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A FoxO1-dependent, but NRF2-independent induction of heme oxygenase-1 during muscle atrophy
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A FoxO1-dependent, but NRF2-independent induction of heme oxygenase-1 during muscle atrophy
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Jione Kang, Mi Gyeong Jeong, Sera Oh, Eun Jung Jang, Hyo Kyeong Kim, Eun Sook Hwang ⇑ College of Pharmacy and Global Top5 Research Program, Ewha Womans University, Seoul 120-750, Republic of Korea
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Article history: Received 5 August 2013 Revised 2 October 2013 Accepted 4 November 2013 Available online xxxx Edited by Berend Wieringa Keywords: Nerve injury MAFbx MuRF1 NRF2 FoxO1
a b s t r a c t Skeletal muscle plays key roles in metabolic homeostasis. Loss of muscle mass, called muscle atrophy exacerbates disease-associated metabolic perturbations. In this study, we characterized the molecular functions and mechanisms underlying regulation of skeletal muscle atrophy induced by denervation. Denervation significantly increased the expression of heme oxygenase-1 (HO-1) and atrogenes in skeletal muscle. Forkhead box protein O1 (FoxO1) drastically increased in atrophied muscle and selectively stimulated HO-1 gene transcription through direct DNA binding. Lack of HO-1 substantially attenuated muscle atrophy, whereas HO-1 overexpression caused muscle damage in vitro and in vivo. Collectively, HO-1 induced by FoxO1 may cause skeletal muscle atrophy. Ó 2013 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
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1. Introduction
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The muscle is an important tissue that stockpiles protein in the body; however, oxidative stress and denervation cause excessive protein degradation and induce skeletal muscle wasting [1]. Proteolytic systems are primarily responsible for eliminating contractile proteins and organelles and for shrinkage of muscle fibers [2]. In particular, two muscle-specific ubiquitin ligases, MAFbx (muscle atrophy F-box) and MuRF1 (muscle-specific RING finger protein 1), cause proteasomal degradation in skeletal muscle and contribute to muscle atrophy [3,4]. MAFbx and MuRF1, called atrogenes, are strongly increased during muscle atrophy through activation of myogenin [5] and forkhead box protein O (FoxO) [6]. Indeed, mice lacking MuRF1 or MAFbx revealed protection from denervation-induced muscle atrophy [3]. FoxO1 transgenic mice showed markedly decreased skeletal muscle mass, and conversely, FoxO1 knockdown blocked MAFbx up-regulation [7]. In addition, inflammatory cytokines and nuclear factor jB (NF-jB) signaling pathways are critically involved in muscle atrophy [8,9] through generation of intracellular reactive oxygen species (ROS) [10]. Dysregulated ROS generation is prominently associated with severe muscle loss [11].
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⇑ Corresponding author. Address: College of Pharmacy, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 120-750, Republic of Korea. Fax: +82 2 3277 3760. E-mail address: [email protected] (E.S. Hwang).
Extensive studies have identified that nuclear factor erythroid 2-related factor 2 (NRF2)/MAF and its cytosolic repressor, kelchlike ECH-associated protein 1 (KEAP1), are indispensable for cellular adaptation to oxidative stress [12]. Oxidative stress dissociates NRF2 from KEAP1 in the cytosol and induces nuclear localization of NRF2. Nuclear NRF2 directly binds to the antioxidant response element (ARE) within the gene promoter of many antioxidants, such as heme oxygenase-1 (HO-1) [13], glutathione S-transferase a (GSTA) 1/2 [14], and NAD(P)H:quinone oxidoreductase 1 (NQO1) [15], and activates their expression, thereby protecting from oxidative stress-induced tissue injury [16]. Despite the key role of NRF2 in protection from oxidative stress, the importance of NRF2 in protection from injury-induced muscle atrophy remains unclear. In this study, we have examined whether NRF2 is involved in protecting skeletal muscle from denervation-induced muscle atrophy and characterized the molecular function during muscle atrophy.
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2. Materials and methods
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2.1. Mice
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C57BL/6 WT and NRF2 KO mice were obtained from Jackson Laboratory (Bar Harbor, ME) and RIKEN BioResource Center (Tsukuba, Japan) and maintained in an animal facility of Ewha Womans University. Animal handling was performed in accordance with the institutional guidelines of and approved by the IACUC (2011-01-027 and 2012-01-035).
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0014-5793/$36.00 Ó 2013 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. http://dx.doi.org/10.1016/j.febslet.2013.11.009
Please cite this article in press as: Kang, J., et al. A FoxO1-dependent, but NRF2-independent induction of heme oxygenase-1 during muscle atrophy. FEBS Lett. (2013), http://dx.doi.org/10.1016/j.febslet.2013.11.009
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2.2. Sciatic nerve injury-induced muscle wasting
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Mice (12 weeks of age, male, 25–30 g) were anesthetized using a constant flow of isoflurane inhalation. A small incision was made below the hip bone, parallel to the sciatic nerve. The nerve was isolated by removing any adherent tissue and tightly ligated with 6-0 silk suture thread around one-third to one-half of the diameter of the sciatic nerve [17]. Separately, the sciatic nerve was exposed, but not ligated in sham-operated mice. The skin incisions were closed with surgical suture and wound clips. The mice were maintained up to 7 days and were sacrificed, followed by analyses of the tibialis anterior (TA) and gastrocnemius (GN) muscles [11].
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2.3. Myogenic differentiation and muscle atrophy in vitro
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C2C12 myoblasts (ATCC, Manassas, VA) were grown to 100% confluence and replaced with myogenic differentiation medium containing 2% horse serum (Invitrogen, Carlsbad, CA) for 6 days. Differentiated myocytes were then treated with dexamethasone (DEX; 500 lM, Sigma–Aldrich, St. Louis, MO) for 24 h to induce muscle atrophy in vitro.
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2.4. Immunoblot analysis
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Cell proteins were harvested and resolved by SDS–PAGE, followed by immunoblotting. Protein blots were incubated with antibody against myosin heavy chain (MyHC; DSHB, Iowa City, IA), FoxO1 (Cell Signaling Technology, Inc., Danvers, MA), MuRF1 (ECM Biosciences LLC, Versailles, KY), HO-1, MAFbx (Abcam, Cambridge, UK), BACH1, and b-actin (Santa Cruz Biotechnology Inc.) and subsequently with horseradish peroxidase-conjugated secondary antibody, followed by detection with enhanced chemiluminescence (Thermo Fisher Scientific, Rockford, IL).
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2.5. Chromatin immunoprecipitation (ChIP)
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ChIP was performed as described previously [18]. Briefly, TA and GN muscles (25 mg) were finely minced in cold PBS and cross-linked with 1% formaldehyde. Muscles were homogenized and immnunoprecipitated using FoxO1 antibody or control IgG. The input DNA and eluted DNA were used for quantitative PCR and real time-PCR. Primers for HO-1 promoter were as follows: HO1-ChIP-FWD 50 -tgtgccatcactacccagaa-30 and HO1-ChIP-REV 50 -agcagggtaaggcttggaat-30 . Relative level was calculated after normalizing to the input samples.
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2.6. Total RNA isolation and real-time PCR
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Total RNA was isolated using TRIzol reagent (Invitrogen) and used for real-time PCR (Applied Biosystems, Foster City, CA). Relative expression level was determined after normalization with GAPDH levels. The primer sets used for real time-PCR were as follows: GAPDH, 50 -tccaccttcgatgccgggg-30 and 50 -agcgctattcattgtcatacc-30 ; GSTA, 50 -ggagattgatgggatgaagc-30 , 50 -aacaccttttcaaaggcagg-30 ; HO1, 50 -accgccttcctgctcaacattgag-30 , 50 -tgttcctctgtcagcatcacc-30 ; IL-1b, 50 -gtgtgccgtctttcattacacag-30 , 50 -ggacagaatatcaaccaagtgata-30 ; IL-6, 50 -gaggataccactcccaacaga-30 , 50 -aagtgcatcatcgttgttcataca-30 ; MAFbx, 50 -aaggctgttggagctgatagca-30 , 50 -cacccacatgttaatgttgcc-30 ; MuRF1, 50 -tgaccacagagggtaaag-30 , 50 -tgtctcactcatctccttcttc-30 ; myogenin, 50 -ccagcccatggtgcccagtg-30 , 50 -gcgtctgtagggtcagccgc-30 ; and NQO1, 50 -gacatgaacgtcattctctg-30 , 50 -ctagctttgatctggttgtc-30 .
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NRF2, FoxO1, and/or BACH1 expression vector. pCMV (Invitrogen) encoding b-galactosidase was cotransfected as an internal control. Cell extracts were harvested 48 h after transfection and assayed using the Luciferase Assay kit (Promega, Madison, WI), and the Galacto-Light b-galactosidase assay kit (Applied Biosystems) according to the manufacturer’s instructions.
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2.8. Immunohistochemistry
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TA muscles were fixed with 10% neutralized formalin for 24 h and embedded in paraffin. Tissue sections (5 lm thick) were prepared and stained with hematoxylin and eosin (HE) solution (Sigma–Aldrich, St. Louis, MO) and antibody against MyHC, NRF2, or FoxO1 (Cell signaling), followed by incubation with fluorescence tagged IgG or peroxide/DAB (Vector Laboratories, Burlingame, CA, USA).
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2.9. Transfection of siRNA
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Fully differentiated myocytes from C2C12 cells were transfected with siRNA using HiPerFect transfection reagents (Qiagen, Hilden, Germany). Transfected cells were additionally treated with Dex for 24 h. At least three different sets of siRNA for FoxO1 and HO-1 were synthesized (Bioneer, Daejeon, South Korea): siCon, 50 -ccuacgccaccaauuucgu-30 ; siFoxO #1, 50 -ccagaccaggauaauuggu-30 ; siFoxO #2, 50 -cuaaugugucucaaacauu-30 ; siFoxO #3, 50 -caucuaugcagccuuguuu30 ; siHO-1, 50 -cagaucagcacuagcucau-30 ; siHO-2, 50 -caccaaggagguacacauc-30 ; and siHO-3, 50 -ccugaaucgagcagaacca-30 .
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2.10. Rotarod test
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Mice (n = 12) were divided into two groups and injected with either vehicle or hemin (25 mg/kg) for 10 days. The latency time it takes the mouse to fall off the rod rotating was measured under continuous acceleration conditions (4, 7, 10, and 20 rpm for 2 min) at days 8, 9, 10, 11 and 12 after injection. The average latency time of each group is expressed as mean ± S.E.M. for 6 mice.
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2.11. Statistical analyses
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Data are presented as mean ± S.E.M. and analyzed by the unpaired Student’s t-test.
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3. Results
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3.1. Sciatic nerve injury induces muscle damage with an increase of NRF2 and FoxO1
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To characterize the molecular mechanisms involved in muscle atrophy, we performed sciatic nerve injury and examined the muscle phenotypes. The weights of GN and TA muscles were significantly decreased and MyHC expression decreased at day 7 after nerve injury (Fig. 1A and B). However, the expression of antioxidants, such as heme oxygenase-1 (HO-1) and catalase was increased (Fig. 1B). Furthermore, the expression of atrogenes, inflammatory cytokines, and myogenin was significantly increased during muscle atrophy (Fig. 1B and C). Simultaneously, NRF2 and FoxO1 expression, but not NF-jB p65, were significantly augmented in atrophied muscle and particularly enriched in the nucleus of the muscle (Fig. 1D and E).
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3.2. NRF2 is not required for induction of muscle atrophy markers and HO-1
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2.7. Reporter gene assay 293T cells were transiently transfected with mouse HO-1 promoter-linked reporter gene (pHO4.3k-luc) [19] together with
Because NRF2 expression is important for protection against oxidative stress by the induction of antioxidants, we examined
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Fig. 1. HO-1 induction in nerve injury-induced muscle atrophy nerve injury was caused in C57BL6 WT mice (NI; n = 15) by tying a tight ligature around the sciatic nerve, and the mice were sacrificed on days 1, 3, and 7 after denervation. Sham-operated mice (sh; n = 15) were used as controls. (A) GN and TA muscles were isolated from the sh and NI groups, and muscle mass was measured at the indicated time points (n = 5 each). Data are presented as mean ± S.E.M. (B) protein extracts were prepared from the TA and GN muscle on days 3 and 7 and were used in SDS–PAGE and immunoblotting analysis (n = 3). (C) Total RNA was collected from the muscle at day 7 after injury and reversetranscribed. The relative transcript level was calculated after normalization to b-actin level. Data are given as mean ± S.E.M. of 5 mice. (D) At day 3, protein extract was harvested and analyzed by immunoblotting (n = 3). (E) TA muscle was fixed and sectioned, followed by HE staining and immunohistochemistry with antibodies against desmin, NRF2, and FoxO1. Scale bars, 200 and 50 lm. Representative images are shown in (B), (D), and (E). ⁄P < 0.05; ⁄⁄P < 0.005; ⁄⁄⁄P < 0.0005.
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whether NRF2 was required for protecting against muscle atrophy. Sciatic nerve injury induced similar extents of muscle wasting of GN and TA muscles in WT and NRF2 KO mice (Fig. 2A). Histological analysis confirmed muscle damage in both WT and KO muscle after denervation (Fig. 2B). MyHC decrease was prominent after nerve injury and induction of MuRF1 and HO-1 was comparable between WT and NRF2 KO mice (Fig. 2C). In addition, muscle atrophy markers and inflammatory factors such as IL-6 and myogenin were similarly expressed in WT and KO muscle after denervation (Fig. 2C and D). As expected, NRF2 target genes like GSTA1/2 and NQO1 were increased in injured muscle but their expression was disappeared in the absence of NRF2 (Fig. 2E). Surprisingly, however, another known target of NRF2, the heme oxygenase-1 (HO1) was expressed in NRF2 deficiency similar to that in WT muscle after injury (Fig. 2E), implying no protective role of NRF2 against muscle atrophy and an NRF2-independent induction of HO-1 in muscle atrophy.
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3.3. FoxO1 is important for the induction of HO-1
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Because another major regulator of oxidative stress, FoxO1 was substantially increased in the nucleus of muscle fibers after injury, we investigated whether FoxO1 could be involved in modulating
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HO-1 activity. We found that FoxO1 was substantially increased after denervation and was comparably expressed in WT and NRF2 KO muscle (Fig. 3A). Immunofluorescence staining showed prominent FoxO1 expression in the nucleus of muscle fibers after injury (Fig. 3B). We then determined whether FoxO1 activated HO-1 promoter activity. Overexpression of either NRF2 or FoxO1 stimulated HO-1 promoter activity in a dose-dependent manner (Fig. 3C); however, no cooperative effect was observed between NRF2 and FoxO1 in the induction of HO-1 promoter activity (Fig. 3D). Direct binding of FoxO1 to the HO-1 gene promoter was stimulated in muscle atrophy, as evidenced by ChIP and quantitative PCR experiments (Fig. 3E and F). However, it remained unclear how FoxO1 substituted NRF2 function to induce HO-1 gene transcription. We investigated the levels of NRF2 suppressors KEAP1 and BACH1. KEAP1 expression was low and unchanged in WT and KO muscle after injury, whereas BACH1 expression was markedly increased in injured muscle, with comparable levels between WT and KO muscle (Fig. 3G). Interestingly, NRF2-induced HO-1 promoter activity was suppressed by BACH1, whereas BACH1 expression had no effect on FoxO1-mediated HO-1 activation (Fig. 3H). These results indicate that the NRF2 suppression by BACH1 instigates FoxO1 to induce HO-1 gene transcription during muscle atrophy.
Please cite this article in press as: Kang, J., et al. A FoxO1-dependent, but NRF2-independent induction of heme oxygenase-1 during muscle atrophy. FEBS Lett. (2013), http://dx.doi.org/10.1016/j.febslet.2013.11.009
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Fig. 2. NRF2-independent expression of HO-1 in muscle atrophy Sciatic nerve injury (NI) and sham operation (sh) were conducted in WT and NRF2 KO mice (n = 6 each), and the mice were sacrificed at day 7 after injury. (A) The weights of GN and TA muscles were measured, and data are expressed as mean ± S.E.M. of 6 mice. (B) TAZ muscle was sectioned and stained with HE. (C) Protein extracts were obtained from TA muscle at day 7 and analyzed by SDS–PAGE and immunoblotting (n = 6). Representative blots of 3 independent experiments are shown. (D and E) Total RNA was collected from the muscles of WT and NRF2 KO mice at day 7 after injury and used for real time-PCR. Relative mRNA level was calculated after normalization to b-actin level. Data are given as mean ± S.E.M. of 6 mice. ns, not significant; ⁄P < 0.05; ⁄⁄P < 0.005; ⁄⁄⁄P < 0.0005.
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3.4. FoxO1 is essential for HO-1 induction in the process of muscle atrophy In order to clarify the importance of FoxO1 in HO-1 induction in muscle atrophy, we established the muscle atrophy system in vitro by treating differentiated myocytes with DEX. DEX treatment induced morphological changes of the differentiated myocytes and decreased expression of MyHC (Fig. 4A). Simultaneously, DEX increased mRNA level of atrogenes and HO-1 in differentiated myocytes (Fig. 4B), mimicking in vivo nerve injury-induced muscle atrophy. We attempted to confirm the importance of FoxO1 by determining HO-1 level by the lack of FoxO1. FoxO1 expression was decreased after transfection with three different types of small interfering RNA (siRNA) against FoxO1 in fully differentiated myocytes (Fig. 4C). The mRNA level of HO-1 was measured in control and FoxO1 knockdown myocytes after treatment with DEX. HO-1 expression was induced by DEX, but was not significantly induced in FoxO1 knockdown cells compared in control cells (Fig. 4D), suggesting a necessity of FoxO1 in HO-1 induction. 3.5. Suppression of HO-1 attenuates DEX-induced expression of muscle atrophy genes We then assessed the effect of HO-1 in muscle atrophy and activity. Knockdown of HO-1 in differentiated myocytes significantly attenuated the expression of atrogenes induced by DEX
treatment (Fig. 5A and B). Conversely, elevated HO-1 expression by treatment with hemin, an HO-1 inducer decreased the level of MyHC but increased MuRF1 through induction of FoxO1 (Fig. 5C). The flattened shape and shrinking of differentiated myocytes and MyHC decline were prominently induced after treatment with hemin (Fig. 5D). More importantly, daily administration of hemin for 7 days caused muscle damage in vivo, as evidenced by HE staining and immunohistochemistry of desmin (Fig. 5E). In addition, physical muscle activity was measured by rotarod test. Latency time to fall was significantly decreased in hemin-injected mice after day 10 when compared in vehicle-treated group (Fig. 5F). These results suggest that sustained HO-1 expression by hemin treatment exerts a deleterious effect on skeletal muscle fibers and physical muscle activity.
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4. Discussion
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Our results showed that HO-1 level increased substantially in muscle atrophy, and its expression was dependent on FoxO1, but not on NRF2. Blockade of HO-1 induction attenuated muscle atrophy gene expression induced by DEX treatment in vitro, and prolonged HO-1 expression per se was sufficiently injurious to the skeletal muscle fibers in vivo. NRF2 is a major protective molecule against oxidative stress [20]. To ensure the timely activity of different antioxidants and detoxifying enzymes, NRF2 activity is tightly controlled by
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Fig. 3. FoxO1-induced HO-1 expression after nerve injury (A and B) WT and NRF2 KO mice were sham-operated (sh; n = 6 each) or injured (NI; n = 6 each) and sacrificed at day 7. Protein extracts were analyzed by immunoblotting (A). Tissue sections were stained with FoxO1 antibody and observed under a fluorescence microscope (B). (C and D) 293T cells were transfected with different amounts of either NRF2 or FoxO1 expression vector together with pHO-luc (C) or with the indicated combinations of NRF2 and FoxO1 (D). pCMVb-gal was cotransfected for normalization of transfection efficiency. Relative luciferase activity was determined after normalization to b-galactosidase activity and expressed as a fold induction normalized to mock control (–). (E and F) TA and GN muscles used for preparing genomic DNA. PCR reactions were carried out 26 cycles (E) and quantitatively determined by real time-PCR (F). (G) protein blots in (A) were incubated with antibody against BACH1, KEAP1, and actin. (H) 293T cells were transfected with expression vector for NRF2, FoxO1, and/or BACH1 together with pHO-luc and pCMVb-gal. Relative activity is expressed as mean ± S.E.M. of 3 independent experiments. Representative images are shown in (A) to (C), (E) and (G). ns, not significant; ⁄P < 0.05; ⁄⁄P < 0.005.
Fig. 4. Necessity of FoxO1 in DEX-induced HO-1 expression in vitro Differentiation of myocytes was induced from C2C12 cells for 6 days and treated with DEX (500 lM) for 24 h. (A) Cells were fixed and incubated with MyHC antibody and DAPI, followed by a microscopic observation. (B) Total RNA was collected and used for determining the relative expression level. (C and D) Differentiated myocytes were transfected with siRNA against control gene (siCon) or FoxO1 (siFoxO1) and treated with DEX for 24 h. Protein extracts (C) and total RNA (D) were analyzed. ns, not significant; ⁄P < 0.05; ⁄⁄⁄P < 0.0005.
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nuclear-cytoplasmic shuttling through KEAP1 interaction in the cytoplasm. NRF2 is also suppressed by BACH1, which competitively binds to ARE and suppresses ARE-regulated gene expression
[21]. NRF2 KO mice showed increased susceptibility to a variety of chemical-induced diseases, such as drug-induced liver injury [22], oxidative stress-induced cardiomyopathy [23], and cigarette
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Fig. 5. Modulation of muscle atrophy by HO-1 in vitro and in vivo (A and B) Differentiated myocytes were transfected with siRNAs and subsequently treated with DEX (500 lM). Protein extract and total RNA were prepared from the cells and analyzed by immunoblotting (A) and real time-PCR (B). Relative expression levels of MuRF1 and MAFbx were calculated after normalization to b-actin level. Data are given as mean ± S.E.M. of 3 independent experiments. ⁄P < 0.05; ⁄⁄P < 0.005; ⁄⁄⁄P < 0.0005. (C and D) Differentiated cells were treated with hemin for 24 h, and protein extracts were harvested for immunoblotting analysis (C). Cells were stained with MyHC antibody and DAPI and observed under a microscope (D). (E) Hemin (30 mg/kg) was daily injected into the mice for 7 days. TA muscle was sectioned and subjected to HE and desmin staining. Scale bars indicate 2000, 200, and 50 lm. Representative images are shown in (A) and (C) to (E). (F) Latency time was determined in veh- or hemin-treated mice by Rota rod test at days 10, 11, 12, and 13 after injection. Data are given as mean ± S.E.M. of 6 mice. ⁄P < 0.05.
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smoke-induced emphysema [24], implicating protective roles of NRF2 against tissue injury. However, abrogation of NRF2 in mice showed muscle wasting, induction of inflammatory and atrophy markers as comparable as in WT mice after injury. Despite a pivotal role of NRF2 in the control of oxidative damage, NRF2 is neither necessary nor sufficient for protecting against nerve injuryinduced muscle atrophy. Interestingly, the expression of GSTA1/2 and NQO1 was impaired in NRF2 deficiency whereas HO-1 was increased in NRF2 KO muscle after injury. Denervation caused an increase in expression level and nuclear localization of NRF2, resulting in the expression of antioxidants such as GSTA1/2, NQO1, and HO-1. However, injured muscle subsequently increased expression of BACH1, an NRF2 competitor, thereby attenuating NRF2 activity to bind to ARE and induce ARE-mediated gene transcription. Although the expression level of GSTA1/2 and NQO1 was reciprocally regulated by NRF2 and BACH1, HO-1 expression was not suppressed by BACH1 but rather increased in denervation-induced muscle atrophy. Our results demonstrated that FoxO1 directly bound to FRE within the HO-1
gene promoter and stimulated HO-1 gene transcription even in the presence of BACH1 (Fig. 6). In addition, FoxO1 strongly increased the expression of MAFbx and caused muscle atrophy. Induced expression of FoxO1 played important role in the induction of not only MAFbx, an atrophy marker but also HO-1 in muscle after denervation. There have been extensive studies on protective roles of HO-1 in many tissues. NRF2-dependent HO-1 expression profoundly attenuated lung injury, skin lesion, and cardiac failure [25]. Furthermore, abrogation of HO-1 in mice developed fulminant renal failure, leading to death [26] and exacerbated atherosclerosis in an apolipoprotein E-deficient genetic background [27]. HO-1 is an indispensable stress response protein. However, HO-1 expression decreased the viability of myocytes [28] and inhibited myoblast differentiation [29]. In addition, HO-1 induction exacerbates early brain injury [30]. Our results also revealed that HO-1 expression was increased by FoxO1 in muscle atrophy in vivo, and suppression of HO-1 level in fully differentiated myocytes attenuated the induction of MAFbx and MuRF1 after DEX treatment.
Please cite this article in press as: Kang, J., et al. A FoxO1-dependent, but NRF2-independent induction of heme oxygenase-1 during muscle atrophy. FEBS Lett. (2013), http://dx.doi.org/10.1016/j.febslet.2013.11.009
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Fig. 6. FoxO1-induced expression of HO-1 and muscle atrophy genes Under basal conditions, nuclear NRF2 is competed by BACH1 for binding to ARE within the promoter of HO-1, GSTA1/2, and NQO1 genes, resulting in low expression of antioxidants. Upon injury, the expression of NRF2 and FoxO1 was increased in muscle. NRF2 is essential for the expression of protective antioxidants such as GSTA1/2 and NQO1 but is not required for HO-1 expression. FoxO1, on the other hand, directly binds to FRE within the promoter of HO-1 and MAFbx genes and promotes gene transcription, thereby inducing muscle atrophy.
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Furthermore, in vivo sustained expression of HO-1 by treatment with hemin was accompanied by a decrease in MyHC and an increase in atrogenes, causing skeletal muscle atrophy. HO-1 thus has a dual and opposing role, it provides protection from oxidative damage in an NRF2-dependent manner and it promotes muscle atrophy through a FoxO1-induced pathway.
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Disclosure of potential conflicts of interest
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No potential conflicts of interest were disclosed.
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Acknowledgments
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We thank Dr. J. Alam (Alton Ochsner Medical Foundation, New Orleans) for providing pHO4.3k-luc vector and Ms. Hyun Jung Kim for helping histological analysis. This work was supported by the Basic research program (2012R1A1B3000603, ESH and for 2011007983 for JOK) of the National Research Foundation funded by Ministry of Education, Science, and Technology.
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[1] Sacheck, J.M. et al. (2007) Rapid disuse and denervation atrophy involve transcriptional changes similar to those of muscle wasting during systemic diseases. FASEB J. 21, 140. [2] Lecker, S.H. et al. (2006) Protein degradation by the ubiquitin-proteasome pathway in normal and disease states. J. Am. Soc. Nephrol. 17, 1807. [3] Bodine, S.C. et al. (2001) Identification of ubiquitin ligases required for skeletal muscle atrophy. Science 294, 1704.
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[4] Gomes, M.D. et al. (2001) Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy. Proc. Natl. Acad. Sci. USA 98, 14440. [5] Moresi, V. et al. (2010) Myogenin and class II HDACs control neurogenic muscle atrophy by inducing E3 ubiquitin ligases. Cell 143, 35. [6] Sandri, M. et al. (2004) Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy. Cell 117, 399. [7] Kamei, Y. et al. (2004) Skeletal muscle FOXO1 (FKHR) transgenic mice have less skeletal muscle mass, down-regulated Type I (slow twitch/red muscle) fiber genes, and impaired glycemic control. J. Biol. Chem. 279, 41114. [8] Li, Y.P. et al. (2000) NF-kappaB mediates the protein loss induced by TNF-alpha in differentiated skeletal muscle myotubes. Am. J. Physiol. Regul. Integr. Comp. Physiol. 279, R1165. [9] Li, Y.P. et al. (2005) TNF-alpha acts via p38 MAPK to stimulate expression of the ubiquitin ligase atrogin1/MAFbx in skeletal muscle. FASEB J. 19, 362. [10] Li, Y.P. et al. (2003) Hydrogen peroxide stimulates ubiquitin-conjugating activity and expression of genes for specific E2 and E3 proteins in skeletal muscle myotubes. Am. J. Physiol. Cell Physiol. 285, C806. [11] Muller, F.L. et al. (2007) Denervation-induced skeletal muscle atrophy is associated with increased mitochondrial ROS production. Am. J. Physiol. Regul. Integr. Comp. Physiol. 293, R1159. [12] Kensler, T.W. et al. (2007) Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annu. Rev. Pharmacol. Toxicol. 47, 89. [13] Rangasamy, T. et al. (2004) Genetic ablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in mice. J. Clin. Invest. 114, 1248. [14] Chanas, S.A. et al. (2002) Loss of the Nrf2 transcription factor causes a marked reduction in constitutive and inducible expression of the glutathione Stransferase Gsta1, Gsta2, Gstm1, Gstm2, Gstm3 and Gstm4 genes in the livers of male and female mice. Biochem. J. 365, 405. [15] McMahon, M. et al. (2001) The Cap‘n’Collar basic leucine zipper transcription factor Nrf2 (NF-E2 p45-related factor 2) controls both constitutive and inducible expression of intestinal detoxification and glutathione biosynthetic enzymes. Cancer Res. 61, 3299. [16] Nguyen, T. et al. (2003) Regulatory mechanisms controlling gene expression mediated by the antioxidant response element. Annu. Rev. Pharmacol. Toxicol. 43, 233. [17] Abbadie, C. et al. (2003) Impaired neuropathic pain responses in mice lacking the chemokine receptor CCR2. Proc. Natl. Acad. Sci. USA 100, 7947. [18] Jeong, H. et al. (2010) TAZ as a novel enhancer of MyoD-mediated myogenic differentiation. FASEB J. 24, 3310. [19] Alam, J. et al. (2000) Mechanism of heme oxygenase-1 gene activation by cadmium in MCF-7 mammary epithelial cells. Role of p38 kinase and Nrf2 transcription factor. J. Biol. Chem. 275, 27694. [20] Ma, Q. (2013) Role of nrf2 in oxidative stress and toxicity. Annu. Rev. Pharmacol. Toxicol. 53, 401. [21] Dhakshinamoorthy, S. et al. (2005) Bach1 competes with Nrf2 leading to negative regulation of the antioxidant response element (ARE)-mediated NAD(P)H:quinone oxidoreductase 1 gene expression and induction in response to antioxidants. J. Biol. Chem. 280, 16891. [22] Bae, S.H. et al. (2011) Concerted action of sulfiredoxin and peroxiredoxin I protects against alcohol-induced oxidative injury in mouse liver. Hepatology 53, 945. [23] Enomoto, A. et al. (2001) High sensitivity of Nrf2 knockout mice to acetaminophen hepatotoxicity associated with decreased expression of AREregulated drug metabolizing enzymes and antioxidant genes. Toxicol. Sci. 59, 169. [24] Iizuka, T. et al. (2005) Nrf2-deficient mice are highly susceptible to cigarette smoke-induced emphysema. Genes Cells 10, 1113. [25] Gozzelino, R. et al. (2010) Mechanisms of cell protection by heme oxygenase1. Annu. Rev. Pharmacol. Toxicol. 50, 323. [26] Nath, K.A. et al. (2000) The indispensability of heme oxygenase-1 in protecting against acute heme protein-induced toxicity in vivo. Am. J. Pathol. 156, 1527. [27] Yet, S.F. et al. (2003) Absence of heme oxygenase-1 exacerbates atherosclerotic lesion formation and vascular remodeling. FASEB J. 17, 1759. [28] Vesely, M.J. et al. (1998) Heme oxygenase-1 induction in skeletal muscle cells: hemin and sodium nitroprusside are regulators in vitro. Am. J. Physiol. 275, C1087. [29] Kozakowska, M. et al. (2012) Heme oxygenase-1 inhibits myoblast differentiation by targeting myomirs. Antioxid. Redox Signal. 16, 113. [30] Wang, J. et al. (2007) Heme oxygenase-1 exacerbates early brain injury after intracerebral haemorrhage. Brain 130, 1643.
Please cite this article in press as: Kang, J., et al. A FoxO1-dependent, but NRF2-independent induction of heme oxygenase-1 during muscle atrophy. FEBS Lett. (2013), http://dx.doi.org/10.1016/j.febslet.2013.11.009
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A FoxO1-dependent, but NRF2-independent induction of heme oxygenase-1 during muscle atrophy
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Jione Kang, Mi Gyeong Jeong, Sera Oh, Eun Jung Jang, Hyo Kyeong Kim, Eun Sook Hwang ⇑ College of Pharmacy and Global Top5 Research Program, Ewha Womans University, Seoul 120-750, Republic of Korea
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Article history: Received 5 August 2013 Revised 2 October 2013 Accepted 4 November 2013 Available online xxxx Edited by Berend Wieringa Keywords: Nerve injury MAFbx MuRF1 NRF2 FoxO1
a b s t r a c t Skeletal muscle plays key roles in metabolic homeostasis. Loss of muscle mass, called muscle atrophy exacerbates disease-associated metabolic perturbations. In this study, we characterized the molecular functions and mechanisms underlying regulation of skeletal muscle atrophy induced by denervation. Denervation significantly increased the expression of heme oxygenase-1 (HO-1) and atrogenes in skeletal muscle. Forkhead box protein O1 (FoxO1) drastically increased in atrophied muscle and selectively stimulated HO-1 gene transcription through direct DNA binding. Lack of HO-1 substantially attenuated muscle atrophy, whereas HO-1 overexpression caused muscle damage in vitro and in vivo. Collectively, HO-1 induced by FoxO1 may cause skeletal muscle atrophy. Ó 2013 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
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1. Introduction
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The muscle is an important tissue that stockpiles protein in the body; however, oxidative stress and denervation cause excessive protein degradation and induce skeletal muscle wasting [1]. Proteolytic systems are primarily responsible for eliminating contractile proteins and organelles and for shrinkage of muscle fibers [2]. In particular, two muscle-specific ubiquitin ligases, MAFbx (muscle atrophy F-box) and MuRF1 (muscle-specific RING finger protein 1), cause proteasomal degradation in skeletal muscle and contribute to muscle atrophy [3,4]. MAFbx and MuRF1, called atrogenes, are strongly increased during muscle atrophy through activation of myogenin [5] and forkhead box protein O (FoxO) [6]. Indeed, mice lacking MuRF1 or MAFbx revealed protection from denervation-induced muscle atrophy [3]. FoxO1 transgenic mice showed markedly decreased skeletal muscle mass, and conversely, FoxO1 knockdown blocked MAFbx up-regulation [7]. In addition, inflammatory cytokines and nuclear factor jB (NF-jB) signaling pathways are critically involved in muscle atrophy [8,9] through generation of intracellular reactive oxygen species (ROS) [10]. Dysregulated ROS generation is prominently associated with severe muscle loss [11].
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⇑ Corresponding author. Address: College of Pharmacy, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 120-750, Republic of Korea. Fax: +82 2 3277 3760. E-mail address: [email protected] (E.S. Hwang).
Extensive studies have identified that nuclear factor erythroid 2-related factor 2 (NRF2)/MAF and its cytosolic repressor, kelchlike ECH-associated protein 1 (KEAP1), are indispensable for cellular adaptation to oxidative stress [12]. Oxidative stress dissociates NRF2 from KEAP1 in the cytosol and induces nuclear localization of NRF2. Nuclear NRF2 directly binds to the antioxidant response element (ARE) within the gene promoter of many antioxidants, such as heme oxygenase-1 (HO-1) [13], glutathione S-transferase a (GSTA) 1/2 [14], and NAD(P)H:quinone oxidoreductase 1 (NQO1) [15], and activates their expression, thereby protecting from oxidative stress-induced tissue injury [16]. Despite the key role of NRF2 in protection from oxidative stress, the importance of NRF2 in protection from injury-induced muscle atrophy remains unclear. In this study, we have examined whether NRF2 is involved in protecting skeletal muscle from denervation-induced muscle atrophy and characterized the molecular function during muscle atrophy.
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2. Materials and methods
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2.1. Mice
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C57BL/6 WT and NRF2 KO mice were obtained from Jackson Laboratory (Bar Harbor, ME) and RIKEN BioResource Center (Tsukuba, Japan) and maintained in an animal facility of Ewha Womans University. Animal handling was performed in accordance with the institutional guidelines of and approved by the IACUC (2011-01-027 and 2012-01-035).
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0014-5793/$36.00 Ó 2013 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. http://dx.doi.org/10.1016/j.febslet.2013.11.009
Please cite this article in press as: Kang, J., et al. A FoxO1-dependent, but NRF2-independent induction of heme oxygenase-1 during muscle atrophy. FEBS Lett. (2013), http://dx.doi.org/10.1016/j.febslet.2013.11.009
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2.2. Sciatic nerve injury-induced muscle wasting
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Mice (12 weeks of age, male, 25–30 g) were anesthetized using a constant flow of isoflurane inhalation. A small incision was made below the hip bone, parallel to the sciatic nerve. The nerve was isolated by removing any adherent tissue and tightly ligated with 6-0 silk suture thread around one-third to one-half of the diameter of the sciatic nerve [17]. Separately, the sciatic nerve was exposed, but not ligated in sham-operated mice. The skin incisions were closed with surgical suture and wound clips. The mice were maintained up to 7 days and were sacrificed, followed by analyses of the tibialis anterior (TA) and gastrocnemius (GN) muscles [11].
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2.3. Myogenic differentiation and muscle atrophy in vitro
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C2C12 myoblasts (ATCC, Manassas, VA) were grown to 100% confluence and replaced with myogenic differentiation medium containing 2% horse serum (Invitrogen, Carlsbad, CA) for 6 days. Differentiated myocytes were then treated with dexamethasone (DEX; 500 lM, Sigma–Aldrich, St. Louis, MO) for 24 h to induce muscle atrophy in vitro.
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2.4. Immunoblot analysis
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Cell proteins were harvested and resolved by SDS–PAGE, followed by immunoblotting. Protein blots were incubated with antibody against myosin heavy chain (MyHC; DSHB, Iowa City, IA), FoxO1 (Cell Signaling Technology, Inc., Danvers, MA), MuRF1 (ECM Biosciences LLC, Versailles, KY), HO-1, MAFbx (Abcam, Cambridge, UK), BACH1, and b-actin (Santa Cruz Biotechnology Inc.) and subsequently with horseradish peroxidase-conjugated secondary antibody, followed by detection with enhanced chemiluminescence (Thermo Fisher Scientific, Rockford, IL).
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2.5. Chromatin immunoprecipitation (ChIP)
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ChIP was performed as described previously [18]. Briefly, TA and GN muscles (25 mg) were finely minced in cold PBS and cross-linked with 1% formaldehyde. Muscles were homogenized and immnunoprecipitated using FoxO1 antibody or control IgG. The input DNA and eluted DNA were used for quantitative PCR and real time-PCR. Primers for HO-1 promoter were as follows: HO1-ChIP-FWD 50 -tgtgccatcactacccagaa-30 and HO1-ChIP-REV 50 -agcagggtaaggcttggaat-30 . Relative level was calculated after normalizing to the input samples.
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2.6. Total RNA isolation and real-time PCR
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Total RNA was isolated using TRIzol reagent (Invitrogen) and used for real-time PCR (Applied Biosystems, Foster City, CA). Relative expression level was determined after normalization with GAPDH levels. The primer sets used for real time-PCR were as follows: GAPDH, 50 -tccaccttcgatgccgggg-30 and 50 -agcgctattcattgtcatacc-30 ; GSTA, 50 -ggagattgatgggatgaagc-30 , 50 -aacaccttttcaaaggcagg-30 ; HO1, 50 -accgccttcctgctcaacattgag-30 , 50 -tgttcctctgtcagcatcacc-30 ; IL-1b, 50 -gtgtgccgtctttcattacacag-30 , 50 -ggacagaatatcaaccaagtgata-30 ; IL-6, 50 -gaggataccactcccaacaga-30 , 50 -aagtgcatcatcgttgttcataca-30 ; MAFbx, 50 -aaggctgttggagctgatagca-30 , 50 -cacccacatgttaatgttgcc-30 ; MuRF1, 50 -tgaccacagagggtaaag-30 , 50 -tgtctcactcatctccttcttc-30 ; myogenin, 50 -ccagcccatggtgcccagtg-30 , 50 -gcgtctgtagggtcagccgc-30 ; and NQO1, 50 -gacatgaacgtcattctctg-30 , 50 -ctagctttgatctggttgtc-30 .
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NRF2, FoxO1, and/or BACH1 expression vector. pCMV (Invitrogen) encoding b-galactosidase was cotransfected as an internal control. Cell extracts were harvested 48 h after transfection and assayed using the Luciferase Assay kit (Promega, Madison, WI), and the Galacto-Light b-galactosidase assay kit (Applied Biosystems) according to the manufacturer’s instructions.
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2.8. Immunohistochemistry
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TA muscles were fixed with 10% neutralized formalin for 24 h and embedded in paraffin. Tissue sections (5 lm thick) were prepared and stained with hematoxylin and eosin (HE) solution (Sigma–Aldrich, St. Louis, MO) and antibody against MyHC, NRF2, or FoxO1 (Cell signaling), followed by incubation with fluorescence tagged IgG or peroxide/DAB (Vector Laboratories, Burlingame, CA, USA).
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2.9. Transfection of siRNA
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Fully differentiated myocytes from C2C12 cells were transfected with siRNA using HiPerFect transfection reagents (Qiagen, Hilden, Germany). Transfected cells were additionally treated with Dex for 24 h. At least three different sets of siRNA for FoxO1 and HO-1 were synthesized (Bioneer, Daejeon, South Korea): siCon, 50 -ccuacgccaccaauuucgu-30 ; siFoxO #1, 50 -ccagaccaggauaauuggu-30 ; siFoxO #2, 50 -cuaaugugucucaaacauu-30 ; siFoxO #3, 50 -caucuaugcagccuuguuu30 ; siHO-1, 50 -cagaucagcacuagcucau-30 ; siHO-2, 50 -caccaaggagguacacauc-30 ; and siHO-3, 50 -ccugaaucgagcagaacca-30 .
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2.10. Rotarod test
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Mice (n = 12) were divided into two groups and injected with either vehicle or hemin (25 mg/kg) for 10 days. The latency time it takes the mouse to fall off the rod rotating was measured under continuous acceleration conditions (4, 7, 10, and 20 rpm for 2 min) at days 8, 9, 10, 11 and 12 after injection. The average latency time of each group is expressed as mean ± S.E.M. for 6 mice.
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2.11. Statistical analyses
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Data are presented as mean ± S.E.M. and analyzed by the unpaired Student’s t-test.
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3. Results
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3.1. Sciatic nerve injury induces muscle damage with an increase of NRF2 and FoxO1
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To characterize the molecular mechanisms involved in muscle atrophy, we performed sciatic nerve injury and examined the muscle phenotypes. The weights of GN and TA muscles were significantly decreased and MyHC expression decreased at day 7 after nerve injury (Fig. 1A and B). However, the expression of antioxidants, such as heme oxygenase-1 (HO-1) and catalase was increased (Fig. 1B). Furthermore, the expression of atrogenes, inflammatory cytokines, and myogenin was significantly increased during muscle atrophy (Fig. 1B and C). Simultaneously, NRF2 and FoxO1 expression, but not NF-jB p65, were significantly augmented in atrophied muscle and particularly enriched in the nucleus of the muscle (Fig. 1D and E).
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3.2. NRF2 is not required for induction of muscle atrophy markers and HO-1
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2.7. Reporter gene assay 293T cells were transiently transfected with mouse HO-1 promoter-linked reporter gene (pHO4.3k-luc) [19] together with
Because NRF2 expression is important for protection against oxidative stress by the induction of antioxidants, we examined
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Fig. 1. HO-1 induction in nerve injury-induced muscle atrophy nerve injury was caused in C57BL6 WT mice (NI; n = 15) by tying a tight ligature around the sciatic nerve, and the mice were sacrificed on days 1, 3, and 7 after denervation. Sham-operated mice (sh; n = 15) were used as controls. (A) GN and TA muscles were isolated from the sh and NI groups, and muscle mass was measured at the indicated time points (n = 5 each). Data are presented as mean ± S.E.M. (B) protein extracts were prepared from the TA and GN muscle on days 3 and 7 and were used in SDS–PAGE and immunoblotting analysis (n = 3). (C) Total RNA was collected from the muscle at day 7 after injury and reversetranscribed. The relative transcript level was calculated after normalization to b-actin level. Data are given as mean ± S.E.M. of 5 mice. (D) At day 3, protein extract was harvested and analyzed by immunoblotting (n = 3). (E) TA muscle was fixed and sectioned, followed by HE staining and immunohistochemistry with antibodies against desmin, NRF2, and FoxO1. Scale bars, 200 and 50 lm. Representative images are shown in (B), (D), and (E). ⁄P < 0.05; ⁄⁄P < 0.005; ⁄⁄⁄P < 0.0005.
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whether NRF2 was required for protecting against muscle atrophy. Sciatic nerve injury induced similar extents of muscle wasting of GN and TA muscles in WT and NRF2 KO mice (Fig. 2A). Histological analysis confirmed muscle damage in both WT and KO muscle after denervation (Fig. 2B). MyHC decrease was prominent after nerve injury and induction of MuRF1 and HO-1 was comparable between WT and NRF2 KO mice (Fig. 2C). In addition, muscle atrophy markers and inflammatory factors such as IL-6 and myogenin were similarly expressed in WT and KO muscle after denervation (Fig. 2C and D). As expected, NRF2 target genes like GSTA1/2 and NQO1 were increased in injured muscle but their expression was disappeared in the absence of NRF2 (Fig. 2E). Surprisingly, however, another known target of NRF2, the heme oxygenase-1 (HO1) was expressed in NRF2 deficiency similar to that in WT muscle after injury (Fig. 2E), implying no protective role of NRF2 against muscle atrophy and an NRF2-independent induction of HO-1 in muscle atrophy.
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3.3. FoxO1 is important for the induction of HO-1
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Because another major regulator of oxidative stress, FoxO1 was substantially increased in the nucleus of muscle fibers after injury, we investigated whether FoxO1 could be involved in modulating
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HO-1 activity. We found that FoxO1 was substantially increased after denervation and was comparably expressed in WT and NRF2 KO muscle (Fig. 3A). Immunofluorescence staining showed prominent FoxO1 expression in the nucleus of muscle fibers after injury (Fig. 3B). We then determined whether FoxO1 activated HO-1 promoter activity. Overexpression of either NRF2 or FoxO1 stimulated HO-1 promoter activity in a dose-dependent manner (Fig. 3C); however, no cooperative effect was observed between NRF2 and FoxO1 in the induction of HO-1 promoter activity (Fig. 3D). Direct binding of FoxO1 to the HO-1 gene promoter was stimulated in muscle atrophy, as evidenced by ChIP and quantitative PCR experiments (Fig. 3E and F). However, it remained unclear how FoxO1 substituted NRF2 function to induce HO-1 gene transcription. We investigated the levels of NRF2 suppressors KEAP1 and BACH1. KEAP1 expression was low and unchanged in WT and KO muscle after injury, whereas BACH1 expression was markedly increased in injured muscle, with comparable levels between WT and KO muscle (Fig. 3G). Interestingly, NRF2-induced HO-1 promoter activity was suppressed by BACH1, whereas BACH1 expression had no effect on FoxO1-mediated HO-1 activation (Fig. 3H). These results indicate that the NRF2 suppression by BACH1 instigates FoxO1 to induce HO-1 gene transcription during muscle atrophy.
Please cite this article in press as: Kang, J., et al. A FoxO1-dependent, but NRF2-independent induction of heme oxygenase-1 during muscle atrophy. FEBS Lett. (2013), http://dx.doi.org/10.1016/j.febslet.2013.11.009
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Fig. 2. NRF2-independent expression of HO-1 in muscle atrophy Sciatic nerve injury (NI) and sham operation (sh) were conducted in WT and NRF2 KO mice (n = 6 each), and the mice were sacrificed at day 7 after injury. (A) The weights of GN and TA muscles were measured, and data are expressed as mean ± S.E.M. of 6 mice. (B) TAZ muscle was sectioned and stained with HE. (C) Protein extracts were obtained from TA muscle at day 7 and analyzed by SDS–PAGE and immunoblotting (n = 6). Representative blots of 3 independent experiments are shown. (D and E) Total RNA was collected from the muscles of WT and NRF2 KO mice at day 7 after injury and used for real time-PCR. Relative mRNA level was calculated after normalization to b-actin level. Data are given as mean ± S.E.M. of 6 mice. ns, not significant; ⁄P < 0.05; ⁄⁄P < 0.005; ⁄⁄⁄P < 0.0005.
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3.4. FoxO1 is essential for HO-1 induction in the process of muscle atrophy In order to clarify the importance of FoxO1 in HO-1 induction in muscle atrophy, we established the muscle atrophy system in vitro by treating differentiated myocytes with DEX. DEX treatment induced morphological changes of the differentiated myocytes and decreased expression of MyHC (Fig. 4A). Simultaneously, DEX increased mRNA level of atrogenes and HO-1 in differentiated myocytes (Fig. 4B), mimicking in vivo nerve injury-induced muscle atrophy. We attempted to confirm the importance of FoxO1 by determining HO-1 level by the lack of FoxO1. FoxO1 expression was decreased after transfection with three different types of small interfering RNA (siRNA) against FoxO1 in fully differentiated myocytes (Fig. 4C). The mRNA level of HO-1 was measured in control and FoxO1 knockdown myocytes after treatment with DEX. HO-1 expression was induced by DEX, but was not significantly induced in FoxO1 knockdown cells compared in control cells (Fig. 4D), suggesting a necessity of FoxO1 in HO-1 induction. 3.5. Suppression of HO-1 attenuates DEX-induced expression of muscle atrophy genes We then assessed the effect of HO-1 in muscle atrophy and activity. Knockdown of HO-1 in differentiated myocytes significantly attenuated the expression of atrogenes induced by DEX
treatment (Fig. 5A and B). Conversely, elevated HO-1 expression by treatment with hemin, an HO-1 inducer decreased the level of MyHC but increased MuRF1 through induction of FoxO1 (Fig. 5C). The flattened shape and shrinking of differentiated myocytes and MyHC decline were prominently induced after treatment with hemin (Fig. 5D). More importantly, daily administration of hemin for 7 days caused muscle damage in vivo, as evidenced by HE staining and immunohistochemistry of desmin (Fig. 5E). In addition, physical muscle activity was measured by rotarod test. Latency time to fall was significantly decreased in hemin-injected mice after day 10 when compared in vehicle-treated group (Fig. 5F). These results suggest that sustained HO-1 expression by hemin treatment exerts a deleterious effect on skeletal muscle fibers and physical muscle activity.
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4. Discussion
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Our results showed that HO-1 level increased substantially in muscle atrophy, and its expression was dependent on FoxO1, but not on NRF2. Blockade of HO-1 induction attenuated muscle atrophy gene expression induced by DEX treatment in vitro, and prolonged HO-1 expression per se was sufficiently injurious to the skeletal muscle fibers in vivo. NRF2 is a major protective molecule against oxidative stress [20]. To ensure the timely activity of different antioxidants and detoxifying enzymes, NRF2 activity is tightly controlled by
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Fig. 3. FoxO1-induced HO-1 expression after nerve injury (A and B) WT and NRF2 KO mice were sham-operated (sh; n = 6 each) or injured (NI; n = 6 each) and sacrificed at day 7. Protein extracts were analyzed by immunoblotting (A). Tissue sections were stained with FoxO1 antibody and observed under a fluorescence microscope (B). (C and D) 293T cells were transfected with different amounts of either NRF2 or FoxO1 expression vector together with pHO-luc (C) or with the indicated combinations of NRF2 and FoxO1 (D). pCMVb-gal was cotransfected for normalization of transfection efficiency. Relative luciferase activity was determined after normalization to b-galactosidase activity and expressed as a fold induction normalized to mock control (–). (E and F) TA and GN muscles used for preparing genomic DNA. PCR reactions were carried out 26 cycles (E) and quantitatively determined by real time-PCR (F). (G) protein blots in (A) were incubated with antibody against BACH1, KEAP1, and actin. (H) 293T cells were transfected with expression vector for NRF2, FoxO1, and/or BACH1 together with pHO-luc and pCMVb-gal. Relative activity is expressed as mean ± S.E.M. of 3 independent experiments. Representative images are shown in (A) to (C), (E) and (G). ns, not significant; ⁄P < 0.05; ⁄⁄P < 0.005.
Fig. 4. Necessity of FoxO1 in DEX-induced HO-1 expression in vitro Differentiation of myocytes was induced from C2C12 cells for 6 days and treated with DEX (500 lM) for 24 h. (A) Cells were fixed and incubated with MyHC antibody and DAPI, followed by a microscopic observation. (B) Total RNA was collected and used for determining the relative expression level. (C and D) Differentiated myocytes were transfected with siRNA against control gene (siCon) or FoxO1 (siFoxO1) and treated with DEX for 24 h. Protein extracts (C) and total RNA (D) were analyzed. ns, not significant; ⁄P < 0.05; ⁄⁄⁄P < 0.0005.
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nuclear-cytoplasmic shuttling through KEAP1 interaction in the cytoplasm. NRF2 is also suppressed by BACH1, which competitively binds to ARE and suppresses ARE-regulated gene expression
[21]. NRF2 KO mice showed increased susceptibility to a variety of chemical-induced diseases, such as drug-induced liver injury [22], oxidative stress-induced cardiomyopathy [23], and cigarette
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Fig. 5. Modulation of muscle atrophy by HO-1 in vitro and in vivo (A and B) Differentiated myocytes were transfected with siRNAs and subsequently treated with DEX (500 lM). Protein extract and total RNA were prepared from the cells and analyzed by immunoblotting (A) and real time-PCR (B). Relative expression levels of MuRF1 and MAFbx were calculated after normalization to b-actin level. Data are given as mean ± S.E.M. of 3 independent experiments. ⁄P < 0.05; ⁄⁄P < 0.005; ⁄⁄⁄P < 0.0005. (C and D) Differentiated cells were treated with hemin for 24 h, and protein extracts were harvested for immunoblotting analysis (C). Cells were stained with MyHC antibody and DAPI and observed under a microscope (D). (E) Hemin (30 mg/kg) was daily injected into the mice for 7 days. TA muscle was sectioned and subjected to HE and desmin staining. Scale bars indicate 2000, 200, and 50 lm. Representative images are shown in (A) and (C) to (E). (F) Latency time was determined in veh- or hemin-treated mice by Rota rod test at days 10, 11, 12, and 13 after injection. Data are given as mean ± S.E.M. of 6 mice. ⁄P < 0.05.
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smoke-induced emphysema [24], implicating protective roles of NRF2 against tissue injury. However, abrogation of NRF2 in mice showed muscle wasting, induction of inflammatory and atrophy markers as comparable as in WT mice after injury. Despite a pivotal role of NRF2 in the control of oxidative damage, NRF2 is neither necessary nor sufficient for protecting against nerve injuryinduced muscle atrophy. Interestingly, the expression of GSTA1/2 and NQO1 was impaired in NRF2 deficiency whereas HO-1 was increased in NRF2 KO muscle after injury. Denervation caused an increase in expression level and nuclear localization of NRF2, resulting in the expression of antioxidants such as GSTA1/2, NQO1, and HO-1. However, injured muscle subsequently increased expression of BACH1, an NRF2 competitor, thereby attenuating NRF2 activity to bind to ARE and induce ARE-mediated gene transcription. Although the expression level of GSTA1/2 and NQO1 was reciprocally regulated by NRF2 and BACH1, HO-1 expression was not suppressed by BACH1 but rather increased in denervation-induced muscle atrophy. Our results demonstrated that FoxO1 directly bound to FRE within the HO-1
gene promoter and stimulated HO-1 gene transcription even in the presence of BACH1 (Fig. 6). In addition, FoxO1 strongly increased the expression of MAFbx and caused muscle atrophy. Induced expression of FoxO1 played important role in the induction of not only MAFbx, an atrophy marker but also HO-1 in muscle after denervation. There have been extensive studies on protective roles of HO-1 in many tissues. NRF2-dependent HO-1 expression profoundly attenuated lung injury, skin lesion, and cardiac failure [25]. Furthermore, abrogation of HO-1 in mice developed fulminant renal failure, leading to death [26] and exacerbated atherosclerosis in an apolipoprotein E-deficient genetic background [27]. HO-1 is an indispensable stress response protein. However, HO-1 expression decreased the viability of myocytes [28] and inhibited myoblast differentiation [29]. In addition, HO-1 induction exacerbates early brain injury [30]. Our results also revealed that HO-1 expression was increased by FoxO1 in muscle atrophy in vivo, and suppression of HO-1 level in fully differentiated myocytes attenuated the induction of MAFbx and MuRF1 after DEX treatment.
Please cite this article in press as: Kang, J., et al. A FoxO1-dependent, but NRF2-independent induction of heme oxygenase-1 during muscle atrophy. FEBS Lett. (2013), http://dx.doi.org/10.1016/j.febslet.2013.11.009
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Fig. 6. FoxO1-induced expression of HO-1 and muscle atrophy genes Under basal conditions, nuclear NRF2 is competed by BACH1 for binding to ARE within the promoter of HO-1, GSTA1/2, and NQO1 genes, resulting in low expression of antioxidants. Upon injury, the expression of NRF2 and FoxO1 was increased in muscle. NRF2 is essential for the expression of protective antioxidants such as GSTA1/2 and NQO1 but is not required for HO-1 expression. FoxO1, on the other hand, directly binds to FRE within the promoter of HO-1 and MAFbx genes and promotes gene transcription, thereby inducing muscle atrophy.
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Furthermore, in vivo sustained expression of HO-1 by treatment with hemin was accompanied by a decrease in MyHC and an increase in atrogenes, causing skeletal muscle atrophy. HO-1 thus has a dual and opposing role, it provides protection from oxidative damage in an NRF2-dependent manner and it promotes muscle atrophy through a FoxO1-induced pathway.
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Disclosure of potential conflicts of interest
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No potential conflicts of interest were disclosed.
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Acknowledgments
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We thank Dr. J. Alam (Alton Ochsner Medical Foundation, New Orleans) for providing pHO4.3k-luc vector and Ms. Hyun Jung Kim for helping histological analysis. This work was supported by the Basic research program (2012R1A1B3000603, ESH and for 2011007983 for JOK) of the National Research Foundation funded by Ministry of Education, Science, and Technology.
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Please cite this article in press as: Kang, J., et al. A FoxO1-dependent, but NRF2-independent induction of heme oxygenase-1 during muscle atrophy. FEBS Lett. (2013), http://dx.doi.org/10.1016/j.febslet.2013.11.009
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