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Endocrinology Vol. 147, No. 11 5217-5227
Copyright © 2006 by The Endocrine Society

The Orphan Nuclear Receptor, NOR-1, Is a Target of ß-Adrenergic Signaling in Skeletal Muscle

Michael A. Pearen, James G. Ryall, Megan A. Maxwell, Naganari Ohkura, Gordon S. Lynch and George E. O. Muscat

Institute for Molecular Bioscience (M.A.P., M.A.M., G.E.O.M.), Division of Molecular Genetics and Development, University of Queensland, St. Lucia, Queensland 4072, Australia; Basic and Clinical Myology Laboratory (J.G.R., G.S.L.), Department of Physiology, The University of Melbourne, Victoria 3010, Australia; and Tumor Endocrinology Project (N.O.), National Cancer Center Research Institute, Tokyo 104-0045, Japan

Address all correspondence and requests for reprints to: George E. O. Muscat, Institute for Molecular Bioscience, Division of Molecular Genetics and Development, University of Queensland, St. Lucia, Queensland 4072, Australia. E-mail: g.muscat{at}imb.uq.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ß-Adrenergic receptor (ß-AR) agonists induce Nur77 mRNA expression in the C2C12 skeletal muscle cell culture model and elicit skeletal muscle hypertrophy. We previously demonstrated that Nur77 (NR4A1) is involved in lipolysis and gene expression associated with the regulation of lipid homeostasis. Subsequently it was demonstrated by another group that ß-AR agonists and cold exposure-induced Nur77 expression in brown adipocytes and brown adipose tissue, respectively. Moreover, NOR-1 (NR4A3) was hyperinduced by cold exposure in the nur77–/– animal model. These studies underscored the importance of understanding the role of NOR-1 in skeletal muscle. In this context we observed 30–480 min of ß-AR agonist treatment significantly and transiently increased expression of the orphan nuclear receptor NOR-1 in both mouse skeletal muscle tissue (plantaris) and C2C12 skeletal muscle cells. Specific ß2- and ß3-AR agonists had similar effects as the pan-agonist and were blocked by the ß-AR antagonist propranolol. Moreover, in agreement with these observations, isoprenaline also significantly increased the activity of the NOR-1 promoter. Stable exogenous expression of a NOR-1 small interfering RNA (but not the negative control small interfering RNA) in skeletal muscle cells significantly repressed endogenous NOR-1 mRNA expression and led to changes in the expression of genes involved in the control of lipid use and muscle mass underscored by a dramatic increase in myostatin mRNA expression. Concordantly the myostatin promoter was repressed by NOR-1 expression. In conclusion, NOR-1 is highly responsive to ß-adrenergic signaling and regulates the expression of genes controlling fatty acid use and muscle mass.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MICE THAT LACK the three known ß-adrenergic receptors (ß-ARs) are very susceptible to diet-induced obesity. These animals completely lack the diet (and cold)-induced thermogenic response, indicating that ß-ARs play a major role in energy expenditure (1). Whereas identification of the target tissues (and genes) relevant to diet-dependent energy expenditure has remained elusive, one such potential target is skeletal muscle. For example, ß-adrenergic signaling is known to elicit hypertrophy in skeletal muscle (2, 3, 4), although the mechanisms that mediate ß-ARs agonist-induced hypertrophy also remain obscure.

Skeletal muscle is a major mass metabolic tissue that accounts for approximately 40% of energy expenditure and total body weight. This peripheral lean tissue is the primary site for fatty acid oxidation and is involved in cholesterol efflux. Furthermore, this tissue is the main target of insulin-stimulated glucose uptake [accounting for approximately 80% of glucose disposal (5)]. Consequently, skeletal muscle has a major role in the blood lipid profile, insulin sensitivity, and energy homeostasis and therefore plays a role in the pathophysiological progression of metabolic disease. Skeletal muscle has a very high metabolic rate and even at rest uses significantly more energy than adipose tissue. Thus, skeletal muscle may play a role in body weight homeostasis through adaptive thermogenesis.

Several lines of evidence indicate skeletal muscle is a target of ß-adrenergic signaling. First, ß-AR agonists induce skeletal muscle hypertrophy through stimulation of ß2-ARs (6), and ß-ARs are involved in skeletal muscle growth and regeneration (7). Second, various studies that examined ß-AR agonists found an increase in resting energy usage, lipolysis, and heat production in skeletal muscle (8, 9, 10). However, the molecular mechanisms responsible for these effects in skeletal muscle in response to ß-AR signaling are currently unclear. Insights into the mode of action may have been provided by the demonstration that ß-AR agonists remarkably increase the mRNA encoding the nuclear hormone receptor (NR), Nur77 (NR4A1), a member of the NR4A subgroup. The specificity of the cross talk between NR and ß-adrenergic signaling was emphasized by the observation that the expression of other NRs was not elicited.

Transfection of a Nur77-specific small interfering (si)RNA into mouse muscle cells attenuated both the gene expression associated with the regulation of lipid use and energy balance [e.g. AMP-activated protein kinase-{gamma}3, uncoupling protein (UCP)-3, GLUT4], and lipolysis (11). The subset of genes identified by the siRNA strategies (11) is consistent with past studies showing that ß-adrenergic signaling increased energy expenditure and lipid catabolism (2, 3, 8, 9, 10). Recently Kanzleiter et al. (12) demonstrated that 30–60 min of ß-AR agonist treatment and cold exposure induced Nur77 expression in brown adipocytes and brown adipose tissue, respectively. Moreover, another member of the NR4A family, NOR-1 (NR4A3) was hyperinduced by cold exposure in the nur77–/– animal model (12). These data were consistent with the observations that high-fat feeding, ß2- and ß3-AR-specific agonists, isoprenaline (ß1–3-AR agonist), and exercise also induce UCP2 and UCP3 mRNA expression in skeletal muscle, adipose, and heart tissue (13, 14, 15, 16, 17).

In the context of understanding ß-adrenergic signaling, two members of the NR4A subgroup noted above, Nur77 and NOR-1, are expressed in skeletal muscle, and other adrenergic target tissues including adipose and heart (18, 19, 20, 21). These observations highlight the importance of elucidating whether NOR-1, the other member of the NR4A subgroup expressed in skeletal muscle, is regulated by ß-adrenergic agonists and is implicated in the regulation of metabolic gene expression.

In the current study, we identified that expression of the mRNA encoding NOR-1 is also strikingly and transiently induced by ß-AR signaling in the C2C12 skeletal muscle cell line and that this involves activation of the NOR-1 promoter. Expression of NOR-1 mRNA was also increased significantly in vivo in mouse skeletal muscle (plantaris) 1 h after treating mice with a ß2-AR agonist.

After siRNA-mediated suppression of NOR-1 mRNA, we observed alterations in the expression of a different (relative to Nur77) subset of genes controlling metabolism and muscle mass. In conclusion, the data suggest that expression of the NOR-1 in skeletal muscle is regulated by ß-AR signaling and is involved in the regulation of fatty acid use.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
C2C12 mouse myoblasts (American Type Culture Collection, Manassas, VA) were expanded in growth medium [DMEM; Invitrogen Australia, Mount Waverley, Victoria, Australia) supplemented with 10% heat-inactivated Serum Supreme (Cambrex Bio Science, Walkersville, MD)] in 7.5% CO2. Confluent myoblasts were differentiated into multinucleated myotubes using DMEM supplemented with 1% heat-inactivated fetal calf serum (Invitrogen Australia) and 1% heat-inactivated Serum Supreme up to 5 d. Cells were harvested at indicated time points.

Cell culture drug treatments
Confluent myoblasts were differentiated over 4 d before all drug treatments. Myotubes were treated with stated concentrations of isoprenaline hydrochloride (Calbiochem, San Diego, CA), 200 nM salbutamol hemisulfate (Sigma-Aldrich, Castle Hill, New South Wales, Australia), 200 nM BRL-37344 sodium (Sigma-Aldrich), 10 µM propranolol hydrochloride (Sigma-Aldrich), both 100 nM isoprenaline hydrochloride and 10 µM propranolol hydrochloride, or vehicle control (ethanol). Myotubes were harvested at indicated times after application of drugs or vehicle.

Animals, ß2-AR agonist administration, and tissue collection
All procedures were approved by the Animal Experimentation Ethics Committee of The University of Melbourne and conformed to the Guidelines for the Care and Use of Experimental Animals described by the National Health and Medical Research Council of Australia. Male C57BL/10 ScSn (C57BL/10, wild-type, 6–7 wk old) mice obtained from the Animal Resource Centre (Canning Vale, Western Australia, Australia) were randomly assigned to either saline control or formoterol-treated groups (n = 4 mice per time point per treatment). The mice were housed in the Biological Research Facility at The University of Melbourne and maintained on a 12 h-light, 12 h-dark cycle, with standard mouse chow and water provided ad libitum.

Treated C57BL/10 mice received a single ip injection of formoterol (100 µg/kg in 0.2 ml saline; Astra-Zeneca, Molndal, Sweden) and control mice (n = 4) received an equivalent volume of sterile saline. We demonstrated previously that this is the most efficacious dose for eliciting skeletal muscle hypertrophy in rats (4). Mice (n = 4 per time point) were anesthetized at 1, 4, 8, and 24 h after formoterol or saline administration and plantaris muscles were surgically excised. Tissue was also removed from anesthetized untreated mice (n = 4) that did not receive an ip injection (zero time point). All samples were snap frozen in liquid nitrogen for RNA extraction, cDNA synthesis, and quantitative real-time PCR (Q-RT-PCR) analysis.

Plasmids
The plasmids pBS-Luc (pGV-B; Wako Pure Chemical Industries, Osaka, Japan) and pNOR {alpha}/–1703 (22), POMC-tkLUC (23), pSG5 (Stratagene, La Jolla, CA), pSG5-NOR-1 (24), pGL3 Basic (Promega, Annandale, New South Wales, Australia), pGL3-B-2.5P (25), and pSilencer 3.1 neomycin negative (siNegative; Ambion, Austin, TX) have been previously described. From the coding region of murine NOR-1 long isoform cDNA (GenBank accession no. AF191211), two target siRNA sequences were chosen using the siRNA target finder on the Ambion Web site (http://www.ambion.com/techlib/misc/siRNA_finder.html). Target sequences were chosen on the basis of low homology with nontarget murine sequences using BLAST searches (26). Oligonucleotides sequences containing the siRNA hairpin were synthesized, annealed, and cloned into the pSilencer 3.1 neomycin expression vectors (Ambion) per the manufacturer’s protocol. Sequence of siRNA hairpin oligonucleotides was: pSilencer 3.1 NOR-1(AB) insert sequence, 5'-GATCCGAGGACGGCCACGCTGACCTTCAAGAGAGGTCAGCGTGGCCGTCCTCTTTTTTGGAAA-3', 5'-AGCTTTTCCAAAAAAGAGGACGGCCACGCTGACCTCTCTTGAAGGTCAGCGTGGCCGTCCTCG-3', pSilencer 3.1 NOR-1(DE) insert sequence [siNOR-1(C)]: 5'-GATCCGAGACGCCGAAACCGATGTTTCAAGAGAACATCGGTTTCGGCGTCTCTTTTTTGGAAA-3', 5'-AGCTTTTCCAAAAAAGAGACGCCGAAACCGATGTTCTCTTGAAACATCGGTTTCGGCGTCTCG-3').

Transient transfection
C2C12 myoblasts were transfected at 50–70% confluence in 24-well plates. Transfections were carried out in growth medium using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol with 0.8 µg of DNA per well. For testing of siRNA constructs on NOR-1-dependent gene expression, a mixture of DOTAP (N-[1-(2,3-dioleoyloxy)-propyl]N,N,N-trimethyl-ammonium methylsulfate; Roche Diagnostics Australia, Castle Hill, New South Wales, Australia) and Metafectene (Biontex Laboratories GmbH, Munich, Germany) at a 4:1 ratio in HEPES-buffered saline [42 mM HEPES, 275 mM NaCl, 10 mM KCl, 0.4 mM Na2HPO4, 11 mM dextrose (pH 7.1)]. For both transfection methods, growth medium was replaced after 14–16 h. After an additional 24 h, cells were either harvested or treated with isoprenaline and then harvested at indicated times. Harvested cells were assayed for luciferase activity using LucLite reagent (PerkinElmer Life Sciences, Boston, MA) according to the manufacturer’s protocol. Luminescence was measured using a Wallac 1450 Microbeta Trilux (PerkinElmer Life Sciences).

Stable C2C12 transfection
C2C12 myoblasts cultured in growth medium were transfected at approximately 40% confluence with 4 µg of siNOR-1(negative), siNOR-1(AB), or siNOR-1(DE) by Lipofectamine 2000 according to the manufacturer’s protocol in 25-cm2 flasks. Cells were selected for 12 d by treatment with 650 µg/ml geneticin (G418; Invitrogen) in growth medium. The polyclonal pool of G418-resistant cells (comprised of > 50 colonies to avoid clonal bias) was maintained in growth medium with 300 µg/ml geneticin. Before freezing cells, the polyclonal population was passaged into three replicates, differentiated into myotubes as described above, and RNA independently extracted from each replicate of cells.

RNA extraction and cDNA synthesis
RNA was extracted from C2C12 cells using TRI-Reagent (Sigma-Aldrich Australia) according to the manufacturer’s protocol. Extracted RNA was treated with 2 U Turbo DNase (Ambion) for 30 min. RNA was further purified using a mini-RNeasy kit (QIAGEN, Doncaster, Victoria, Australia) according to the manufacturer’s instructions. cDNA was synthesized from 3 µg total RNA (normalized via UV spectroscopy) using Superscript III primed by random hexamers, according to the manufacturer’s instructions (Invitrogen).

Q-RT-PCR
Target cDNA levels were compared by Q-RT-PCR in 25-µl reactions containing either 1x SYBR green (Applied Biosystems, Warrington, UK) or Taqman PCR master mix (Applied Biosystems, Foster City, CA), 100 µM of each forward and reverse primers for SYBR green, or 1x Assay-on-Demand Taqman primers (Applied Biosystems) and the equivalent of 0.3 µl cDNA. Using an ABI Prism 7500 sequence detection system (Applied Biosystems), PCR was conducted over 45 cycles of 95 C for 15 sec and 60 C for 1 min, preceded by an initial 95 C for 10 min. Results were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) unless otherwise indicated and compared by relative expression and the delta-delta-cycle threshold method for fold change calculations.

Primers
Primers for Q-RT-PCR analysis of the mRNA populations using SYBR green have been described in detail (11, 28, 36) except for primers used to detect NOR-1 (forward: 5'-GATCACAGAGCGACATGGGTTA-3'; reverse: 5'-GAGCCTGTCCCTTC CTCTGG-3'). All primers were designed using Primer Express (Applied Biosystems). Assay-on-Demand Taqman primer/probe sets were used to assay fatty acid binding protein (FABP) 4 and myostatin expression.

Western blot analysis
Whole-cell lysates were prepared from cells disrupted in lysis buffer [50 mM Tris HCl, 75 mM NaCl, 5 mM MgCl2, 1% Nonidet-P40, 1 mM dithiothreitol and complete protease inhibitors (Roche Diagnostics Australia)] resolved on a 10% SDS-PAGE gel. Proteins were transferred to a polyvinyl difluoride membrane (Immobilon-P; Millipore, North Ryde, New South Wales, Australia), blocked with 5% skim milk powder in Tris-buffered saline with 0.1% Tween 20, and probed with mouse anticaveolin (CAV)-3 (BD Biosciences, Lexington, KY) at 1:500, rabbit anti-GAPDH (R&D Systems, Minneapolis. MN) at 1:2000, or mouse antimyosin heavy chain (Sigma-Aldrich). Secondary antibodies were used at 1:2000 for both antirabbit-horseradish peroxidase (Pierce Biotechnology, Rockford, IL) and antimouse-HRP (Zymed Laboratories, San Francisco, CA). Horseradish peroxidase localization was detected with Supersignal West Pico Substrate (Pierce Biotechnology) according to the manufacturer’s instructions and visualized by autoradiography.

Cholesterol assay
Total cholesterol was measured using the Amplex Red cholesterol assay kit (Invitrogen) according to the manufacturer’s protocol. Protein was assayed via the method of Bradford (29) using Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NOR-1 mRNA is expressed in C2C12 skeletal muscle cells and induced during skeletal myogenesis
NOR-1 mRNA is expressed in human (21, 22) and murine skeletal muscle (30); however, expression in the C2C12 skeletal muscle cell line has not been ascertained to date. This study used the C2C12 skeletal muscle cell line as an in vitro model of skeletal muscle differentiation (myogenesis). During this process proliferating myoblasts differentiate into postmitotic multinucleated myotubes.

We harvested RNA for Q-RT-PCR analysis from C2C12 proliferating myoblasts (PMBs), confluent myoblasts (CMBs), and myotubes after 1–4 d of differentiation (MT1–4) after serum withdrawal.

We observed that the expression of NOR-1 mRNA was markedly increased during myogenesis (Fig. 1AGo). We confirmed that the cells had biochemically differentiated by assaying the expression of mRNAs that encode muscle-specific differentiation-dependent markers. For example, we observed the expected dramatic induction of myogenin mRNA (Fig. 1BGo) that encodes a key hierarchical basic helix-loop-helix transcription factor responsible for the activation of contractile-specific gene expression. Furthermore, we observed a similarly striking induction in the mRNA encoding type 1 (slow twitch) troponin I (TNNI1; Fig. 1CGo), and type 2 (fast twitch) troponin I (TNNI2; Fig. 1DGo), respectively. These data confirmed that the C2C12 cells had terminally differentiated.


Figure 1
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  		FIG. 1. NOR-1 mRNA is expressed during myogenesis in C2C12 cells. C2C12 cells were harvested during myogenesis as PMBs, CMBs, and MT1–4 for Q-RT-PCR analysis. Gene-specific primers were used to examine mRNA expression of NOR-1 (A), myogenin (B), TNNI1 (C), and TNNI2 (D) relative to GAPDH. Each data point is expressed as the mean of three replicates ± SD.

 
In summary, these data demonstrate that the mRNA transcript encoding NOR-1 was expressed in C2C12 cells in a differentiation-dependent manner, concomitant with the acquisition of a contractile and metabolic phenotype.

The ß-AR agonist isoprenaline significantly and transiently induces NOR-1 mRNA expression in C2C12 cells
We used the C2C12 skeletal muscle cell culture model to examine whether regulatory cross talk occurred between the ß-AR and NOR-1 signaling pathways in skeletal muscle. The induction of Nur77 by the ß-AR agonist isoprenaline in skeletal muscle cells has been reported previously (11, 18). Therefore, to investigate whether NOR-1 mRNA was induced by ß-AR signaling, we treated differentiated C2C12 skeletal muscle myotubes with 100 nM of the ß-AR agonist, isoprenaline (and the vehicle, ethanol) for 30, 60, 90, 120, 240, 480, and 1440 min and subsequently isolated total RNA. Q-RT-PCR was used to examine the expression of mRNA encoding NOR-1. Interestingly, we observed that the mRNA encoding NOR-1 (relative to 18S rRNA) was dramatically induced (>30-fold) after a 60-min isoprenaline treatment (Fig. 2AGo) normalized against the vehicle control at all times. The increases in mRNA expression were strikingly transient and declined after 60 min. Each time point between 30 and 240 min was significantly (P < 0.01) above the vehicle control. The ß-adrenergic induction of NOR-1 mRNA trails Nur77 mRNA activation by 15–30 min (data not shown). We have previously shown that the expression of many other mRNAs encoding the nuclear hormone receptors that have been demonstrated to regulate lipid and carbohydrate metabolism, including liver X receptors-{alpha} and -ß, peroxisome proliferator activated receptors-{alpha}, -ß/{delta}, and -{gamma}, estrogen-related receptor-{alpha}, and retinoid-related orphan receptor-{alpha}1, did not respond to isoprenaline treatment in skeletal muscle cells (11).


Figure 2
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  		FIG. 2. ß-Adrenergic agonists increase NOR-1 mRNA expression and promoter activity. Q-RT-PCR was used to assay the expression of NOR-1 in differentiated C2C12 myotubes in the following treatment. A, Treatment for 30–1440 min with 100 nM isoprenaline and vehicle. Cells were harvested over 24 h and RNA extracted. Q-RT-PCR results were normalized against 18S and converted to fold induction relative to vehicle controls at each time point. Data are expressed as the mean of three independent treatments/experiments (n = 3), each assayed in triplicate ± SD. B, Treatment with increasing concentrations of isoprenaline after 60 min. Q-RT-PCR results were normalized against 18S and converted to fold induction relative to vehicle controls at each time point. Data from each point are derived from the mean of three replicates ± SD. C, Treatment for 60 min with vehicle, salbutamol (ß2-AR agonist), BRL-37344 (ß3-AR agonist), isoprenaline (ß-AR agonist), or propranolol (ß-AR antagonist) or cotreated with both isoprenaline and propranolol. Cells were harvested after 60 min. Q-RT-PCR results were normalized against 18S and converted to fold induction relative to vehicle controls. Data are expressed as the mean of three independent treatments/experiments (n = 3), each assayed in triplicate ± SD. D, Q-RT-PCR was used to assay the expression of NOR-1 mRNA in mouse plantaris muscle removed at 60, 240, 480, and 1440 min after a single ip injection of the ß2-AR agonist formoterol or vehicle (n = 4 mice per time point for each treatment). Results were normalized against 18S and converted to fold induction relative to vehicle controls (saline) at each time point. Data are expressed as the mean of four animals (n = 4 per time point) ± SD. E, The promoter activity of NOR-1 in response to isoprenaline was also examined by transfecting undifferentiated C2C12 myoblasts with either pBS-Luc or pNOR {alpha}/-1703 (NOR-1 promoter) overnight followed by treatment with vehicle or 100 nM isoprenaline for 24 h. Values are expressed as relative light units (RLU) ± SD and derived from three independent transfections (n = 3); each transfection was comprised of three replicates. F, Analysis of increasing concentrations of isoprenaline and vehicle on the activity of the NOR-1 promoter. Each value is expressed as relative light units (RLU) ± SD and derived from six replicates. Statistical significance in experiments A, C, D, and E was assessed using an unpaired Student’s t test where P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).

 
In addition, we examined the induction of NOR-1 mRNA at 60 min after treatment using increasing concentrations of isoprenaline from 10 nM to 10 µM and observed that 100 nM was sufficient to induce maximal induction of NOR-1 mRNA expression (Fig. 2BGo).

Selective ß2- and ß3-AR agonists activate NOR-1 mRNA expression
ß2-ARs are abundant in skeletal muscle; however, ß1- and ß3-ARs are also present (31, 32, 33). Moreover, the ß-AR agonist, isoprenaline, activates all three ß-AR subtypes. We used the selective and specific ß2-, and ß3-AR agonists, salbutamol and BRL-37344, respectively, to examine which subtype mediated the induction of the mRNAs encoding NOR-1. NOR-1 mRNA was significantly induced at 60 min after treatment of MT4 myotubes with both salbutamol (P < 0.001) and BRL-37344 (P < 0.005), compared with vehicle (Fig. 2CGo). In summary, these data demonstrate that various ß-AR agonists strikingly activate the expression of mRNA encoding NOR-1.

To confirm and validate that isoprenaline-mediated induction of NOR-1 mRNA expression specifically involved ß-AR signaling, we used propranolol, an antagonist of ß-AR-mediated signaling. Differentiated C2C12 muscle cells were treated with isoprenaline and propranolol and also cotreated with isoprenaline and propranolol. The antagonist significantly (P < 0.001) attenuated the induction of NOR-1 mRNA expression (relative to 18S) observed after a 60-min isoprenaline treatment, compared with the vehicle control (Fig. 2CGo).

NOR-1 mRNA is significantly induced by ß2-AR agonists in murine plantaris muscle
To confirm whether ß-adrenergic agonists induce NOR-1 mRNA in vivo, we treated groups of mice with either the ß2-AR agonist formoterol or vehicle (saline) control and assayed NOR-1 mRNA expression in plantaris muscle. Supporting the cell culture data, we observed a significant dramatic induction (>100-fold; P < 0.005) of the mRNA encoding NOR-1 (normalized to 18S rRNA) after 60 min (relative to the vehicle control) after ß2-AR treatment (Fig. 2DGo). The significant increase in NOR-1 mRNA above the vehicle control was also observed at 4 h (P < 0.001) and 8 h (P < 0.01) but not 24 h after treatment.

Isoprenaline significantly increases the activity of the NOR-1 promoter
To examine whether isoprenaline activated the NOR-1 promoter, we transfected C2C12 myoblasts with either the NOR-1 promoter [pNOR {alpha}/-1703 vector (22)] or pBS-Luc (vector control) and treated with 100 nM isoprenaline for 24 h. Luciferase expression in the pNOR {alpha}/-1703 vector is driven by the region 1–1703 nucleotides upstream of the NOR-1{alpha} transcription start site (22). Isoprenaline treatment resulted in a significant (P < 0.005) activation of the NOR-1 promoter (Fig. 2EGo). In addition, we examined the activity of the promoter in the presence of increasing concentrations of isoprenaline from 10 nM to 10 µM. Again we observed that 100 nM was sufficient to induce maximal induction (Fig. 2FGo).

Expression of NOR-1 siRNA attenuates NOR-1-dependent gene expression and represses levels of endogenous NOR-1 mRNA
To elucidate the biological role of NOR-1 in skeletal muscle lipid and energy homeostasis and discover the metabolic target genes of this orphan receptor in muscle cells, we examined the effect of attenuating NOR-1 expression/function. This provided a tool for elucidating the consequences of ß-AR-mediated induction of NOR-1.

We used RNA interference to achieve targeted silencing of NOR-1 using double-stranded siRNA expression as the triggering agent. We designed two small interfering (siRNA) expression vectors [siNOR-1(AB) and siNOR-1(DE)] targeted to NOR-1 mRNA at the activation domain (AB) and ligand binding domain (DE). The location and sequence of these targets is shown in Fig. 3AGo.


Figure 3
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  		FIG. 3. Expression of NOR-1 siRNA attenuates NOR-1-dependent gene expression and endogenous NOR-1 expression. A, Location and sequences of siRNA targets on the primary structure of NOR-1. B, Cotransfection of POMC-tkLUC reporter with pSG5 or pSG5-NOR-1 (for exogenous NOR-1 expression) and siRNA constructs [siNEG, siNOR-1(AB) or siNOR-1(DE)]. Mean luciferase activity is expressed as relative light units (RLU) ± SD of six replicates. C, Q-RT-PCR was used to assay NOR-1 mRNA expression in differentiated stable cell lines expressing NOR-1 siRNA [C2C12-siNOR-1(DE)], compared with stable cell lines expressing negative control siRNA (C2C12-siNEG). Q-RT-PCR results were normalized against 18S. The data represent the mean ± SD of three independent cell preparations (n = 3) assayed in triplicate. ***, Statistical significance was assessed using an unpaired Student’s t test where P < 0.001.

 
We analyzed the efficacy of the siRNA vectors by their ability to attenuate transactivation of the native NOR-1-dependent heterologous reporter gene (POMC-tkLUC, Fig. 3BGo) in the presence of cotransfected/ectopic and overexpressed NOR-1. The NR4A response element in this reporter is efficiently bound and transactivated by NOR-1 homodimers (23). C2C12 skeletal muscle myoblasts were cotransfected with the reporter construct, POMC-tkLUC and pSG5-NOR-1 (in the presence and absence of the pSilencer vectors). This demonstrated that the pSilencer 3.1 plasmid driving the siRNA targeting the C-terminal DE region successfully suppressed NOR-1-dependent gene expression (Fig. 3BGo).

We stably transfected C2C12 cells with pSilencer 3.1-negative control siRNA (a sequence not found in the mouse, rat, or human genomes), pSilencer 3.1-siNOR-1(AB), and pSilencer 3.1-siNOR-1(DE) vectors. Cells were selected with G418, differentiated, and total RNA extracted. Q-RT-PCR on three independent RNA preparations from each cell line was then performed to measure the expression of NOR-1 mRNA normalized against GAPDH. In these stable cell lines, we observed a highly significant (~3-fold; P < 0.001) attenuation of NOR-1 mRNA from pSilencer 3.1-siNOR-1(DE), compared with pSilencer 3.1 neomycin-negative control (henceforth denoted as C2C12-siNOR-1 and C2C12-siNEG, respectively; Fig. 3CGo). The stable pSilencer 3.1-siNOR-1(AB) cell line did not display a significant attenuation of NOR-1 mRNA (data not shown).

Expression of NOR-1 siRNA results in differential regulation of genes controlling lipid homeostasis
In previous studies, the C2C12 skeletal muscle cell line has provided an effective model for the investigation of lipid homeostasis in skeletal muscle (11, 28, 34, 35, 36, 37, 38). Moreover, the physiological validation of the cell culture model with respect to the control of lipid metabolism by liver X receptor and peroxisomal proliferator-activated receptors in the mouse substantiates the utility of this model system (11, 28, 35).

Hence, we proceeded to examine the effect of attenuating NOR-1 mRNA expression on the expression of genes involved in lipid and energy homeostasis. We compared expression in the siNOR-1-expressing stable cell line relative to the pSilencer-negative control cell line. RNA was harvested from both cells lines after differentiation in culture for Q-RT-PCR analysis (both cells line were phenotypically similar). To identify changes in the expression of metabolic genes after suppression of NOR-1, we used Q-RT-PCR to examine relative mRNA expression of various genes in differentiated C2C12-siNOR-1 relative to C2C12-siNEG (normalized against GAPDH). We examined a panel of several dozen genes involved in skeletal muscle metabolism. For the sake of brevity, the graphical data for only a select number of genes that were affected by NOR-1 inhibition are presented. For example, the expression of mRNA encoding the genes FABP3, apolipoprotein-E, and stearoyl-CoA desaturase 1 was not significantly affected by NOR-1 attenuation (data not shown).

In the context of lipid and fatty acid homeostasis, we observed significantly increased expression (~3-fold, P < 0.01) of the mRNA encoding FABP4 (Fig. 4AGo), in contrast to a less than 2-fold repression of FABP3 (data not shown). Changes in mRNA expression less than 2-fold obtained from the stable cell lines were not considered important; this issue is addressed in detail in the next section. The mRNA encoding CAV3, which has been implicated in insulin resistance in knockout mice (39) and lipid transport and storage in vitro (40, 41), was significantly suppressed (~3-fold, Fig. 4BGo) in the C2C12-siNOR-1 cell line relative to C2C12-siNEG control cells. This change appeared more prominent at the protein level via Western blotting (Fig. 4CGo). No changes were evident at the protein level in GAPDH or myosin heavy chain (a major thick filament contractile protein of muscle; Fig. 4CGo). Because CAV3 may regulate cholesterol efflux, we examined total cholesterol (normalized to total protein) present in both cells lines and found no significant change in total cholesterol between the cell lines (Fig. 4DGo).


Figure 4
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  		FIG. 4. Expression of NOR-1 siRNA results in differential changes in the expression of mRNAs encoding genes that regulate lipid use. Using Q-RT-PCR, the expression of various metabolic genes was compared in both C2C12-siNEG and C2C12-siNOR-1 myotubes. FABP4 (A) and CAV3 (B) are shown graphically relative to GAPDH. C, The CAV3 result was validated at the protein level via Western blotting with GAPDH and myosin heavy chain (MHC). D, Total cellular cholesterol was examined between the cell lines relative to total protein. Values are expressed as mean ± SD (n = 6). Using Q-RT-PCR, the expression of UCP2 (E) and UCP3 (F) was also compared in both C2C12-siNEG and C2C12-siNOR-1 myotubes relative to GAPDH. G, Q-RT-PCR was used to assay UCP2 in differentiated C2C12 myotubes after treatment with 100 nM isoprenaline or vehicle for 60–1440 min. Cells were harvested over 24 h and RNA extracted for Q-RT-PCR analysis. RT-PCR results were normalized against 18S and converted to fold induction relative to vehicle controls at each time point. All Q-RT-PCR data above represent the mean ± SD of three independent cell preparations/treatments (n = 3) assayed. H, Q-RT-PCR was used to assay the expression of UCP2 mRNA in mouse plantaris muscle removed at various time points over 24 h after a single ip injection of formoterol or vehicle control (saline). Results were normalized against 18S and converted to fold induction relative to vehicle controls at each time point. Data are expressed as the mean of four animals (n = 4 per time point for each treatment) ± SD. Statistical significance was assessed using an unpaired Student’s t test where P < 0.05 (*), P < 0.01 (**); P < 0.001 (***) and P > 0.05 (NS).

 
Subsequently we analyzed the expression of a panel of genes involved in the regulation of lipid use (and energy expenditure) and found the expression of UCP2 mRNA (Fig. 4EGo) was significantly repressed (~7-fold; P < 0.001). In contrast, UCP3 mRNA was induced in the cell line expressing siNOR-1 RNA relative to the C2C12-siNEG control cell line (Fig. 4FGo).

Because UCP2 expression was repressed in C2C12-siNOR-1 cells, we were interested in examining UCP2 expression in cells that have increased NOR-1 mRNA expression due to isoprenaline treatment. Differentiated C2C12 skeletal muscle myotubes were treated with 100 nM isoprenaline or vehicle (ethanol) for 60, 120, 480, and 1440 min and harvested for isolation of total RNA. Q-RT-PCR was used to examine the expression of mRNA encoding UCP2, and it was observed that the expression was subtly but significantly (P < 0.02) induced at all time points from 60 to 1440 min after isoprenaline treatment when normalized to the vehicle control (see Fig. 4GGo).

To confirm whether ß-adrenergic agonists induce UCP2 mRNA in vivo, groups of mice treated with either the ß2-AR agonist formoterol or saline control for 60, 240, 480, and 1440 min were assayed for UCP2 mRNA expression in plantaris muscle. The expression of UCP2 mirrored cell culture data with a significant induction in this transcript above the vehicle controls at 4 and 8 h after treatment (Fig. 4HGo).

The effect of NOR-1 repression on lipid homeostasis is independent of differentiation
To evaluate whether the changes observed in the NOR-1-siRNA stable cell lines were due to aberrant differentiation, the mRNA levels of the biochemical markers of differentiation (myogenin) and contraction [(TNNI1 and TNNI2), i.e. the acquisition of the contractile phenotype] were analyzed in both the C2C12-siNEG and the NOR-1-siRNA stable cell lines.

Minimal changes (1.4- to 1.8-fold) in the expression of the mRNA encoding the skeletal muscle differentiation markers myogenin (Fig. 5AGo), TNNI1 (Fig. 5BGo) and TNNI2 (Fig. 5CGo) were observed relative to the dramatic changes observed in the metabolic genes reported above. Because the changes in the muscle markers were in the range of 1.4- to 1.8-fold, we chose to focus on genes modulated greater than 2-fold; this threshold ensured the changes were independent of differentiation effects.


Figure 5
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  		FIG. 5. The effects of NOR-1 repression on gene expression are independent of differentiation. Using Q-RT-PCR, the expression levels of myogenin (A), TNNI1 (B), and TNNI2 (C) were compared between C2C12-siNEG and C2C12-siNOR-1 myotubes relative to GAPDH. Data represent the mean ± SD of three independent cell preparations/treatments (n = 3) assayed in triplicate. All three genes were nonsignificant when assessed using an unpaired Student’s t test (P > 0.05). For gene expression during myogenesis, PMBs, CMBs, and MT1–4 were harvested for Q-RT-PCR analysis. Gene-specific primers were used to examine mRNA expression of FABP4 (D), CAV3 (E), UCP2 (F), UCP3 (G), and myostatin (H) relative to GAPDH. D–H represent the mean ± SD of one experiment assayed in triplicate (n = 1).

 
We observed both an induction (e.g. myostatin, FABP4, and UCP3) and repression (e.g. CAV3 and UCP2) of gene expression in the stable cell lines that were all induced during the normal differentiation process (Fig. 5Go, D–I). These data emphasize that the observed phenomenon are not differentiation dependent and thus validate that the modulating effects of NOR-1 attenuation on metabolic gene expression were not due to aberrant differentiation.

Expression of NOR-1 siRNA results in the marked induction of myostatin (a regulator of muscle mass)
Interestingly, we observed a dramatic (>65-fold, P < 0.001) increase in the expression of myostatin mRNA (that encodes a negative regulator of muscle mass and hypertrophy) in the C2C12-siNOR-1 cell line relative to the C2C12-siNEG control cell line (Fig. 6AGo). This was a very striking change in mRNA expression; hence, we validated this by making another independent set of stable cell lines with the NOR-1 siRNA relative to the negative control vector and observed a similar increase in myostatin mRNA expression (data not shown).


Figure 6
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  		FIG. 6. NOR-1 modulates myostatin mRNA expression and the activity of the myostatin promoter. Using Q-RT-PCR, the expression of myostatin (A) was compared in both C2C12-siNEG and C2C12-siNOR-1 myotubes relative to GAPDH. Data represent the mean ± SD of three independent cell preparations (n = 3) assayed in triplicate. B, C2C12 myoblasts were cotransfected with pGL3-B or pGL3-B-2.5P (myostatin promoter) and NOR-1 expression vector (pSG5-NOR-1) or empty vector (pSG5). Values are expressed as mean relative light units (RLU) ± SD of three individual liposome/DNA mixtures assayed in triplicate (n = 3). C, Also varying concentrations of pSG5-NOR-1 balanced with pSG5 were examined. Values are expressed as mean relative light units (RLU) ± SD of six replicates. Statistical significance was assessed using an unpaired Student’s t test where applicable: *, P < 0.05; ***, P < 0.001.

 
We had observed that NOR-1 attenuation led to increased myostatin expression; therefore, we examined whether NOR-1 expression regulated the activity of the myostatin promoter. When C2C12 myoblasts were cotransfected with the myostatin promoter (pGL3-B-2.5P) and a NOR-1 expression vector (pSG5-NOR-1), the activity of the myostatin promoter was significantly and reproducibly repressed by 2-fold (Fig. 6BGo; P < 0.05), compared with the vector control. The effects of NOR-1 expression on the myostatin promoter were also examined using an increasing amount of pSG5-NOR-1 (balanced with pSG5). Increasing the amount of pSG5-NOR-1 in the transfection (and therefore NOR-1 expression) repressed the activity of myostatin promoter (pGL3-B-2.5P) in a dose-dependent manner but had no effect on the vector control (pGL3 basic; Fig. 6CGo).

In summary, the activation of myostatin mRNA expression after NOR-1 siRNA expression correlates with the repression of the myostatin promoter by NOR-1 expression. Furthermore, these data are consistent with ß-adrenergic induction of NOR-1 expression, and hypertrophy.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We used the C2C12 in vitro skeletal muscle cell culture system and exogenous expression of a NOR-1 siRNA to investigate the role of this orphan nuclear receptor in ß-adrenergic signaling and the gene expression associated with the regulation of lipid metabolism. Here we report that ß-AR agonist treatment strikingly induced NOR-1 mRNA in both mouse skeletal muscle cells and tissue (plantaris). Moreover, we demonstrated that attenuation of NOR-1 expression in skeletal muscle cells alters the expression of genes involved in the control of lipid homeostasis and muscle mass. These observations were underscored by the significant increase in myostatin mRNA expression after stable expression of NOR-1 siRNA.

The ß-adrenergic-mediated induction of NOR-1 is consistent with a recent study from our group that demonstrated a dramatic induction of Nur77 (NR4A1) mRNA expression after treatment of skeletal muscle cells with ß-AR agonists. Moreover, in this previous study, attenuation of Nur77 expression (and function) led to the aberrant expression of a different subset of genes involved in lipid homeostasis [CD36, AMP-activated protein kinase-{gamma}3, GLUT4, CAV3, and UCP3 (11)]. After our investigation, another study demonstrated that 30–60 min of ß-AR agonist treatment induced Nur77 expression in brown adipocytes, and cold exposure also induced Nur77 expression in murine brown adipose tissue. In addition, NOR-1 was hyperinduced in brown adipose tissue by cold exposure in the nur77–/– animal model, suggesting that the NR4A family is involved in thermogenesis, and raised the scenario of functional redundancy (12). Therefore, in this study we set out to elucidate whether NOR-1 mRNA expression was increased by ß-adrenergic agonists in skeletal muscle cells and tissue. We used siRNA targeting to identify genes/pathways that are targeted in skeletal muscle cells.

Specifically we demonstrated that the pan-ß-AR agonist, isoprenaline, the ß2- and the ß3-specific ß-AR agonists strikingly (and transiently) induced NOR-1 mRNA expression in skeletal muscle cells. Importantly, we demonstrated that NOR-1 mRNA expression was significantly induced (>100-fold) by a ß2-AR agonist in vivo. This is consistent with predominant expression of these ß-AR isotypes in skeletal muscle (10, 42). This induction of expression involved the activation of the NOR-1 promoter and suggested a direct transcriptional effect. Activation of the NOR-1 promoter by ß-AR signaling is not surprising because the promoter contains three cAMP response elements (22). Previous studies have demonstrated that the cAMP response element-binding protein directly transactivates the NOR-1 promoter (43, 44, 45). Our demonstration that ß-AR agonists activate the NOR-1 promoter is consistent with these studies because cAMP response element-binding protein is activated by cAMP in response to adrenergic signaling (46).

In this study, the ß-AR-specific antagonist, propranolol, blocked the induction of NOR-1 mRNA by isoprenaline, clearly indicating that NOR-1 mRNA is responsive to ß-AR signaling. In contrast, we previously demonstrated that the expression of mRNAs encoding the other subgroups of nuclear receptors are not sensitive to ß-AR agonist treatment in skeletal muscle cells (11). This indicates that the NR4A subgroup is selectively induced by ß-AR signaling, suggesting that cross talk between these signaling pathways will have specific and unique consequences.

The specific effects of NOR-1 (and Nur77) perturbation on the expression of genes involved in lipid homeostasis have distinct and overlapping effects. For example, in regard to lipid absorption/use, attenuation of Nur77 expression affects CD36 and FABP3 expression, whereas in contrast, NOR-1 perturbation affects FABP4 mRNA expression, a result that is consistent with the down-regulation of FABP4 mRNA expression (47). FABP4 is a cytosolic protein expressed in adipocytes, macrophages, and skeletal muscle, and is involved in the absorption/transport of fatty acids and other lipid-soluble molecules (48).

Expression of CAV3 mRNA was suppressed when both NOR-1 and Nur77 expression were suppressed (11). CAV3 is the primary structural protein of skeletal muscle caveolae and is selectively expressed in muscle. Lisanti and colleagues (39) recently demonstrated genetic ablation of CAV3 in mice results in hyperinsulinemia, insulin resistance, and glucose intolerance. Interestingly the ß2-AR is localized to the caveolae, and this localization seems to be required for function of the ß2-AR (49). Other studies have also shown down-regulation of CAV1 and -3 via ß-AR signaling (50) and cAMP (51), thus highlighting cross talk between caveolins and ß-AR signaling pathways.

Nur77 siRNA expression represses UCP3 mRNA expression, whereas NOR-1 siRNA expression inhibits UCP2 expression. Both of these proteins may be involved in lipid use, mitochondrial uncoupling, and perhaps energy expenditure (although this remains controversial). The subset of genes identified by the siRNA strategies (11) is consistent with past studies showing that ß-AR signaling increased energy usage, lipolysis, and heat production (2, 3, 8, 9, 10).

The major findings of the present study are that: 1) the NR4A subgroup is hypersensitive to ß-AR stimulation in skeletal muscle in vivo and in vitro; 2) there is selective and specific cross talk between ß-AR and nuclear hormone signaling; 3) attenuation of NOR-1 and Nur77 led to the suppression of UCP2 and UCP3, respectively (11); and 4) the weak but statistically significant induction of UCP2 mRNA by ß-AR agonists observed in both skeletal muscle and C2C12 skeletal muscle cells appears to be consistent with the phenotype of ß1–3-AR null mice. These mice develop severe obesity on a high-fat diet due to aberrant thermogenesis and do not demonstrate increased oxygen consumption in response to diet, temperature (cold), or ß-AR agonists, as observed in wild-type mice (1). Supporting this, previous studies have also shown specific ß2- and ß3-AR agonists and isoprenaline induce UCP2 and -3 mRNA expression in skeletal muscle (tissue and cultured cells), adipose, and heart tissue (13, 14, 15, 16, 17).

Because UCP2 and UCP3 have been linked to mitochondrial uncoupling (52, 53, 54, 55) and lipid metabolism (38, 56, 57, 58), it is possible that NR4A signaling is part of the regulatory cascade that controls the expression of UCP2 and -3 in response to ß-AR signaling. However, the function of UCP2 and -3 in regard to energy expenditure remains controversial because knockout mice for these genes do not exhibit a failure of adaptive thermogenesis (59, 60, 61). Because UCP2 and -3 mRNA expression in skeletal muscle and adipose is associated with increased levels of free fatty acid, it has been suggested that UCP2 and UCP3 may be regulators of fatty acid/lipid use (62, 63). In the current study, attenuation of NOR-1 mRNA expression in C2C12 skeletal muscle cells led to increased expression of UCP3 mRNA. However, this result may be a compensatory response associated with the reduction in UCP2 expression. For example, it has been demonstrated that UCP3 mRNA is induced in a UCP1 null mouse model, perhaps a compensatory adaptation to the loss of UCP1-mediated thermogenesis (64, 65).

The most notable and significant change observed in mRNA after NOR-1 inhibition (in vitro) was the increase in myostatin expression. This finding may provide an insight into the molecular mechanisms that mediate the metabolic and anabolic changes associated with chronic ß2-adrenoceptor agonist-induced skeletal muscle hypertrophy in rodent animal models (2, 3, 4). Skeletal muscle expresses myostatin, a negative regulator of mass (66, 67, 68) that also appears to be a positive regulator of adiposity (68, 69). Lack of and/or repression of myostatin correlates with muscle hypertrophy and an increase in muscle force producing capacity (66, 67, 68, 70). We noted that siRNA-mediated NOR-1 mRNA inhibition in C2C12 skeletal muscle cells led to an increase in the expression of myostatin mRNA, whereas exogenous NOR-1 expression decreased the activity of the myostatin promoter. This suggests that NOR-1 and ß-adrenergic signaling are important components of the regulatory pathways that modulate myostatin expression. It is interesting to note that myostatin knockout mice appear resistant to the anabolic effects of ß2-AR agonists on skeletal muscle, suggesting that the anabolic effects of ß2-AR agonists may involve modulation of myostatin expression (27).

In conclusion, we demonstrated that NOR-1 influences the expression of genes in skeletal muscle that regulate lipid use and muscle mass. We speculate that NOR-1 (hypersensitive to ß-AR stimulation) is involved in the signaling cascade associated with ß-AR-induced muscle hypertrophy.


   Acknowledgments
 
The IMB is an Australian Research Council Special Research Centre in Functional and Applied Genomics. The authors thank Rachel Burow and Shayama Wijedasa for technical assistance, Ravi Kambadur and Mridula Sharma for the myostatin promoter, and Annika Stark and Rob Parton for the CAV-3 antibody.


   Footnotes
 
This work was supported by a National Health and Medical Research Council of Australia (NHMRC) project grant. G.E.O.M. is a Principal Research Fellow of the NHMRC.

Disclosure Statement: M.A.P., J.G.R., M.A.M., N.O., G.S.L., and G.E.O.M. have nothing to declare.

First Published Online August 10, 2006

Abbreviations: ß-AR, ß-Adrenergic receptor; CAV, caveolin; CMB, confluent myoblast; FABP, fatty acid binding protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MT1–4, myotubes after 1–4 d of differentiation; NOR-1, NR4A3; NR, nuclear hormone receptor; PMB, proliferating myoblast; Q-RT-PCR, quantitative real-time PCR; si, small interfering; TNNI1, type 1 (slow twitch) troponin I; TNNI2, type 2 (fast twitch) troponin I; UCP, uncoupling protein.

Received April 10, 2006.

Accepted for publication July 31, 2006.


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 Introduction
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 Results
 Discussion
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