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A new orphan member of the nuclear hormone receptor superfamily closely related to Rev-Erb
Dumas, B.; Harding, H.P.; Choi, H.S.; Lehmann, K.A.; Chung, M.; Lazar, M.A.; Moore, D.D., 1994: A new orphan member of the nuclear hormone receptor superfamily closely related to Rev-Erb. Molecular Endocrinology 8(8): 996-1005
We have isolated complementary DNA clones encoding a novel orphan member of the nuclear receptor superfamily, termed BD73. This protein shows strong amino acid sequence similarity to the previously described Rev-ErbA alpha. Unlike Rev-Erb, in which the opposite strand of the C-terminal coding region encodes the C-terminal portion of a variant thyroid hormone receptor isoform, the opposite strand of the C-terminal coding region of BD73 does not have any extensive open reading frames. BD73 messenger RNA is expressed in a wide variety of tissues and cell lines. In quiescent HepG2 cells, BD73 messenger RNA levels are strongly induced by planar aromatic antioxidants. Like Rev-Erb, BD73 binds as a monomer to a DNA sequence which consists of a specific A/T-rich sequence upstream of the consensus hexameric half-site specified by the P box of the DNA-binding domain. Amino acid sequence comparisons suggest that the A box sequence, which has been suggested to mediate monomer binding by other superfamily members, lies closer to the DNA-binding domain in BD73 and Rev-Erb than in other receptors. Under the conditions examined, neither BD73 nor Rev-Erb activated reporters containing multiple copies of their common binding site. Thus, these two orphans may require an as yet unidentified ligand or other signal for such activation. Together, BD73 and Rev-Erb define a subgroup of orphan receptors that bind as monomers to a half-site flanked by a specific and extended A/T-rich sequence.
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Related references科研 | Science：肠道微生物群通过生物钟和NFIL3调节机体组成
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科研 | Science：肠道微生物群通过生物钟和NFIL3调节机体组成
导读全球性流行的肥胖症呈现出紧迫的公共卫生危机。全世界超过21亿人超重或肥胖，每年约有340万人死于肥胖相关疾病。肠道微生物群是显着影响哺乳动物机体组成的环境因素。微生物群可促进脂肪组织中的能量储存。但是，微生物如何调控宿主代谢途径，如何影响能量储存和身体组成，知之甚少。许多宿主代谢途径通过生物钟与昼夜光周期保持同步。哺乳动物的昼夜节律是机体所有细胞中的转录因子网络，其驱动基因的节律性表达（~24小时波动）。研究表明，微生物群以多种方式与生物钟相互作用，对宿主代谢产生深远影响，破坏二者间的相互作用可能导致肥胖和其他代谢疾病。然而，微生物与生物钟相互作用的机制以及这些相互作用如何改变宿主的新陈代谢，知之甚少。论文ID原名：The intestinal microbiota regulates body composition through NFIL3 and the circadian clock译名：肠道微生物群通过NFIL3和生物钟调节机体组成期刊：ScienceIF：37.205发表时间：2017年通信作者：Lora V. Hooper通信作者单位：The University of TexasSouthwestern Medical Center综述内容1.&NFIL3在肠上皮细胞中的生理功能NFIL3是一种转录因子，可在多种免疫细胞中表达，并可根据细胞类型调控免疫功能。研究发现小肠上皮细胞也表达NFIL3，并且无菌小鼠中NFIL3表达显着降低（图1A），提示上皮NFIL3可能调节响应于肠道微生物群的生理活性。正常膳食饲养的Nfil3△IEC小鼠体重比Nfil3fl/fl同窝小鼠轻（图1B），高脂膳食（HFD）饲喂10周时，Nfil3fl/fl和Nfil3△IEC小鼠的体重均增加（图1B），然而Nfil3△IEC小鼠体重较低（图1B），体脂百分比较低（图1C）。Nfil3△IEC小鼠的附睾脂肪垫重量较低（图1D），并且免受血液甘油三酯升高（图1E），肝脏脂肪堆积（图1F），葡萄糖耐量降低（图1D）和胰岛素抵抗增加（图1G）等现象。结论：上皮细胞表达的NFIL3调节小鼠的脂质储存和机体组成。同时，抗生素处理的Nfil3fl/fl小鼠的脂肪减少和瘦体重增加，且与Nfil3△IEC小鼠的机体组成没有显着差异（图1H）。因此，HFD诱导的体脂累积同时需要NFIL3和微生物群。图 1&Nfil3△IEC小鼠抵抗高脂诱导的肥胖2．微生物群调控NFIL3表达的机制Nfil3转录由生物钟控制。研究发现在小肠上皮细胞群中，Nfil3转录丰度随昼夜波动（图2A）。野生型无菌小鼠和常规小鼠的对比研究发现，微生物群介导Nfil3的最大程度的表达，并且调节Nfil3转录节律性的幅度（图2A）。Nfil3的蛋白质表达水平与转录水平趋势一致（图2B）。昼夜节律转录阻遏物REV-ERBα直接调节Nfil3的表达。在肠上皮细胞中，Rev-erbα转录和蛋白质丰度也随昼夜波动，且在无菌小鼠的转录和翻译水平比常规小鼠的高（图2C和D）。提示REV-ERBa调控Nfil3的昼夜表达，并且微生物群通过抑制Rev-erbα表达来诱导Nfil3表达。通过染色质免疫共沉淀（ChIP）发现，REV-ERBα直接结合于肠上皮细胞中的Nfil3启动子（图2E）。此外，抗生素处理的Rev-erbα-/-小鼠与常规野生型小鼠的Nfil3表达水平相似（图2F）。结论：微生物调节Nfil3表达依赖于REV-ERBα，微生物群通过抑制Rev-erbα表达提高Nfil3表达。图 23.&DC-ILC3通路将微生物群信号传递至上皮细胞以调节Nfil3表达肠上皮细胞通过Toll样受体（TLRs）及MyD88来识别微生物群，调控关键基因的表达。研究发现，在Myd88-/-小鼠中，上皮细胞Rev-erbα表达增加，Nfil3表达降低至无菌小鼠的表达水平（图2G），表明微生物调节Rev-erbα-Nfil3&级联信号需要MyD88。然而，CD11c+细胞群体（包括部分树突状细胞），而不是上皮细胞的Myd88显著抑制Rev-erba表达和诱导Nfil3表达（图2G）。并且用白喉毒素处理，选择性消除CD11c+细胞导致Rev-erba表达增加，并且Nfil3表达减少（图2H），表明微生物群诱导Nfil3表达需要DCs。细菌激活DCs中的TLR-MyD88信号，并通过IL-23从DC传递到3型先天淋巴细胞（ILC3）。然后，ILC3通过IL-22向上皮细胞传递信号。研究表明，在缺乏所有已知的ILC亚型的ID2缺陷型小鼠（Id2gfp/gfp）中，小肠上皮细胞Rev-erbα表达增加，Nfil3表达随之降低（图2H）。此外，缺乏Th17和ILC3的RORδγ缺陷小鼠（Rorcgfp/gfp）的Rev-erbα和Nfil3的昼夜表达模式（图2I和J）与无菌小鼠类似（图2A和B）。用重组IL-23或IL-22处理Myd88-/-小鼠可使上皮Rev-erbα和Nfil3表达恢复至野生型常规小鼠的水平（图2K）。结论：上皮下的DC-ILC3通路将微生物群信号传递至上皮细胞以调节Nfil3表达。图 24.&鉴定调节Rev-erbα和Nfil3表达的上皮细胞固有信号途径最近，研究表明，中枢神经系统（CNS）可以调节啮齿动物的肝脏和肠道脂质和脂蛋白分泌。转录因子STAT3是IL-22受体（IL-22R）下游的关键反应元件。肠上皮细胞的ChIP分析显示，STAT3与Rev-erbα启动子结合（图3A和B）。无菌小鼠的上皮细胞中STAT3结合显着降低（图3A和B）。共转染编码STAT3的载体，其荧光素酶报告基因与Rev-erba启动子融合，导致HEK-293T细胞中的荧光素酶活性降低（图3C），并且STAT3主要活性成分的表达进一步抑制萤光素酶活性（图3C）。因此，STAT3与Rev-erba启动子结合并抑制其转录。向肠道类器官培养模型中添加重组IL-22导致STAT3磷酸化（图3D）。同时IL-22处理降低了Rev-erba的表达并且增加了Nfil3的表达（图3E和F）。相反地，向类器官培养物中添加STAT3磷酸化抑制剂（Stattic）（22）时，重组IL-22不能使STAT3磷酸化（图3D），并且Rev-erbα和Nfil3的表达水平与对照组相似（图3E和F）。研究证明STAT3是Rev-erbα的转录抑制子。相比于Stat3fl/fl同窝小鼠，上皮细胞特异性Stat3缺失小鼠（Stat3△IEC）的肠上皮细胞Rev-erbα表达增加，Nfil3表达降低（图3G和H）。结论：NFIL3表达的昼夜节律是由生物钟通过REV-ERBα产生的，而节律的振幅由微生物群通过STAT3微调。图 3&STAT3与Rev-erbα启动子结合抑制其转录5.&上皮细胞NFIL3调节脂肪储存和机体组成的机制小肠具有长时间储存膳食脂质的能力。餐后，脂质作为细胞内CLDs和可能作为预先形成的脂蛋白颗粒，在肠道中停留。Nfil3fl/fl和Nfil3△IEC小鼠上皮细胞的转录组鉴定了33个差异表达的转录本，并发现部分基因在Nfil3fl/fl小鼠中的表达具有昼夜节律性（图4A）。在Nfil3△IEC小鼠中，生物钟基因Bmal1（Arnt1），Per2和Nr1d1（Rev-erbα）依然保持节律表达（图4A），表明其核心生物钟机制保持完整。有17个转录本编码在脂质摄取和代谢中起作用的蛋白质（图4A），包括Cd36、Scd1、Cyp2e1和Fabp4。通过qRT-PCR分析发现，Cd36和Scd1的表达依赖于上皮细胞NFIL3和微生物群（图4B）。Western blot分析显示，在无菌小鼠和Nfil3△IEC小鼠中CD36蛋白水平降低（图4C）。此外，在Id2gfp/gfp和Stat3△IEC小鼠中，Cd36和Scd1的表达降低（图4D和E）。同时，研究发现：Nfil3fl/fl小鼠的肠上皮细胞含有丰富的脂质（图4F）。相比之下，Nfil3△IEC小鼠上皮细胞中脂质较少（图4F）。此外，与Nfil3fl/fl小鼠相比，Nfil3△IEC小鼠的肠上皮细胞的脂质浓度也较低（图4G），但粪便中的脂质浓度较高（图4H）。结论：微生物群调节NFIL3依赖性的脂质代谢程序是肠道上皮细胞固有途径，且上皮细胞NFIL3调节上皮细胞的脂质吸收和输出。图 4&上皮细胞NFIL3调节脂肪储存和机体组成实验结论本研究发现，NFIL3是微生物群，昼夜节律和宿主新陈代谢之间的重要分子链接。研究结果表明，微生物群通过NFIL3调节脂质摄取和储存，从而阐明了肠道微生物群调节宿主代谢和机体组成的机制。此外，研究表明，ILC3-STAT3信号传递衔接了微生物群与上皮生物钟，因此确定了微生物群与生物钟相互作用的关键分子通路。这些结果可能为微生物与生物钟相互作用的紊乱导致代谢疾病的机制提供更深入的了解。本研究阐明了昼夜节律紊乱（由于轮班工作或国际旅行引）与代谢疾病（包括肥胖，糖尿病和心血管疾病）增加的相关性。点评本研究的发现可能为治疗代谢性疾病提供了新策略，例如靶向NFIL3，STAT3，微生物群或生物钟。本文由莫孞编译，莫秋芬、江舜尧编辑。
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Rev-erb Regulates the Expression of Genes Involved in Lipid Absorption in Skeletal Muscle Cells
作者：Sathiya N. Ramakrishnan,Patrick Lau,Les J. Burke, George E. O. Muscat&&&&作者单位：Institute for Molecular Bioscience, Division of Molecular Genetics and Development, University of Queensland, St. Lucia, Queensland 407 Australia
Rev-erb is an orphan nuclear receptor that selectively blocks trans-activation mediated by the retinoic acid-related orphan receptor- (ROR). ROR has been implicated in the regulation of high density lipoprotein cholesterol, lipid homeostasis, and inflammation. Reverb and ROR are expressed in similar tissues, inclu however, the pathophysiological function of Rev-erb has remained obscure. We hypothesize from the similar expression patterns, target genes, and overlapping cognate sequences of these nuclear receptors that Rev-erb regulates lipid metabolism 30% of total body weight and 50% of energy expenditure. Moreover, this metabolically demanding tissue is a primary site of glucose disposal, fatty acid oxidation, and cholesterol efflux. Consequently, muscle has a significant role in insulin sensitivity, obesity, and the blood-lipid profile. We utilize ectopic expression in skeletal muscle cells to understand the regulatory role of Rev-erb in this major mass peripheral tissue. Exogenous expression of a dominant negative version of mouse Rev-erb decreases the expression of many genes involved in fatty acid/lipid absorption (including Cd36, and Fabp-3 and -4). Interestingly, we observed a robust 15-fold) in mRNA expression of interleukin-6, an &exercise-induced myokine& that regulates energy expenditure and inflammation. Furthermore, we observed the dramatic repression 20-fold) of myostatin mRNA, another myokine that is a negative regulator of muscle hypertrophy and hyperplasia that impacts on body fat accumulation. This study implicates Rev-erb in the control of lipid and energy homoeostasis in skeletal muscle. In conclusion, we speculate that selective modulators of Rev-erb may have therapeutic utility in the treatment of dyslipidemia and regulation of muscle growth.
【关键词】& Regulates Expression Involved Absorption Skeletal
INTRODUCTION
Members of the nuclear receptor (NR)1 superfamily bind to specific DNA elements and function as transcriptional regulators (1, 2). In addition to the ligand-activated NRs, many members within this superfamily have no known ligand, and are referred to as "orphan NRs" (3). The orphan receptor Rev-erb (NR1D2, also known as Rev-erb-related receptor, RVR) belongs to the family of "Reverbs" that also contain Rev-erb (4, 5). The primary structure of these two receptors together with retinoic acid-related orphan receptor- (ROR) and the Drosophila orphan receptor, E75A, is very similar especially in the DNA-binding domain and the putative ligand-binding domain (6).
Two Rev-erb genes
Rev-erb1 and Rev-erb2, which are alternatively spliced products of the Rev-erb gene (7). The mRNA expression data shows that Rev-erb is abundantly expressed in most tissues, although higher levels of expression are observed in skeletal muscle, brain, kidney, and liver (Refs. 4 and 8 and references therein).
Rev-erb, Rev-erb, and ROR bind as monomers to the nuclear receptor half-site motif, PuGGTCA flanked 5' by an AT-rich sequence ((A/T)6PuGGTCA). Although these receptors are closely related, and bind to the same motif, they function in an opposing manner. Whereas ROR activates gene transcription, Rev-erb and Rev-erb mediate transcriptional repression, and can repress trans-activation mediated by ROR (6, 9-12). The inter-relationship between these nuclear receptors is underscored by the evidence that demonstrates ROR trans-activates the Rev-erb promoter (13).
Loss of function studies in cell culture and in animal models has demonstrated a role of ROR in lipid metabolism. ROR-deficient staggerer mice are predisposed to the development of atherosclerosis. The staggerer mice possess an aberrant blood-lipid profile with low circulating levels of major lipoproteins such as HDL cholesterol, apolipoprotein (apo) C-III, and plasma triglycerides. Furthermore, decreases in specific apolipoprotein compartments, namely Apoa-I, the major constituent of HDL, and Apoa-II that lead to hypo--lipoproteinemia have been reported in staggerer mice (15). Moreover, our recent studies demonstrate that ROR regulates the expression of genes involved in lipid homeostasis and energy balance in skeletal muscle cells (16).
Several reports have demonstrated the role of Rev-erb in lipid metabolism and inflammation. In the context of lipid metabolism, Rev-erb, together with peroxisome proliferator activated receptor- (PPAR-) has been shown to regulate the expression of ApoA1 in a species-specific manner (17). Apoa1 has an important role in the formation of nascent HDL particles and apolipoprotein-mediated cholesterol efflux in mice (18, 19). Furthermore, Rev-erb has also been shown to regulate the expression of the ApoC-III gene that plays a key role in maintaining serum triglyceride levels (20, 21). The inter-relationship between Rev-erb and lipid metabolism has not been investigated. Within inflammatory pathways, Rev-erb and ROR act opposingly on the NFB pathway. ROR has been shown to directly up-regulate the expression of IB, the major inhibitor of the NFB signaling pathway by binding to the IB promoter (22). The functional role of Rev-erb in NFB-mediated inflammation still remains obscure.
Rev-erb is abundantly expressed in skeletal muscle and brown fat. Skeletal muscle is one of the most metabolically demanding major mass peripheral tissues that accounts for 50% of energy expenditure. Moreover, this lean tissue relies heavily on fatty acids as an energy source, accounts for 75% of glucose disposal, and is involved in cholesterol efflux. Consequently, muscle has a significant role in insulin sensitivity and the blood-lipid profile. However, the role of Rev-erb in skeletal muscle lipid and energy homeostasis has not been well studied.
The C2C12 in vitro cell culture model system has been used to investigate the regulation of lipid metabolism and cholesterol homeostasis by nuclear receptors such as LXR (23), PPAR, -/, - (24-28), and ROR (16). Selective and synthetic agonists to these nuclear receptors induce similar effects on mRNAs encoding Abca1/g1, ApoE, sterol regulatory element-binding protein (Srebp)-1c, Scd-1, Fabp3, fatty acid synthase, lipoprotein lipase, Glut5, Ucp-2, and Ucp-3 in differentiated C2C12-myotubes and Mus musculus skeletal muscle tissue (23-25). The physiological validation of the cell culture model with respect to lipid homeostasis in the mouse corroborates the utility of this model system. This cell culture model provides an ideal platform to identify the Rev-erb-dependent regulation of genes involved in metabolism.
We have examined the regulation of gene expression involved in lipid homeostasis by Rev-erb "loss of function" in skeletal muscle cell culture model. Our investigation demonstrates that Rev-erb controls the expression of genes involved in lipid absorption. Moreover, we observed the regulation of genes encoding critical myokines that influence energy expenditure and inflammation.
MATERIALS AND METHODS
Cell Culture-Mouse myogenic C2C12 cells were cultured in growth medium (Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated Serum Supreme (BioWhittaker, Edward Keller Pty. Ltd., Hallam, Victoria, Australia)) in 6% CO2. Myoblasts were differentiated into post-mitotic multinucleated myotubes by 4 days of serum withdrawal (i.e. cultured in Dulbecco's modified Eagle's medium supplemented with 1% fetal calf serum + 1% serum supreme). Cells were harvested 96 h (4 days) after mitogen withdrawal.
C2C12 Transfections-Each well of a 24-well plate of C2C12 cells (50% confluence) was transfected with DNA using the liposome-mediated transfection procedure as described previously (16). Cells were transfected using a DOTAP and Metafectene (Biontex Laboratories GmbH, Munich, Germany) liposome mixture in 1x HBS (HEPES-buffered saline (42 mM HEPES, 275 mM NaCl, 10 mM KCl, 0.4 mM Na2HPO4, 11 mM dextrose (pH 7.1)). The DNA/DOTAP/Metafectene mixture was added to the cells in 0.6 ml of Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum for experiments with ROR or 10% serum supreme. After 16-24 h, the culture medium was changed and after a further 24 h the cells were harvested for the assay of luciferase activity. Luciferase activity was measured as described previously (16).
C2:Rev-erbE Stably Transfected Cell Line-The transfection of pSG5-Rev-erbE into mouse myogenic C2C12 cells and the selection of stably transfected polyclonal cells using G418 has been described previously (29). The cells were cultured in growth medium with G418, and differentiated into post-mitotic multinucleated myotubes by 4 days of serum withdrawal.
RNA Extraction and cDNA Synthesis-Total RNA was extracted from C2C12 cells using Tri-Reagent (Sigma) according to the manufacturer's protocol. After treatment with 2 units of Turbo DNase (Ambion, Austin, TX) for 60 min, DNase was inactivated by heating to 75 &C for 10 min. RNA for quantitative real time (Q-RT)-PCR was further purified using the RNeasy RNA extraction kit (Qiagen, Clifton Hill, Victoria, Australia) according to the manufacturer's instructions.
Q-RT-PCR-RNA was normalized using UV spectrometry and agarose gel electrophoresis. Complementary DNA was synthesized from 3 &g of total RNA using Superscript III Reverse Transcriptase (Invitrogen Australia Pty. Ltd., Mulgrave, Victoria, Australia) and random hexamers according to the manufacturer's instructions. Target cDNA levels were analyzed by Q-RT-PCR in 25-&l reactions containing either SYBR green (ABI, Warrington, UK) or Taqman PCR master mix (ABI, Branchburg, NJ), 200 nM each of forward and reverse primers or Assays-on-Demand Taqman primers (ABI, Foster City, CA), and cDNA (derived from 50 nanograms of RNA) by using an ABI Prism 7000 Sequence Detector system. PCR was conducted over 45 cycles at 95 &C for 15 s and 60 &C for a 1-min two-step thermal cycling preceded by an initial 95 &C for 10 min for activation of Amplitaq Gold DNA polymerase.
Primers-Primers used for the Q-RT-PCR analysis of the mRNA populations have been described in detail (16), with the exception of primers designed for the detection of Rev-erbE (pSG5-RVR-F, TGGGCAACGTGCTGGTTA; pSG5-RVR-R, CCATGGTGGGATCCGAATT), Mcad (MCAD-F, CAGCCAATGATGTGTGCTTACTG; MCAD-R, ATAACATACTCGTCACCCTTCTTCTCT), Err (ERR-F, CTCTGGCTACCACTACGGTGTG; ERR-R, AGCTGTACTCGATGCTCCCCT), IL-6 (IL-6-F, AGCCAGAGTCCTTCAGA; IL-6-R, GGTCCTTAGCCACTCCT) using SYBR green. Rev-erb, Fabp4, NFB (relA), IB, Cox-2, IL-15, and myostatin were detected using Assay-on-demand primer/probe sets.
Plasmids-pSG5, pSG5-Rev-erb, and pSG5-Rev-erbE have been described previously (29). The reporters containing the human Rev-erbA promoter (pRev-erbAWT) or four copies of the mouse Purkinje cell protein-2 RORE (mPCP-2tk LUC) linked to the luciferase gene have been described previously (9, 16).
Rev-erb mRNA Is Expressed in Skeletal Muscle Cells and Repressed during Myogenic Differentiation-Many studies indicate that the C2C12 cell line is an excellent cell culture model to investigate myogenesis, and the NR-mediated regulation of lipid homeostasis in skeletal muscle (23-26). In this culture system, proliferating C2C12 myoblasts can be induced to biochemically and morphologically differentiate into post-mitotic multinucleated myotubes by serum withdrawal in culture over a 48-96-h period. This transition from a non-muscle phenotype to a contractile phenotype is associated with activation/expression of a structurally diverse group of genes responsible for contraction and the extreme metabolic demands on this tissue.
Initially, total RNA was isolated from proliferating myoblasts and post-mitotic myotubes after 4 days of serum withdrawal, converted to cDNA for the analysis of mRNA expression by Q-RT-PCR. We utilized the GenBankTM sequences of mouse Rev-erb to design specific primers for detection of mouse Rev-erb by Q-RT-PCR.
We observed that Rev-erb is expressed in proliferating myoblasts and the transcript was decreased 2-3-fold as the cells exit the cell cycle and form differentiated multinucleated myotubes (Fig. 1A). Concomitant with this decrease in Rev-erb mRNA was the striking induction of expression of mRNA that encodes the slow (type I, Tnni1) and fast (type II, Tnni2) isoforms of the contractile protein, troponin I, and myogenin that encodes the hierarchical basic helix loop helix regulator (Fig. 1, B-D). These data confirmed that these cells had terminally differentiated and had acquired a contractile phenotype.
Q-RT-PCR analysis of mRNA expression during skeletal muscle myogenesis. Total RNA from wild type C2C12 proliferating myoblasts (PMB), and myotubes after 4 days of serum withdrawal (MT4) was reverse transcribed to cDNA and analyzed by Q-RT-PCR. Gene-specific primers were used to examine the expression of: A, R B, Tnni1; C, Tnni2; D, E, F, ROR; G, Fat/Cd36; H, Fabp3; I, Fabp4; J, Scd-1; K, Ucp-3; L, Ucp-2; M, Srebp1c; and N, AdipoR2. Normalized expression is relative to the expression of 18S rRNA determined with the same cDNA then multiplied by 10,000. All reactions were performed in triplicate and results are shown as average & S.D.
Furthermore, we analyzed the expression of several genes that encode major metabolic enzymes and regulators that are involved in the regulation of energy expenditure and lipid homeostasis (as described in Table I). Acquisition of the muscle-specific phenotype saw an increase in expression of mRNAs encoding lipoprotein lipase (Fig. 1E), ROR (Fig. 1F), fatty acid translocase/CD36 (Fat/Cd36, Fig. 1G), fatty acid binding proteins 3 and 4 (Fig. 1, H and I), and the uncoupling proteins (Ucp2 and -3, Fig. 1, K and L). Finally, there was no change in the expression of the mRNAs encoding Srebp-1c (Fig. 1M), stearoyl-CoA desaturase 1 (Scd-1, Fig. 1J), and adiponectin receptor 2 (adipoR2, Fig. 1N).
Key target genes in this study
In summary, these data demonstrate that the mRNA transcript encoding Rev-erb was expressed during myogenesis. Second, during the acquisition of a contractile and metabolic phenotype (consistent with the increased utilization of lipids in skeletal muscle) we observed an 2.5-fold repression of this orphan NR. However, we note that Rev-erb is still expressed at a significant level in differentiated cells, in concordance with the expression of this orphan NR in adult muscle tissue.
Deletion of the Rev-erb Ligand Binding Domain Compromises Its Ability to Repress ROR-mediated Trans-activation-To understand the metabolic role of Rev-erb in skeletal muscle lipid and energy homeostasis, and to identify target genes of this orphan receptor in muscle cells, we examined the effect of perturbing Rev-erb function. To disrupt Rev-erb-mediated trans-repression of gene expression, we utilized Rev-erbE, that encodes amino acids 1-394 but lacks the entire E region that encodes the putative ligand binding domain of Rev-erb. Deletion of this region has been reported to have a dominant negative effect on Rev-erb-mediated trans-repression of gene expression (29). Moreover, this region has been implicated in associating with the nuclear receptor co-repressors (30). In transient transfection assays in C2C12 skeletal muscle cells, deletion of the E region ablated the ability of Rev-erb to trans-repress the human Rev-erbA promoter (Fig. 2A). Moreover, the capacity of Rev-erb to repress ROR-mediated transcription is diminished when the E region is absent (Fig. 2, B and C). This demonstrates that deletion of the E-region that encodes the ligand binding domain of Rev-erb compromises the capacity of this orphan NR to trans-repress the Rev-erb promoter, and to attenuate ROR-mediated trans-activation.
Deletion of the E region of Rev-erb compromises its trans-repression activity. A, C2C12 cells were co-transfected with 0.5 &g of the reporter, pRev-erbAWT, and 0.16 &g each of pSG5, pSG5-Rev-erb, and pSG5-Rev-erbE. -Fold repression is expressed relative to luciferase activity obtained after co-transfection of the reporter and pSG5 vector only, arbitrarily set at 1. All transfections comprised six replicates and results are shown as average & S.D. B, C2C12 cells were co-transfected with 0.33 &g of the ROR responsive reporter, mPCP-2tk-LUC, and 0.48 &g of pSG5 or 0.16 &g of pSG5-ROR and increasing amounts (0.05, 0.1, and 0.2 &g) of pSG5-Rev-erb or pSG5-Rev-erbE. pSG5 was added to normalize the final amount of DNA transfected to 0.63 &g in each well. All transfections comprised six replicates and results are shown as average & S.D. C, data from B are represented in terms of -fold repression. -Fold repression is expressed relative to luciferase activity obtained after co-transfection of the reporter and pSG5-ROR, arbitrarily set at 1.
Ectopic Dominant Negative (Rev-erbE) Expression Induces Precocious Differentiation and ROR mRNA Expression-The stable C2C12 cell line expressing Rev-erbE (C2:Rev-erbE) has been described previously (29). Prior to more extensive analysis of this cell line, we decided to ascertain the Rev-erb status of this cell line. We designed a SYBR green-mediated Q-RT-PCR assay to selectively detect the ectopically expressed Rev-erbE transcript. We observed that the C2:Rev-erbE cell line specifically expresses the ectopically introduced mRNA in myoblasts and myotubes (Fig. 3, A and B). The expression of the exogenous transcript is constitutive (Fig. 3, A and B) in contrast to the endogenous transcript that is down-regulated during differentiation of proliferating myoblasts to postmitotic myotubes (Fig. 1A). Moreover, the steady state levels of myogenin mRNA were elevated in myotubes from the C2:Rev-erbE cell line relative to wild-type C2C12 cells (Fig. 3C). This is consistent with the previously reported precocious biochemical and morphological differentiation of the C2:ReverbE cell line relative to wild-type C2C12 cells (29). For example, Burke et al. (29) reported induction of differentiation upon serum withdrawal leads to accelerated activation and expression of the myogenin, and p21Cip-1/Waf-1 mRNAs. Moreover, reduced expression of cyclin D1 was observed, which correlated with the increased expression of the cdk inhibitor, p21 (29). In addition, the levels of the non-muscle cytoskeletal -actin are repressed in the C2:Rev-erbE cell line, relative to wild-type C2C12 cells (Fig. 3D) in concordance with the elevated myogenin mRNA and precocious differentiative capacity of this cell line (29).
Ectopic expression of the dominant negative Rev-erbE accelerates myogenesis and induces expression of ROR. Total RNA was extracted from the wild type (C2C12) and the dominant negative (C2:Rev-erbE) cell lines from proliferating myoblasts (PMB), and myotubes after 4 days of serum withdrawal (MT4), reverse transcribed to cDNA, and analyzed by Q-RT-PCR. Gene-specific primers were used to examine the expression of: A and B, Rev-erbE; C, D, -actin E, Rev- F, ROR. Normalized expression is calculated relative to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (B) or 18 S rRNA (A, C-F) determined with the same cDNA. All reactions were performed in triplicate and results are shown as average & S.D.
We used Q-RT-PCR with an ABI Taqman primer set that does not discriminate between ectopic Rev-erbE and endogenous Rev-erb mRNA expression to determine the total quantity of Rev-erb mRNA in the stable cell line. We observed that the total pool of Rev-erb mRNA is increased by 20-30% in myotubes from the C2:Rev-erbE cell line relative to wild type C2C12s (data not shown). Considering the efficient expression of the exogenous Rev-erbE mRNA transcript (Fig. 3, A and B), this data suggests that the endogenous transcript expression is reduced, in concordance with the previous analysis of this cell line (29). The down-regulation of the endogenous transcript is not surprising. During myogenesis, mRNA pool sizes in muscle tissue are under strict control (31) and mechanisms in skeletal muscle exist that sense total output from exogenous and endogenous genes (32). Furthermore, exogenous expression of a number of different contractile protein transgenes in the mouse (e.g. myosin light chain 2, troponin I fast, skeletal and cardiac actin) results in the decline of the expression of the corresponding endogenous gene (32-36).
Ectopic Rev-erbE mRNA expression does not affect the mRNA expression level of the other member of the Rev-erb family, Rev-erbA (Fig. 3E). Interestingly, the closely related but opposingly acting orphan receptor, ROR, was dramatically increased 6-fold (Fig. 3F) in the C2:Rev-erbE cell line. However, the levels of ROR- remain unchanged (see Table IV). These data indicate that there is cross-talk between these two related, but opposingly acting, orphan receptors in skeletal muscle cells.
Relative changes in the expression of the nuclear hormone receptors in skeletal muscle cells constitutively expressing Rev-erbE
Dominant Negative Rev-erb Regulates the Expression of Genes Involved in Lipid Absorption and Energy Expenditure- The expression of many metabolic markers is altered during skeletal muscle differentiation and acquisition of the muscle-specific and contractile phenotype (Fig. 1). We therefore analyzed the expression of the genes involved in differentiation and skeletal muscle metabolism in the C2:Rev-erbE cell line, relative to wild-type C2C12 cells (see Table I and II). Before analysis of the mRNAs encoding the metabolic enzymes we first characterized the key markers of differentiation in the C2:Rev-erbE cell line to determine a threshold of significance with which to assess putative changes in a relevant context.
Relative changes in the expression of genes involved in lipid metabolism in skeletal muscle cells constitutively expressing Rev-erbE
We observed that after mitogen withdrawal, the C2:Rev-erbE cells formed post-mitotic multinucleated myotubes and thus had morphologically differentiated. The induction and activation of myogenin (Fig. 3C) demonstrated that the cells had also acquired a muscle-specific phenotype. Transcripts for the contractile proteins troponin I slow ((Tnni1) Fig. 4A), and troponin I fast ((Tnni2) (Fig. 4B)) were also induced after serum withdrawal demonstrating that the C2:Rev-erbE cell line had acquired a contractile phenotype and was biochemically differentiated. Analysis of the relative expression of these biochemical markers in wild type C2C12 versus C2:Rev-erbE myotubes after 4 days of serum withdrawal revealed that myogenin (Fig. 3C) and fast type II (Tnni2) mRNA expression (Fig. 4, C and D) were increased 1.5-2-fold. The TNNI1 mRNA encoding the slow type I isoform was actually slightly reduced.
The C2:Rev-erbE cell line acquires contractile markers of skeletal muscle. A and B, expression of troponin I type I ( Tnni1) and II ( Tnni2) mRNA levels, respectively, in the C2:Rev-erbE cell line. Total RNA was extracted from proliferating myoblasts (PMB) or myotubes were harvested after 4 days of serum withdrawal (MT4), reverse transcribed to cDNA, and analyzed by Q-RT-PCR. Gene-specific primers were used to examine the expression of Tnni1 (A) and Tnni2 (B). C and D, total RNA was extracted from wild type (C2C12) and dominant negative (C2:Rev-erbE) cell lines from myotubes after 4 days of serum withdrawal, reverse transcribed to cDNA, and analyzed by Q-RT-PCR. Gene-specific primers were used to examine the expression of Tnni1 (C) and Tnni2 (D). Normalized expression was calculated relative to the expression of 18S rRNA determined with the same cDNA. All reactions were performed in triplicate and results are shown as average & S.D.
This indicated that any major changes in the expression of metabolic markers were not because of compromised morphological and/or biochemical differentiation. Moreover, these data provide a threshold of significance whereby altered expression of genes involved in skeletal muscle metabolism would only be considered to be of importance if it varied more than 2-fold.
Analysis of the expression of the genes involved in skeletal muscle metabolism in these cell lines revealed that attenuation of Rev-erb had a number of significant effects in C2C12 cells (Table II). In the context of lipid and fatty acid absorption we observed repression of the mRNAs encoding Fabp-3 and -4 (5 and 24-fold) and Fat/Cd36 (5-fold) (Fig. 5, A-C). Analysis of genes involved in the regulation of energy expenditure and lipid utilization showed that the expression of Ucp3 mRNA (Fig. 5D) was significantly repressed (7.5-fold). In contrast, Ucp2 mRNA showed minimum changes in the cell line overexpressing Rev-erbE, relative to the 2-fold changes observed in the markers of differentiation (Table II). Moreover, the mRNA encoding Ampk3 (preferentially expressed in glycolytic fast twitch muscle fibers), a regulator of glucose uptake, and energy balance was unchanged by Rev-erbE expression. The expression of Scd-1, a key enzyme involved in adiposity, is repressed 4-fold in the cell line overexpressing Rev-erbE (Fig. 5E). In contrast, the expression of mRNA encoding the Scd-2 was relatively unaffected. Surprisingly, Srebp-1c mRNA expression increased 2.8-fold (Fig. 5F, Table II).
Rev-erb controls genes that regulate lipid metabolism and energy expenditure. Total RNA was extracted from wild type (C2C12) and dominant negative (C2:Rev-erbE) cell lines from myotubes after 4 days of serum withdrawal, reverse transcribed to cDNA, and analyzed by Q-RT-PCR. Gene-specific primers were used to examine the expression of: A, Fabp-3; B, Fabp-4; C, Cd36; D, Ucp-3; E, Scd-1; and F, Srebp1c. Relative expression is shown as -fold repression and -fold activation calculated relative to the expression in the wild type C2C12 cell line, which was arbitrarily set at 1. All reactions were performed in triplicate and results are shown as average & S.D.
The expression of the transcripts encoding Cpt1, Mcad, and Acs-4 that are involved in lipid catabolism and preferential fuel utilization were not affected in the Rev-erbE expressing cell line (Table II). Additionally, we did not observe any changes in the expression of mRNAs encoding ABCA1, ABCA8/G1, apoE, Cav-3, and ADRP, which are involved in cholesterol homeostasis and lipid storage (Table II). Analysis of the expression of many other nuclear hormone receptors that have been demonstrated to regulate lipid and carbohydrate metabolism, including ERR, PPAR-, -/, and -, LXR and -, Rev-erb, and Nur77 demonstrated that Rev-erbE expression did not affect the expression of the mRNAs encoding these NRs (see Table IV).
Rev-erb Modulates Myokine Expression-Muscle cytokines (myokines) include IL-6, IL-15, and myostatin control inflammation, energy expenditure, muscle growth, and fat deposition (see Table I). The Rev-erb nuclear receptor has been implicated in the control of inflammation, and the modulation of the NFB-dependent pathway. Therefore, we decided to investigate the expression of several myokines and inflammatory markers/regulators in the wild-type and C2C12 cells expressing Rev-erbE.
Initially, we examined the expression of IL-6, IL-15, myostatin, and IB (the regulator of NFB) during differentiation of native C2C12 cells. We isolated total RNA from wild type C2C12 proliferating myoblasts, and post-mitotic multinucleated myotubes after 4 days of serum withdrawal, and analyzed the expression levels of mRNAs by Q-RT-PCR. First, we observed that these myokines, and modulators of the inflammatory process, were expressed in wild type C2C12 cells (Fig. 6). Second, we observed that IL-15 and myostatin were induced (31- and 25-fold, respectively), whereas IB and IL-6 were repressed (2.5- and 5.5-fold, respectively) during myogenic differentiation (Fig. 6, A-D).
Rev-erb regulates myokine expression. Total RNA was isolated from wild type C2C12 proliferating myoblasts (PMB) and post-mitotic multinucleated myotubes after 4 days of serum withdrawal (MT4), A-D, or wild type (C2C12) and dominant negative (C2:Rev-erbE) cell lines from myotubes after 4 days of serum withdrawal (E-H). RNA was reverse transcribed to cDNA and analyzed by Q-RT-PCR. Gene-specific primers were used to examine the expression of: A and E, IB; B and F, IL-6; C and G, IL-15; and D and H, myostatin. Relative expression is shown as -fold repression and -fold activation calculated relative to the expression in the wild type C2C12 cell line that was arbitrarily set at 1. All reactions were performed with at least triplicates and results are shown as average & S.D.
Subsequently, we examined the effect of Rev-erbE on the expression of the myokines, and inflammatory regulators in C2:Rev-erbE cells after 4 days of serum withdrawal, relative to similarly differentiated native C2C12 cells by Q-RT-PCR (see Table III). Interestingly, we observed that ectopic expression of Rev-erbE leads to an induction in the inhibitor of NFB-mediated gene expression, IB (Fig. 6E). Consistent with increased expression of IB, mRNAs encoding the NFB and tumor necrosis factor target genes IL-15 (Fig. 6G) and Cox-2 (Table III) were not induced in cells expressing Rev-erbE.
Relative changes in the expression of genes encoding myokines and inflammatory markers in skeletal muscle cells constitutively expressing Rev-erbE
Curiously, we found that IL-6 mRNA is robustly increased (15- Fig. 6F), and myostatin mRNA is dramatically reduced (24- Fig. 6H) in the cells expressing Rev-erbE, concordant with the precocious differentiation of the C2:Rev-erbE cell line. In summary, this demonstrates that Rev-erb regulates myokine expression, and as such is a critical modulator of muscle growth and inflammation in this tissue.
DISCUSSION
We have utilized the C2C12 in vitro cell culture model system and ectopic overexpression of dominant negative Rev-erb to investigate the role of this orphan receptor in skeletal muscle lipid, and energy homeostasis. Here we report that perturbation of Rev-erb function decreases the expression of genes involved in lipid absorption. Additionally, we observed dramatic induction and repression of two myokines, IL-6, and myostatin, respectively, which are involved in energy expenditure, inflammation, muscle hypertrophy and hyperplasia, and the accumulation of body fat.
These observations are consistent with genetic and molecular studies that demonstrate that the NR1D subfamily of nuclear receptors (Reverbs) has a direct role in lipid homeostasis. For example, the NR1D subfamily of orphan receptors regulate the expression of ApoC-III (20, 21), a major component of triglyceride-rich remnant lipoprotein and associated with hypertriglyceridemia (37). In addition, Rev-erb -/- mice have elevated levels of ApoC-III and very low density lipoprotein triglycerides. Moreover, Rev-erb regulates ApoA1 gene expression in rodents treated with the hypolipidemic fibrate drugs. Finally, Rev-erb is preferentially expressed in fast twitch/type IIB glycolytic, and intermediate type IIA oxidative fibers and null mutations lead to a transition to type I fibers (38). This suggests that this orphan NR subgroup regulates muscle fiber type, which can influence insulin sensitivity and the utilization of aerobic/anaerobic metabolism for the generation of ATP.
Specifically, we demonstrated that attenuation of Rev-erb-mediated gene expression suppresses the expression of a subgroup of genes that include Fabp-3 and -4, Cd36, Ucp-3, and Scd-1 that are involved in lipid absorption and utilization. In this context, it is interesting to note that mice with a targeted disruption of SCD-1, a key target for Rev-erb, had lower levels of very low density lipoprotein, impaired triglyceride and cholesterol ester biosynthesis, and a lean phenotype (39). The Scd-1 -/- phenotype is very similar to the natural mutation called staggerer (RORsg/sg) in an obese mouse strain, which leads to a functional knockout of ROR. The staggerer mice exhibit an aberrant blood-lipid profile with lower circulating plasma levels of HDL-C, apoC-III, and plasma triglycerides. It should be noted that a dominant negative ROR represses both Rev-erb and - mRNA expression in skeletal muscle (16). Moreover, lack of Fabp4, a key target for Rev-erb in our model, protected the mice deficient in ApoE against atherosclerosis (40).
Interestingly, we observed a suppression in the expression of genes involved in lipid absorption, and in Scd-1 that is involved in the formation of cholesterol esters. Moreover the activity of Scd-1 has also been linked to adiposity. Curiously, we observed an increase in Srebp-1c expression, the master regulator of fatty acid metabolism. This apparent contradiction is in concordance with several reports in the literature. For example, Raspe et al. (15), showed that staggerer mice have reduced plasma triglycerides, and Lau et al. (16) demonstrated that a dominant negative ROR expression in muscle reduced Srebp-1c mRNA expression. The increase 5-fold increase in ROR mRNA expression in the C2:Rev-erbE cell line. Second, in most tissues SREBP-1c and SCD-1 mRNA are coordinately expressed and regulated. However, it has been previously reported in skeletal muscle cells, and tissue treated with LXR agonists, that Srebp-1c and Scd-1 mRNA expression are uncoupled, and not co-ordinately regulated (23).
Our studies also demonstrate that Rev-erb has a crucial role in regulating UCP3. Abundant expression of this gene correlates with preferential lipid utilization, and increased energy expenditure. The role of uncoupling proteins in regulating energy balance have been extensively investigated through transgenic mice and cell culture studies (41-43). Repression of UCP3 mRNA also correlates with the decreased expression of genes involved in lipid absorption and utilization, in concordance with the observations derived from this study.
Recent studies (22, 44, 45) have demonstrated a role of ROR in the inflammation process. For example, ROR has an anti-inflammatory role by inhibiting tumor necrosis factor--induced gene expression (22). The molecular basis of this modulation involves the direct induction of IB transcription, thereby inhibiting the NFB signaling cascade. Conversely, Rev-erb induces the NFB-mediated activation of the inflammatory cascade, for example, Cox-2 (44). Our studies show that attenuation of Rev-erb-mediated gene regulation leads to elevated ROR and IB expression in skeletal muscle cells. This correlates with no change in expression of the NFB/tumor necrosis factor target genes, Cox-2 and IL-15.
Surprisingly, these studies have revealed a dramatic induction (15-fold) of interleukin-6, in the presence of increased IB expression. However, IL-6 is an exercised induced myokine that induces lipolysis in adipose tissue, and suppresses tumor necrosis factor production. The increased expression of IL-6 is consistent with decreased myostatin expression. For example, it has been reported that IL-6 deficiency leads to late-onset obesity (46), whereas myostatin deficiency leads to the reduced accumulation of body fat (47). Therefore, the inverse correlation between IL-6 and myostatin expression in our Rev-erb cell line is in concordance with the phenotypic effects of these myokines on metabolism (48-50). The study has implicated Rev-erb in the regulatory cascade controlling genes involved in lipid homeostasis. Furthermore, the significant impact on myokine expression that regulates inflammation, lipolysis, muscle growth, and the accumulation of body fat underscores the potential therapeutic utility of Rev-erb modulation by inverse agonists/antagonists. However, identification of small molecule regulators will be hindered by the lack of a significant pocket in the ligand binding domain in the Rev-erb/NR1D subgroup of orphan nuclear receptors (51).
It is becoming increasingly apparent that skeletal muscle is a critical target tissue in the fight against metabolic disorders associated with diet, lifestyle, and metabolism. For example, LXR, PPAR, -/, and - in skeletal muscle have been shown to be involved in enhancing the insulin-stimulated glucose disposal rate, decreasing triglycerides, increasing energy expenditure, and increasing lipid catabolism, cholesterol efflux, and plasma HDL-C levels (14, 23-28).
Activation of these NRs in muscle has lead to increased insulin sensitivity, resistance to diet-induced obesity, and atherosclerosis. Hence, orphan nuclear receptors (for example, Rev-erb) that regulate lipid homeostasis and inflammation in skeletal muscle have enormous homeostatic ramifications. As discussed, skeletal muscle is a major mass peripheral tissue, and this lean tissue accounts for 40% of the total body mass (36% for females, and 42% for males) and 30-50% of the energy expenditure. These results indicate significant homeostatic interrelationships between the muscular and other body systems including the cardiovascular, endocrine, and lymphatic networks for the treatment of dyslipidemia, syndrome X, and inflammation. In conclusion, we suggest that in skeletal muscle cells, Rev-erb programs a cascade of gene expression that controls the regulatory cross-talk between lipid metabolism and inflammation.
ACKNOWLEDGMENTS
We thank Rachel Burow and Shayama Wijedasa for technical assistance with cell culture and plasmid preparation.
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