REDD1 mRNA Research Articles

Non-canonical STAT3 function reduces REDD1 transcription.

The transcription factor STAT3 is a potent activator of transcription, but evidence exists that STAT3 can also repress gene expression. However, little is known about the molecular mechanisms involved in STAT3-dependent gene repression. Notably, STAT3 reduces the expression of the stress-induced mTOR inhibitor REDD1 by reducing REDD1 mRNA transcription. Here, we determined the functional domains of STAT3 responsible for the reduction of REDD1 mRNA and protein expression. Within STAT3, the N-terminal domain and tyrosine 705 are crucial for STAT3-dependent reduction of REDD1 expression. Interestingly, binding of STAT3 to canonical STAT-binding sides within the REDD1 promoter is not necessary for STAT3-mediated reduction of REDD1 expression. Still, STAT3 is recruited to the REDD1 promoter upon stimulation with IL-6, and reduces REDD1 promoter activity. The reduction of REDD1 expression is specific for STAT3, as neither expression nor activation of STAT1 reduces REDD1 mRNA and protein expression. In summary, we present a novel, non-canonical STAT3-dependent mechanism for reducing gene expression. This transcriptional repression increases the functions of STAT3 proteins beyond classical transcriptional activation of cytokine-regulated target genes to a more complex function in modulating gene expression in immunity and cellular stress.

Activation of Disulfide Redox Switch in REDD1 Promotes Oxidative Stress Under Hyperglycemic Conditions.

The stress response protein regulated in development and DNA damage response 1 (REDD1) has been implicated in visual deficits in patients with diabetes. The aim here was to investigate the mechanism responsible for the increase in retinal REDD1 protein content that is observed with diabetes. We found that REDD1 protein expression was increased in the retina of streptozotocin-induced diabetic mice in the absence of a change in REDD1 mRNA abundance or ribosome association. Oral antioxidant supplementation reduced retinal oxidative stress and suppressed REDD1 protein expression in the retina of diabetic mice. In human retinal Müller cell cultures, hyperglycemic conditions increased oxidative stress, enhanced REDD1 expression, and inhibited REDD1 degradation independently of the proteasome. Hyperglycemic conditions promoted a redox-sensitive cross-strand disulfide bond in REDD1 at C150/C157 that was required for reduced REDD1 degradation. Discrete molecular dynamics simulations of REDD1 structure revealed allosteric regulation of a degron upon formation of the disulfide bond that disrupted lysosomal proteolysis of REDD1. REDD1 acetylation at K129 was required for REDD1 recognition by the cytosolic chaperone HSC70 and degradation by chaperone-mediated autophagy. Disruption of REDD1 allostery upon C150/C157 disulfide bond formation prevented the suppressive effect of hyperglycemic conditions on REDD1 degradation and reduced oxidative stress in cells exposed to hyperglycemic conditions. The results reveal redox regulation of REDD1 and demonstrate the role of a REDD1 disulfide switch in development of oxidative stress.

Human pancreatic tumour organoid-derived factors enhance myogenic differentiation.

BackgroundMost patients with pancreatic cancer develop cachexia, which is characterized by progressive muscle loss. The mechanisms underlying muscle loss in cancer cachexia remain elusive. Pancreatic tumour organoids are 3D cell culture models that retain key characteristics of the parent tumour. We aimed to investigate the effect of pancreatic tumour organoid‐derived factors on processes that determine skeletal muscle mass, including the regulation of muscle protein turnover and myogenesis.MethodsConditioned medium (CM) was collected from human pancreatic cancer cell lines (PK‐45H, PANC‐1, PK‐1, and KLM‐1), pancreatic tumour organoid cultures from a severely cachectic (PANCO‐9a) and a non‐cachectic patient (PANCO‐12a), and a normal pancreas organoid culture. Differentiating C2C12 myoblasts and mature C2C12 myotubes were exposed to CM for 24 h or maintained in control medium. In myotubes, NF‐kB activation was monitored using a NF‐κB luciferase reporter construct, and mRNA expression of E3‐ubiquitin ligases and REDD1 was analysed by RT‐qPCR. C2C12 myoblast proliferation and differentiation were monitored by live cell imaging and myogenic markers and myosin heavy chain (MyHC) isoforms were assessed by RT‐qPCR.ResultsWhereas CM from PK‐1 and KLM‐1 cells significantly induced NF‐κB activation in C2C12 myotubes (PK‐1: 3.1‐fold, P < 0.001; KLM‐1: 2.1‐fold, P = 0.01), Atrogin‐1/MAFbx and MuRF1 mRNA were only minimally and inconsistently upregulated by the CM of pancreatic cancer cell lines. Similarly, E3‐ubiquitin ligases and REDD1 mRNA expression in myotubes were not altered by exposure to pancreatic tumour organoid CM. Compared with the control condition, CM from both PANCO‐9a and PANCO‐12a tumour organoids increased proliferation of myoblasts, which was accompanied by significant downregulation of the satellite cell marker paired‐box 7 (PAX7) (PANCO‐9a: −2.1‐fold, P < 0.001; PANCO‐12a: −2.0‐fold, P < 0.001) and myogenic factor 5 (MYF5) (PANCO‐9a: −2.1‐fold, P < 0.001; PANCO‐12a: −1.8‐fold, P < 0.001) after 48 h of differentiation. Live cell imaging revealed accelerated alignment and fusion of myoblasts exposed to CM from PANCO‐9a and PANCO‐12a, which was in line with significantly increased Myomaker mRNA expression levels (PANCO‐9a: 2.4‐fold, P = 0.001; PANCO‐12a: 2.2‐fold, P = 0.004). These morphological and transcriptional alterations were accompanied by increased expression of muscle differentiation markers such as MyHC‐IIB (PANCO‐9a: 2.5‐fold, P = 0.04; PANCO‐12a: 3.1‐fold, P = 0.006). Although the impact of organoid CM on myogenesis was not associated with the cachexia phenotype of the donor patients, it was specific for tumour organoids, as CM of control pancreas organoids did not modulate myogenic fusion.ConclusionsThese data show that pancreatic tumour organoid‐derived factors alter the kinetics of myogenesis, which may eventually contribute to impaired muscle mass maintenance in cancer cachexia.

Loss of REDD1 prevents chemotherapy-induced muscle atrophy and weakness in mice.

BackgroundChemotherapy is an essential treatment to combat solid tumours and mitigate metastasis. Chemotherapy causes side effects including muscle wasting and weakness. Regulated in Development and DNA Damage Response 1 (REDD1) is a stress‐response protein that represses the mechanistic target of rapamycin (mTOR) in complex 1 (mTORC1), and its expression is increased in models of muscle wasting. The aim of this study was to determine if deletion of REDD1 is sufficient to attenuate chemotherapy‐induced muscle wasting and weakness in mice.MethodsC2C12 myotubes were treated with carboplatin, and changes in myotube diameter were measured. Protein synthesis was measured by puromycin incorporation, and REDD1 mRNA and protein expression were analysed in myotubes treated with carboplatin. Markers of mTORC1 signalling were measured by western blot. REDD1 global knockout mice and wild‐type mice were treated with a single dose of carboplatin and euthanized 7 days later. Body weight, hindlimb muscle weights, forelimb grip strength, and extensor digitorum longus whole muscle contractility were measured in all groups. Thirty minutes prior to euthanasia, mice were injected with puromycin to measure puromycin incorporation in skeletal muscle.ResultsC2C12 myotube diameter was decreased at 24 (P = 0.0002) and 48 h (P < 0.0001) after carboplatin treatment. Puromycin incorporation was decreased in myotubes treated with carboplatin for 24 (P = 0.0068) and 48 h (P = 0.0008). REDD1 mRNA and protein expression were increased with carboplatin treatment (P = 0.0267 and P = 0.0015, respectively), and this was accompanied by decreased phosphorylation of Akt T308 (P < 0.0001) and S473 (P = 0.0006), p70S6K T389 (P = 0.0002), and 4E‐binding protein 1 S65 (P = 0.0341), all markers of mTORC1 activity. REDD1 mRNA expression was increased in muscles from mice treated with carboplatin (P = 0.0295). Loss of REDD1 reduced carboplatin‐induced body weight loss (P = 0.0013) and prevented muscle atrophy in mice. REDD1 deletion prevented carboplatin‐induced decrease of protein synthesis (P = 0.7626) and prevented muscle weakness.ConclusionsCarboplatin caused loss of body weight, muscle atrophy, muscle weakness, and inhibition of protein synthesis. Loss of REDD1 attenuates muscle atrophy and weakness in mice treated with carboplatin. Our study illustrates the importance of REDD1 in the regulation of muscle mass with chemotherapy treatment and may be an attractive therapeutic target to combat cachexia.

REDD1 attenuates hepatic stellate cell activation and liver fibrosis via inhibiting of TGF-β/Smad signaling pathway

Liver fibrosis is caused by repetitive hepatic injury. Regulated in development and DNA damage response 1 (REDD1) gene is induced by various stresses and has been studied in cell proliferation and survival. However, the role of REDD1 in hepatic stellate cell activation and hepatic fibrogenesis has not yet been investigated. In the current study, we examined the effect of REDD1 on hepatic fibrogenesis and the underlying molecular mechanism. REDD1 protein was upregulated in the activated primary hepatic stellate cells and transforming growth factor-β (TGF-β)-treated LX-2 cells. REDD1 mRNA levels were also elevated by TGF-β treatment. TGF-β signaling is primarily transduced via the activation of the Smad transcription factor. However, TGF-β-mediated REDD1 induction was not Smad-dependent. Thus, we investigated the transcription factors that influence the REDD1 expression by TGF-β. We found that c-JUN, a component of AP-1, upregulated the REDD1 expression that was specifically suppressed by p38 inhibitor. In silico analysis of the REDD1 promoter region showed putative AP-1-binding sites; additionally, its deletion mutants demonstrated that the AP-1-binding site between −716 and −587 bp within the REDD1 promoter is critical for TGF-β-mediated REDD1 induction. Moreover, REDD1 overexpression markedly inhibited TGF-β-induced plasminogen activator inhibitor-1 (PAI-1) expression and Smad phosphorylation. REDD1 adenovirus infection inhibited CCl4-induced hepatic injury in mice, which was demonstrated by reduced ALT/AST levels and collagen accumulation. In addition, we observed that REDD1 inhibited CCl4-induced fibrogenic gene induction and restored GSH and malondialdehyde levels. Our findings implied that REDD1 has the potential to inhibit HSC activation and protect against liver fibrosis.

REDD1 upregulation via AP‐1 alleviates oxidative stress‐mediated liver injury

Regulated in development and DNA damage responses 1 (REDD1) is an inducible gene in response to various stresses, which functions as a negative regulator of the mammalian target of rapamycin protein kinase in complex 1. In the present study, we identified the role of REDD1 under the oxidative stress‐mediated hepatocyte injury and its regulatory mechanism. REDD1 protein was increased in H2O2 or tert‐butylhydroperoxide (t‐BHP)‐treated hepatocytes· H2O2 also elevated REDD1 mRNA levels. This event was inhibited by antioxidants such as diphenyleneiodonium chloride, N‐acetyl‐L‐cysteine, or butylated hydroxy anisole. Interestingly, we found that H2O2‐mediated REDD1 induction was transcriptionally regulated by activator protein‐1 (AP‐1), and that overexpression of c‐Jun increased REDD1 protein levels and REDD1 promoter‐driven luciferase activity. Deletion of the putative AP‐1 binding site in proximal region of the human REDD1 promoter significantly abolished REDD1 transactivation by c‐Jun. A NF‐E2‐related factor 2 activator, tert‐butylhydroquinone treatment also elevated REDD1 levels, but it was independent on NF‐E2‐related factor 2 activation. Furthermore, we observed that REDD1 overexpression attenuated H2O2 or t‐BHP‐derived reactive oxygen species formation as well as cytotoxicity. Conversely, siRNA against REDD1 aggravated t‐BHP‐induced reactive oxygen species generation and cell death. In addition, we showed that REDD1 was induced by in vitro or in vivo ischemia/reperfusion model. Our results demonstrate that REDD1 induction by oxidative stress is mainly transcriptionally regulated by AP‐1, and protects oxidative stress‐mediated hepatocyte injury. These findings suggest REDD1 as a novel molecule that reduced susceptibility to oxidant‐induced liver injury.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Induction of REDD1 via AP-1 prevents oxidative stress-mediated injury in hepatocytes

Regulated in development and DNA damage responses 1 (REDD1) is an inducible gene in response to various stresses, which functions as a negative regulator of the mammalian target of rapamycin protein kinase in complex 1. In the present study, we identified the role of REDD1 under the oxidative stress-mediated hepatocyte injury and its regulatory mechanism. REDD1 protein was increased in H2O2 or tert-butylhydroperoxide (t-BHP)-treated hepatocytes· H2O2 also elevated REDD1 mRNA levels. This event was inhibited by antioxidants such as diphenyleneiodonium chloride, N-acetyl-L-cysteine, or butylated hydroxy anisole. Interestingly, we found that H2O2-mediated REDD1 induction was transcriptionally regulated by activator protein-1 (AP-1), and that overexpression of c-Jun increased REDD1 protein levels and REDD1 promoter-driven luciferase activity. Deletion of the putative AP-1 binding site in proximal region of the human REDD1 promoter significantly abolished REDD1 transactivation by c-Jun. A NF-E2-related factor 2 activator, tert-butylhydroquinone treatment also elevated REDD1 levels, but it was independent on NF-E2-related factor 2 activation. Furthermore, we observed that REDD1 overexpression attenuated H2O2 or t-BHP-derived reactive oxygen species formation as well as cytotoxicity. Conversely, siRNA against REDD1 aggravated t-BHP-induced reactive oxygen species generation and cell death. In addition, we showed that REDD1 was induced by in vitro or in vivo ischemia/reperfusion model. Our results demonstrate that REDD1 induction by oxidative stress is mainly transcriptionally regulated by AP-1, and protects oxidative stress-mediated hepatocyte injury. These findings suggest REDD1 as a novel molecule that reduced susceptibility to oxidant-induced liver injury.

Anabolic signaling responses to exercise and recovery whey protein are suppressed at high altitude

Lowlanders sojourning to high‐altitude (HA) often experience muscle mass losses that are attributable to negative energy balance and potential dysregulation of intramuscular anabolic signaling. The combined effects of exercise and high‐quality protein ingestion can ameliorate the effects of short‐term, moderate negative energy balance (10‐d, ≤ 40 % total energy requirements) on intramuscular anabolic signaling at sea level (SL); however, the ability to sustain this effect at HA is unknown. This study examined the combined effects of HA (4300 m) and severe negative energy balance (21‐d, 70% of SL total energy requirements) on mTORC1 signaling responses after aerobic exercise and recovery whey protein (WP) ingestion in recreationally active, native lowlanders (n = 8 males). Total body mass and fat‐free mass were assessed using dual energy x‐ray absorptiometry before and after the 21‐d energy deficit at HA. Muscle biopsies of the vastus lateralis were obtained at SL, acute HA (3‐h HA exposure, day 0), and chronic HA (day 21), before (BASE) and after completing 80‐min of steady‐state aerobic exercise followed by a 2 mile time trial (POST), and 3 h into recovery (REC). In each trial, 25 g WP was consumed as a drink immediately after the POST biopsy. Intramuscular anabolic signaling was determined using Western blot and RT‐qPCR. Participants lost 7.5 ± 0.8 kg of total body mass (4.3 ± 1.0 kg of fat‐free mass, P &lt; 0.05). POST and REC p‐mTORSer2448, p‐70S6KSer424/421 and p‐rpS6Ser235/236 were 2‐, 23‐, and 7‐fold higher (P &lt; 0.05) than BASE at SL. At acute HA, POST p‐mTORSer2448, and POST and REC p‐70S6KSer424/421 were not different (P &gt; 0.05) from BASE and were 1‐ and 20‐fold lower than SL (P &lt; 0.05). However, POST and REC p‐rpS6Ser235/236 at acute HA was 4‐fold higher (P &lt; 0.05) than BASE and not different from SL. At chronic HA, POST and REC p‐mTORSer2448 and p‐70S6KSer424/421 were not different from BASE and were 1‐ and 19‐fold lower (P &lt; 0.05) than SL. POST and REC p‐rpS6Ser235/236 at chronic HA was 2‐fold higher (P &lt; 0.05) than BASE, but overall phosphorylation was 4‐fold lower (P &lt; 0.05) than SL. BASE HIF‐1α mRNA expression tended to be higher (P = 0.07) at acute HA than SL, whereas REC HIF‐1α mRNA expression at acute and chronic HA were lower (P &lt; 0.05) than SL. REDD1 mRNA expression and REDD1/14‐3‐3 coupling were not affected by study phase. These findings indicate that acute HA exposure induces intramuscular anabolic resistance that is further impaired with chronic HA exposure concomitant with severe negative energy balance, suggesting a mechanism for muscle loss at HA.Support or Funding InformationThis material is based on the work supported by US Army Medical Research and Material Command and the US Army Natick Soldier Research Development and Engineering Center. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the US Army.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Exercise Protects Skeletal Muscle during Chronic Doxorubicin Administration.

This study aimed to assess the ability for exercise training performed before and during biweekly doxorubicin (DOX) administration to attenuate adverse effects of DOX on skeletal muscle. We hypothesized that DOX treatment would increase REDD1, impair mammalian target of rapamycin (mTOR) signaling, and reduce muscle fiber size, and that exercise training would attenuate these responses. Eight-week-old ovariectomized female Sprague-Dawley rats were randomized to one of four treatments: exercise + DOX (Ex-Dox), Ex + vehicle (Ex-Veh), sedentary + DOX (Sed-Dox), and Sed + Veh (Sed-Veh). DOX (4 mg·kg) or vehicle (saline) intraperitoneal injections were performed biweekly for a total of three injections (cumulative dose, 12 mg·kg). Ex animals performed interval exercise (4 × 4 min, 85%-90% V˙O2peak) 5 d·wk starting 1 wk before the first injection and continued throughout study duration. Animals were euthanized ~5 d after the last injection, during which the soleus muscle was dissected and prepared for immunoblot and immunohistochemical analyses. REDD1 mRNA and protein were increased only in Sed-Dox (P < 0.05). The phosphorylation of mTOR and 4E-BP1 and MHC I and MHC IIa fiber size were lower in Sed-Dox versus Sed-Veh (P < 0.05). By contrast, REDD1 mRNA and protein, mTOR, 4E-BP1, and MHC I fiber size were not different between Ex-Dox and Ex-Veh (P > 0.05). LC3BI was higher, and the LC3BII/I ratio was lower in Sed-Dox versus Sed-Veh (P < 0.05) but not between Ex-Dox and Ex-Veh (P > 0.05). These data suggest that DOX may inhibit mTORC1 activity and reduce MHCI and MHCIIa fiber size, potentially through elevated REDD1, and that exercise may provide a therapeutic strategy to preserve skeletal muscle size during chronic DOX treatment.

Chronic stress inhibits growth and induces proteolytic mechanisms through two different nonoverlapping pathways in the skeletal muscle of a teleost fish.

Chronic stress detrimentally affects animal health and homeostasis, with somatic growth, and thus skeletal muscle, being particularly affected. A detailed understanding of the underlying endocrine and molecular mechanisms of how chronic stress affects skeletal muscle growth remains lacking. To address this issue, the present study assessed primary (plasma cortisol), secondary (key components of the GH/IGF system, muscular proteolytic pathways, and apoptosis), and tertiary (growth performance) stress responses in fine flounder ( Paralichthys adspersus) exposed to crowding chronic stress. Levels of plasma cortisol, glucocorticoid receptor 2 ( gr2), and its target genes ( klf15 and redd1) mRNA increased significantly only at 4 wk of crowding ( P < 0.05). The components of the GH/IGF system, including ligands, receptors, and their signaling pathways, were significantly downregulated at 7 wk of crowding ( P < 0.05). Interestingly, chronic stress upregulated the ubiquitin-proteasome pathway and the intrinsic apoptosis pathways at 4wk ( P < 0.01), whereas autophagy was only significantly activated at 7 wk ( P < 0.05), and meanwhile the ubiquitin-proteasome and the apoptosis pathways returned to control levels. Overall growth was inhibited in fish in the 7-wk chronic stress trial ( P < 0.05). In conclusion, chronic stress directly affects muscle growth and downregulates the GH/IGF system, an action through which muscular catabolic mechanisms are promoted by two different and nonoverlapping proteolytic pathways. These findings provide new information on molecular mechanisms involved in the negative effects that chronic stress has on muscle anabolic/catabolic signaling balance.

Interleukin-6 influences stress-signalling by reducing the expression of the mTOR-inhibitor REDD1 in a STAT3-dependent manner

Interleukin 6 (IL-6) is a pleiotropic cytokine and a strong activator of Mammalian Target of Rapamycin (mTOR). In contrast, mTOR activity is negatively regulated by Regulated in Development and DNA Damage Responses 1 (REDD1). Expression of REDD1 is induced by cellular stressors such as glucocorticoids and DNA damaging agents. We show that the expression of basal as well as stress-induced REDD1 is reduced by IL-6. The reduction of REDD1 expression by IL-6 is independent of proteasomal or caspase-mediated degradation of REDD1 protein. Instead, induction of REDD1 mRNA is reduced by IL-6. The regulation of REDD1 expression by IL-6 is independent of Phosphatidylinositide-3-Kinase (PI3K) and Mitogen-Activated Protein Kinase (MAPK) signalling but depends on the expression and activation of Signal Transducer and Activator of Transcription 3 (STAT3). Furthermore, the reduction of basal REDD1 expression by IL-6 correlates with IL-6-induced activation of mTOR signalling. Inhibition of STAT3 activation blocks IL-6-induced mTOR activation. In summary, we present a novel STAT3-dependent mechanism of both IL-6-induced activation of mTOR and IL-6-dependent reversion of stress-induced inhibition of mTOR activity.

ID: 74: Interleukin-6 globally influences stress-signaling by reducing the expression of the mTOR-Inhibitor REDD1 in a STAT3-dependent manner

Mammalian target of rapamycin (mTOR) is a central mediator of cellular growth and survival which is potently activated by interleukin 6 (IL-6). However, the molecular mechanisms of IL-6-induced mTOR activation are still poorly understood. mTOR activity is negatively regulated by regulated in development and DNA damage response 1 (REDD1). Expression of REDD1 is induced by cellular stressors such as DNA damaging agents. Stress-induced REDD1 expression facilitates cellular damage repair by temporarily blocking mTOR-induced signaling. However, reduced REDD1 expression has been associated with proliferative diseases such as liver cancer. We show that REDD1 expression is reduced by IL-6. The reduction of REDD1 protein by IL-6 is independent of proteasomal or caspase-mediated degradation of REDD1 but occurs on the level of REDD1 mRNA. IL-6-induced reduction of REDD1 follows the same kinetics as mTOR activation, suggesting that it contributes to IL-6-induced mTOR activation. The decrease in REDD1 expression is independent of phosphatidylinositide-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling but depends on the expression and activation of signal transducer and activator of transcription 3 (STAT3). Consequently, inhibition of STAT3 blocks IL-6-induced mTOR activation. Of note, beside basal REDD1 expression also stress-induced REDD1 expression is reduced by IL-6 enabling IL-6 to globally interfere in stress-signaling. In summary, we present a novel STAT3-dependent mechanism of both IL-6-induced activation of mTOR and IL-6-dependent reversion of stress-induced inhibition of mTOR (through repression of REDD1 expression) that might contribute to the development of proliferative diseases in inflammatory conditions.

Effects of tail suspension on serum testosterone and molecular targets regulating muscle mass

The contribution of reduced testosterone levels to tail suspension (TS)-induced muscle atrophy remains equivocal. The molecular mechanism by which testosterone regulates muscle mass during TS has not been investigated. Effects of TS on serum testosterone levels, muscle mass, and expression of muscle atrophy- and hypertrophy-inducing targets were measured in soleus (SOL) and extensor digitorum longus (EDL) muscles after testosterone administration during 1, 5, and 14 days of TS in male mice. TS produced an increase followed by a transient drop in testosterone levels. Muscle atrophy was associated with downregulation of Igf1 and upregulation of Mstn, Redd1, Atrogin-1, and MuRF1 mRNA with clear differences in Igf1, Mstn, and MAFbx/Atrogin-1 gene expression between SOL and EDL. Testosterone supplementation did not affect muscle mass or protein expression levels during TS. Conclusions The known anabolic effects of testosterone are not sufficient to ameliorate loss of muscle mass during TS.

Endurance exercise induces REDD1 expression and transiently decreases mTORC1 signaling in rat skeletal muscle.

Working muscle conserves adenosine triphosphate (ATP) for muscle contraction by attenuating protein synthesis through several different pathways. Regulated in development and DNA damage response 1 (REDD1) is one candidate protein that can itself attenuate muscle protein synthesis during muscle contraction. In this study, we investigated whether endurance exercise induces REDD1 expression in association with decreased mammalian target of rapamycin (mTOR) complex I (mTORC1) signaling and global protein synthesis in rat skeletal muscle. After overnight fasting, rats ran on a treadmill at a speed of 28 m/min for 60 min, and were killed before and immediately, 1, 3, 6, 12, and 24 h after exercise. REDD1 mRNA and corresponding protein levels increased rapidly immediately after exercise, and gradually decreased back to the basal level over a period of 6 h in the gastrocnemius muscle. Phosphorylation of mTOR Ser2448 and S6K1 Thr389 increased with the exercise, but diminished in 1–3 h into the recovery period after cessation of exercise. The rate of protein synthesis, as determined by the surface sensing of translation (SUnSET) method, was not altered by exercise in fasted muscle. These results suggest that REDD1 attenuates exercise‐induced mTORC1 signaling. This may be one mechanism responsible for blunting muscle protein synthesis during exercise and in the early postexercise recovery period.

REDD2 expression in rat skeletal muscle correlates with nutrient-induced activation of mTORC1: responses to aging, immobilization, and remobilization.

In a previous study (Kelleher AR, Kimball SR, Dennis MD, Schilder RJ, and Jefferson LS. Am J Physiol Endocrinol Metab 304: E229-236, 2013.), we observed a rapid (i.e., 1-3 days) immobilization-induced repression of mechanistic target of rapamycin complex 1 (mTORC1) signaling in hindlimb skeletal muscle of young (2-mo-old) rats that was associated with elevated expression of regulated in development and DNA-damage response (REDD) 1 and REDD2. The present study extends that observation to include an assessment of those parameters in soleus muscle of the immobilized hindlimb of various-aged rats as well as in response to remobilization. Male Sprague-Dawley rats aged 2, 9, and 18 mo were subjected to unilateral hindlimb immobilization for 7 days, whereas one group of the 9-mo-old animals underwent 7 days of remobilization. Soleus muscle mass-to-body mass ratio declined with age, with the loss of muscle mass following hindlimb immobilization being inversely proportional to age. Compared with 2-mo-old rats, the older rats exhibited reduced mTORC1 signaling in the nonimmobilized limb in association with elevated REDD2, but not REDD1, mRNA expression. In the 2-mo-old rats, 7 days of hindlimb immobilization attenuated mTORC1 signaling and induced REDD2, but not REDD1, mRNA expression. In contrast, hindlimb immobilization did not further attenuate the age-related reduction in mTORC1 signaling nor further enhance the age-related induction of REDD2 mRNA expression in 9- and 18-mo-old rats. Across ages, REDD1 mRNA was not impacted by immobilization. Finally, remobilization elevated mTORC1 signaling and lowered REDD2 mRNA expression, with no impact on REDD1 gene expression. In conclusion, changes in mTORC1 signaling associated with aging, immobilization, and remobilization were inversely proportional to alterations in REDD2 mRNA expression.

Nuclear factor of activated T-cells 5 increases intestinal goblet cell differentiation through an mTOR/Notch signaling pathway.

The intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis that is regulated by multiple signaling pathways. Previously, we have shown that the nuclear factor of activated T-cells 5 (NFAT5) is involved in the regulation of intestinal enterocyte differentiation. Here we show that treatment with sodium chloride (NaCl), which activates NFAT5 signaling, increased mTORC1 repressor regulated in development and DNA damage response 1 (REDD1) protein expression and inhibited mTOR signaling; these alterations were attenuated by knockdown of NFAT5. Knockdown of NFAT5 activated mammalian target of rapamycin (mTOR) signaling and significantly inhibited REDD1 mRNA expression and protein expression. Consistently, overexpression of NFAT5 increased REDD1 expression. In addition, knockdown of REDD1 activated mTOR and Notch signaling, whereas treatment with mTOR inhibitor rapamycin repressed Notch signaling and increased the expression of the goblet cell differentiation marker mucin 2 (MUC2). Moreover, knockdown of NFAT5 activated Notch signaling and decreased MUC2 expression, while overexpression of NFAT5 inhibited Notch signaling and increased MUC2 expression. Our results demonstrate a role for NFAT5 in the regulation of mTOR signaling in intestinal cells. Importantly, these data suggest that NFAT5 participates in the regulation of intestinal homeostasis via the suppression of mTORC1/Notch signaling pathway.

Changes in REDD1, REDD2, and atrogene mRNA expression are prevented in skeletal muscle fixed in a stretched position during hindlimb immobilization.

Immobilized skeletal muscle fixed in a shortened position displays disuse atrophy, whereas when fixed in a stretched position it does not (Goldspink, D. F. (1977) J Physiol 264, 267–282). Although significant advances have been made in our understanding of mechanisms involved in development of atrophy in muscle fixed in a shortened position, little is known about why mass is maintained when muscle is immobilized in a stretched position. In the present study, we hypothesized that skeletal muscle immobilized in a stretched position would be protected from gene expression changes known to be associated with disuse atrophy. To test the hypothesis, male Sprague‐Dawley rats were anesthetized using isoflurane and subjected to unilateral hindlimb immobilization for 3 days with the soleus fixed in either a shortened or stretched position. All comparisons were made to the contralateral nonimmobilized muscle. Soleus immobilized in a shortened position exhibited disuse atrophy, attenuated rates of protein synthesis, attenuated mTORC1 signaling, and induced expression of genes for REDD1, REDD2, MAFbx, and MuRF1. In contrast, immobilization of the soleus in a stretched position prevented these changes as it exhibited no difference in muscle mass, rates of protein synthesis, mTORC1 signaling, or expression of genes encoding REDD1, MAFbx or MuRF1, with REDD2 expression being reduced compared to control. In conclusion, fixed muscle length plays a major role in immobilization‐induced skeletal muscle atrophy whereby placing muscle in a shortened position leads to induction of gene expression for REDD1, REDD2, and atrogenes.

Rapid induction of REDD1 gene expression in macrophages in response to stress-related catecholamines

In the present study, we examined the effect of stress-related catecholamines adrenaline and noradrenaline on macrophage expression of a new host defense factor REDD1 using murine macrophage cell line RAW264.7 and murine peritoneal macrophages. Short-term adrenaline exposure (15–60min) upregulated REDD1 mRNA expression and its protein synthesis in macrophages. This adrenaline-induced REDD1 expression was completely blocked by β2-adrenoceptor selective antagonist ICI 118,551, whereas β2-adrenoceptor specific agonist salmeterol markedly enhanced REDD1 expression. Moreover, noradrenaline increased REDD1 mRNA expression at doses higher than the effective doses of adrenaline. The effect of adrenaline on REDD1 mRNA expression was mimicked by treatment with membrane-permeable cAMP analog 8-Br-cAMP. Thus, increased intracellular cAMP level resulting from β2-adrenoceptor stimulation appeared to be responsible for adrenaline-induced REDD1 mRNA expression. However, inhibiting protein kinase A (PKA) activity had no significant effect on REDD1 mRNA expression after β2-adrenoceptor stimulation. In addition, exchange protein activated by cAMP (Epac) agonist 8-CPT-20-O-Me-cAMP had no effect on REDD1 mRNA expression. Thus, β2-adrenoceptor-mediated increase in cAMP levels seems to induce REDD1 mRNA expression in macrophages through a PKA- and Epac-independent pathway.

Effect of acute environmental hypoxia on protein metabolism in human skeletal muscle

Hypoxia-induced muscle wasting has been observed in several environmental and pathological conditions. However, the molecular mechanisms behind this loss of muscle mass are far from being completely elucidated, certainly in vivo. When studying the regulation of muscle mass by environmental hypoxia, many confounding factors have to be taken into account, such as decreased protein ingestion, sleep deprivation or reduced physical activity, which make difficult to know whether hypoxia per se causes a reduction in muscle mass. We hypothesized that acute exposure to normobaric hypoxia (11% O2 ) would repress the activation of the mTOR pathway usually observed after a meal and would activate the proteolytic pathways in skeletal muscle. Fifteen subjects were exposed passively for 4h to normoxic and hypoxic conditions in a random order after consumption of a light breakfast. A muscle biopsy and a blood sample were taken before, after 1 and 4h of exposure. After 4h, plasma insulin concentration and the phosphorylation state of PKB and S6K1 in skeletal muscle were higher in hypoxia than in normoxia (P<0.05). At the same time, Redd1 mRNA level was upregulated (P<0.05), whilst MAFbx mRNA decreased (P<0.05) in hypoxia compared with normoxia. Proteasome, cathepsin L and calpain activities were not altered by environmental hypoxia. Contrary to our hypothesis and despite an increase in the mRNA level of Redd1, an inhibitor of the mTORC1 pathway, short-term acute environmental hypoxia induced a higher response of PKB and S6K1 to a meal, which may be due to increased plasma insulin concentration.

The Stress-Response Gene redd1 Regulates Dorsoventral Patterning by Antagonizing Wnt/β-catenin Activity in Zebrafish

REDD1/redd1 is a stress-response gene that is induced under various stressful conditions such as hypoxia, DNA damage, and energy stress. The increased REDD1 inhibits mTOR signaling and cell growth. Here we report an unexpected role of Redd1 in regulating dorsoventral patterning in zebrafish embryos and the underlying mechanisms. Zebrafish redd1 mRNA is maternally deposited. Although it is ubiquitously detected in many adult tissues, its expression is highly tissue-specific and dynamic during early development. Hypoxia and heat shock strongly induce redd1 expression in zebrafish embryos. Knockdown of Redd1 using two independent morpholinos results in dorsalized embryos and this effect can be rescued by injecting redd1 mRNA. Forced expression of Redd1 ventralizes embryos. Co-expression of Redd1 with Wnt3a or a constitutively active form of β-catenin suggests that Redd1 alters dorsoventral patterning by antagonizing the Wnt/β-catenin signaling pathway. These findings have unraveled a novel role of Redd1 in early development by antagonizing Wnt/β-catenin signaling.