2018 年 65 巻 12 号 p. 1209-1218
Estrogen deficiency has been known to associate with musculoskeletal diseases in women, based on the clinical observations of frequent susceptibility to osteoporosis and sarcopenia among postmenopausal women. In skeletal muscles, estrogen has been assumed to play physiological roles in maintaining muscle mass and strength, although its precise molecular mechanism remains to be elucidated. We have previously shown that estrogen regulates energy metabolism through the downregulation of mitochondrial uncoupling protein 3 (UCP3) in skeletal muscles, which may contribute to the prolonged exercise endurance in female mice. In the present study, we investigated the effects of estrogen on the expression levels of all members of the nuclear receptor superfamily. Microarray analysis showed that the mRNA level of nuclear receptor subfamily 4 group A member 1 (Nr4a1) was upregulated by the transduction of a recombinant adenovirus expressing constitutively active estrogen receptor α (caERα) in differentiated myoblastic C2C12 cells. Thus we assumed that NR4A1 may be an estrogen-inducible gene in myoblastic cells. We also demonstrated that caERα increases the cellular ATP content along with an increase in mitochondrial DNA content in differentiated myoblastic C2C12 cells. In contrast, the knockdown of Nr4a1 using siRNA exhibited reduced ATP generation as well as a decrease in mitochondrial DNA content. Overall, the present study indicates a crosstalk between estrogen signaling and NR4A1 in skeletal muscle cells. We consider that estrogen-dependent NR4A1 upregulation could increase efficient ATP generation in skeletal muscle cells partly through enhancing mitochondrial functions.
ESTROGEN is one of the sex steroid hormones and it modulates physiological functions in various organs/tissues including bone and skeletal muscle [1, 2]. Its deficiency closely relates to musculoskeletal diseases such as osteoporosis and sarcopenia [3]. Several studies reported that perimenopausal women often exhibit an accelerated decline in muscle strength whereas the muscle strength of aging men seems to decrease gradually [4]. The physiological relevance of estrogen is also revealed as estrogen replacement therapy can prevent muscle strength loss in postmenopausal women [5]. Accumulating evidence implies that estrogen signaling may directly contribute to the maintenance of muscle strength and/or volume, although the precise mechanisms remain to be elucidated. We previously showed that estrogen deficiency in female mice decreased exercise endurance, and the administration of estrogen to ovariectomized (OVX) mice reversed this phenomenon [6]. In that study, the skeletal muscle mass of OVX mice, however, was not substantially different from that of control mice. Thus, we assumed that the estrogen-dependent alteration of exercise endurance would be due to other factors rather than muscle morphology. We performed a microarray analysis on mouse soleus muscle and found that the expression levels of Ucp3 were upregulated by OVX whereas they were downregulated by estrogen administration. Downregulated Ucp3 along with an increase in cellular ATP content was also observed in differentiated myoblastic mouse C2C12 cells adenovirally transduced with constitutively active estrogen receptor α (Ad-caERα) [6, 7].
In the present study, we further focus on the function of nuclear receptor superfamily [8] in estrogen signaling. Steroid hormones such as estrogens, androgens, and glucocorticoids are assumed to play an important role in controlling muscle mass, strength, and metabolism [9, 10]. The receptors (estrogen receptor [ER], androgen receptor [AR], and glucocorticoid receptor [GR]) for these steroid hormones constitute a nuclear receptor superfamily that acts as a ligand-dependent transcription factor, and their transcriptional activation is structurally and functionally controlled by some common molecular mechanisms. For example, transcriptional coactivators (SRC-1, 2, 3; PGC1α, β; and CBP/p300) and corepressors (NCOR-1 and 2) can act commonly on nuclear receptors [11, 12]. It is also known that AR, GR, mineral corticosteroid receptor (MR) and progesterone receptor (PGR) can recognize a common consensus sequence for DNA binding. Furthermore, nuclear receptors regulate the expression of other nuclear receptors. Indeed, PGR is one of the representative estrogen responsive genes in reproductive organs. From this viewpoint, it is assumed that the control network and crosstalk by nuclear receptors play a role in the muscle; however this has not been fully clarified.
In the present study, we identify NR4A1 as an estrogen-inducible gene in differentiated C2C12 myoblasts. Overexpression of caERα upregulates NR4A1 mRNA expression and protein level in the cells. Notably, knockdown of Nr4a1 in the differentiated C2C12 cells expressing caERα reduced cellular ATP and mitochondrial DNA contents. These findings reveal a link between ER and NR4A1 in the regulation of energy metabolism in the skeletal muscle system.
Mouse C2C12 myoblasts (ATCC, Manassas, VA, USA) were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and penicillin/streptomycin (growth medium) at 37°C in 5% CO2. When the cells reached confluence, the medium was switched to high-glucose DMEM supplemented with 2% horse serum and penicillin/streptomycin (differentiation medium) to induce C2C12 myotubes for 7 days and replaced with the fresh differentiation medium every 2 days. Infection of differentiated myoblastic C2C12 cells with Ad-caERα or fluorescent protein DsRed (Ad-DsRed) at m.o.i.30 (n = 3 for each group) was performed as described previously [6, 7]. Then, cells were treated with 1 μM ICI 182,780 (ICI) (Nacalai tesque, Kyoto, Japan) or vehicle for 48 h. Prior to estrogen treatment (10 nM 17β-estradiol (E2) (Sigma-Aldrich, St. Louis, MO, USA)), cells were maintained in phenol red-free DMEM containing 2% dextran-coated charcoal-stripped serum for 48 h and treated with combination of 1 μM ICI and 10 μg/mL puromycin (Sigma-Aldrich, St. Louis, MO, USA) for 4 h (n = 3 for each group) [13].
Luciferase assayDifferentiated myoblastic C2C12 cells on 24-well plates were transfected with an estrogen response element (ERE)-driven firefly luciferase reporter, ERE-tk-Luc (0.4 μg) [7] and a control Renilla luciferase reporter, pRL-CMV (0.1 μg) (Promega, Madison, WI), in phenol red-free DMEM containing 2% dextran-coated charcoal-stripped serum using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA) for 24 h. After transfection, medium was removed and cells were infected with Ad-caERα or Ad-DsRed at m.o.i. 30 for 24 h as described previously [6, 7]. Then, cells were treated with or without 10 nM E2 (Sigma-Aldrich, St. Louis, MO, USA) or 1 μM ICI for 24 h and luciferase assay was performed (n = 3 for each group). The results were calculated as the quotient of firefly/Renilla luciferase activities.
Microarray analysisTotal RNA was extracted from differentiated myoblastic C2C12 cells transduced with Ad-caERα or control Ad-DsRed using ISOGEN reagent (Nippon Gene, Toyama, Japan) and subjected to microarray analysis using Affymetrix GeneChip (Mouse Gene 1.0 ST Array) according to the manufacturer’s instructions. We registered the full microarray data with the Gene Expression Omnibus (GEO). The GEO accession number is GSE111367.
Small interfering RNA transfectionSmall interfering RNA (siRNA) duplexes, siNr4a1 #1 (5'-CGCCUGGCAUACCGAUCUAAA-3' and 5'-UAGAUCGGUAUGCCAGGCGGA-3') and siNr4a1 #2 (5'-CCAUUAGAUGAGACCCUAUCC-3' and 5'-AUAGGGUCUCAUCUAAUGGGC-3'), both of which target Nr4a1, were synthesized using an algorithm that minimizes off-target effects [14]. A negative control siRNA (siControl) with no homology to known gene targets in mammalian cells was obtained from RNAi Inc. (Tokyo, Japan). Differentiated myoblastic C2C12 cells were transfected with siRNA at a final concentration of 50 nM using Lipofectamine RNAiMAX (Invitrogen; Carlsbad, CA, USA). Knockdown efficiency of siRNA was determined by quantitative real time RT-PCR (qRT-PCR) and Western blot analysis using RNA and protein prepared from the cells 24 h after transfection, respectively, as described below.
qRT-PCRqRT-PCR analysis was performed using RNAs isolated from the differentiated C2C12 cells as described previously [15]. The sequences of the PCR primers used were as follows: human ERα forward, 5'-TCTGCCAAGGAGACTCGCTACT-3'; human ERα reverse, 5'-CTCCCCCGTGATGTAATACT-3'; mouse Nr4a1 forward, 5'-CGCTCTGGTCCTCATCACTG-3'; mouse Nr4a1 reverse, 5'-GTCTCCTGCCACGGTAGCC-3'; mouse Greb1 forward, 5'-CCTCCAATACCATAGCCATCG-3'; mouse Greb1 reverse, 5'-GATGTTTAAGGAGTGGGGCCT-3'; mouse Ucp3 forward, 5'-TTGGCCTCTAC-GACTCTGTCAA-3'; mouse Ucp3 reverse, 5'-ATGGCTTGAAATCGGACCTTC-3'; mouse Myog1 forward, 5'-AGAAGCGCAGGCTCAAGAAA-3'; mouse Myog1 reverse, 5'-ATCTCCACTTTAGGCAGCCG-3'; mouse Myh1 forward, 5'-GGCCAGGGTCCGTGAAC-3'; mouse Myh1 reverse; 5'-GCGCAGACCCTTGATAGCTT-3'; mouse Tpm1 forward, 5'-ATCATCGAGAGCGACCTGGAACG-3'; mouse Tpm1 reverse, 5'-CTTCTTTGGCATGGGCCACTTTC-3' and mouse Gapdh forward, 5'-GCATGGCCTTCCGTGTTC-3'; mouse Gapdh reverse, 5'-TGTCATCATACTTGGCAGGTTTCT-3'. The comparison of PCR product amounts was carried out by the comparative cycle threshold (CT) method, using Gapdh as a control. The experiments were independently performed in triplicate.
Western blot analysisDifferentiated myoblastic C2C12 cells were transfected with siNr4a1 #1, siNr4a1 #2 or siControl (50 nM each) for 24 h. After transfection, medium was removed and cells were infected with Ad-caERα or Ad-DsRed at m.o.i. 30 for 48 h [6, 7]. Total cell protein was extracted by RIPA buffer. Extracts were boiled at 100°C for 5 min after adding sample buffer and subjected to 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to polyvinylidene difluoride membrane (Millipore). The membranes were probed with rabbit anti-NR4A1 monoclonal antibody (EPR3209; abcam, Cambridge, UK) and then incubated with anti-rabbit IgG, horseradish peroxidase-linked whole antibody (NA934; GE Healthcare, Buckinghamshire, UK). The signals on the blots were detected with an enhanced chemiluminescence system (GE Healthcare, Buckinghamshire, UK). Finally, the membranes were stripped and reprobed with anti-β-actin (AC-74; Sigma-Aldrich, St. Louis, MO, USA) as a loading control. Signal intensities of Nr4a1 and β-actin proteins on the blots were densitometrically quantified using ImageJ software (https://imagej.nih.gov/ij/). Densitometry value of each Nr4a1 signal was normalized to the corresponding β-actin signal and presented as the relative fold difference from Ad-DsRed sample.
Mitochondrial DNA contentRelative mitochondrial DNA content was estimated using a quantitative real-time PCR (qPCR) by determining the ratio of the 16S ribosomal RNA mitochondrial gene to nuclear reference gene (Tert). Genomic DNA was extracted from differentiated myoblastic C2C12 cells transduced with Ad-caERα or control Ad-DsRed and subjected to qPCR using KAPA SYBR Fast qPCR Kit (Kapa Biosystems, Woburn, MA, USA). The sequences of the PCR primers used were as follows: 16S ribosomal RNA forward, 5'-ACTTCTAACTAAAAGAATTACAGC-3'; 16S ribosomal RNA reverse, 5'-TAGACGAGTTGATTCATAAAATTG-3'; Tert forward, 5'-CCTCAAGCATTCACCTCTTCTTTG-3'; Tert reverse, 5'-CCAAGGACCTGCTCGATGA-3' [16].
ATP contentDifferentiated myoblastic C2C12 cells transduced with Ad-caERα or control Ad-DsRed were triturated in TE buffer (100 mM Tris-HCL, pH 7.5, 4 mM EDTA) on ice and boiled at 95°C for 7 min. The cell lysates were centrifuged at 14,000 rpm for 3 min to remove debris. Then, the supernatants were subjected to ATP measurement using ATP-assay kit (TOYO B-Net, Tokyo, Japan) and the fluorescence were measured using MicroLumat Plus (Berthold Technologies, Bad Wildbad, Germany).
Statistical analysisData are presented as means ± SD. Unpaired Student’s t-test was used to compare two groups. For comparison of more than two groups, the analysis of variance (ANOVA) with Tukey–Kramer HSD post hoc test was performed. Particularly, a two-way ANOVA with Tukey–Kramer HSD post hoc test was used for comparison of means between groups that contain the two independent variables, i.e., adenovirus infection status and ICI treatment status. All statistical analyses were conducted on JMP software. Differences were considered significant when p < 0.05.
To investigate estrogen signaling in muscle cells, we used a recombinant adenovirus expressing caERα that was previously generated [7]. A luciferase assay using an ERE-driven reporter plasmid showed that caERα enhanced the ERE-driven transcription regardless of the presence or absence of estrogen in differentiated C2C12 myoblastic cells though an ERα antagonist ICI could suppress the activity of caERα (Fig. 1). We then performed microarray analysis using differentiated C2C12 myoblastic cells transduced with Ad-caERα or control Ad-DsRed (Table 1). In terms of the alteration of nuclear receptor expression, Nr3c3, also known as Pgr, was upregulated >5-fold by Ad-caERα when compared to that in cells infected with the control virus. We assumed that the substantial upregulation of mouse estrogen receptor (Esr1) may result from the cross reactivity of exogenous human ERα to the probe of its mouse ortholog. Besides Pgr, we identified Nr4a1 as an estrogen-inducible gene in myoblastic cells because it was upregulated >1.5-fold by caERα overexpression. A qRT-PCR analysis confirmed that Nr4a1 expression was upregulated by Ad-caERα transduction but repressed by ERα antagonist ICI treatment (Fig. 2a). We examined whether ICI and puromycin, a protein synthesis inhibitor, would attenuate the estrogen induction of Nr4a1 mRNA in C2C12 cells (Fig. 2b and c). As shown in Fig. 2b, the expression level of Nr4a1 was increased by estrogen but the induction was impaired by ICI, indicating that it was mediated by ER. Moreover, although the expression level of Nr4a1 mRNA was decreased by puromycin in control vehicle medium, it was still elevated in response to E2 (Fig. 2c), suggesting that new protein synthesis would not be a prerequisite for its induction.
Transcription activity of caERα. Differentiated myoblastic C2C12 cells were transfected with the ERE-driven firefly luciferase reporter (ERE-tk-Luc) and the Renilla luciferase reporter as an internal control (pRL-CMV). After transfection, the cells were infected with Ad-caERα or Ad-DsRed and treated with or without 10 nM E2 or 1 μM ICI for 24 h, and then luciferase assays were performed. Data are represented as the mean ± SD of three independent experiments. Between-group comparison was done using one-way ANOVA with Tukey-Kramer HSD post hoc test; **p < 0.01.
| Gene | Nuclear Receptor | Fold change |
|---|---|---|
| Nr1a1 | Thra | 1.19 |
| Nr1a2 | Thrb | 1.00 |
| Nr1b1 | Rara | 1.03 |
| Nr1b2 | Rarb | 0.84 |
| Nr1b3 | Rarg | 1.15 |
| Nr1c1 | Ppara | 1.00 |
| Nr1c2 | Pparb/d | 0.98 |
| Nr1c3 | Pparg | 0.96 |
| Nr1d1 | Rev-Erba | 1.05 |
| Nr1d2 | Rev-Erbb | 0.84 |
| Nr1f1 | Rora | 0.91 |
| Nr1f2 | Rorb | 1.02 |
| Nr1f3 | Rorg | 0.87 |
| Nr1h3 | Lxra | 0.95 |
| Nr1h2 | Lxrb | 1.17 |
| Nr1h4 | Fxra | 1.15 |
| Nr1h5 | Fxrb | 0.88 |
| Nr1i1 | Vdr | 1.41 |
| Nr1i2 | Pxr | 0.90 |
| Nr1i3 | Car | 0.97 |
| Nr2a1 | Hnf4a | 1.01 |
| Nr2a2 | Hnf4g | 0.88 |
| Nr2b1 | Rxra | 1.19 |
| Nr2b2 | Rxrb | 1.07 |
| Nr2b3 | Rxrg | 0.96 |
| Nr2c1 | Tr2 | 1.11 |
| Nr2c2 | Tr4 | 0.93 |
| Nr2e1 | Tlx | 1.15 |
| Nr2e3 | Pnr | 0.80 |
| Nr2f1 | Coup-TfI | 1.24 |
| Nr2f2 | Coup-TfII | 0.74 |
| Nr2f6 | Ear2 | 1.08 |
| Nr3a1 | Esr1 | 2.08 |
| Nr3a2 | Esr2 | 0.93 |
| Nr3b1 | Esrra | 1.11 |
| Nr3b2 | Esrrb | 1.00 |
| Nr3b3 | Esrrg | 0.83 |
| Nr3c1 | Gr | 1.03 |
| Nr3c2 | Mr | 0.83 |
| Nr3c3 | Pgr | 5.52 |
| Nr3c4 | Ar | 1.01 |
| Nr4a1 | Ngfib | 1.93 |
| Nr4a2 | Nurr1 | 0.94 |
| Nr4a3 | Nor1 | 1.31 |
| Nr5a1 | Sf1 | 0.88 |
| Nr5a2 | Lrh1 | 1.05 |
| Nr6a1 | Gcnf | 0.87 |
| Nr0b1 | Dax1 | 1.14 |
| Nr0b2 | Shp | 1.04 |
Nr4a1 as an estrogen responsive gene. (a) Differentiated myoblastic C2C12 cells were infected with Ad-caERα or Ad-DsRed and simultaneously treated with or without 1 μM ICI for 48 h. The qRT-PCR analyses were performed using primers for amplifying mouse Nr4a1. (b, c) Differentiated myoblastic C2C12 cells treated with 10 nM E2 for 4 h in the presence or absence of 1 μM ICI (b) or 10 μg/mL puromycin, a protein synthesis inhibitor (c). The qRT-PCR analyses were performed using primers for amplifying mouse Nr4a1. Relative mRNA levels are expressed as means ± SD, by normalizing to Gapdh level of each sample. Results are shown as means ± SD. Between-group comparison was done using two-way ANOVA with Tukey-Kramer HSD post hoc test; **p < 0.01.
To assess whether NR4A1 plays a role in estrogen signaling associated with energy production, we next performed experiments using siRNA. The expression of human caERα was confirmed by qRT-PCR in the Ad-caERα-transduced C2C12 myoblastic cells (Supplementary Fig. 1a). The upregulation of a prototypic estrogen-inducible gene growth regulation by estrogen in breast cancer 1 (Greb1) was observed in cells treated with Ad-caERα (Supplementary Fig. 1b). In this cell system, we observed the upregulation of Nr4a1 expression by Ad-caERα transduction, which was repressed by siNr4a1 transfection (Fig. 3a). We subsequently assessed protein levels of NR4A1 by western blotting (Fig. 3b). Ad-caERα markedly increased the expression level of NR4A1 protein compared to Ad-DsRed. We found that NR4A1 protein levels were significantly decreased to the same level by transfection of siNr4a1 #1 and #2 (Fig. 3b and Supplementary Fig. 2). Estrogen has been previously shown to repress myogenesis in C2C12 myoblastic cells [17]. In our model, however, the expression levels of myogenic markers (Myog1, Myh1, and Tpm1) were similar in both Ad-caERα- and Ad-DsRed-transduced cells, suggesting a dependence on the experimental condition (Supplementary Fig. 3a–c).
Differentiated myoblastic C2C12 cells were infected with Ad-caERα or Ad-DsRed for 48 h. The qRT-PCR analyses were performed using primers for amplifying human ERα (a) and mouse Greb1 (b). Relative mRNA levels are expressed as means ± SD, by normalizing to Gapdh level of each sample. Results are shown as means ± SD. **p < 0.01 using Student’s t test.
Nr4a1 modulates cellular ATP content and mitochondrial DNA content. (a) Differentiated myoblastic C2C12 cells were treated with vehicle or indicated siRNAs. Then, cells were infected with Ad-caERα or Ad-DsRed. The qRT-PCR analyses were performed using primers for amplifying mouse Nr4a1. Relative mRNA levels are expressed as means ± SD, by normalizing to Gapdh level of each sample. (b) Cell extracts prepared as described in (a) were subjected to SDS-PAGE and western blot analysis using the indicated antibodies. A representative blot was shown (upper panel). Signal intensities for Nr4a1 were quantified by densitometry using ImageJ software, normalized to the corresponding β-actin signals, and presented as the relative fold difference from Ad-DsRed sample. The relative ratios of Nr4a1/β-actin were calculated from three replicates (lower panel). (c, d) Cellular ATP content (nmol mg–1 protein) (c) and relative mitochondrial DNA (mtDNA) content (d) were determined using the cell extracts described in (b). Results are shown as means ± SD. Between-group comparison was done using one-way ANOVA with Tukey-Kramer HSD post hoc test; *p < 0.05, **p < 0.01.
Differentiated myoblastic C2C12 cells were treated with vehicle or the indicated siRNAs, and then infected with Ad-caERα or Ad-DsRed. Cell extracts were subjected to SDS-PAGE and western blot analysis using the indicated antibodies. Signal intensities for Nr4a1 were quantified by densitometry using ImageJ software, normalized to the corresponding β-actin signals, and presented as the relative fold difference from Ad-DsRed sample.
Differentiated myoblastic C2C12 cells were treated with the indicated siRNAs or control siRNA (siControl) (50 nM each). After 24 h, cells were infected with Ad-caERα or Ad-DsRed for 48 h. The qRT-PCR analyses were performed using primers for amplifying mouse Myog1 (a), mouse Myh1 (b), mouse Tpm1 (c) and mouse Ucp3 (d). Relative mRNA levels are expressed as means ± SD, by normalizing to Gapdh level of each sample. Results are shown as means ± SD. Between-group comparison was done using one-way ANOVA with Tukey-Kramer HSD post hoc test; **p < 0.01.
Previously we showed that the cellular ATP content in differentiated C2C12 cells expressing caERα was substantially increased whereas it was repressed by ICI treatment [6]. In this study, we then examined the effects of NR4A1 on cellular ATP content in C2C12 cells. Notably, siNr4a1 transfection attenuated the caERα-dependent increase in cellular ATP content (Fig. 3c). We also analyzed Ucp3 mRNA expression levels in differentiated C2C12 myoblastic cells that were transduced with Ad-caERα and treated with siRNAs targeting Nr4a1 (Supplementary Fig. 3d). The result showed that Nr4a1 knockdown had no effect on Ucp3 expression. Therefore, we speculated that cellular ATP content was regulated by at least two independent mechanisms through UCP3 [6] and NR4A1 in the differentiated C2C12 myotubes.
Considering that enhanced cellular ATP content is often associated with an increase in mitochondrial DNA content, we questioned whether estrogen signaling also modulates mitochondrial DNA content. We assumed that estrogen signaling may be potentially involved in the maintenance of mitochondrial DNA content, as relative mitochondrial DNA content was increased in the differentiated C2C12 cells expressing caERα compared with that of control adenovirus-infected cells (Supplementary Fig. 4). Besides, siNr4a1 substantially repressed relative mitochondrial DNA content in caERα-expressing C2C12 myoblasts expressing caERα (Fig. 3d), indicating that NR4A1 could impair the estrogen signaling-mediated increase of mitochondrial DNA content.
Differentiated myoblastic C2C12 cells were infected with Ad-caERα or Ad-DsRed and simultaneously treated with or without 1 μM ICI for 48 h. Relative mtDNA content was determined. Results are shown as means ± SD. Between-group comparison was done using two-way ANOVA with Tukey-Kramer HSD post hoc test; **p < 0.01.
In the present study, we investigated the potential mechanisms of estrogen signaling on myocyte energy metabolism. We performed microarray analysis and revealed that the mRNA level of Nr4a1 was upregulated by the transduction of Ad-caERα in differentiated myoblastic C2C12 cells. Thus, we assumed that Nr4a1 will be an estrogen-inducible gene in myoblastic cells. We also demonstrated that ER signaling increases the cellular ATP content along with an increase in mitochondrial DNA content in differentiated myoblastic C2C12 cells whereas these effects were abrogated by Nr4a1 knockdown. These results suggest that estrogen may increase the efficiency of ATP generation partly through enhancing mitochondrial functions and that the increase in energy production will improve exercise ability.
Several reports have shown the involvement of ER and NR4A1 in mitochondrial respiration capacity. Ribas V et al. reported that soleus muscle fiber from muscle-specific ERα knockout (MERKO) mice fatigued more quickly compared with control mice in muscular endurance analysis [18]. They also showed that C2C12 myotubes with ERα knockdown exhibited a reduction in basal and maximally stimulated cellular oxygen consumption and ATP synthesis [18]. Although mitochondrial DNA content in MERKO muscle demonstrated no difference compared with wild type mice, ERα deficiency impaired mitochondrial DNA replication and altered mitochondrial morphology. In addition, C2C12 myotubes with ERα-knockdown altered the morphology of mitochondria, similar to that observed in MERKO muscle. Chao LC et al. showed that NR4A1 expression stimulated the intrinsic respiratory capacity of mitochondria isolated from skeletal muscle [19]. Together with our results that caERα increases NR4A1 expression, these reports support our notion that ER would regulate mitochondrial function through NR4A1.
It has been previously reported that NR4A1 regulates glucose and lipid metabolism in skeletal muscle [20-22]. Mouse skeletal muscles expressing transgene NR4A1 had enhanced mitochondrial DNA content and oxidative metabolism while exhibiting a fatigue-resistant phenotype [19]. In terms of muscle morphology, a previous report showed that mouse skeletal muscles tissue-specifically expressing NR4A1 exhibit an increase in myofiber size, whereas mice with muscle-specific or global NR4A1 deficiency have reduced myofiber size and muscle mass [23]. NR4A1 has been demonstrated to be an important mediator of muscle growth during physiological muscle accretion in adulthood [24]. Moreover, NR4A nuclear receptor subfamily was also described as potential therapeutic targets for the treatment of age-related disorders [25].
The expression level of Nr4a1 was increased by estrogen but the induction was impaired by ICI. Moreover, the induction was maintained in the presence of puromycin. In addition, analysis using the MotifMap database (http://motifmap.ics.uci.edu/) revealed that a potential ERE was located 9,858 bp upstream of the Nr4a1 gene in the mouse genome (data not shown). These findings suggest that Nr4a1 expression would be directly mediated by ER as a primary responsive gene.
In conclusion, our present data in C2C12 myoblastic cells together with the above observations concerning ER knockout and NR4A1 transgenic studies suggest that estrogen-dependent NR4A1 upregulation will contribute to the enhancement of exercise performance. Further studies will elucidate the precise role of estrogen in the regulation of muscle activities.
This study was supported in part by Grants of “Support Project of Strategic Research Center in Private Universities” and “Supported Program for the Strategic Research Foundation at Private Universities” from the MEXT, Japan; and by Grants from Mitsukoshi Health and Welfare Foundation, and Mitsui Life Social Welfare Foundation.
All authors had no conflict of interest regarding this manuscript.
