expansion, which appears to be an essential aspect of adipogenesis in 3T3-L1 cell line. After one to two rounds of mitosis, growth arrest occurs again, and early adipogenic transcription factors such as CCAAT enhancer binding protein (C/EBP)β and C/EBPδ are expressed (4, 5). These two proteins subsequently induce the up-regulation of peroxisome proliferator-activated receptor (PPAR)γ, the master transcriptional regulator of adipogenesis (6, 7). PPARγ drives the expression of C/EBPα, which in a positive feedback loop further amplifies the expression of the former. These factors contribute to the phenotype of triglyceride accumulation and the expression of differentiation-dependent metabolic genes such as fatty acid binding protein 4 (also known as aP2), adiponectin, phosphoenol pyruvate carboxykinase, and the insulin-sensitive glucose transporter 4.

Among transcription factors that regulate metabolism, nuclear receptors are particularly important in coordinating changes in environmental milieu with downstream metabolic pathways in insulin-sensitive tissues. A newcomer to this group is the orphan nuclear receptor 4A (NR4A) subfamily. The NR4A subfamily consists of three isotypes, commonly known as Nur77, Nurr1, and NOR1 (NR4A1, 2, and 3). These receptors have pleotropic functions ranging from suppression of leukemogenesis to dopaminergic neuron development (8–11). In recent years, the NR4A receptors have also been established as transcriptional regulators of hepatic gluconeogenesis (12). In skeletal muscle, these receptors regulate the expression of glucose utilization as well as oxidative phosphorylation genes (13, 14). However, the function of NR4A receptors in adipocytes is relatively poorly understood.

Recently, Fu et al. (15) reported that preadipocyte expression of NR4A receptors was induced within 30 min of treatment by either standard differentiation cocktail [dexamethasone, isobutylmethylxanthine (IBMX), and insulin] or the PPARγ ligand rosiglitazone. Others have reported, however, that induction of NR4A receptors occurs some 24–48 h after the addition of the PPARγ ligands troglitazone or pioglitazone to fully differentiated adipocytes (16). On the other hand, Au et al. (17) have suggested that NR4A receptors are not required for adipogenesis. Collectively, these studies raise the possibility that the NR4As may be involved in the regulation of adipocyte gene expression during differentiation. However, no target genes for NR4As in adipocytes have yet been described. Moreover, the precise function of NR4A receptors in adipogenesis remains unclear.

To address NR4A functions in adipogenesis, we generated stable 3T3-L1 and 3T3-F442A preadipocyte cell lines overexpressing each of the three NR4A receptors. Unexpectedly, expression of the NR4A receptors strongly inhibited differentiation. Furthermore, NR4A-mediated inhibition of adipogenesis could not be rescued by overexpression of PPARγ or by treatment with a PPARγ ligand. We also identified and validated two potential mediators of this process: gap-junction protein, α1 (Gja1) and tolloid-like 1 (Tll1). Finally, we showed that NR4A1 expression disrupted mitotic clonal expansion in 3T3-L1 cells, providing another mechanism by which NR4A receptors inhibit adipogenesis.

RESULTS

Nur77 Expression during Preadipocyte Differentiation

Previous work on NR4A receptors in adipogenesis suggested that these receptors are rapidly induced upon differentiation, in response to either standard differentiation cocktail (DMI) consisting of dexamethasone, IBMX, and insulin, or a PPARγ ligand (15). As NR4A receptors are positively regulated by cAMP-responsive element binding protein (18, 19), the rapid and transient up-regulation of NR4A in response to adipogenic cocktail likely reflects increased extracellular cAMP concentration induced by the phosphodiesterase inhibitor IBMX. Furthermore, NR4A receptors are inducible by various growth factors and may be responsive to changes in media (20, 21).

We examined the expression of Nur77 in response to the standard differentiation cocktail (DMI) or PPARγ ligand GW7845 and insulin in 3T3-L1 preadipocytes. To avoid fluctuations in Nur77 expression due to serum refreshment, chemicals were diluted in serum-free DMEM and added to cells in volumes less than 1% of total media volume. As shown in Fig. 1A, Nur77 mRNA was rapidly but transiently induced in response to DMI within 1 h of differentiation. Nur77 protein level peaked at 2 h and remained elevated 8 h after addition of 8-bromo-cAMP (8-Br-cAMP) (Fig. 1B). The addition of GW7845 and insulin resulted in minimal change in Nur77 expression. We also examined the expression of Nur77 in 3T3-F442A preadipocytes, which requires only insulin for adipogenesis. As Fig. 1C shows, Nur77 is not up-regulated within the early hours of differentiation in 3T3-F442A cells. Expression of the fatty acid binding protein aP2 is shown as a marker of adipocyte differentiation for both 3T3-L1 and 3T3-F442A cells.

Fig. 1.

Induction of Nur77 Expression by cAMP Signaling during Adipogenesis A, 3T3-L1 preadipocytes were grown to confluence (d 0) and then differentiated as described elsewhere (53 ), except that all chemicals were added directly to the cells without media refreshment. B, cAMP induction of Nur77 protein in 3T3-L1 preadipocytes. Nuclear extract was immunoblotted with Nur77 and HMG-1. Time represents hours after 8-Br-cAMP (1 mm) treatment. Controls for Nur77 protein (first two lanes) were total cell lysates from HEK-293T cells transfected with control or CMX-Nur77 plasmid. C, 3T3-F442A preadipocytes were differentiated with 0.5 μg/ml insulin. D, Confluent 3T3-L1 preadipocytes were treated with PBS, 1 μm dexamethasone (Dex), 0.5 mm IBMX, or 0.5 μg/ml insulin. Chemicals were added directly without media refreshment. E, Confluent 3T3-L1 preadipocytes were treated with vehicle control, 8-Br-cAMP (1 mm), PPARγ agonist GW7845 (100 nm), PPARγ antagonist GW9662 (1 μm), BRL (1 μm rosiglitazone), or combinations thereof. D and E, Cells harvested with Trizol (Invitrogen) 1 h after chemical addition. *, P < 0.05; **, P < 0.01 vs. time zero (panels A and C) vs. PBS (panel D) vs. dimethylsulfoxide (DMSO) (panel E). Ctrl, Control.

To further delineate the component in DMI that may account for the induction of Nur77 observed in Fig. 1A, 3T3-L1 preadipocytes were treated for 1 h with PBS, 0.5 μg/ml insulin, 1 μm dexamethasone, or 0.5 mm IBMX. As suspected, the rapid induction of Nur77 as well as the other NR4A isotypes in differentiating 3T3-L1 cells occurred in response to IBMX (Fig. 1D). Of note, Nur77 expression was also slightly increased by insulin, which likely accounts for the increase in Nur77 in 3T3-L1 cells treated with GW7845/insulin. To confirm that PPARγ ligands do not induce NR4A expression, 3T3-L1 preadipocytes were incubated for 1 h with various combinations of PPARγ agonists (GW7845, BRL/rosiglitazone) and antagonist (GW9662). As shown in Fig. 1E, none of the PPARγ ligands was capable of inducing NR4A receptor expression. 8-Br-cAMP was included as a positive control. These data demonstrate that early up-regulation of NR4A receptors observed during adipocyte differentiation reflects cAMP-mediated regulation of these receptors. Furthermore, although insulin may contribute to the increase in Nur77 expression during adipogenesis, PPARγ activation alone has no significant effect on Nur77 expression.

Nur77 Is Induced by Inflammatory Signals in Adipocytes

Inflammatory signals such as TNFα and lipopolysaccharide (LPS) are known inhibitory factors in adipogenesis (22, 23). In addition, TNFα, LPS, and other proinflammatory stimuli have been shown to be potent inducers of Nur77 in macrophages (24, 25). We therefore examined whether these inflammatory triggers can induce Nur77 expression in adipocytes. In 3T3-L1 preadipocytes, various inflammatory stimuli were added directly to the culture media, to minimize serum-induced fluctuation of Nur77. As shown in Fig. 2A, Nur77 was induced approximately 2-fold by TNFα and LPS in 3T3-L1 preadipocytes. To determine whether Nur77 is similarly up-regulated in vivo, wild-type C57Bl/6 mice were injected with PBS or LPS (5 mg/kg). Epididymal white adipose tissue was isolated and flash frozen 1 h after injection. Total RNA preparation from the adipose tissue revealed that, compared with PBS-injected controls, LPS robustly up-regulated Nur77 expression (Fig. 2B). This finding suggests that one plausible physiological function of Nur77 is to mediate inflammation-induced inhibition of lipid accumulation and adipogenesis.

Fig. 2.

NR4A Expression Is Induced by Inflammatory Stimuli in Vitro and in Vivo A, Confluent 3T3-L1 preadipocytes were treated with: PBS, mTNFα (25 ng/ml), interferon (IFN)γ (100 U/ml), mIL1β (20 ng/ml), murine epidermal growth factor (mEGF) (10 ng/ml), LPS (1 μg/ml), LTA (100 ng/ml), polyinosinic acid (polyIC) (2 μg/ml), CpG (100 nm). Total RNA was isolated 1 h later. B, Six age-matched nonfasted wild-type male C57Bl/6 mice were injected with PBS or LPS, 5 mg/kg ip. Epididymal white adipose tissue was isolated and flash frozen exactly 1 h after injection. Total RNA was prepared as described in Materials and Methods. *, P < 0.05; **, P < 0.01 vs. PBS. LTA, Lipoteichoic acid; CpG, CpG oligonucleotide.

Fasting Induces Expression of NR4A Receptors

We have previously demonstrated that fasting potently induces hepatic NR4A receptor expression to enhance gluconeogenesis (12). To determine whether NR4A receptors are similarly up-regulated in white adipose tissue in response to fasting, we measured NR4A expression of epididymal fat pads from fasted and fasted/refed wild-type and ob/ob mice. As shown in Fig. 3, Nur77 and NOR1 expression was markedly reduced in the fed state. Nurr1 expression did not appear to be down-regulated by refeeding. Interestingly, ob/ob mice appeared to lack the feeding-regulated suppression of Nur77 and NOR1.

Fig. 3.

NR4A Expression in White Adipose Tissue Male wild-type (WT) C57Bl/6 and ob/ob mice (n = 8–10) were fasted for 24 h. A subgroup was refed for 8 h. *, P < 0.05; **, P < 0.01, WT vs. ob/ob. ‡, P < 0.05, ‡‡, P < 0.01, fasting vs. refed.

NR4A Overexpression Inhibits Adipogenesis

NR4A receptors have been shown to mediate various metabolic processes in insulin-sensitive tissues such as liver and skeletal muscle (12–14, 26, 27). To determine the function of NR4A receptors in adipocytes, we first tested their roles in differentiation. Using retroviral transduction, each of the three NR4A isotypes was stably overexpressed in 3T3-L1 preadipocytes. As an additional control, we also tested the effect of Nur77–898, an N-terminally truncated dominant-negative receptor (deletion of the first 897 nucleotides of Nur77). Deletion of the N-terminal transactivation domain has previously been shown to antagonize the activity of all three NR4A isotypes (12, 17, 28). After puromycin selection of large mixed pool of cells (not single clones), each cell line was differentiated in standard differentiation cocktail for 7 d (DMI for 2 d, followed by insulin alone for 5 d). As shown in Fig. 4, expression of each of the NR4A receptors, but not Nur77–898, strongly inhibited adipogenesis of 3T3-L1 preadipocytes (Fig. 4, top row). Similar results were obtained with multiple independent sets of stable lines.

Fig. 4.

NR4A Receptors Inhibit the Differentiation of 3T3 Preadipocytes Stable cell lines were generated by retroviral transduction as described in Materials and Methods. 3T3-L1 cells were induced to differentiate with DMI alone (top row) or DMI and GW7845 (third row). 3T3-L1 cells expressing both PPARγ and NR4A receptors were differentiated with DMI (bottom row). Induction media was replaced 2 d later with 10% FBS containing insulin, with or without GW7845. 3T3-F442A cell lines were differentiated with 10% FBS containing insulin (second row). Cells were stained with Oil Red O as described in Materials and Methods.

Our finding that NR4A receptors inhibit adipogenesis was reproduced in 3T3-F442A cells (Fig. 4, second row) as well as in C3H10T1/2 cells (data not shown). Even more striking was the observation that treatment of the NR4A-expressing 3T3-L1 preadipocytes with the PPARγ ligand GW7845 (in addition to the standard DMI cocktail) was unable to rescue adipogenesis (Fig. 4, third row). Likewise, NR4A-mediated inhibition of adipogenesis in 3T3-L1 preadipocytes could not be overcome even when PPARγ was ectopically expressed (Fig. 4, bottom row). These data identify NR4A receptors as regulators of adipogenesis. The observation that PPARγ activation is unable to rescue adipogenesis in NR4A-expressing 3T3-L1 preadipocytes suggests that NR4A receptors regulate a PPARγ-independent pathway in adipogenesis (see below).

Examination of the expression of early adipogenic transcription factors revealed that C/EBPβ and C/EBPδ were expressed at lower levels in 3T3-L1 preadipocytes overexpressing Nur77 (Fig. 5A). We observed an increase in expression of GATA2, a known inhibitor of adipogenesis (29), 24 h after DMI induction in Nur77-expressing cells. There was also a subtle increase in expression of PPARγ 24 h after DMI treatment. Given the minimal expression level of PPARγ at this time point, the change is likely of little biological significance. Expression of Pref-1, another inhibitor of adipogenesis (30, 31), was unaffected (Fig. 5B). The changes in expression of these early adipocyte transcription factors suggest that Nur77 is acting within 24 h of induction of differentiation.

Fig. 5.

Expression of Adipogenic (A) and Antiadipogenic (B) Transcription Factors in 3T3-L1 Cells Overexpressing Nur77 Total RNA was harvested at confluence (time zero) or 24 h after DMI/GW7845 treatment. Each condition represents four to six separate wells. *, P < 0.05; **, P < 0.01 vs. empty vector control. §, P <0.01 vs. empty vector control.

Acute Expression of Nur77 Does Not Cause Dedifferentiation of Adipocytes

Having demonstrated that chronic overexpression of NR4A receptors inhibits the differentiation of preadipocytes, we proceeded to determine whether ectopic Nur77 expression in mature adipocytes would lead to dedifferentiation. 3T3-L1 cells that express the human (h) coxsackie-adenovirus receptor (hCAR; courtesy of B. Spiegelman) were differentiated into mature adipocytes in the presence of standard adipogenic cocktail and GW7845 for 6 d. After differentiation, cells were maintained in 10% fetal bovine serum (FBS)/DMEM and 0.5 μg/ml insulin. Adipocytes were then infected overnight with adenovirus expressing either the green fluorescent protein (GFP) control or Nur77. Cells were harvested on d 5, 10, or 15 after infection for RNA isolation. As shown in Fig. 6, the expression of adipocyte markers such as PPARγ and aP2 was unaffected by acute ectopic expression of Nur77. This finding indicates that although Nur77 inhibits preadipocyte differentiation, it has no apparent effect on maintaining the phenotypic markers of mature adipocytes.

Fig. 6.

Acute Nur77 Expression Does Not Cause Dedifferentiation of 3T3-L1 Adipocytes Differentiated 3T3-L1 hCAR adipocytes were infected with adenovirus encoding GFP or Nur77 overnight. Total RNA was harvested on d 5, 10, or 15 after infection.

Identification of Nur77-Target Genes that Inhibit Adipogenesis

To identify putative Nur77 targets that may mediate its inhibitory effect on adipogenesis, we performed expression profiling of control (retrovirally transduced with empty vector) or Nur77-expressing 3T3-L1 preadipocytes. Total RNA was harvested at confluence or 24 h after DMI/GW7845 treatment. Genes induced greater than 5-fold by Nur77 are shown in supplemental Table 1 published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org. Several genes that are induced greater than 2-fold and are involved in adipogenesis are also included in this list. Induction of selected genes relevant to adipogenesis was verified by quantitative real-time PCR (supplemental Table 1). Among the genes shown in supplemental Table 1, gap junction protein, α1 (Gja1), tolloid-like 1 (Tll1), and WNT1-inducible signaling pathway protein 2 (Wisp2) were three particularly interesting genes that were induced by Nur77 in stable 3T3-L1 transformants both at baseline as well as at 24 h after differentiation induction. Gja1 is a member of the connexin family and is the major gap junction protein in the heart involved in synchronous contraction of the myocardium (32, 33). It has also been shown to influence mitotic clonal expansion or 3T3-L1 preadipocytes (34). Tll1, an astacin-like metalloprotease with structural similarity to bone morphogenetic protein-1, is crucial for normal cardiac septation in mice (35). Although it is among the most highly induced gene in our microarray, Tll1 was not previously described to have a function in adipose tissue. Wisp2, a known downstream target of Wnt signaling (36), was also a plausible candidate to effect Nur77-driven inhibition of adipogenesis. Wisp2 expression was previously shown to be down-regulated during adipogenic differentiation of human bone marrow-derived mesenchymal stem cells (37).

As shown in Fig. 7A, we verified with real-time PCR that Gja1, Tll1, and Wisp2 were up-regulated by Nur77 in stable 3T3-L1 transformants. To determine whether the induction in gene expression was responsive to acute as well as chronic overexpression of Nur77, the expression of Gja1, Tll1, and Wisp2 was further analyzed in 3T3-L1 hCAR adipocytes acutely infected with GFP- or Nur77-expressing adenovirus. The data of Fig. 7B confirmed that these genes were robustly up-regulated by Nur77, suggesting that they are likely direct targets of the receptor.

Fig. 7.

Nur77 Induces the Expression of Gja1, Tll1, and Wisp2 A, Total RNA was harvested from vector-control and Nur77-expressing 3T3-L1 cells at confluence (time zero) or 24 h post-DMI/GW7845 treatment. B, 3T3-L1 hCAR adipocytes were infected with Ad-GFP or Ad-Nur77. Total RNA was harvested 3 d after infection. **, P < 0.01 vs. GFP control.

We next determined whether ectopic expression of Gja1, Tll1, and Wisp2 was sufficient to inhibit adipogenesis in 3T3-L1 preadipocytes. The cDNA encoding each of these genes was PCR amplified and inserted into vectors for retroviral transduction of 3T3-L1 cells. After puromycin selection, cells were differentiated using standard adipogenic cocktail for 7 d. Oil red O staining revealed that ectopic expression of Gja1 and Tll1 inhibited adipogenesis, whereas Wisp2 overexpression had no effect (Fig. 8A). The paucity of lipid droplet accumulation in Gja1- and Tll1-expressing cells was accompanied by diminished expression of adipogenic markers such as aP2, adipsin, lipoprotein lipase, and PPARγ (Fig. 8B). Similar results were obtained with multiple independent sets of stable lines. Although neither Gja1 nor Tll1 diminished differentiation of 3T3-L1 cells to the same extent as Nur77, their ability to independently inhibit adipogenesis strongly suggests that they contribute to Nur77’s inhibitory activity on adipogenesis.

Fig. 8.

Expression of Gja1 or Tll1 Inhibits Adipogenesis A, 3T3-L1 preadipocytes were retrovirally transduced with MSCV-vector expressing Nur77, Gja1, Tll1, or Wisp2. Stable transformants were induced to differentiate by DMI. Cells were stained with Oil Red O 7 d after the addition of adipogenic cocktail. The two rows of cells represent two separate experiments. B, Expression of adipocyte markers from 3T3-L1 stable transformants. Total RNA was harvested on d 7 after differentiation. *, P < 0.05; **, P < 0.01 vs. empty vector control. LPL, Lipoprotein lipase.

Nur77 Inhibits Mitotic Clonal Expansion

A prerequisite for 3T3-L1 adipocyte differentiation appears to be mitotic clonal expansion, during which quiescent cells synchronously cross the G₁/S checkpoint and undergo mitosis within 24–60 h after differentiation cocktail treatment (38). Our gene expression data of 3T3-L1 Nur77-expressing cells showed that changes in adipogenic transcriptional programs were already underway by 24 h after DMI treatment (Fig. 5), suggesting that Nur77 imparts its inhibitory effect on adipogenesis early during the differentiation process. We therefore tested the hypothesis that Nur77 may inhibit adipogenesis, in part, by preventing mitotic clonal expansion.

To first determine whether Nur77 alters proliferation of actively dividing 3T3-L1 cells, 50,000 cells were plated in one well of a six-well dish. Total cell number was determined 20 h after plating. As Fig. 9A shows, control and Nur77-expressing 3T3-L1 cells both doubled their cell number, indicative of approximately one round of cell division. However, 42 h after DMI treatment, Nur77-expressing 3T3-L1 cells had only half the number of cells as controls, consistent with diminished mitosis. Gja1 expression had no effect on DMI-induced cell division (Fig. 9B). We next performed flow cytometric analysis at different time points after DMI treatment to more precisely determine the percentage of cells acquiring diploid (2n) DNA content. At confluence, approximately 27% of empty vector- and Nur77–898- (N terminus-truncated Nur77) expressing 3T3-L1 cells acquired 2n DNA content, whereas 23% of Nur77-expressing cells contained 2n DNA (Fig. 9C). As expected, 13 h after DMI treatment, the percentage of cells acquiring 2n DNA content had increased to 45% for control cells (empty vector only and Nur77–898). Remarkably, Nur77 cells seemingly failed to escape from G₀/G₁ interphase, as the percentage of cells with 2n DNA persisted at approximately 25%. All three cell lines exited cell cycle by 40 h after DMI induction, with only 20–25% of cells having 2n DNA content. Our data thus suggest that one mechanism by which Nur77 inhibits adipogenesis in 3T3-L1 cells is by arresting mitotic clonal expansion.

Fig. 9.

Nur77 Inhibits Mitotic Clonal Expansion A, Undifferentiated 3T3-L1 stable transformants were plated at a density of 50,000 cells per well. Cells were trypsinized and counted 20 h after plating. B, Cells were plated as in panel A. DMI was added at time of confluence. Cells were trypsinized and counted 42 h after the addition of DMI. C, DNA content was determined by propidium iodide staining. 2n DNA status was determined by sum of percentage of cells in S- and G₂/M phases of cell cycle. Time represents hours after DMI addition. **, P < 0.01 vs. empty vector control.

DISCUSSION

Recent studies have identified NR4A receptors as regulators of metabolism in insulin-sensitive tissues such as liver and skeletal muscle (12, 13, 26). Here we outline a function for NR4A receptors as inhibitors of adipogenesis. We demonstrated that the previously reported acute induction of NR4A expression early in 3T3-L1 adipogenesis occurs in response to cAMP signaling, but not to PPARγ ligand. We also presented data showing that in white adipose tissue, NR4A expression is responsive to both inflammatory signals as well as fasting/refeeding. In addition, we showed that stable NR4A expression in several preadipocyte cell lines inhibits adipogenesis, even in the presence of exogenous PPARγ activation or ectopic PPARγ expression. We identified Gja1 and Tll1 as two putative NR4A target genes that may mediate NR4A’s inhibitory effect on adipose differentiation. In addition to the effect imposed by these two genes, Nur77 also inhibited adipogenesis by limiting mitotic clonal expansion.

Fu et al. (15) have reported that NR4A receptors are induced within 30 min of treating 3T3-L1 preadipocytes with either standard differentiation cocktail or PPARγ ligand, suggesting that these receptors are positive regulators of adipogenesis. Our findings demonstrate that whereas NR4A receptors are up-regulated in 3T3-L1 cells within 60 min of receiving standard adipogenic cocktail, induction occurs in response to the increase in cAMP signaling triggered by the adipogenic cocktail and may not be an intrinsic aspect of adipogenesis per se. This is supported by our data that Nur77 expression was not significantly altered in 3T3-L1 cells differentiated with insulin and PPARγ ligand, whereas 8-Br-cAMP or IBMX treatment caused an acute rise in Nur77 expression. Furthermore, PPARγ ligand treatment had no acute effect on the expression of NR4A receptors. NR4A receptor expression was also not induced during the differentiation of 3T3-F442A cells. Our observations are consistent with the time frame required for nuclear receptors to be activated by ligand and to exert downstream transcriptional regulation—likely hours rather than minutes. Because NR4A receptors are immediate-early genes inducible by various mitogens, it is plausible that previous observations that PPARγ ligands acutely inducing NR4A expression may be explained by the effects of serum refreshment.

Our laboratory previously showed that inflammatory signals rapidly up-regulate the expression of NR4A receptors in macrophages (24, 25). In the present work we have extended this observation to 3T3-L1 preadipocytes as well as white adipose tissue isolated from wild-type mice. Because inflammation-mediated inhibition of adipose differentiation is a well-described process (22, 23, 39), we speculate that one physiological function of NR4A receptors in adipose tissue may be to mediate the metabolic and/or antitrophic effects of inflammation on adipogenesis. Because the three NR4A receptors exhibit functional redundancy, this hypothesis cannot be easily studied in vivo in single NR4A knockout mouse models. Adipose-specific NR4A knockout or transgenic mouse models will likely be required to test this hypothesis.

Similar to our previous findings in the liver, we showed here that Nur77 and NOR1 were up-regulated in adipose tissue in the fasted state. That expression of NR4A receptors responds to nutritional status is consistent with our theory that these receptors mediate physiological processes to increase energy delivery to the organism (13). It is thus reasonable to posit that NR4A receptors may mediate lipolysis in adipose tissue. In fact, the lack of fasting/feeding-induced changes in NR4A expression in ob/ob mice we observed is akin to the impaired lipolysis exhibited by these mice (40–42). Using the dominant-negative Nur77 receptor, we examined whether disruption of NR4A function would prevent lipolysis in vitro. At least in 3T3-L1 adipocytes, NR4A function appears to be dispensable for isoproterenol- and TNFα-mediated lipolysis (supplemental Fig. 1 published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org), suggesting that NR4A function is not necessary for β3-adrenergic receptor- or TNFα-induced lipolysis.

In contrast to previous suggestions that NR4A signaling may promote adipocyte differentiation, we find that NR4A receptors are potent inhibitors of adipogenesis. That NR4A receptors are induced by cAMP response element binding protein, and cAMP response element binding protein signaling is required for adipogenesis, are not necessarily contradictory processes (43, 44). We showed that Nur77 expression led to early changes in the expression of several transcriptional factors such as C/EBPβ, C/EBPδ, and GATA2. This finding suggests that NR4A receptors exert their inhibitory effect on adipogenesis at an early time point, and there may be a window of opportunity beyond which NR4A expression no longer impacts the differentiation process.

One mechanism by which Nur77 may inhibit adipogenesis is by preventing mitotic clonal expansion (Fig. 9). At present, however, it remains unclear whether Nur77 affects clonal expansion by altering the expression and/or activity of early adipogenic transcription factors, the expression of gap junction proteins, or through other unknown mechanisms. Previous studies showed that disruption of C/EBPβ activity blocks mitotic clonal expansion in 3T3-L1 cells (45–47). The observation that Nur77 overexpression reduced C/EBPβ and C/EBPδ transcript levels by 24 h after hormonal induction suggests that interruption of clonal expansion may reflect diminished promoter activity of C/EBPβ or C/EBPδ. One also cannot exclude that possibility that Nur77 directly alters C/EBPβ and C/EBPδ activity. Additional work is ongoing to investigate these scenarios.

Previous studies of cell-to-cell communication of 3T3-L1 preadipocytes have shown that gap junction activity decreases during adipogenesis (34, 48). This decline in cell-to-cell communication appears to occur at the time when spindle-shaped fibroblasts become more rounded and begin lipid accumulation (48). Our observation that expression of the Nur77-responsive gene Gja1 inhibited adipogenesis is thus consistent with the previously published observation that unremitting gap junction activity prevents adipogenesis. Interestingly, Yanagiya and colleagues (34) showed that abrogation of Gja1 expression by small interfering RNA reduced mitotic clonal expansion as well as adipogenesis. This finding suggests that, at least in 3T3-L1 fibroblasts, there may be an optimal window during which gap junction proteins are critical for clonal expansion, such that either constitutive loss- or gain-of-function interrupts normal mitotic clonal expansion and adipogenic programming.

Although inhibition of mitotic clonal expansion represents one pathway by which Nur77 may negatively regulate adipogenesis, there are likely other mechanisms involved. Modulation of extracellular matrix through metalloproteases (49, 50) or cell-cell signaling molecules such as integrins (51, 52) has been shown to regulate adipose differentiation. One might thus hypothesize that Nur77-mediated induction of Tll1 may inhibit adipogenesis by affecting the remodeling of extracellular matrix. In addition, although we stably overexpressed a select set of genes induced by Nur77 in 3T3-L1 preadipocytes, there may be additional Nur77 targets that we have not tested, which may also affect adipogenesis. Notably, this list (supplemental Table 1) includes many extracellular matrix proteins as well as adhesion molecules. Future studies are necessary to further define Nur77-regulated pathways that may impact adipogenesis.

MATERIALS AND METHODS

Reagents

Synthetic ligands GW7845 and GW9662 were provided by Jon Collins and Timothy Willson (GlaxoSmithKline, Research Triangle Park, NC). Ligands were dissolved in DMSO before use in cell culture.

Cell Culture

We purchased human embryonic kidney (HEK)293T and 3T3-L1 cells from American Type Culture Collection (Manassas, VA). 3T3-F442A cells were a gift from E. Saez. HEK293T cells were maintained in 10% FBS (Omega Scientific, Stamford, CT) in DMEM at 8% CO₂. 3T3-L1 and 3T3-F442A cells were maintained and differentiated as previously described (53). 3T3-L1 hCAR cells (gift from B. Spiegelman) were differentiated as described for 3T3-L1 cells, except that 100 nm GW7845 was added to the culture media during differentiation. For adenovirus infection, cells were cultured with adenovirus at a multiplicity of infection of 250 in 10% FBS/0.5 μg/ml insulin overnight before media refreshment the next day. Oil Red O staining was performed as described (7).

Stable Cell Lines

Full-length cDNA of murine Nur77, Nurr1, NOR1, Nur77–898 (cDNA starts at nucleotide +898), Gja1, Tll1, Wisp2 genes was PCR amplified and cloned into MSCV-pac plasmid. Expression plasmids along with ecotropic helper plasmid were transiently transfected into HEK293T cells using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). Supernatant was sterile filtered 2 d after transfection and added to either 3T3-L1 or 3T3-F442A cells. Puromycin (4 μg/ml) in 10% calf serum was used for selection of stable clones. 3T3-L1 cells overexpressing PPARγ were selected with hygromycin (50 μg/ml).

Quantitative Real-Time PCR

Total RNA was isolated with Trizol (Invitrogen). cDNA was synthesized using reverse transcriptase from Applied Biosystems (Foster City, CA). Real-time PCR was performed as described previously (13). See supplemental Table 2 for a complete list of primer sequences used in real-time PCR.

Microarrays

RNA from control (empty vector) or Nur77-expressing 3T3-L1 cells was harvested at confluence (time zero) or 24 h after DMI/GW7845 (100 nm) treatment. Total RNA prepared by Trizol was further purified through RNEasy columns (QIAGEN, Chatsworth, CA). Each condition was done with replicate arrays, each representing three pooled wells. cRNA preparation and hybridization to Affymetrix Mouse Genome Arrays 430 version 2.0 was performed by the UCLA Microarray Core, and data were analyzed using GeneSpring GX7.3.1. We included only genes with raw data signal greater than 500 for at least one condition for analysis.

Cell Cycle Analysis

Cells (50,000 per well) were plated on gelatin-coated six-well plates. Cells were trypsinized before cell counting and staining. Using the Z1 Coulter Counter (Beckman Coulter, Inc., San Jose, CA), we averaged the mean of cell counts from two independent aliquots per well. Staining of DNA content was performed as previously described (54). Briefly, we resuspended PBS-washed cells in PBS containing 0.1% sodium citrate (wt/vol) (Sigma Chemical Co., St. Louis, MO), 0.3% Triton X-100 (vol/vol) (Sigma), 0.01% propidium iodide (wt/vol) (Calbiochem, La Jolla, CA), and 0.002% ribonuclease A (wt/vol) (Sigma), and incubated the cells for 30 min at 20–25 C. We acquired cell cycle data on a FACS Calibur (BD Biosciences, Palo Alto, CA). Data were analyzed using FloJo Software (Tree Star, Inc., Ashland, OR).

Animals

We purchased age-matched C57Bl/6 wild-type and ob/ob male mice from The Jackson Laboratory (Bar Harbor, ME). Mice were provided with water and standard mouse chow ad libitum and were maintained on 12-h light cycle. PBS or lipopolysaccharide (5 mg/kg) was injected into the peritoneum. Mice were euthanized by isoflurane exactly 1 h after injection. Epididymal adipose tissue was isolated and flash frozen in liquid nitrogen. Total RNA was prepared as described previously (13).

Lipolysis Assay

3T3-L1 hCAR cells were differentiated as described above for 5–7 d. Adipocytes were then infected with adenovirus expressing either GFP or Nur77–898 in 10% FBS/DMEM at a multiplicity of infection of 250, before media refreshment the next day (10% FBS/DMEM). Lipolysis assay was performed as described elsewhere (55).

Immunoblot

3T3-L1 preadipocytes were grown to confluence and treated with 1 mm 8-Br-cAMP. Nuclear extract was prepared as follows: cells were scraped and pelleted in PBS, and washed in cold buffer A (10 mm HEPES, pH 7.9; 10 mm KCl; 0.1 mm EGTA; 0.1 mm EDTA; 1 mm dithiothreitol; 0.5 mm phenylmethylsulfonylfluoride; protease inhibitor) and resuspended in buffer A. After 15 min on ice, Nonidet P-40 was added to the cells to a final concentration of 5.9%. After vigorous vortexing, nuclei were pelleted and resuspended in buffer C (20 mm HEPES, pH 7.9; 420 mm NaCl; 1.5 mm MgCl₂; 0.2 mm EDTA; 25% glycerol; 0.5 mm phenylmethylsulfonylfluoride; 1 mm dithiothreitol; protease inhibitor). Samples were then agitated intermittently, kept on ice for 20 min, and spun down. Nuclear extract (supernatant) was loaded on 4–12% Invitrogen Nupage gel. Nur77 antibody (Cell Signaling Technology, Beverly, MA) was used at 1:1000 dilution and HMG-1 (BD Pharmingen, Franklin Lakes, NJ) at 1:5000.

Statistics

We performed statistical analysis using the Student’s t test, with significance determined a priori at P < 0.05.

Acknowledgments

We thank K. W. Park and S. Hummasti for discussions. We also thank B. Spiegelman for providing 3T3-L1 hCAR cells and E. Saez for providing 3T3-F442A cells.

Disclosure Statement: The authors have nothing to disclose.

This work was supported by National Institutes of Health Grant HL30568 (to P.T.). L.C.C. was a Fellow of the Pediatric Scientist Development Program (National Institute of Child Health and Human Development Grant Award K12-HD00850). P.T. is an Investigator of the Howard Hughes Medical Institute.

Abbreviations

 
• 8-Br-cAMP

  8-Bromo-cAMP;

 
• CAR

  coxsackie-adenovirus receptor;

 
• C/EBP

  CCAAT enhancer binding protein;

 
• DMI cocktail

  dexamethasone, isobutylmethylxanthine, and insulin cocktail;

 
• FBS

  fetal bovine serum;

 
• GFP

  green fluorescent protein;

 
• HEK

  human embryonic kidney;

 
• IBMX

  isobutylmethylxanthine;

 
• LPS

  lipopolysaccharide;

 
• 2n

  diploid;

 
• NR4A

  nuclear receptor 4A;

 
• PPAR

  peroxisome proliferator-activated receptor.