An essential role for Ewing sarcoma gene (EWS) in early white adipogenesis
Funding agencies: This research was supported by start-up funds from Tulane University (S.B.L.).
Disclosure: The authors declared no conflict of interest
Author contributions: Jun Hong Park and Sean Bong Lee designed experiments, analyzed data, and wrote the manuscript. Both were involved in writing the paper and had final approval of the submitted and published versions.
Abstract
Objective
White adipose tissue is important for mammalian energy homeostasis and metabolism. It was previously demonstrated that Ewing sarcoma gene (EWS) is essential for early classical brown fat lineage determination, but its role in white adipocyte differentiation is not known.
Methods
Mouse embryonic fibroblasts (MEFs) lacking Ews and shRNA-mediated silencing of Ews in 3T3L1 preadipocytes were used to investigate the role of EWS in adipogenesis. White fat differentiation was determined by analyzing the expression of key adipogenic genes and by Oil red O staining.
Results
Following adipogenic stimulation, Ews expression arose rapidly in 3T3L1 cells during early induction period. Ews-null MEFs and 3T3L1 cells with reduced Ews expression failed to undergo adipogenesis. This was accompanied by significant reduction in the expression of critical early adipogenic regulators, Bmp2, Bmp4 (bone morphogenic protein 2 and 4), Cebpβ, and Cebpδ (CCAAT/enhancer binding protein β and δ). Complementation of recombinant BMP2 or BMP4 partially rescued adipogenesis in Ews-depleted 3T3L1 cells.
Conclusions
These results demonstrate that EWS is essential during the early steps of white adipocyte differentiation, at least in part through its regulation of BMP2 and BMP4 expression.
Introduction
Excessive food intake and sedentary lifestyle have contributed to the growing obesity pandemic and metabolic comorbidities such as diabetes, hypertension, cardiovascular disease, and cancer (1, 2). White adipocytes are critical for energy homeostasis by storing unutilized excess nutrients as single large lipid droplets and mobilizing the stored lipids when nutrients become scarce. This is coordinated with secretion of hormones such as leptin and adiponectin by white adipocytes to communicate with other tissues to control appetite, glucose homeostasis, and fatty acid oxidation (3, 4). Recent studies have shown that some white adipocytes also contribute to thermogenesis by transdifferentiation of mature white fat cells into brown fat-like cells (termed brite or beige) (5, 6), or by differentiation of bipotent progenitor cells residing in white fat depots (7, 8) in response to chronic cold exposure or β-adrenergic stimulation.
In the past two decades, many studies have focused on the transcriptional control of adipocyte differentiation using well-established in vitro models (9-11). As a result, a number of key transcriptional regulators of adipogenesis, such as peroxisome proliferator activated receptor gamma (PPARγ) (12), CCAAT/enhancer binding protein beta (Cebpβ) (13), delta (Cebpδ) (14), alpha (Cebpα) (15), and growth factors, such as bone morphogenic protein family (BMP) (16, 17), have been identified. While PPARγ and CEBP-family transcription factors are critical for both white and brown adipocyte differentiation, different members of BMP-family proteins have distinctive roles in adipogenesis. BMP2 and BMP4 have been shown to promote differentiation of multi-potent mesenchymal progenitor C3H10T1/2 cells to white adipocytes (16-18) whereas BMP7 is critical for development of brown adipogenesis (19) and energy expenditure (20).
Ewing sarcoma gene, EWSR1 (herein termed EWS), and two of its close homologs, FUS/TLS (fused in Sarcoma/translocated in Liposarcoma) and TAF15 (TATA-binding protein-associated factor 15), encode a RNA/ssDNA binding protein and together form the FET (or TET)-family proteins (21, 22). Initially, EWS was thought to function in basic transcription and splicing (23, 24), but generation of an Ews-deficient mouse demonstrated that EWS encodes a multifunctional protein with roles in pre-B lymphocyte development, meiosis, prevention of premature cellular senescence in fibroblasts (25), and hematopoietic stem cells (26) and regulation of microRNAs (27). EWS has also been shown to regulate mitosis (28) and alternative splicing in response to DNA-damage (29, 30). Recently, we have uncovered yet another role of EWS in early brown fat lineage determination during development (31). We found that following adipogenic stimulation, EWS rapidly forms a complex with YBX1 (Y-box binding protein 1) and the EWS-YBX1 complex activates an early brown fat determination factor, BMP7, during embryonic brown adipose tissue development. As a result, classic brown adipose tissue development is completely arrested in Ews-null embryos and newborns as well as in brown preadipocytes in vitro. However, the role of EWS in white adipose tissue development has not been determined. In this study, we examined the role of EWS in white adipocyte differentiation.
Methods
Cell culture and lentivirus vectors
Mouse embryonic fibroblasts (MEFs) and 3T3L1 cells were cultured in Dulbecco's modified Eagles's medium (DMEM) supplemented with 10% fetal bovine serum or 10% heat-inactivated bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM l-glutamine at 5% CO2. All cell culture or differentiation reagents were purchased from Invitrogen or Sigma. Lentivirus expressing (shRNAs) against mouse Ews and scrambled control were purchased from Sigma. 3T3L1 cells were transduced with lentivirus expressing shRNAs against Ews or scrambled control and selected with puromycin to generate stable cell lines.
Adipocyte differentiation
To induce adipogenic differentiation into mature adipocytes, MEFs and 3T3L1 cells were cultured for 2 days in differentiation media supplemented with 850 nM insulin and 1 nM triiodothyronine (T3), followed by 2-day incubation with induction media supplemented with 850 nM insulin, 1 nM T3, 3 uM troglitazone, 125 nM indomethacin, 1 mM dexamethasone, and 0.5 mM isobutylmethylxanthine (IBMX) (this is referred to as the adipogenic stimulation). After induction, cells were cultured in differentiation media for 6 days. Subsequently, cells were washed twice in phosphate buffered saline, fixed with 4% formalin, and stained for 2 h with 0.5% Oil red O staining solution. For the rescue experiments, Ews-depleted 3T3L1 preadipocytes were sequentially treated with induction and differentiation media containing 100 ng/ml of recombinant BMP2 or BMP4 (R&D systems) for 2 days in each media.
Gene expression analysis
Total RNAs were isolated using Trizol (Invitrogen) and quantified using Nanodrop (Nanodrop Technology). cDNAs were synthesized using iScript Reverse Transcription Supermix (Bio-Rad) and analyzed by quantitative real-time PCR with SYBR Green (Bio-Rad). The relative quantity of each transcript was calculated by the comparative Ct method normalized against β-actin. The reference value (0 time point) of each gene was set to the expression levels at the beginning of the 2-day induction period. The oligonucleotide primers were as follows: Bmp4 (forward) 5′-GAG GAG TTT CCA TCA CGA AG-3′, (reverse) 5′-ATT CTC TGG GAT GCT GCT G-3′; Bmp2 (forward) 5′-AAA CAG TAG TTT CCA GCA CC-3′, (reverse) 5′-TCT CCC ACT GAC TTG TGT TC-3′. The other primer sequences have been described previously (31).
Western blot analysis
3T3L1 preadipocytes transduced with lentivirus expressing shRNAs against Ews or scrambled control were harvested, and Western blotting was performed with antibodies against EWS (25) or anti-actin (Sigma). Similarly, expression of endogenous EWS in 3T3L1 cells following adipogenic stimulation for 12 h was examined by Western blotting as above. EWS-YBX1 interaction was detected by immunoprecipitation with IgG or anti-YBX1 antibody followed by Western blotting with anti-EWS antibody.
Statistical analysis
Statistical analysis was performed by ANOVA or Student's t-test using Graph Prism 5 software (GraphPad Software). Data are represented as mean ± SEM, and significance was set at P < 0.05.
Results
Depletion of EWS arrests adipogenesis in MEFs or 3T3L1 preadipocytes
To determine whether EWS is required for white adipogenesis, we first examined Ews wild type or null MEFs treated with adipogenic stimulation. Wild type Ews MEFs readily differentiated into lipid-containing adipocytes, but mutant MEFs failed to undergo adipogenesis as determined by Oil red O staining (Figure 1A). To further confirm this result, we depleted Ews in 3T3L1 cells using lentivirus expressing shRNAs against Ews or a scrambled control. Two independent shRNAs efficiently reduced EWS expression in undifferentiated 3T3L1 cells compared to control as revealed by immunoblotting (Figure 1B). 3T3L1 cells transduced with a control shRNA differentiated robustly following adipogenic stimulation, but cells with reduced EWS expression were greatly impaired in white fat differentiation as shown by Oil red O staining (Figure 1C). Expression of key adipogenic genes, Pparγ, Adipoq, and Lep, was significantly repressed in Ews-depleted 3T3L1 cells (Figure 1D). These results clearly demonstrate that EWS is required for adipogenesis of MEFs and 3T3L1 cells.
EWS is required for white adipocyte differentiation. (A) Oil red O staining for MEFs after 8 days of in vitro adipogenesis. (B) 3T3L1 cells were transduced with lentivirus expressing shRNAs against Ews (two independent) or scrambled control, and cells were induced to undergo adipocyte differentiation. Successful knockdown of Ews was confirmed by Western blot analysis using anti-EWS and anti-actin antibodies. (C) 3T3L1 cells expressing shRNAs against Ews or control were stimulated to undergo adipocyte differentiation for 8 days and stained with Oil red O. (D) qRT-PCR analysis of Ews, Pparγ, Adipoq, and Lep in differentiated 3T3L1 cells expressing scrambled control or Ews shRNAs. Three independent experiments were performed and data represented as mean ± SEM. One-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001.
EWS expression oscillates during white adipocyte differentiation
To understand the role of EWS in white adipogenesis, we examined EWS expression during 3T3L1 preadipocyte differentiation following adipogenic stimulation. Adipogenesis was assessed by the expression of critical regulators of adipogenesis, Pparγ, Cebpα, and fatty acid binding protein 4 (Fabp4, also known as aP2). Following adipogenic stimulation, expression of Pparγ and Cebpα increased gradually, reaching maximal levels at day 4, and decreased as 3T3L1 cells differentiated to mature adipocytes (Figure 2A). Expression of Fabp4 continued to rise until the last day of differentiation. Interestingly, expression of Ews rose modestly during the first few days, then gradually declined below its pre-differentiation level, followed by a modest increase above its pre-differentiation level on the last day (day 8). These results illustrate the dynamic expression pattern of Ews during adipogenesis.
Expression of Ews and adipogenic genes during adipogenesis in 3T3L1 cells. (A) 3T3L1 cells were induced to undergo adipocyte differentiation for 8 days, and total RNA was isolated daily and analyzed for expression of Cebpα, Pparγ, Ews, and Fabp2 by qRT-PCR. Three independent experiments were performed. (B) 3T3L1 preadipocytes were stimulated with adipogenic cocktail, and at indicated times (from 0 to 24 h), expression of Ews, Cebpβ, and Cebpδ mRNA was measured by qRT-PCR. Three independent experiments were performed and data represented as mean ± SEM. One-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001. (C) Western blot analysis of Ews in 3T3L1 cells with or without adipogenic stimulation (12 h). (D) EWS-YBX1 interaction was examined without (upper) or with (lower) adipogenic stimulation (6 h) in 3T3L1 cells by immunoprecipitation (IP) with either lgG or anti-YBX1 followed by immunoblot (IB) with anti-EWS.
We previously showed that EWS has an essential role during the earliest period of brown adipogenesis in vitro (31). Thus, we examined the expression of Ews during the first 24 h of white adipocyte differentiation following adipogenic stimulation. As shown in Figure 2B, expression of Ews increased rapidly within 2-4 h following adipogenic stimulation and reached the highest level (5-fold) at 12 h before declining at 24 h. Expression of early adipogenic regulators, Cebpβ and Cebpδ, also increased very early, peaking at 4 h and gradually declined. To confirm this, we examined EWS protein levels following 12 h of adipogenic stimulation. EWS protein expression was significantly increased compared to unstimulated 3T3L1 cells (Figure 2C). We previously showed that EWS forms a complex with YBX1 (Y-box binding protein 1, also called YB1) during the early hours of brown adipogenesis (31). Thus, we examined the EWS-YBX1 interaction in 3T3L1 cells before or after adipogenic stimulation. Before adipogenic stimulation, the EWS-YBX1 interaction was barely detectable (Figure 2D, upper panel), but following short adipogenic stimulation (6 h), EWS interaction with YBX1 was remarkably increased (Figure 2D lower panel). These results suggest that the EWS-YBX1 complex might have an important role during the earliest stage of white adipocyte differentiation.
Depletion of EWS leads to loss of early adipogenic regulators
Several studies have shown that BMP signaling is important during early adipogenesis or adipocyte commitment stage (32). BMP7 is required for the commitment to brown adipogenesis, while BMP2 and BMP4 enhance white adipogenesis in the presence of adipogenic stimulation (16, 17, 19). BMP2/4 was shown to increase the expression of PPARγ and the CEBP-family proteins (16-18). We previously showed that EWS activates BMP7 expression during early brown adipogenesis (31). Thus, to determine whether EWS regulates the expression of BMP2 or BMP4 during early adipogenesis, we examined the expression level of various early adipogenic regulators, including BMP2 and BMP4, in Ews-depleted 3T3L1 cells following adipogenic stimulation using qRT-PCR analysis. Adipogenic stimulation rapidly increased the expression levels of Bmp2 and Bmp4 as well as early adipogenic transcriptional regulators, Cebpβ and Cebpδ, in control shRNA expressing 3T3L1 cells (Figure 3A). Bmp2 and Bmp4 expression levels reached maximum at 4 h and declined precipitously by 8 h following adipogenic stimulation. In contrast, silencing Ews blocked Bmp2 and Bmp4 expression as well as Cebpβ and Cebpδ in 3T3L1 cells treated with adipogenic stimulation. Expression of an upstream inhibitor of adipogenesis, wingless-type MMTV integration site family member 10A (Wnt10a), was not altered by depletion of Ews. Expression of Pparγ, a downstream master regulator of adipogenesis, was also significantly reduced at 24 h and 48 h in Ews-depleted 3T3L1 cells (Figure 3B). These results demonstrate that loss of EWS leads to an early inhibition of white adipogenesis due to loss of Bmp2, Bmp4, Cebpβ, and Cebpδ expression.
Ews is required for expression of Bmp2, Bmp4, Cebpβ, Cebpδ, and Pparγ during early adipogenesis in 3T3L1 cells. qRT-PCR analysis of 3T3L1 cells expressing shRNAs against Ews or control at the indicated hours (0–24 h) following adipogenic stimulation. Expression of (A) Bmp2, Bmp4, Cebpβ, Cebpδ, Wnt10a and (B) Pparγ transcripts was analyzed. Three independent experiments were performed and data represented as mean ± SEM. Two-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001.
Complementation of BMP2 or BMP4 restores adipogenesis
Since BMP2 and BMP4 are secreted proteins capable of inducing adipogenesis (16-18), we examined whether restoring BMP2 or BMP4 activity could rescue adipogenesis in cells lacking EWS. To this end, Ews-depleted 3T3L1 cells were grown in the absence or presence of recombinant BMP2 or BMP4 and analyzed for adipocyte differentiation. Addition of BMP2 (Figure 4A, shRNA-EWS+BMP2) or BMP4 (Figure 4A, shRNA-EWS+BMP4) along with adipogenic cocktail in Ews-depleted 3T3L1 cells partially rescued adipogenesis, whereas the Ews-depleted 3T3L1 cells grown in the adipogenic cocktail without BMP2 or BMP4 failed to differentiate as determined by Oil red O staining (Figure 4A-b). Recombinant BMP2 or BMP4 also partially restored the expression of essential adipogenic genes, Pparγ, Cebpα, Adipoq, and Fabp4 in Ews-depleted 3T3L1 cells (Figure 4B). Recombinant BMP4 was slightly more effective in restoring adipogenesis in Ews-depleted 3T3L1 cells than recombinant BMP2 as evidenced by Oil red O staining and gene expression analysis (Figure 4B). Expression of Ews was unaffected by either BMP2 or BMP4. These results strongly suggest that EWS is required for early white adipogenesis, at least partially, through its regulation of BMP2 and BMP4 expression.
Complementation of BMP2 or BMP4 restores white adipocyte differentiation in Ews-depleted 3T3L1 cells. 3T3L1 cells expressing shRNA against Ews or control were cultured in differentiation and induction media containing adipogenic cocktail with or without BMP2 or BMP4 for 4 days and continuously cultured in adipogenic differentiation media for 6 more days. Differentiated cells were either (A) stained with Oil red O (scale bar = 50 μm) or (B) analyzed for adipogenic gene expression by qRT-PCR analysis. Three independent experiments were performed and data represented as mean ± SEM. One-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Discussion
Over the past decades, the transcriptional cascade controlling adipocyte differentiation has been worked out in great detail. Studies have shown that adipocyte differentiation is driven by critical transcription factors such as PPARγ and CEBPα, and cofactors. Expression of these transcription factors and early adipocyte differentiation are controlled by another set of transcription factors, CEBPβ and CEBPδ, and by a family of BMP growth factors, respectively (33, 34). However, upstream factors that regulate early adipogenic factors such as BMP proteins, CEBPβ, and CEBPδ during early adipogenesis have not yet been fully elucidated.
Our study revealed that a multifunctional protein EWS is important for the expression of these early regulators during white fat differentiation. Notably, adipogenic stimulation rapidly and robustly induced EWS expression within 4 h, indicating an important role for EWS during the early stage of white adipocyte differentiation. Silencing Ews led to significant inhibition of Bmp2 and Bmp4 expression during early adipogenesis. Our complementation experiments showed that recombinant BMP2 or BMP4 partially restored adipogenesis in Ews-depleted 3T3L1 cells, suggesting that EWS is an upstream regulator of BMP2 and BMP4 expression during the early white fat differentiation. We previously showed that the EWS-YBX1 complex was critical for controlling BMP7 expression during early brown adipocyte differentiation (31). Similarly, formation of the EWS-YBX1 complex was dependent on adipogenic stimulation in 3T3L1 cells. Based on these results, we propose that YBX1 likely plays an important role in regulating BMP2/4 expression during white fat differentiation. Since expression of Cebpβ and Cebpδ was also affected by EWS, it will be intriguing to examine whether EWS has a direct role in controlling the expression of Cebpβ and Cebpδ. Notably, a recent study showed that adipogenic stimulation induced nuclear localization of EWS in 3T3L1 cells by increasing its O-GlcNAc glycosylation (35). It is tempting to speculate that O-GlcNAc glycosylation of EWS is required for the expression of Bmp2, Bmp4, Cebpβ, and Cebpδ during early adipocyte differentiation.
White adipose tissues play essential roles in mammalian energy homeostasis by secreting a number of peptide hormones that regulate food intake, insulin sensitivity, blood pressure, and inflammation (4, 36). Thus, alterations in white adipose tissue mass will not only affect obesity, but could have pleiotrophic effects on immune response, blood pressure control, hemostasis, bone mass, thyroid, and reproductive functions (37). A recent study has shown that long-term survivors of pediatric sarcoma (24/32 were Ewing sarcoma patients) had an increased prevalence of metabolic syndrome such as hypertension and hypertriglyceridemia (38). Given our present findings of EWS in adipogenesis and the fact that Ewing sarcoma patients are haploinsufficient for EWS due to a balanced chromosomal translocation, it will be intriguing to examine the prevalence of metabolic syndrome in sarcoma patients that involve EWS translocation. Furthermore, it will be interesting to explore whether and how EWS regulates adipogenesis and energy homeostasis in humans.
Acknowledgments
Authors thank Dr. Hong Jun Kang for technical assistance.
