STAT 3 promotes IFN γ/ TNF α‐induced muscle wasting in an NF ‐κB‐dependent and IL ‐6‐independent manner
2017; Springer Nature; Volume: 9; Issue: 5 Linguagem: Inglês
10.15252/emmm.201607052
ISSN1757-4684
AutoresF. Jennifer, Brenda Janice Sánchez, Derek Hall, Anne‐Marie K Tremblay, Sergio Di Marco, Imed‐Eddine Gallouzi,
Tópico(s)Viral Infections and Immunology Research
ResumoResearch Article6 March 2017Open Access Source DataTransparent process STAT3 promotes IFNγ/TNFα-induced muscle wasting in an NF-κB-dependent and IL-6-independent manner Jennifer F Ma Jennifer F Ma Department of Biochemistry, Rosalind and Morris Goodman Cancer Centre, McGill University, Montreal, QC, Canada Search for more papers by this author Brenda J Sanchez Brenda J Sanchez Department of Biochemistry, Rosalind and Morris Goodman Cancer Centre, McGill University, Montreal, QC, Canada Search for more papers by this author Derek T Hall Derek T Hall Department of Biochemistry, Rosalind and Morris Goodman Cancer Centre, McGill University, Montreal, QC, Canada Search for more papers by this author Anne-Marie K Tremblay Anne-Marie K Tremblay Department of Biochemistry, Rosalind and Morris Goodman Cancer Centre, McGill University, Montreal, QC, Canada Search for more papers by this author Sergio Di Marco Sergio Di Marco Department of Biochemistry, Rosalind and Morris Goodman Cancer Centre, McGill University, Montreal, QC, Canada Search for more papers by this author Imed-Eddine Gallouzi Corresponding Author Imed-Eddine Gallouzi [email protected] [email protected] orcid.org/0000-0003-4758-4835 Department of Biochemistry, Rosalind and Morris Goodman Cancer Centre, McGill University, Montreal, QC, Canada Life Sciences Division, College of Science and Engineering, Hamad Bin Khalifa University (HBKU), Education City, Doha, Qatar Search for more papers by this author Jennifer F Ma Jennifer F Ma Department of Biochemistry, Rosalind and Morris Goodman Cancer Centre, McGill University, Montreal, QC, Canada Search for more papers by this author Brenda J Sanchez Brenda J Sanchez Department of Biochemistry, Rosalind and Morris Goodman Cancer Centre, McGill University, Montreal, QC, Canada Search for more papers by this author Derek T Hall Derek T Hall Department of Biochemistry, Rosalind and Morris Goodman Cancer Centre, McGill University, Montreal, QC, Canada Search for more papers by this author Anne-Marie K Tremblay Anne-Marie K Tremblay Department of Biochemistry, Rosalind and Morris Goodman Cancer Centre, McGill University, Montreal, QC, Canada Search for more papers by this author Sergio Di Marco Sergio Di Marco Department of Biochemistry, Rosalind and Morris Goodman Cancer Centre, McGill University, Montreal, QC, Canada Search for more papers by this author Imed-Eddine Gallouzi Corresponding Author Imed-Eddine Gallouzi [email protected] [email protected] orcid.org/0000-0003-4758-4835 Department of Biochemistry, Rosalind and Morris Goodman Cancer Centre, McGill University, Montreal, QC, Canada Life Sciences Division, College of Science and Engineering, Hamad Bin Khalifa University (HBKU), Education City, Doha, Qatar Search for more papers by this author Author Information Jennifer F Ma1, Brenda J Sanchez1, Derek T Hall1, Anne-Marie K Tremblay1, Sergio Di Marco1 and Imed-Eddine Gallouzi *,*,1,2 1Department of Biochemistry, Rosalind and Morris Goodman Cancer Centre, McGill University, Montreal, QC, Canada 2Life Sciences Division, College of Science and Engineering, Hamad Bin Khalifa University (HBKU), Education City, Doha, Qatar *Corresponding author. Tel: +1 514 398 4537, +97444546416; Fax: +1 51 398 7384; E-mails: [email protected]; [email protected] EMBO Mol Med (2017)9:622-637https://doi.org/10.15252/emmm.201607052 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Cachexia is a debilitating syndrome characterized by involuntary muscle wasting that is triggered at the late stage of many cancers. While the multifactorial nature of this syndrome and the implication of cytokines such as IL-6, IFNγ, and TNFα is well established, we still do not know how various effector pathways collaborate together to trigger muscle atrophy. Here, we show that IFNγ/TNFα promotes the phosphorylation of STAT3 on Y705 residue in the cytoplasm of muscle fibers by activating JAK kinases. Unexpectedly, this effect occurs both in vitro and in vivo independently of IL-6, which is considered as one of the main triggers of STAT3-mediated muscle wasting. pY-STAT3 forms a complex with NF-κB that is rapidly imported to the nucleus where it is recruited to the promoter of the iNos gene to activate the iNOS/NO pathway, a well-known downstream effector of IFNγ/TNFα-induced muscle loss. Together, these findings show that STAT3 and NF-κB respond to the same upstream signal and cooperate to promote the expression of pro-cachectic genes, the identification of which could provide effective targets to combat this deadly syndrome. Synopsis Proinflammatory cytokines such as IFNγ and TNFα are known to be key mediators of cancer cachexia-induced muscle loss. Here, they are shown to promote muscle atrophy by directly activating the transcription factor STAT3. IFNγ/TNFα activate the JAKs/STAT3 signaling pathway in an IL-6-independent manner in skeletal muscle fibers. IFNγ/TNFα trigger the formation of an NF-κB/STAT3 complex. NF-κB is required for the translocation of STAT3 to the nucleus. STAT3 mediates the expression of iNOS to promote IFNγ/TNFα-induced muscle atrophy. Introduction Cancer-related cachexia is a debilitating syndrome characterized by the progressive loss of body weight that is triggered in part by an involuntary loss of skeletal muscle mass, referred to as muscle wasting (Dodson et al, 2011; Ma et al, 2012; Argiles et al, 2016). Cachexia is associated with anorexia, fatigue, skeletal muscle weakness, and an overall reduced quality of life (Tisdale, 2009; Argiles et al, 2014a,b). Unlike muscle atrophy caused by starvation or physical inactivity, muscle wasting in cachectic patients cannot be reversed by nutritional supplementation (Fearon, 2011; Argiles et al, 2013). Furthermore, the development of cachexia is a strong predictor of poor treatment outcome and mortality for individuals afflicted with cancer, and is estimated to be a direct cause of 20% of patient death (Muscaritoli et al, 2015). Cancer cachexia is largely considered to be an end-of-life condition. Despite this, there is no standard treatment option available to prevent or treat cachexia. This highlights the importance of delineating the pro-cachectic mechanism of action in order to identify targets to combat this deadly syndrome. The primary catabolic mediators of cachexia include proinflammatory cytokines such as tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), and interleukin-6 (IL-6) (Hall et al, 2011; Argiles et al, 2013; Cohen et al, 2015). Numerous studies have been dedicated to develop pharmacological inhibitors to interfere with their pro-cachectic effects both in animals and humans. However, the targeted inhibition of these cytokines, specifically in human, was not efficacious. For example, the use of anti-TNFα inhibitors not only had limited effect on the progression of this condition but also has been associated with unwanted side effects (Wiedenmann et al, 2008; Jatoi et al, 2010). Additionally, recent clinical trials using a monoclonal anti-IL-6 antibody on patients with lung cancer-induced muscle wasting showed a reversal of anorexia, fatigue, and anemia, but did not prevent the loss of lean body mass (Bayliss et al, 2011). While no anti-IFNγ therapy has been tested in humans so far, some studies using antibodies against this cytokine have shown some successes in interfering with cancer-induced muscle loss in mice (Langstein et al, 1991; Matthys et al, 1991). Inhibition of cytokines may also impinge on their primary roles in the immune response. Indeed, it has been reported that the prolonged suppression of cytokines such as IL-6 or TNFα can result in increased risk of infection and delayed wound healing (McFarland-Mancini et al, 2010; Jones et al, 2011). The limited success of these mono-therapeutic approaches underscores the multifactorial nature of cachexia and highlights the possibility that its pathology could result from the combined activation of common downstream effector pathways. During the last few decades, several downstream signaling pathways and their effectors have been identified and linked to cytokine-induced muscle loss (Cohen et al, 2015). It has been shown that TNFα in collaboration with IFNγ mediates its pro-cachectic effects through the activation of the NF-κB (Nuclear transcription Factor kappa B) pathway, which promotes the expression of several target genes (Dahlman et al, 2010; Grivennikov & Karin, 2010; Hall et al, 2011; Fearon et al, 2012; Ma et al, 2012; Argiles et al, 2016). Others and we have demonstrated that the inducible nitric oxide (NO) synthase (iNOS) enzyme is one of the main effectors of the TNFα/NF-κB-induced muscle wasting (Buck & Chojkier, 1996; Di Marco et al, 2005, 2012). The upregulation of the iNOS/NO pathway correlates with a dramatic decrease in general translation and the loss of promyogenic factor such as MyoD and Myogenin (Hall et al, 2011; Ma et al, 2012). More recently, several studies have indicated that IL-6 triggers muscle atrophy by activating the STAT3 (Signal transducer and activator of transcription 3) pathway (Bonetto et al, 2011, 2012; Sala & Sacco, 2016; Zimmers et al, 2016). IL-6 activates signal transduction by binding to the IL-6 receptor alpha-chain and the common receptor subunit gp130. The gp130-associated Janus kinases (JAKs) become activated and mediate the phosphorylation of STAT3 protein on its key tyrosine 705 (Y705) residues. This JAK-mediated phosphorylation allows STAT3 to dimerize and bind to DNA to promote the transcription of several pro-cachectic genes (Zhang et al, 2009; Bonetto et al, 2011, 2012; Sala & Sacco, 2016; Zimmers et al, 2016). Interestingly, IL-6 is not the only activator of STAT3 during muscle wasting. Treating Il6−/− mice with lipopolysaccharides (LPS), a component of Gram-negative bacteria, also leads to phosphorylation of STAT3 (Bonetto et al, 2012). While these observations raise the possibility that STAT3 can trigger muscle wasting independently of IL-6, a STAT3-mediated and IL-6-independent molecular mechanism remain elusive. Several studies have shown that NF-κB and STAT3 can collaborate together to mediate cell response to various extracellular challenges. For example, unphosphorylated STAT3 was shown to associate with the p65 subunit of NF-κB to promote the transcription of the serum amyloid A (SSA) gene, the expression of which is associated with serious complications of inflammatory diseases, such as rheumatoid (RA) arthritis and juvenile inflammatory arthritis (Gillmore et al, 2001; Hagihara et al, 2005). It has also been shown that TNFα activates STAT3 in various cell systems in an NF-κB-dependent manner (Guo et al, 1998; Lee et al, 2013; Snyder et al, 2014). TNFα stimulates metastatic pathways in breast cancer cells by triggering the formation of the STAT3-NF-κB complex, which in turn, upregulates the transcription of the actin-bundling protein fascin (Snyder et al, 2014). Therefore, it is possible that the failure of the anti-TNFα and -IL-6 therapies to prevent muscle wasting is due to the fact that both cytokines redundantly activate a common downstream effector such as STAT3. In this work, we show that STAT3 is required for the IFNγ/TNFα-induced muscle wasting and that these effects depend on the collaboration between STAT3 and NF-κB pathways. Interestingly, while this STAT3-NF-κB-mediated effect occurs independently of IL-6, NF-κB is required for the translocation of STAT3 to the nucleus as well as for the activation of the iNOS/NO pathway, one of the main effectors of IFNγ/TNFα-induced muscle wasting. Results TNFα and IFNγ activate STAT3 in muscle fibers in an IL-6-independent manner As a first step in assessing the role of the STAT3 pathway in IFNγ and TNFα-induced muscle wasting, we treated fully differentiated muscle fibers with these two cytokines over various period of times and assessed muscle wasting as well as STAT3 activation (Fig 1). While as expected (Guttridge et al, 2000) IFNγ or TNFα separately did not promote muscle wasting of C2C12 myotubes (Fig 1C), both cytokines together did trigger a dramatic loss of muscle fibers within 72 h of treatment (Fig 1A and B; Di Marco et al, 2012, 2005). The observed IFNγ/TNFα-induced muscle wasting was not due to cell death by apoptosis, since these cytokines failed to trigger the cleavage of caspase-3, a well-known marker of caspase-mediated apoptosis (Beauchamp et al, 2010; von Roretz et al, 2013), in C2C12 myotubes even after 72 h of treatment (Appendix Fig S1). Next, we assessed the activation of STAT3 in muscle fibers exposed to both cytokines as described above. We followed the phosphorylation status of the tyrosine (Y) 705 and serine (S) 727 residues, two well-characterized phosphorylation sites of STAT3 (Grivennikov & Karin, 2010). While IFNγ and TNFα did not affect the level of pS-STAT3 when compared to untreated muscle fibers, a 30-min treatment with these two cytokines was sufficient to significantly enhance the levels of pY-STAT3 in C2C12 myotubes (Fig 1D and E). Similar results were obtained when we assessed pY-STAT3 levels in primary muscle fibers treated with IFNγ/TNFα (Appendix Fig S2). Together, these observations raise the possibility that the phosphorylation of STAT3 on its Y705 residue is an integral part of the downstream signaling pathway used by IFNγ/TNFα to trigger muscle wasting. Figure 1. STAT3 is phosphorylated on residue Tyr705 during IFNγ/TNFα-induced muscle wastingC2C12 cells were grown to confluency then induced for differentiation for 4 days to form fully differentiated myotubes. These myotubes were exposed or not to IFNγ/TNFα for 72 or 96 h. Phase contrast images of cultured C2C12 myotubes treated with or without IFNγ/TNFα for 72 h. Scale bar = 200 μm. Images shown are representative of n = 4 independent experiments. Immunofluorescence (IF) images of C2C12 myotubes either untreated or treated with IFNγ/TNFα for 72 h were stained with anti-myosin heavy-chain (MyHC; green) and anti-myoglobin (red) antibodies. Scale bar = 50 μm. Images shown are representative of n = 4 independent experiments. IF images of C2C12 myotubes treated for 72 h (panels 1–3) or 96 h (panels 4–6) with either TNFα (panels 2 and 5) or IFNγ (panels 3 and 6). Untreated (panels 1 and 4) and treated myotubes were stained with myosin heavy chain (green) and myoglobin (red) as marker for differentiated muscle cells. Images shown are representatives of n = 2 independent experiments. Scale bar = 50 μm. Total extracts of C2C12 myotubes treated with or without IFNγ/TNFα were used for Western blot analysis with antibodies against pY-STAT3, pS-STAT3, total STAT3, and α-tubulin. The blot shown is a representative of n = 4 independent experiments. Densitometric quantification of pY-STAT3 signal relative to total STAT3 signal in panel (D). Data represented as mean ± SEM (n = 4) with *P-value = 0.0135 by one-way ANOVA with Dunnett's post hoc test. Source data are available online for this figure. Source Data for Figure 1 [emmm201607052-sup-0002-SDataFig1.pptx] Download figure Download PowerPoint Work from several groups has established pY-STAT3 as a one of the key mediators in the IL-6-induced muscle atrophy (Bonetto et al, 2011, 2012; Zimmers et al, 2016). Since IFNγ/TNFα have been demonstrated to induce IL-6 expression and secretion in various cell lines (Sanceau et al, 1991; Alvarez et al, 2002), we wanted to verify whether their ability to activate STAT3 in muscle fibers depends on IL-6 expression and secretion. As a first step, we analyzed the levels of IL-6 secreted by muscle fibers treated with IFNγ/TNFα as described above. Using ELISA assay, we detected a dramatic increase in IL-6 protein levels 24 h and 48 h post-treatment with amounts ranging from ~2 to ~14 ng/ml (Fig 2A). Next, we tested whether these levels of IL-6 are sufficient to induce STAT3 phosphorylation in muscle fibers. These fibers were treated with various concentration of murine recombinant IL-6 (rIL-6, ranging from 1 to 20 ng/ml) that reflected the approximate concentrations detected by ELISA during the IFNγ/TNFα treatment. Western blot analysis using the anti-pY-STAT3 antibody indicated no change in the levels of pY-STAT3 with any of these concentrations of recombinant IL-6 (Fig 2B and C). However, pY-STAT3 was detected only when C2C12 muscle fibers or macrophages (used as positive control) were treated with 100 ng/ml of IL-6 (Appendix Fig S3). Therefore, the failure of low doses of rIL-6 (20 ng/ml or lower) to phosphorylate STAT3 in myotubes might be due to instability or inactivity of our rIL-6. To further explore the effect of IL-6 on STAT3 phosphorylation, we assessed pY-STAT3 levels in the muscle of IL-6 knockout (KO) and wild-type mice, which were intramuscularly injected with IFNγ/TNFα or saline for 5 days as described (Di Marco et al, 2012). Interestingly, we observed a significant induction of pY-STAT3 in the gastrocnemius muscle of both wild-type and IL-6 KO animals (Fig 2D and E). However, unlike in wild-type mice, IFNγ/TNFα failed to trigger the wasting of skeletal muscle of IL-6 KO mice as evidenced by the weight and cross-sectional analyses of the tibialis anterior of these animals (Fig 2F and H). Therefore, our data demonstrate that in muscle fibers IFNγ/TNFα activate STAT3 by triggering the phosphorylation of its Y705 residue in an IL-6-independent manner. Figure 2. IFNγ/TNFα induces STAT3 phosphorylation in an IL-6-independent manner Supernatant was collected from C2C12 myotubes treated with or without IFNγ/TNFα and analyzed by ELISA to determine the concentration of IL-6. Data represented as mean ± SEM (n = 2). Total cell extracts of C2C12 myotubes treated with or without recombinant murine IL-6 (rIL-6) were used for Western blot analysis with antibodies against pY-STAT3, total STAT3, and α-tubulin. The blot shown is a representative of n = 3. Densitometric quantification of pY-STAT3 signal relative to total STAT3 signal from Western blots in panel (B). Data represented as mean ± SEM (n = 3) with P-value = N.S. by two-way ANOVA with a Tukey post hoc test. Wild-type (WT) and IL-6 KO mice were intramuscularly injected with IFNγ/TNFα for five consecutive days and sacrificed on the sixth day. Gastrocnemius muscle was homogenized and used for Western blot analysis with antibodies against pY-STAT3, total STAT3, iNOS, and α-tubulin. The blot shown is a representative of n = 3 mice. Densitometric quantification of pY-STAT3 signal relative to total STAT3 signal from Western blots in panel (D). Data represented as mean ± SEM (n = 3) with **P-value = 0.0033 (WT) and **P-value = 0.0091 (IL-6 KO) by two-way ANOVA with a Tukey post hoc test. The tibialis anterior (TA) muscle weight significantly decreased in WT animals but not in IL-6 KO animals. Data represented as mean ± SEM for n = 6 (WT PBS), 4 (WT IFNγ/TNFα), 4 (IL-6 KO PBS), and 4 (IL-6 KO IFNγ/TNFα) mice with **P-value = 0.0081 by two-way ANOVA with a Tukey post hoc test. Image of a representative section of the TA muscle from wild-type and IL-6 KO mice stained with hematoxylin and eosin. Scale bar = 100 μm. The cross-sectional areas (CSA) of TA muscle from panel (G) are represented as a frequency histogram from n = 2 mice. Nine hundred fibers were analyzed for each animal. The mean CSA ± SD is indicated in the legend of the histogram. Source data are available online for this figure. Source Data for Figure 2 [emmm201607052-sup-0003-SDataFig2.pptx] Download figure Download PowerPoint IFNγ/TNFα use the JAK signaling pathway to phosphorylate STAT3 and induce muscle wasting Next, we assessed the possibility that STAT3 activation could play an important part in IFNγ/TNFα-induced muscle wasting. Since it is well established that knocking down STAT3 expression prevents myogenesis in vitro (Sun et al, 2007; Wang et al, 2008), to assess this possibility, we used the STAT3 inhibitor S3I-201 on the C2C12 myotubes. As has been shown in other cell lines (Pang et al, 2010), we observed that this inhibitor was effective in interfering with IL-6-mediated phosphorylation of STAT3 on its Y705 residue in muscle cells (Fig 3A and B). Phase contrast images as well as immunofluorescence experiments on muscle fibers exposed or not to IFNγ/TNFα for 72 h showed that S3I-201 significantly reduced the muscle fiber loss that is normally observed with these two cytokines (Fig 3C–E). To identify the signaling pathway behind the IFNγ/TNFα-mediated activation of STAT3, we assessed the involvement of the Janus kinases (JAK's), an upstream non-receptor tyrosine kinases that, in response to IL-6 and other stimuli, phosphorylate STAT3 on its Y705 residue (Grivennikov & Karin, 2010). We observed that the Jak kinase inhibitor 1 (Pedranzini et al, 2006) was sufficient to prevent both STAT3 phosphorylation (Fig 3F) and the IFNγ/TNFα-induced muscle atrophy (Fig 3G and H). The widths of the muscle fibers simultaneously treated with a Jak inhibitor and IFNγ/TNFα were also significantly higher than the IFNγ/TNFα-treated DMSO control fibers (Fig 3I). These results indicate that IFNγ/TNFα trigger muscle wasting by activating the JAK signaling pathway, which in turn phosphorylates STAT3 on its Y705 residue. Figure 3. Inhibition of the Jak/STAT3 pathway prevents IFNγ/TNFα-induced muscle wasting A. Total cell extracts from proliferating C2C12 treated with STAT3 inhibitor S3I-201 then with recombinant murine IL-6 for 30 min were used for Western blot analysis with antibodies against pY-STAT3, total STAT3, and α-tubulin. The blot shown is a representative of n = 3 experiments. B. Densitometric quantification of pY-STAT3 signal relative to total STAT3 signal of Western blots in panel (A). Data represented as mean ± SEM (n = 3) with a ***P-value = 0.00439 by two-tailed, unpaired Student's t-test. C, D. Phase contrast (C) and IF images (D) of C2C12 myotubes treated with or without IFNγ/TNFα and with STAT3 inhibitor S3I-201 or DMSO as a control. IF of C2C12 myotubes were stained with antibodies against endogenous myosin heavy chain (green), myoglobin (red), and counter stained with DAPI for nuclei (blue). Scale bar is 200 μm and 50 μm for images in (C) and (D), respectively. Images shown are representatives of n = 3 experiments. E. Quantification of average myotube width from panel (D). The average myotube widths are presented as percentage change ± SEM (n = 3) relative to the untreated, with ***P-value = 5.6 × 10−5 (DMSO) and ***P-value = 7.56 × 10−4 (S3I-201) by two-way ANOVA with Tukey post-test. F. Total extracts from C2C12 myotubes pre-treated with a Jak inhibitor then with IFNγ/TNFα for 30 min was used for Western blotting analysis with antibodies against pY-STAT3, total STAT3, and α-tubulin. The blot shown is a representative of n = 3 experiments. G, H. Phase contrast images (G) and IF (H) of C2C12 myotubes treated with or without a Jak inhibitor or DMSO as a control and with or without IFNγ/TNFα for 72 h. IF images (H) of cultured C2C12 myotubes stained for myosin heavy chain (green), myoglobin (red), and counter stained with DAPI for nuclei (blue). Scale bar is 200 μm and 50 μm for images in (G) and (H), respectively. Images shown are representative of n = 3 experiments. I. Quantification of C2C12 myotube widths from panel (H) were determined as described in panel (D). Data represented as average ± SEM (n = 3), with ***P-value = 1.8 × 10−5 (DMSO) and ***P-value = 2.3 × 10−5 (Jak inhibitor) by two-way ANOVA with Tukey post-test. Source data are available online for this figure. Source Data for Figure 3 [emmm201607052-sup-0004-SDataFig3.pptx] Download figure Download PowerPoint pY-STAT3 promotes iNOS expression during IFNγ/TNFα-mediated muscle wasting Previously, iNOS/NO pathway was shown as an important mediator of IFNγ/TNFα-induced muscle wasting (Di Marco et al, 2005, 2012). In addition, work from various groups has suggested that STAT3 modulates iNOS expression in non-muscle cells (Yu et al, 2002b; Ziesche et al, 2007; Park et al, 2010). Therefore, we assessed the involvement of STAT3 in IFNγ/TNFα-mediated iNOS expression in C2C12 muscle fibers. First, as expected (Di Marco et al, 2005), when used separately, TNFα but not IFNγ was able to slightly induce iNOS expression in C2C12 myotubes when used for 48 h or more. However, when both cytokines were used together for the same period of time, they synergistically induced a high expression level of iNOS in these muscle fibers (Appendix Fig S4). Next, we observed that S3I-201 completely inhibited iNOS expression and NO production in muscle fibers treated with IFNγ/TNFα (Fig 4A–C and Appendix Fig S5). Moreover, an shRNA that reduced STAT3 expression by ~40% also decreased iNOS protein levels by > 35% in muscle fibers treated with IFNγ/TNFα (Fig 4D). To further explore the importance of STAT3 phosphorylation, we assessed the effect of two STAT3 mutants on iNOS expression. We observed that while expressing a constitutively active isoform of STAT3 (STAT3-C) (Bromberg & Darnell, 1999) in C2C12 cells failed, on its own, to promote iNOS expression (Fig 4E, lanes 5 and 6), this isoform dramatically enhanced the level of IFNγ/TNFα-induced iNOS protein. However, the expression of a Tyr705 to Phe (Y705F)-STAT3 mutant (Wen & Darnell, 1997) dramatically decreased iNOS expression under these conditions (Fig 4E). Additionally, using iNOS KO mice, we observed that although these animals are protected against IFNγ/TNFα-induced loss of muscle mass (Fig 4F–I), a high level of pY-STAT3 was detected in their gastrocnemius muscle (Fig 4G). These data show that while activating STAT3 alone is not sufficient to trigger iNOS expression, STAT3 collaborates with other signaling pathways to promote IFNγ/TNFα-induced iNOS and muscle atrophy. Moreover, these observations also indicate that iNOS is a key downstream effector of the IFNγ/TNFα-induced muscle wasting in vivo. Figure 4. STAT3 promotes the expression of iNOS in IFNγ/TNFα-treated myotubes A. C2C12 myotubes treated with or without IFNγ/TNFα and with STAT3 inhibitor S3I-201 or DMSO as a control. Total cell extracts from these muscle fibers were used for Western blot analysis with antibodies against iNOS, STAT3, and α-tubulin as a loading control. The blot shown is representative of n = 3 experiments. B. NO levels were measured in supernatant from the myotubes described in panel (A). Data represented as mean ± SEM (n = 3) with **P-value = 0.0013 and ***P-value = 0.0006 by two-tailed, unpaired Student's t-test. C. Total mRNA from C2C12 myotubes treated with IFNγ/TNFα and with the STAT3 inhibitor S3I-201 or DMSO as a control was used for RT–qPCR analysis of iNOS (Nos2) and Rpl32 mRNA. Data represented as mean ± SEM (n = 3) with ***P-value = 0.0007 by two-tailed, unpaired Student's t-test. D. C2C12 cells were transfected with a plasmid expressing shRNA against STAT3 or a scramble control. (Left) Total cell extract was used for Western blot analysis with antibodies against pY-STAT3, total STAT3, iNOS, and α-tubulin. Densitometric quantification of STAT3 (middle panel) and iNOS (right panel) signals relative to α-tubulin of Western blots from left panel. Data represented as mean ± SD (n = 2) with *P-value = 0.0123 by two-tailed, unpaired Student's t-test. E. C2C12 cells were transfected with an empty vector or a STAT3 constitutively active (STAT3-C) mutant or a Tyr705 to Phe (Y705F) mutant-expressing plasmid and followed by treatment with TNFα alone, IFNγ alone, or both for 24 h. Total cell extracts were used for Western blot analysis using antibodies against pY-STAT3, total STAT3, iNOS, and α-tubulin. The blot shown is a representative of n = 2 experiments. F–I. Wild-type (WT) and iNOS KO mice were intramuscularly injected with IFNγ/TNFα for five consecutive days and sacrificed on the sixth day. F. Wild-type (WT) and iNOS KO mice were intramuscularly injected with IFNγ/TNFα for five consecutive days and sacrificed on the sixth day. The gastrocnemius muscle weight significantly decreased in WT animals but not in iNOS KO animals treated with IFNγ/TNFα. Data represented as mean ± SEM (n = 7 (WT, PBS), 4 (WT, IT), 6 (iNOS KO, PBS), and 5 (iNOS KO, IT) mice) with *P-value = 0.0210 by two-tailed, unpaired Student's t-test. G. Wild-type (WT) and iNOS KO mice were intramuscularly injected with IFNγ/TNFα for five consecutive days and sacrificed on the sixth day. Gastrocnemius muscle was homogenized and used for Western blot analysis with antibodies against pY-STAT3, total STAT3, iNOS, and α-tubulin. The blot shown is a representative of n = 3 (WT PBS), 2 (WT IFNγ/TNFα), 3 (iNOS KO PBS), and 2 (iNOS KO IFNγ/TNFα) mice. H. Wild-type (WT) and iNOS KO mice were intramuscularly injected with IFNγ/TNFα for five consecutive days and sacrificed on the sixth day. Image of representative sections of the gastrocnemius muscle from wild-type and iNOS KO mice stained with hematoxylin and eosin. Scale bar = 100 μm. The image shown is a representative of gastrocnemius muscles from each group (n = 2). I. Wild-type (WT) and iNOS KO mice were intramuscularly injected with IFNγ/TNFα for five consecutive days and sacrificed on the sixth day. The CSA of gastrocnemius muscles from panel (G) is represented as a frequency histogram. One thousand fibers were analyzed for each group (n = 2). The mean CSA ± SD is indicated in the legend of the histogram. Source data are available online for this figure. Source Data
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