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Modulation of adipogenesis-related gene expression by estrogen-related receptor γ during adipocytic differentiation
Biochimica et Biophysica Acta 1789 (2009) 71–77
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a g r m
Modulation of adipogenesis-related gene expression by estrogen-related receptor γ during adipocytic differentiation Mayumi Kubo a,b, Nobuhiro Ijichi a, Kazuhiro Ikeda a, Kuniko Horie-Inoue a, Satoru Takeda b, Satoshi Inoue a,c,⁎ a b c
Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama, Japan Department of Obstetrics and Gynecology, Juntendo University School of Medicine, Tokyo, Japan Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
a r t i c l e
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Article history: Received 1 April 2008 Received in revised form 5 August 2008 Accepted 25 August 2008 Available online 6 September 2008 Keywords: Estrogen-related receptor γ (ERRγ) Preadipocyte 3T3-L1 Pluripotent mesenchymal cell Adipocyte differentiation
a b s t r a c t Estrogen-related receptor γ (ERRγ) is an orphan nuclear receptor that regulates cellular energy metabolism by modulating gene expression involved in oxidative metabolism and mitochondrial biogenesis in brown adipose tissue and heart. However, the physiological role of ERRγ in adipogenesis and the development of white adipose tissue has not been well studied. Here we show that ERRγ was up-regulated in murine mesenchyme-derived cells, especially in ST2 and C3H10T1/2 cells, at mRNA levels under the adipogenic differentiation condition including the inducer of cAMP, glucocorticoid, and insulin. The up-regulation of ERRγ mRNA was also observed in inguinal white adipose and brown adipose tissues of mice fed a high-fat diet. Gene knockdown by ERRγ-specific siRNA results in mRNA down-regulation of adipogenic marker genes including fatty acid binding protein 4, PPARγ, and PGC-1β in a preadipocyte cell line 3T3-L1 preadipocytes and mesenchymal ST2 and C3H10T1/2 cells in the adipogenesis medium. In contrast, stable expression of ERRγ in 3T3-L1 cells resulted in up-regulation of these adipogenic marker genes under the adipogenic condition. These results suggest that ERRγ positively regulate the adipocyte differentiation with modulating the expression of various adipogenesis-related genes. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Adipose tissue is a structure for storage of energy as lipid. Obesity corresponding to excess accumulation of lipid is one of the major public health concerns because it has been recognized as a significant risk factor for various metabolic diseases, including type2 diabetes, insulin resistance, and osteoarthritis [1]. Obesity increases adipose tissue mass resulting from hypertrophy and/or hyperplasia of adipocytes [2]. Adipose tissue itself has been found as an essential regulator of whole-body energy homeostasis [3], by such as secreting growth factors and cytokines, currently so-called as ‘adipokines’ [4]. Thus, understanding of developmental mechanism and physiological property of adipocyte are important for the treatment of obesityrelated diseases. Estrogen-related receptors (ERRs) have been recently revealed as regulators of cellular energy metabolism. Although ERRs share sequence homology to the estrogen receptors, ERα and ERβ, these receptors are not activated by natural estrogens and are classified as orphan nuclear receptors [5–7]. Among three subtypes of the
⁎ Corresponding author. Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Tel.: +81 3 5800 8652; fax: +81 42 984 4541. E-mail address: [email protected] (S. Inoue). 1874-9399/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagrm.2008.08.012
receptors, ERRα and ERRγ are abundantly expressed in mitochondria-rich tissues, such as heart, brown adipose, and slow-twitch skeletal muscle [7–9]. ERRα has been shown to stimulate the expression of genes associated with mitochondrial biogenesis and energy production [8], and participate in the development of white adipose tissue since ERRα-deficient mice have reduction of fat mass and the resistance to high-fat diet-induced obesity [10]. We have previously showed that ERRα is up-regulated in preadipocyte cells and pluripotent mesenchymal cells under the adipogenic condition, and positively regulates lipid accumulation in preadipocyte cells [11]. ERRγ has been characterized to play an essential role in the development as ERRγ null mice exhibit early postnatal death due to abnormal heart function [12]. Yet the physiological role of ERRγ in the adipogenesis remains to be examined. In the ERR-mediated transcriptional activation, coactivators are required in the interaction between receptors and the basal transcriptional apparatus. Among such coactivators, peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1 α and β (PGC-1α and PGC-1β) have been revealed to play important roles in the ERR-mediated transcription and mitochondrial functions [13–16]. To understand the functional role of ERRγ in adipogenesis, here we employ cell culture models including preadipocyte 3T3-L1 cells, pluripotent mesenchymal C3H10T1/2 cells, and stromal ST2 cells, and obesity mouse models fed a high-fat diet. ERRγ mRNA is shown to be
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fatty acid binding protein 4 (aP2), PPARγ and PGC-1β in 3T3-L1 cells treated with the standard adipogenic cocktail comprised IBMX, dexamethasone, and insulin (MDI mixture). In addition, stable expression of ERRγ in 3T3-L1 cells promotes the induction of adipogenic gene expression and lipid accumulation during differentiation. The present study suggests a role of ERRγ in the regulation of adipogenesis. 2. Materials and methods 2.1. Cell culture and adipocyte differentiation
Fig. 1. Regulation of ERRγ mRNA in mesenchyme-derived cell lines under the adipogenic condition. 3T3-L1 (A), ST2 (B), and C3H10T1/2 (C) cells were started to culture under the adipogenic condition of a MDI mixture (0.5 mM IBMX, 1 μM dexamethasone and 10 μg/ml insulin) at day 0. Medium was changed at day 2 and day 5. ERRγ mRNA levels at indicated time points were examined by qPCR and the results were shown as fold change over the expression level at day 0. ⁎p b 0.05; ⁎⁎p b 0.01; ⁎⁎⁎p b 0.001 (by Student's t test).
regulated in these cell lines in response to the adipogenic induction and also in adipose tissues in mice fed high-fat diet. Gene knockdown of ERRγ using a specific small interfering RNA (siRNA) repressed the induction of adipogenesis-related genes including
3T3-L1, ST2, and C3H10T1/2 cells were obtained from American Type Culture Collection (Manassas, VA). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (3T3-L1 and C3H10T1/ 2) or α-MEM (ST2) supplemented with 10% fetal bovine serum (FBS), 50 U/ml penicillin, and 50 μg/ml streptomycin. For adipocyte differentiation, the cells were grown to confluency (day 2), and differentiation was induced 2 days post-confluence (day 0) by changing the medium to the differentiation medium, standard medium containing 10% FBS together with the mixture of 0.5 mM isobutylmethylxanthine (IBMX, Sigma), 1 μM dexamethasone (Dex, Sigma) and 10 μg/ml bovine insulin (Sigma) (MDI mixture). The medium was changed at day 2 to the post-differentiation medium, the standard medium containing 10% FBS and 10 μg/ml insulin. The postdifferentiation medium was also changed at day 5, and the cultivation was continued up to 8 days after the induction (day 8). 2.2. Oil-Red-O staining Lipid accumulation of 3T3-L1 cells in the time course of adipogenic induction was evaluated by staining with Oil-Red-O as described
Fig. 2. Expression of ERRγ and adipogenesis-related genes in mouse adipose tissues. Total RNA was extracted from brown adipose tissue (BAT), inguinal white adipose tissue, and epididymal white adipose tissue of male mice fed with normal chow or a high-fat diet for 10 weeks. Data are relative mRNA levels normalized to GAPDH levels. ⁎p b 0.05; ⁎⁎p b 0.01; ⁎⁎⁎p b 0.001 (by Student's t test). (A) ERRγ mRNA levels examined by qPCR. (B) Fatty acid binding protein 4 (aP2) mRNA levels. (C) Peroxisome proliferator-activated receptor γ (PPARγ) mRNA levels. (D) PPARγ coactivator-1 β (PGC-1β) mRNA levels.
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Fig. 3. Knockdown of ERRγ mRNA expression reduces induction levels of adipogenic marker genes in 3T3-L1 preadipocytes and mesenchymal ST2, and C3H10T1/2 cells under the adipogenesis condition. (A, E, and I) Reduction of ERRγ expression by siRNA targeting for ERRγ (siERRγ). 3T3-L1 (A), ST2 (E), and C3H10T1/2 (I) cells were transfected with siERRγ or control siRNA against firefly luciferase (siLuc) for 48 h as described in the Materials and methods. mRNA levels were evaluated by qPCR. Data are shown as fold change in siERRγtreated cells over control siLuc-treated cells. ⁎p b 0.05; ⁎⁎⁎p b 0.001 (by Student's t test). (B–D, F–H, and J–L) Repression of adipogenic marker genes in siERRγ-treated cells under the adipogenesis condition. 3T3-L1 (B–D), ST2 (F–H), and C3H10T1/2 (J–L) cells were induced to differentiate as described in Fig. 1 except for transfection with siERRγ or siLuc 48 h prior to confluency. mRNA levels of aP2 (B, F, and J), PPARγ (C, G, and K) and PGC-1β (D, H, and L) genes at indicated time points were examined by qPCR. Data are shown as fold change over the mRNA level of each gene at day 0. ⁎p b 0.05; ⁎⁎p b 0.01; ⁎⁎⁎p b 0.001 (by Student's t test).
previously [17]. Briefly, cells were washed twice with phosphate buffered saline (PBS), fixed with 10% formalin in PBS for 30 min, washed twice again with PBS, and stained with the filtered Oil-Red-O solution (120 mg of Oil-Red-O dissolved in 40 ml of isopropyl alcohol) for 1 h. Lipid accumulation was observed by light phase contrast microscopy. All Oil-Red-O staining images were captured at the same settings to allow direct quantitative comparison of staining patterns. Quantification of the staining intensity was performed on a Macintosh computer using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). 2.3. Animal experiments All animal experiments were approved by the Institutional Animal Care and Use Committee. C57BL/6J mice were purchased from CLEA Japan. Mice were maintained in a temperature-controlled (23 °C) facility with a 12 h light/dark cycle. For the chronic high-fat feeding study, 10-week-old male mice were randomly divided into 2 groups (n = 5) and started to feed a normal chow diet (CE2, CLEA Japan) or a high-fat diet (HFD32, CLEA Japan) for additional 10 weeks, having ad
libitum access to water and either chow diet. Then, adipose tissues were harvested, quickly frozen in liquid nitrogen, and stored at −80 °C until further processing. 2.4. Quantitative real-time RT-PCR Total RNAs were extracted from mouse adipose tissues or cells at the indicated times during the period of adipogenic induction using the ISOGEN reagent (Nippon Gene, Tokyo, Japan). Quantitative realtime RT-PCR (qPCR) was performed as described previously [18]. Briefly, first strand cDNA was generated from 2 μg of total RNA by using the SuperScript II reverse transcriptase (Invitrogen) and oligo(dT)20 primer. mRNAs were quantified by real-time PCR using SYBR green PCR master mix (Applied Biosystems) and the ABI Prism 7000 system (Applied Biosystems) based on SYBR Green I fluorescence. The sequences of PCR primers are as follows: aP2 forward, 5′-GCGTGGAATTCGATGAAATCA-3′; aP2 reverse, 5′-CCCGCCATCTAGGGTTATGA-3′; PPARγ forward, 5′-TTCCGAAGAACCATCCGATT-3′; PPARγ reverse, 5′TTTGTGGATCCGGCAGTTAAG-3′; PGC-1β forward, 5′-CATCTGGGAAAAGCAAGTACGA-3′; PGC-1β reverse, 5′-CCTCGAAGGTTAAGGCTGATATCA-3′; ERRγ forward, 5′-GACCCTA-CTGTCCCCGACAGT-3′; ERRγ
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reverse, 5′-AACTCTCGGTCAGCCAAGTCA-3′. The comparison of PCR product amounts among differentiation stages was carried out by the comparative cycle threshold (CT) method, using GAPDH as a control. The experiments were independently repeated at least three times, each performed in triplicate. 2.5. siRNA transfection Synthetic small interfering RNA (siRNA) duplexes targeted for mouse ERRγ gene (a smart pool of double-stranded siRNA; ESRRGNM_011935) and for the luciferase reporter plasmid pGL2 (Luciferase GL2 Duplex) were purchased from Dharmacon (Lafayette, CO). 3T3-L1
Fig. 5. Stable expression of ERRγ up-regulates adipogenic marker genes during 3T3-L1 differentiation. 3T3-L1 clones stably expressing control vector (3T3-L1-vector) (C3 and C5) or FLAG-hERRγ (G1 and G4) were maintained in the differentiate medium as described in Fig. 1. mRNA levels of the aP2 (A), PPARγ (B), and PGC-1β (C) in each clone at indicated time points were examined by qPCR. Results are shown as fold change over the mRNA level in each clone at day 0. ⁎p b 0.05; ⁎⁎p b 0.01; ⁎⁎⁎p b 0.001 (by Student's t test).
cells were incubated with 400 nM siRNA against either ERRγ (siERRγ) or the luciferase gene (siLuc) using Lipofectamine 2000 (Invitrogen) for 48 h. ST2 and C3H10T1/2 cells were incubated with 100 nM and 5 nM siRNAs, respectively, using HiPerFect transfection reagent (QIAGEN) for 48 h. Transfected cells were subjected to adipogenic induction as described above. Fig. 4. Stable expression of ERRγ increases lipid accumulation during 3T3-L1 differentiation. (A) Generation of 3T3-L1 cells stably expressing FLAG-hERRγ (3T3-L1hERRγ). Control vector-expressing clones (C3 and C5) and FLAG-hERRγ-expressing clones (G1 and G4) were isolated by G418 selection. Expression of exogenous hERRγ mRNA in 3T3-L1 clones was validated by qPCR. (B) Oil-Red-O staining of 3T3-L1 clones. 3T3-L1 clones were maintained in the differentiate medium as described in Fig. 1 by day 8. (C) Quantification of Oil-Red-O staining of 3T3-L1 clones. Relative densities of OilRed-O staining at day 8 were determined using NIH Image program. Data were shown as fold change over the staining density in vector-expressing C5 clone. Differences between 3T3-L1-hERRγ clones (G1 and G4) and 3T3-L1-vector clones (C3 and C5) were analyzed by Student's t test. ⁎⁎⁎p b 0.001.
2.6. Plasmid construction Human ERRγ cDNA (hERRγ amino acids 2–458) was cloned from human prostate cDNA (Clontech) by RT-PCR using the following primers: forward, 5′-CGCGAATTCGATTCGGTAGAACTTTGCCT-3′; reverse, 5′-ATACTCGAGTCAGACCTTGGCCTCCAACA-3′. The PCR product was N-terminally tagged with FLAG and cloned into blunted EcoRI sites of pCXN2 [19] (pCXN2-FLAG-hERRγ). DNA sequence of plasmid was confirmed by ABI PRISM 377 Sequencer (Applied Biosystems).
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2.7. Generation of stable cell lines 3T3-L1 cells were transfected with pCXN2-FLAG-human ERRγ or empty pCXN2 plasmid using the Lipofectamine 2000 reagent (Invitrogen) and neo-resistant clones were isolated by G418 selection (0.8 mg/ml). Expression levels of FLAG-human ERRγ mRNA were measured by qPCR using following primers: forward, 5′-GACTACAAGGACGATGATGACAAG-3′ and reverse, 5′-TCGTAGTGCAGGGAAAAAGATTC-3′. 2.8. Statistical analysis Differences between two groups were analyzed using two-sample, two-tailed Student's t test. A p value less than 0.05 was considered to be significant. All data are presented in the text and figures as the mean ± S.D.
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siERRγ attenuated the time-dependent induction of aP2 by about 50% in days 2–8 after adipogenic stimulation compared to siLuc (Fig. 3B). In terms of PPARγ and PGC-1β mRNA, siERRγ repressed the induction of these mRNAs by 50–75% at day 5 and day 8 after the stimulation (Fig. 3C and D). We further investigated the knockdown effect of ERRγ on the expression of adipogenesis-related genes in ST2 and C3H10T1/ 2 cells under the adipogenic condition. siERRγ reduced ERRγ mRNA levels by 50% and 40% in ST2 and C3H10T1/2 cells, respectively (Fig. 3E and I). In ST2 cells, siERRγ moderately repressed the induction of aP2 and PPARγ mRNAs (Fig. 3F and G), while the PGC-1β induction was substantially repressed particularly on day 2 under the adipogenic condition (Fig. 3H). In C3H10T1/2 cells, the effect of siERRγ on the PPARγ induction was rather subtle (Fig. 3K), whereas prominent repression of aP2 and PGC-1β expression was observed on day 5 and day 8 of the differentiation (Fig. 3J and L). These results indicate that ERRγ could be a modulator of the expression of adipogenesis-related genes.
3. Results 3.1. Regulation of ERRγ mRNA during adipocyte differentiation of mesenchyme-derived cell lines and in adipose tissues of high-fat fed mice Mouse 3T3-L1 preadipocytes are known to provide a well characterized model for the study of adipogenesis. 3T3-L1 cells could exhibit terminal adipocyte differentiation after 4–6 days of confluent culture treated with the standard adipogenic cocktail comprised of IBMX, Dex and insulin (MDI mixture) [20,21]. In addition, mesenchyme-derived cell lines including ST2 stromal cells and mesenchymal cell line C3H10T1/2 [22,23] are also shown to differentiate into adipocytes by the same method. Using these cell culture models, we investigated the expression of ERRγ gene by qPCR under adipogenic condition. Expression of ERRγ mRNA in 3T3-L1, ST2, and C3H10T1/2 cells was significantly induced at day 0 compared to the day of confluency (day 2) during the adipogenic differentiation (1.6-, 5.0-, and 2.4-fold, respectively) (Fig. 1). Significant up-regulation of ERRγ mRNA was observed in ST2 and C3H10T1/2 cells at day 2 under the same condition compared to day 0 (2- and 3-fold, respectively) (Fig. 1B and C). mRNA expression of adipocyte differentiation markers, aP2 and PPARγ, was also time-dependently elevated in all the cells examined following the adipogenic induction (data not shown). To examine the function of ERRγ in the adipogenesis in vivo, we employed a diet-induced obesity model in which mice were fed a high-fat diet or normal chow for 10 weeks. Feeding a high-fat diet caused significant weight gain in white adipose tissues (WAT) including inguinal, and epididymal depots, as well as in interscapular brown fat tissue (BAT). We observed up-regulation of ERRγ mRNA in inguinal WAT and BAT in mice fed a high-fat diet compared to control mice fed normal chow (Fig. 2A). mRNA expression of adipocyte differentiation markers, aP2 and PPARγ, and PGC-1β was significantly up-regulated in inguinal WAT and BAT in fed a high-fat diet compared to control mice fed normal chow (Fig. 2B–D). These results suggest that ERRγ mRNA is up-regulated during adipogenesis. 3.2. Knockdown of ERRγ mRNA by siRNA results in reduced induction of adipocyte differentiation markers in adipogenic condition To assess the role of ERRγ in adipocyte differentiation, we investigated the effect of ERRγ knockdown on the expression of adipogenesis-related genes. 3T3-L1 preadipocytes, which were transfected with a specific siRNA targeted for ERRγ mRNA (siERRγ) 48 h prior to confluency, were subjected to adipogenic stimulation. The expression of adipogenesis-related genes at indicated time points was evaluated by qPCR (Fig. 3A–D). siERRγ reduced ERRγ mRNA levels by about 50% in 3T3-L1 cells compared to control siLuc, an siRNA targeted for the luciferase gene (Fig. 3A). In the adipogenic condition,
3.3. Stable expression of ERRγ up-regulates adipogenesis-related genes and increases lipid accumulation during adipocyte differentiation To further characterize the function of ERRγ in adipocyte differentiation, we generated 3T3-L1 clones stably expressing FLAG-hERRγ (3T3-L1-hERRγ) or empty vector (3T3-L1-vector). Exogenous expression of hERRγ mRNA was confirmed by qPCR in 3T3-L1-hERRγ clones, G1 and G4, but not in 3T3-L1-vector clones, C3 and C5 (Fig. 4A). Using these 3T3-L1 clones, lipid accumulation was investigated by Oil-Red-O staining during MDI-induced differentiation. Oil-Red-O staining revealed that the levels of lipid accumulation in 3T3-L1-hERRγ cells were increased compared to 3T3-L1-vector cells during differentiation (Fig. 4B). The relative density of Oil-Red-O staining was significantly increased in 3T3-L1-hERRγ clones compared to that in 3T3-L1-vector clones at day 8 after adipogenic stimulation (Fig. 4C). Furthermore, the alteration of adipogenesis-related gene expression was investigated by qPCR. aP2 mRNA levels at day 5 and PGC-1β mRNA levels at day 5 were significantly up-regulated in 3T3-L1-hERRγ clones compared to control clones (Fig. 5A and C). Expression of PPARγ in hERRγ-expressing clones was also elevated at day 8 compared to control clones (Fig. 5B). Taken together, loss- and gain-of-function studies for ERRγ reveal that this nuclear receptor positively regulates the expression of adipogenesis-related genes including its coactivator PGC-1β, and functions as a promoting factor for 3T3-L1 differentiation. 4. Discussion In the present study, we showed that ERRγ mRNA expression was regulated in mesenchyme-derived cells in response to the adipogenic induction. Up-regulation of ERRγ mRNA was significant especially in ST2 and C3H10T1/2 cells, and also observed in inguinal WAT and BAT of high-fat fed mice. ERRγ knockdown by the specific siRNA repressed the induction of adipogenesis-related genes such as aP2, PPARγ, and PGC-1β in 3T3-L1 preadipocyte cells. Furthermore, stable expression of ERRγ in 3T3-L1 cells enhanced the up-regulation of adipogenesisrelated genes and lipid accumulation. Our results suggest that ERRγ promotes the adipocyte differentiation by modulating the expression of adipogenic genes. ERRγ has been considered to play a role in the regulation of mitochondrial functions including mitochondrial biogenesis, oxidative phosphorylation, and β-oxidation of fatty acids [24,25]. In addition, it is recently revealed that ERRγ regulates metabolic switch from a predominant dependence on carbohydrates during fetal life to a greater dependence on postnatal oxidative metabolism in heart mitochondria [12]. Although the precise signification of ERRγ function in adipose tissues in vivo remains elusive, these findings and our results suggest that ERRγ would control energy production and lipid accumulation in the development and differentiation of both WAT and BAT.
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It has been characterized that ERRs do not have any endogenous small lipophilic ligand [26–30]. Instead, coactivators PGC-1α and PGC1β have been identified as potential protein ligands for ERR family [24,31–33]. We have previously shown that ERRα could be a critical gene in the differentiation of adipocytes [11]. ERRγ is closely related to ERRα in terms of its recognition pattern of DNA motifs, its interaction with coactivators [33–35], and its target genes in the heart [36], suggesting that ERRγ and ERRα may cooperatively participate in a common adipogenic context. Interestingly, it has been shown that ERRγ can transactivate PGC-1α and ERRα promoters [37,38]. Therefore, the positive role of ERRγ on adipocyte differentiation may be partially mediated by up-regulation of ERRα and PGC-1α. In the commitment and differentiation of the adipocyte lineage, adipocyte-fated progenitor cells derived from pluripotent mesenchymal stem cells proceed to the stage of preadipocytes, further induce mitotic clonal expansion and progress into final differentiation as lipid-laden mature adipocytes [23]. In our data, up-regulation of ERRγ was observed at day 0 in 3T3-L1, ST2, and C3H10T1/2 cells compared with day 2, and at day 2 in ST2 and C3H10T1/2 cells compared with day 0 under adipogenic condition, whereas ERRα mRNA level was time-dependently up-regulated following the induction [11]. Since the expression profiles of ERRα and ERRγ are different in adipogenic differentiation but both have been revealed to be important for adipogenesis, ERRα and ERRγ would have each own function in the development of adipose tissues. We have previously shown that ERRβ mRNA levels were also induced in a preadipocyte DFAT-D1 cells and C3H10T1/2 cells [11]. ERRβ null mice are midgestationally lethal due to placental defects, thus its functional significance in vivo has not been well understood [5,39]. Yet, Yu et al. recently reported that prostate cancer LNCaP cells stably expressing ERRβ exhibited significant accumulation of lipid droplets [40]. Further study will reveal the precise roles of ERR family during adipocyte differentiation. In summary, the present study shows that ERRγ is a novel adipogenic marker that may regulate the expression of adipogenesisrelated genes. These findings may have physiological relevance that ERRγ could be potentially used as a molecular target for the treatment of obesity-related diseases. Acknowledgements We thank for Dr. T. Katagiri (Saitama Medical University) for the technical advice. We are also grateful to W. Satoh for his technical assistance. This work was supported in part by Grants-in-Aid from the Ministry of Health, Labor and Welfare; from the Japan Society for the Promotion of Science; from The Promotion and Mutual Aid Corporation for Private Schools of Japan; from the ONO Medical Research Foundation. This work was supported in part by grants of the Genome Network Project and the DECODE from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a g r m
Modulation of adipogenesis-related gene expression by estrogen-related receptor γ during adipocytic differentiation Mayumi Kubo a,b, Nobuhiro Ijichi a, Kazuhiro Ikeda a, Kuniko Horie-Inoue a, Satoru Takeda b, Satoshi Inoue a,c,⁎ a b c
Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama, Japan Department of Obstetrics and Gynecology, Juntendo University School of Medicine, Tokyo, Japan Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
a r t i c l e
i n f o
Article history: Received 1 April 2008 Received in revised form 5 August 2008 Accepted 25 August 2008 Available online 6 September 2008 Keywords: Estrogen-related receptor γ (ERRγ) Preadipocyte 3T3-L1 Pluripotent mesenchymal cell Adipocyte differentiation
a b s t r a c t Estrogen-related receptor γ (ERRγ) is an orphan nuclear receptor that regulates cellular energy metabolism by modulating gene expression involved in oxidative metabolism and mitochondrial biogenesis in brown adipose tissue and heart. However, the physiological role of ERRγ in adipogenesis and the development of white adipose tissue has not been well studied. Here we show that ERRγ was up-regulated in murine mesenchyme-derived cells, especially in ST2 and C3H10T1/2 cells, at mRNA levels under the adipogenic differentiation condition including the inducer of cAMP, glucocorticoid, and insulin. The up-regulation of ERRγ mRNA was also observed in inguinal white adipose and brown adipose tissues of mice fed a high-fat diet. Gene knockdown by ERRγ-specific siRNA results in mRNA down-regulation of adipogenic marker genes including fatty acid binding protein 4, PPARγ, and PGC-1β in a preadipocyte cell line 3T3-L1 preadipocytes and mesenchymal ST2 and C3H10T1/2 cells in the adipogenesis medium. In contrast, stable expression of ERRγ in 3T3-L1 cells resulted in up-regulation of these adipogenic marker genes under the adipogenic condition. These results suggest that ERRγ positively regulate the adipocyte differentiation with modulating the expression of various adipogenesis-related genes. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Adipose tissue is a structure for storage of energy as lipid. Obesity corresponding to excess accumulation of lipid is one of the major public health concerns because it has been recognized as a significant risk factor for various metabolic diseases, including type2 diabetes, insulin resistance, and osteoarthritis [1]. Obesity increases adipose tissue mass resulting from hypertrophy and/or hyperplasia of adipocytes [2]. Adipose tissue itself has been found as an essential regulator of whole-body energy homeostasis [3], by such as secreting growth factors and cytokines, currently so-called as ‘adipokines’ [4]. Thus, understanding of developmental mechanism and physiological property of adipocyte are important for the treatment of obesityrelated diseases. Estrogen-related receptors (ERRs) have been recently revealed as regulators of cellular energy metabolism. Although ERRs share sequence homology to the estrogen receptors, ERα and ERβ, these receptors are not activated by natural estrogens and are classified as orphan nuclear receptors [5–7]. Among three subtypes of the
⁎ Corresponding author. Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Tel.: +81 3 5800 8652; fax: +81 42 984 4541. E-mail address: [email protected] (S. Inoue). 1874-9399/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagrm.2008.08.012
receptors, ERRα and ERRγ are abundantly expressed in mitochondria-rich tissues, such as heart, brown adipose, and slow-twitch skeletal muscle [7–9]. ERRα has been shown to stimulate the expression of genes associated with mitochondrial biogenesis and energy production [8], and participate in the development of white adipose tissue since ERRα-deficient mice have reduction of fat mass and the resistance to high-fat diet-induced obesity [10]. We have previously showed that ERRα is up-regulated in preadipocyte cells and pluripotent mesenchymal cells under the adipogenic condition, and positively regulates lipid accumulation in preadipocyte cells [11]. ERRγ has been characterized to play an essential role in the development as ERRγ null mice exhibit early postnatal death due to abnormal heart function [12]. Yet the physiological role of ERRγ in the adipogenesis remains to be examined. In the ERR-mediated transcriptional activation, coactivators are required in the interaction between receptors and the basal transcriptional apparatus. Among such coactivators, peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1 α and β (PGC-1α and PGC-1β) have been revealed to play important roles in the ERR-mediated transcription and mitochondrial functions [13–16]. To understand the functional role of ERRγ in adipogenesis, here we employ cell culture models including preadipocyte 3T3-L1 cells, pluripotent mesenchymal C3H10T1/2 cells, and stromal ST2 cells, and obesity mouse models fed a high-fat diet. ERRγ mRNA is shown to be
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fatty acid binding protein 4 (aP2), PPARγ and PGC-1β in 3T3-L1 cells treated with the standard adipogenic cocktail comprised IBMX, dexamethasone, and insulin (MDI mixture). In addition, stable expression of ERRγ in 3T3-L1 cells promotes the induction of adipogenic gene expression and lipid accumulation during differentiation. The present study suggests a role of ERRγ in the regulation of adipogenesis. 2. Materials and methods 2.1. Cell culture and adipocyte differentiation
Fig. 1. Regulation of ERRγ mRNA in mesenchyme-derived cell lines under the adipogenic condition. 3T3-L1 (A), ST2 (B), and C3H10T1/2 (C) cells were started to culture under the adipogenic condition of a MDI mixture (0.5 mM IBMX, 1 μM dexamethasone and 10 μg/ml insulin) at day 0. Medium was changed at day 2 and day 5. ERRγ mRNA levels at indicated time points were examined by qPCR and the results were shown as fold change over the expression level at day 0. ⁎p b 0.05; ⁎⁎p b 0.01; ⁎⁎⁎p b 0.001 (by Student's t test).
regulated in these cell lines in response to the adipogenic induction and also in adipose tissues in mice fed high-fat diet. Gene knockdown of ERRγ using a specific small interfering RNA (siRNA) repressed the induction of adipogenesis-related genes including
3T3-L1, ST2, and C3H10T1/2 cells were obtained from American Type Culture Collection (Manassas, VA). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (3T3-L1 and C3H10T1/ 2) or α-MEM (ST2) supplemented with 10% fetal bovine serum (FBS), 50 U/ml penicillin, and 50 μg/ml streptomycin. For adipocyte differentiation, the cells were grown to confluency (day 2), and differentiation was induced 2 days post-confluence (day 0) by changing the medium to the differentiation medium, standard medium containing 10% FBS together with the mixture of 0.5 mM isobutylmethylxanthine (IBMX, Sigma), 1 μM dexamethasone (Dex, Sigma) and 10 μg/ml bovine insulin (Sigma) (MDI mixture). The medium was changed at day 2 to the post-differentiation medium, the standard medium containing 10% FBS and 10 μg/ml insulin. The postdifferentiation medium was also changed at day 5, and the cultivation was continued up to 8 days after the induction (day 8). 2.2. Oil-Red-O staining Lipid accumulation of 3T3-L1 cells in the time course of adipogenic induction was evaluated by staining with Oil-Red-O as described
Fig. 2. Expression of ERRγ and adipogenesis-related genes in mouse adipose tissues. Total RNA was extracted from brown adipose tissue (BAT), inguinal white adipose tissue, and epididymal white adipose tissue of male mice fed with normal chow or a high-fat diet for 10 weeks. Data are relative mRNA levels normalized to GAPDH levels. ⁎p b 0.05; ⁎⁎p b 0.01; ⁎⁎⁎p b 0.001 (by Student's t test). (A) ERRγ mRNA levels examined by qPCR. (B) Fatty acid binding protein 4 (aP2) mRNA levels. (C) Peroxisome proliferator-activated receptor γ (PPARγ) mRNA levels. (D) PPARγ coactivator-1 β (PGC-1β) mRNA levels.
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Fig. 3. Knockdown of ERRγ mRNA expression reduces induction levels of adipogenic marker genes in 3T3-L1 preadipocytes and mesenchymal ST2, and C3H10T1/2 cells under the adipogenesis condition. (A, E, and I) Reduction of ERRγ expression by siRNA targeting for ERRγ (siERRγ). 3T3-L1 (A), ST2 (E), and C3H10T1/2 (I) cells were transfected with siERRγ or control siRNA against firefly luciferase (siLuc) for 48 h as described in the Materials and methods. mRNA levels were evaluated by qPCR. Data are shown as fold change in siERRγtreated cells over control siLuc-treated cells. ⁎p b 0.05; ⁎⁎⁎p b 0.001 (by Student's t test). (B–D, F–H, and J–L) Repression of adipogenic marker genes in siERRγ-treated cells under the adipogenesis condition. 3T3-L1 (B–D), ST2 (F–H), and C3H10T1/2 (J–L) cells were induced to differentiate as described in Fig. 1 except for transfection with siERRγ or siLuc 48 h prior to confluency. mRNA levels of aP2 (B, F, and J), PPARγ (C, G, and K) and PGC-1β (D, H, and L) genes at indicated time points were examined by qPCR. Data are shown as fold change over the mRNA level of each gene at day 0. ⁎p b 0.05; ⁎⁎p b 0.01; ⁎⁎⁎p b 0.001 (by Student's t test).
previously [17]. Briefly, cells were washed twice with phosphate buffered saline (PBS), fixed with 10% formalin in PBS for 30 min, washed twice again with PBS, and stained with the filtered Oil-Red-O solution (120 mg of Oil-Red-O dissolved in 40 ml of isopropyl alcohol) for 1 h. Lipid accumulation was observed by light phase contrast microscopy. All Oil-Red-O staining images were captured at the same settings to allow direct quantitative comparison of staining patterns. Quantification of the staining intensity was performed on a Macintosh computer using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). 2.3. Animal experiments All animal experiments were approved by the Institutional Animal Care and Use Committee. C57BL/6J mice were purchased from CLEA Japan. Mice were maintained in a temperature-controlled (23 °C) facility with a 12 h light/dark cycle. For the chronic high-fat feeding study, 10-week-old male mice were randomly divided into 2 groups (n = 5) and started to feed a normal chow diet (CE2, CLEA Japan) or a high-fat diet (HFD32, CLEA Japan) for additional 10 weeks, having ad
libitum access to water and either chow diet. Then, adipose tissues were harvested, quickly frozen in liquid nitrogen, and stored at −80 °C until further processing. 2.4. Quantitative real-time RT-PCR Total RNAs were extracted from mouse adipose tissues or cells at the indicated times during the period of adipogenic induction using the ISOGEN reagent (Nippon Gene, Tokyo, Japan). Quantitative realtime RT-PCR (qPCR) was performed as described previously [18]. Briefly, first strand cDNA was generated from 2 μg of total RNA by using the SuperScript II reverse transcriptase (Invitrogen) and oligo(dT)20 primer. mRNAs were quantified by real-time PCR using SYBR green PCR master mix (Applied Biosystems) and the ABI Prism 7000 system (Applied Biosystems) based on SYBR Green I fluorescence. The sequences of PCR primers are as follows: aP2 forward, 5′-GCGTGGAATTCGATGAAATCA-3′; aP2 reverse, 5′-CCCGCCATCTAGGGTTATGA-3′; PPARγ forward, 5′-TTCCGAAGAACCATCCGATT-3′; PPARγ reverse, 5′TTTGTGGATCCGGCAGTTAAG-3′; PGC-1β forward, 5′-CATCTGGGAAAAGCAAGTACGA-3′; PGC-1β reverse, 5′-CCTCGAAGGTTAAGGCTGATATCA-3′; ERRγ forward, 5′-GACCCTA-CTGTCCCCGACAGT-3′; ERRγ
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reverse, 5′-AACTCTCGGTCAGCCAAGTCA-3′. The comparison of PCR product amounts among differentiation stages was carried out by the comparative cycle threshold (CT) method, using GAPDH as a control. The experiments were independently repeated at least three times, each performed in triplicate. 2.5. siRNA transfection Synthetic small interfering RNA (siRNA) duplexes targeted for mouse ERRγ gene (a smart pool of double-stranded siRNA; ESRRGNM_011935) and for the luciferase reporter plasmid pGL2 (Luciferase GL2 Duplex) were purchased from Dharmacon (Lafayette, CO). 3T3-L1
Fig. 5. Stable expression of ERRγ up-regulates adipogenic marker genes during 3T3-L1 differentiation. 3T3-L1 clones stably expressing control vector (3T3-L1-vector) (C3 and C5) or FLAG-hERRγ (G1 and G4) were maintained in the differentiate medium as described in Fig. 1. mRNA levels of the aP2 (A), PPARγ (B), and PGC-1β (C) in each clone at indicated time points were examined by qPCR. Results are shown as fold change over the mRNA level in each clone at day 0. ⁎p b 0.05; ⁎⁎p b 0.01; ⁎⁎⁎p b 0.001 (by Student's t test).
cells were incubated with 400 nM siRNA against either ERRγ (siERRγ) or the luciferase gene (siLuc) using Lipofectamine 2000 (Invitrogen) for 48 h. ST2 and C3H10T1/2 cells were incubated with 100 nM and 5 nM siRNAs, respectively, using HiPerFect transfection reagent (QIAGEN) for 48 h. Transfected cells were subjected to adipogenic induction as described above. Fig. 4. Stable expression of ERRγ increases lipid accumulation during 3T3-L1 differentiation. (A) Generation of 3T3-L1 cells stably expressing FLAG-hERRγ (3T3-L1hERRγ). Control vector-expressing clones (C3 and C5) and FLAG-hERRγ-expressing clones (G1 and G4) were isolated by G418 selection. Expression of exogenous hERRγ mRNA in 3T3-L1 clones was validated by qPCR. (B) Oil-Red-O staining of 3T3-L1 clones. 3T3-L1 clones were maintained in the differentiate medium as described in Fig. 1 by day 8. (C) Quantification of Oil-Red-O staining of 3T3-L1 clones. Relative densities of OilRed-O staining at day 8 were determined using NIH Image program. Data were shown as fold change over the staining density in vector-expressing C5 clone. Differences between 3T3-L1-hERRγ clones (G1 and G4) and 3T3-L1-vector clones (C3 and C5) were analyzed by Student's t test. ⁎⁎⁎p b 0.001.
2.6. Plasmid construction Human ERRγ cDNA (hERRγ amino acids 2–458) was cloned from human prostate cDNA (Clontech) by RT-PCR using the following primers: forward, 5′-CGCGAATTCGATTCGGTAGAACTTTGCCT-3′; reverse, 5′-ATACTCGAGTCAGACCTTGGCCTCCAACA-3′. The PCR product was N-terminally tagged with FLAG and cloned into blunted EcoRI sites of pCXN2 [19] (pCXN2-FLAG-hERRγ). DNA sequence of plasmid was confirmed by ABI PRISM 377 Sequencer (Applied Biosystems).
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2.7. Generation of stable cell lines 3T3-L1 cells were transfected with pCXN2-FLAG-human ERRγ or empty pCXN2 plasmid using the Lipofectamine 2000 reagent (Invitrogen) and neo-resistant clones were isolated by G418 selection (0.8 mg/ml). Expression levels of FLAG-human ERRγ mRNA were measured by qPCR using following primers: forward, 5′-GACTACAAGGACGATGATGACAAG-3′ and reverse, 5′-TCGTAGTGCAGGGAAAAAGATTC-3′. 2.8. Statistical analysis Differences between two groups were analyzed using two-sample, two-tailed Student's t test. A p value less than 0.05 was considered to be significant. All data are presented in the text and figures as the mean ± S.D.
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siERRγ attenuated the time-dependent induction of aP2 by about 50% in days 2–8 after adipogenic stimulation compared to siLuc (Fig. 3B). In terms of PPARγ and PGC-1β mRNA, siERRγ repressed the induction of these mRNAs by 50–75% at day 5 and day 8 after the stimulation (Fig. 3C and D). We further investigated the knockdown effect of ERRγ on the expression of adipogenesis-related genes in ST2 and C3H10T1/ 2 cells under the adipogenic condition. siERRγ reduced ERRγ mRNA levels by 50% and 40% in ST2 and C3H10T1/2 cells, respectively (Fig. 3E and I). In ST2 cells, siERRγ moderately repressed the induction of aP2 and PPARγ mRNAs (Fig. 3F and G), while the PGC-1β induction was substantially repressed particularly on day 2 under the adipogenic condition (Fig. 3H). In C3H10T1/2 cells, the effect of siERRγ on the PPARγ induction was rather subtle (Fig. 3K), whereas prominent repression of aP2 and PGC-1β expression was observed on day 5 and day 8 of the differentiation (Fig. 3J and L). These results indicate that ERRγ could be a modulator of the expression of adipogenesis-related genes.
3. Results 3.1. Regulation of ERRγ mRNA during adipocyte differentiation of mesenchyme-derived cell lines and in adipose tissues of high-fat fed mice Mouse 3T3-L1 preadipocytes are known to provide a well characterized model for the study of adipogenesis. 3T3-L1 cells could exhibit terminal adipocyte differentiation after 4–6 days of confluent culture treated with the standard adipogenic cocktail comprised of IBMX, Dex and insulin (MDI mixture) [20,21]. In addition, mesenchyme-derived cell lines including ST2 stromal cells and mesenchymal cell line C3H10T1/2 [22,23] are also shown to differentiate into adipocytes by the same method. Using these cell culture models, we investigated the expression of ERRγ gene by qPCR under adipogenic condition. Expression of ERRγ mRNA in 3T3-L1, ST2, and C3H10T1/2 cells was significantly induced at day 0 compared to the day of confluency (day 2) during the adipogenic differentiation (1.6-, 5.0-, and 2.4-fold, respectively) (Fig. 1). Significant up-regulation of ERRγ mRNA was observed in ST2 and C3H10T1/2 cells at day 2 under the same condition compared to day 0 (2- and 3-fold, respectively) (Fig. 1B and C). mRNA expression of adipocyte differentiation markers, aP2 and PPARγ, was also time-dependently elevated in all the cells examined following the adipogenic induction (data not shown). To examine the function of ERRγ in the adipogenesis in vivo, we employed a diet-induced obesity model in which mice were fed a high-fat diet or normal chow for 10 weeks. Feeding a high-fat diet caused significant weight gain in white adipose tissues (WAT) including inguinal, and epididymal depots, as well as in interscapular brown fat tissue (BAT). We observed up-regulation of ERRγ mRNA in inguinal WAT and BAT in mice fed a high-fat diet compared to control mice fed normal chow (Fig. 2A). mRNA expression of adipocyte differentiation markers, aP2 and PPARγ, and PGC-1β was significantly up-regulated in inguinal WAT and BAT in fed a high-fat diet compared to control mice fed normal chow (Fig. 2B–D). These results suggest that ERRγ mRNA is up-regulated during adipogenesis. 3.2. Knockdown of ERRγ mRNA by siRNA results in reduced induction of adipocyte differentiation markers in adipogenic condition To assess the role of ERRγ in adipocyte differentiation, we investigated the effect of ERRγ knockdown on the expression of adipogenesis-related genes. 3T3-L1 preadipocytes, which were transfected with a specific siRNA targeted for ERRγ mRNA (siERRγ) 48 h prior to confluency, were subjected to adipogenic stimulation. The expression of adipogenesis-related genes at indicated time points was evaluated by qPCR (Fig. 3A–D). siERRγ reduced ERRγ mRNA levels by about 50% in 3T3-L1 cells compared to control siLuc, an siRNA targeted for the luciferase gene (Fig. 3A). In the adipogenic condition,
3.3. Stable expression of ERRγ up-regulates adipogenesis-related genes and increases lipid accumulation during adipocyte differentiation To further characterize the function of ERRγ in adipocyte differentiation, we generated 3T3-L1 clones stably expressing FLAG-hERRγ (3T3-L1-hERRγ) or empty vector (3T3-L1-vector). Exogenous expression of hERRγ mRNA was confirmed by qPCR in 3T3-L1-hERRγ clones, G1 and G4, but not in 3T3-L1-vector clones, C3 and C5 (Fig. 4A). Using these 3T3-L1 clones, lipid accumulation was investigated by Oil-Red-O staining during MDI-induced differentiation. Oil-Red-O staining revealed that the levels of lipid accumulation in 3T3-L1-hERRγ cells were increased compared to 3T3-L1-vector cells during differentiation (Fig. 4B). The relative density of Oil-Red-O staining was significantly increased in 3T3-L1-hERRγ clones compared to that in 3T3-L1-vector clones at day 8 after adipogenic stimulation (Fig. 4C). Furthermore, the alteration of adipogenesis-related gene expression was investigated by qPCR. aP2 mRNA levels at day 5 and PGC-1β mRNA levels at day 5 were significantly up-regulated in 3T3-L1-hERRγ clones compared to control clones (Fig. 5A and C). Expression of PPARγ in hERRγ-expressing clones was also elevated at day 8 compared to control clones (Fig. 5B). Taken together, loss- and gain-of-function studies for ERRγ reveal that this nuclear receptor positively regulates the expression of adipogenesis-related genes including its coactivator PGC-1β, and functions as a promoting factor for 3T3-L1 differentiation. 4. Discussion In the present study, we showed that ERRγ mRNA expression was regulated in mesenchyme-derived cells in response to the adipogenic induction. Up-regulation of ERRγ mRNA was significant especially in ST2 and C3H10T1/2 cells, and also observed in inguinal WAT and BAT of high-fat fed mice. ERRγ knockdown by the specific siRNA repressed the induction of adipogenesis-related genes such as aP2, PPARγ, and PGC-1β in 3T3-L1 preadipocyte cells. Furthermore, stable expression of ERRγ in 3T3-L1 cells enhanced the up-regulation of adipogenesisrelated genes and lipid accumulation. Our results suggest that ERRγ promotes the adipocyte differentiation by modulating the expression of adipogenic genes. ERRγ has been considered to play a role in the regulation of mitochondrial functions including mitochondrial biogenesis, oxidative phosphorylation, and β-oxidation of fatty acids [24,25]. In addition, it is recently revealed that ERRγ regulates metabolic switch from a predominant dependence on carbohydrates during fetal life to a greater dependence on postnatal oxidative metabolism in heart mitochondria [12]. Although the precise signification of ERRγ function in adipose tissues in vivo remains elusive, these findings and our results suggest that ERRγ would control energy production and lipid accumulation in the development and differentiation of both WAT and BAT.
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It has been characterized that ERRs do not have any endogenous small lipophilic ligand [26–30]. Instead, coactivators PGC-1α and PGC1β have been identified as potential protein ligands for ERR family [24,31–33]. We have previously shown that ERRα could be a critical gene in the differentiation of adipocytes [11]. ERRγ is closely related to ERRα in terms of its recognition pattern of DNA motifs, its interaction with coactivators [33–35], and its target genes in the heart [36], suggesting that ERRγ and ERRα may cooperatively participate in a common adipogenic context. Interestingly, it has been shown that ERRγ can transactivate PGC-1α and ERRα promoters [37,38]. Therefore, the positive role of ERRγ on adipocyte differentiation may be partially mediated by up-regulation of ERRα and PGC-1α. In the commitment and differentiation of the adipocyte lineage, adipocyte-fated progenitor cells derived from pluripotent mesenchymal stem cells proceed to the stage of preadipocytes, further induce mitotic clonal expansion and progress into final differentiation as lipid-laden mature adipocytes [23]. In our data, up-regulation of ERRγ was observed at day 0 in 3T3-L1, ST2, and C3H10T1/2 cells compared with day 2, and at day 2 in ST2 and C3H10T1/2 cells compared with day 0 under adipogenic condition, whereas ERRα mRNA level was time-dependently up-regulated following the induction [11]. Since the expression profiles of ERRα and ERRγ are different in adipogenic differentiation but both have been revealed to be important for adipogenesis, ERRα and ERRγ would have each own function in the development of adipose tissues. We have previously shown that ERRβ mRNA levels were also induced in a preadipocyte DFAT-D1 cells and C3H10T1/2 cells [11]. ERRβ null mice are midgestationally lethal due to placental defects, thus its functional significance in vivo has not been well understood [5,39]. Yet, Yu et al. recently reported that prostate cancer LNCaP cells stably expressing ERRβ exhibited significant accumulation of lipid droplets [40]. Further study will reveal the precise roles of ERR family during adipocyte differentiation. In summary, the present study shows that ERRγ is a novel adipogenic marker that may regulate the expression of adipogenesisrelated genes. These findings may have physiological relevance that ERRγ could be potentially used as a molecular target for the treatment of obesity-related diseases. Acknowledgements We thank for Dr. T. Katagiri (Saitama Medical University) for the technical advice. We are also grateful to W. Satoh for his technical assistance. This work was supported in part by Grants-in-Aid from the Ministry of Health, Labor and Welfare; from the Japan Society for the Promotion of Science; from The Promotion and Mutual Aid Corporation for Private Schools of Japan; from the ONO Medical Research Foundation. This work was supported in part by grants of the Genome Network Project and the DECODE from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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