REDD1 Expression Research Articles

REDD1 knockdown ameliorates endothelial cell senescence through repressing TXNIP-mediated oxidative stress

Endothelial cell senescence characterized by reactive oxygen species (ROS) accumulation and chronic inflammation is widely recognized as a key contributor to atherosclerosis (AS). Regulated in development and DNA damage response 1 (REDD1), a conserved stress-response protein that regulates ROS production, is involved in the pathogenesis of various age-related diseases. However, the role of REDD1 in endothelial cell senescence is still unclear. Here, we screened REDD1 as a differentially expressed senescence-related gene in the AS progression using bioinformatics methods, and validated the upregulation of REDD1 expression in AS plaques, senescent endothelial cells, and aging aorta by constructing AS mice, D-galactose (DG)-induced senescent endothelial cells and DG-induced accelerated aging mice, respectively. siRNA against REDD1 could improve DG-induced premature senescence of endothelial cells and inhibit ROS accumulation, similar to antioxidant N-Acetylcysteine (NAC) treatment. Meanwhile, NAC reduced the upregulation of REDD1 induced by DG, supporting the positive feedback loop between REDD1 and ROS contributes to endothelial cell senescence. Mechanistically, the regulatory effect of REDD1 on ROS might be related to the TXNIP-REDD1 interaction in DG-induced endothelial cell senescence. Collectively, experiments above provide evidence that REDD1 participates in endothelial cell senescence through repressing TXNIP-mediated oxidative stress, which may be involved in the progression of atherosclerosis.

Lipoteichoic acid restrains macrophage senescence via β-catenin/FOXO1/REDD1 pathway in age-related osteoporosis.

Osteoporosis and its related fractures are common causes of morbidity and mortality in older adults, but its underlying molecular and cellular mechanisms remain largely unknown. In this study, we found that lipoteichoic acid (LTA) treatment could ameliorate age-related bone degeneration and attenuate intramedullary macrophage senescence. FOXO1 signaling, which was downregulated and deactivated in aging macrophages, played a key role in the process. Blocking FOXO1 signaling caused decreased REDD1 expression and increased phosphorylation level of mTOR, a major driver of aging, as well as aggravated bone loss and deteriorated macrophage senescence. Moreover, LTA elevated FOXO1 signaling through β-catenin pathway while β-catenin inhibition significantly suppressed FOXO1 signaling, promoted senescence-related protein expression, and accelerated bone degeneration and macrophage senescence. Our findings indicated that β-catenin/FOXO1/REDD1 signaling plays a physiologically significant role that protecting macrophages from senescence during aging.

Cyanidin-3-O-glucoside from blueberry anthocyanin extracts protects ARPE-19 cells against high glucose damage via REDD1/GSK3β pathway

The retinal pigment epithelium (RPE) serves dual roles: it acts as a selective barrier and also as a regulator for the photoreceptor layer situated above it. The RPE is considered a crucial target for halting the development and progression of diabetic retinopathy (DR). This study focuses on understanding the protective role of blueberry anthocyanin extracts (BAEs), particularly its anti-oxidant constituent, cyanidin-3-O-glucoside (C3G), against damage to RPE cells induced by high glucose (HG) levels. The findings revealed that the BAEs mitigated damage to HG-induced ARPE-19 cells by decreasing the intracellular reactive oxygen species (ROS) levels, which improved cell morphology and cell viability (p < 0.01). Importantly, these cytoprotective effects were linked to the C3G rich in BAEs. The underlying mechanism may involve C3G interrupting the ROS generation feedback loop by downregulating REDD1 expression, which in turn enhanced the retinal Nrf2 antioxidant response to HG through the promotion of the GSK3β phosphorylation at Ser-9. Moreover, C3G suppressed VEGFA expression by inhibiting REDD1 levels in HG-induced ARPE-19 cells, subsequently preventing an increase in vascular permeability. In conclusion, this study suggested that the C3G relieved HG-induced oxidative stress damage in ARPE-19 cells via REDD1/GSK3β pathway and inhibited an increase in vascular permeability by downregulating VEGFA expression. These insights could shed light on how this natural bioactive compound can help manage HG-associated eye disorders like DR.

Articular Cartilage Regeneration via Induced Chondrocyte Autophagy by Sustained Release of Leptin Inhibitor from Thermo-Sensitive Hydrogel through STAT3/REDD1/mTORC1 Cascade.

The pathophysiology of osteoarthritis (OA) is closely linked to autophagy abnormalities in articular chondrocytes, the sole mature cell type in healthy cartilage. Nevertheless, the precise molecular mechanism remains uncertain. Previous research has demonstrated that leptin activates mTORC1, thereby inhibiting chondrocyte autophagy during the progression of OA. In this study, it is demonstrated that the presence of leptin induces a substantial increase in the expression of STAT3, leading to a notable decrease in REDD1 expression and subsequent phosphorylation of p70S6K, a recognized downstream effector of mTORC1. Conversely, inhibition of leptin yields contrasting effects. Additionally, the potential advantages of utilizing a sustained intra-articular release of a leptin inhibitor (LI) via an injectable, thermosensitive poly(D,L-lactide)-poly(ethylene glycol)-poly(D,L-lactide) (PDLLA-PEG-PDLLA: PLEL) hydrogel delivery system for the purpose of investigating its impact on cartilage repair are explored. The study conducted on LI-loaded PLEL (PLEL@LI) demonstrates remarkable efficacy in inhibiting OA and displays encouraging therapeutic advantages in the restoration of subchondral bone and cartilage. These findings establish a solid foundation for the advancement of a pioneering treatment approach utilizing PLEL@LI for OA.

REDD1-dependent GSK3β dephosphorylation promotes NF-κB activation and macrophage infiltration in the retina of diabetic mice

Increasing evidence supports a role for inflammation in the early development and progression of retinal complications caused by diabetes. We recently demonstrated that the stress response protein regulated in development and DNA damage response 1 (REDD1) promotes diabetes-induced retinal inflammation by sustaining canonical activation of nuclear transcription factor, NF-κB. The studies here were designed to identify signaling events whereby REDD1 promotes NF-κB activation in the retina of diabetic mice. We observed increased REDD1 expression in the retina of mice after 16 weeks of streptozotocin (STZ)-induced diabetes and found that REDD1 was essential for diabetes to suppress inhibitory phosphorylation of glycogen synthase kinase 3β (GSK3β) at S9. In human retinal MIO-M1 Müller cell cultures, REDD1 deletion prevented dephosphorylation of GSK3β and increased NF-κB activation in response to hyperglycemic conditions. Expression of a constitutively active GSK3β variant restored NF-κB activation in cells deficient for REDD1. In cells exposed to hyperglycemic conditions, GSK3β knockdown inhibited NF-κB activation and proinflammatory cytokine expression by preventing inhibitor of κB kinase complex autophosphorylation and inhibitor of κB degradation. In both the retina of STZ-diabetic mice and in Müller cells exposed to hyperglycemic conditions, GSK3 inhibition reduced NF-κB activity and prevented an increase in proinflammatory cytokine expression. In contrast with STZ-diabetic mice receiving a vehicle control, macrophage infiltration was not observed in the retina of STZ-diabetic mice treated with GSK3 inhibitor. Collectively, the findings support a model wherein diabetes enhances REDD1-dependent activation of GSK3β to promote canonical NF-κB signaling and the development of retinal inflammation.

YAP inhibition by CO promotes ferroptosis via ATF4-dependent REDD1 increase in melanoma

Abstract Ferroptotic cell death is a recently recognized form of non-apoptotic regulated cell death and exerts cytotoxicity in cancer cells. Carbon monoxide (CO) as an endogenous physiological mediator confers therapeutic potential in various cancer, while the therapeutic effects of CO on melanoma and its mechanism remain elusive. In this study, we suggest that CO induces ferroptosis via YAP inhibition and ATF4 activation in melanoma. YAP phosphorylation on serine 127 by CO-releasing molecule 2 (CORM2) caused cytoplasmic retention of YAP in B16F10 mouse melanoma cells, resulting in inhibition of YAP activity for transcription of target genes. Thus, CO-mediated YAP inhibition decreased SLC7A11 expression in melanoma cell lines, leading to ferroptosis. Moreover, CO increased REDD1 expression through PERK-ROS-ATF4 pathway. ATF4-REDD expression increased by CO became vulnerable to ferroptosis in melanoma. Thus, CO-induced YAP inhibition can induce ferroptosis viaATF4-mediated REDD1 increase and inhibition of SLC7A11. Furthermore, CO gas inhalation significantly inhibited B16F10 tumor size in xenograft mouse models resulting from ferroptosis induction via down-regulation of SLC7A11 and up-regulation of REDD1. These results suggest that CO acts as a potent inducer of ferroptosis in melanoma and that CO critically depends on YAP inhibition and ATF4 activation to promote ferroptosis.

Decreasing REDD1 expression protects against high glucose-induced apoptosis, oxidative stress and inflammatory injury in podocytes through regulation of the AKT/GSK-3β/Nrf2 pathway

Objective Our goal in this work was to investigate the possible role and mechanism of regulated in development and DNA damage response 1 (REDD1) in mediating high glucose (HG)-induced podocyte injury in vitro. Materials and methods Mouse podocytes were stimulated with HG to establish HG injury model. Protein expression was examined by Western blotting. Cell viability was measured by cell counting kit-8 assay. Cell apoptosis was assessed by annexin V‐FITC/propidium iodide and TUNEL apoptotic assays. Levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GPx) were quantified by commercial kits. Concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β were measured by ELISA. Results A marked increase in REDD1 expression was observed in podocytes stimulated with HG. Reduced REDD1 expression strikingly restrained HG-induced increases in apoptosis, oxidative stress, and inflammation response in cultured podocytes. Decreasing REDD1 expression enhanced nuclear factor erythroid 2-related factor 2 (Nrf2) activation in HG-exposed podocytes via regulation of the AKT/glycogen synthase kinase-3 beta (GSK-3β) pathway. Inhibition of AKT or reactivation of GSK-3β prominently abolished Nrf2 activation induced by decreasing REDD1 expression. Pharmacological repression of Nrf2 markedly reversed the protective effects of decreasing REDD1 expression in HG-injured podocytes. Conclusion Our data demonstrate that decreasing REDD1 expression protects cultured podocytes from HG-induced injuries by potentiating Nrf2 signaling through regulation of the AKT/GSK-3β pathway. Our work underscores the potential role of REDD1-mediated podocyte injury during the development of diabetic kidney disease.

Chronic Stress Induces Type 2b Skeletal Muscle Atrophy via the Inhibition of mTORC1 Signaling in Mice

Chronic stress induces psychological and physiological changes that may have negative sequelae for health and well-being. In this study, the skeletal muscles of male C57BL/6 mice subjected to repetitive water-immersion restraint stress to model chronic stress were examined. In chronically stressed mice, serum corticosterone levels significantly increased, whereas thymus volume and bone mineral density decreased. Further, body weight, skeletal muscle mass, and grip strength were significantly decreased. Histochemical analysis of the soleus muscles revealed a significant decrease in the cross-sectional area of type 2b muscle fibers. Although type 2a fibers also tended to decrease, chronic stress had no impact on type 1 muscle fibers. Chronic stress increased the expression of REDD1, FoxO1, FoxO3, KLF15, Atrogin1, and FKBP5, but did not affect the expression of myostatin or myogenin. In contrast, chronic stress resulted in a decrease in p-S6 and p-4E-BP1 levels in the soleus muscle. Taken together, these results indicate that chronic stress promotes muscle atrophy by inhibiting mammalian targets of rapamycin complex 1 activity due to the upregulation of its inhibitor, REDD1.

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.

GATA2 promotes oxidative stress to aggravate renal ischemia-reperfusion injury by up-regulating Redd1

Renal ischemia-reperfusion injury (RIRI) is a common pathophysiological process, and it is also an important cause of acute renal failure. Therefore, finding an effective therapeutic target for RIRI is extremely urgent. In our study, we constructed hypoxia-reoxygenation (HR) model in vitro and a renal ischemia-reperfusion (IR) model in vivo. Elevated levels of serum creatinine (Cr), blood urea nitrogen (BUN) tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and malondialdehyde (MDA) along with the decreased levels of superoxide dismutase (SOD) and glutathione (GSH) proved that kidney function was damaged after IR, and pathological changes of renal tissues were observed using HE staining and TUNEL staining. The protein of Redd1 expression level was detected to be upregulated after IR by western blot (WB). However, transfection of short hairpin RNA of Redd1 (sh-Redd1) alleviated the HR injury on LLC-PK1 cells, as evidenced by increased cell viability, proliferation and decreased cell apoptosis; additionally, the accumulation of ROS was inhibited. Sh-Redd1 also alleviated IR injury in the mouse model. Subsequently, GATA2 was proved to be upregulated in IR and HR models and was the transcription factor of Redd1. Knockdown of GATA2 efficiently mitigated the oxidative stress induced damages in vivo and in vitro, while these mitigations were reversed by transfection of Redd1 overexpression plasmid. In conclusion, our study clarified the possible underlying mechanism of protecting RIRI.

PERK/ATF4-dependent expression of the stress response protein REDD1 promotes proinflammatory cytokine expression in the heart of obese mice.

Endoplasmic reticulum (ER) stress and inflammation are hallmarks of myocardial impairment. Here, we investigated the role of the stress response protein regulated in development and DNA damage 1 (REDD1) as a molecular link between ER stress and inflammation in cardiomyocytes. In mice fed a high-fat high-sucrose (HFHS, 42% kcal fat, 34% sucrose by weight) diet for 12 wk, REDD1 expression in the heart was increased in coordination with markers of ER stress and inflammation. In human AC16 cardiomyocytes exposed to either hyperglycemic conditions or the saturated fatty acid palmitate, REDD1 expression was increased coincident with ER stress and upregulated expression of the proinflammatory cytokines IL-1β, IL-6, and TNFα. In cardiomyocytes exposed to hyperglycemic/hyperlipidemic conditions, pharmacological inhibition of the ER kinase protein kinase RNA-like endoplasmic reticulum kinase (PERK) or knockdown of the transcription factor ATF4 prevented the increase in REDD1 expression. REDD1 deletion reduced proinflammatory cytokine expression in both cardiomyocytes exposed to hyperglycemic/hyperlipidemic conditions and in the hearts of obese mice. Overall, the findings support a model wherein HFHS diet contributes to the development of inflammation in cardiomyocytes by promoting REDD1 expression via activation of a PERK/ATF4 signaling axis.NEW & NOTEWORTHY Interplay between endoplasmic reticulum stress and inflammation contributes to cardiovascular disease progression. The studies here identify the stress response protein known as REDD1 as a missing molecular link that connects the development of endoplasmic reticulum stress with increased production of proinflammatory cytokines in the hearts of obese mice.

Diabetes-Induced Renal Inflammation Is Facilitated by Expression of the Stress Response Protein REDD1

Diabetes-Induced Renal Inflammation Is Facilitated by Expression of the Stress Response Protein REDD1

Stress response protein REDD1 promotes diabetes-induced retinal inflammation by sustaining canonical NF-κB signaling

Inflammation contributes to the progression of retinal pathology caused by diabetes. Here, we investigated a role for the stress response protein regulated in development and DNA damage response 1 (REDD1) in the development of retinal inflammation. Increased REDD1 expression was observed in the retina of mice after 16-weeks of streptozotocin (STZ)-induced diabetes, and REDD1 was essential for diabetes-induced pro-inflammatory cytokine expression. In human retinal MIO-M1 Müller cell cultures, REDD1 deletion prevented increased pro-inflammatory cytokine expression in response to hyperglycemic conditions. REDD1 deletion promoted nuclear factor erythroid-2-related factor 2 (Nrf2) hyperactivation; however, Nrf2 was not required for reduced inflammatory cytokine expression in REDD1-deficient cells. Rather, REDD1 enhanced inflammatory cytokine expression by promoting activation of nuclear transcription factor κB (NF-κB). In WT cells exposed to tumor necrosis factor α (TNFα), inflammatory cytokine expression was increased in coordination with activating transcription factor 4 (ATF4)-dependent REDD1 expression and sustained activation of NF-κB. In both Müller cell cultures exposed to TNFα and in the retina of STZ-diabetic mice, REDD1 deletion promoted inhibitor of κB (IκB) expression and reduced NF-κB DNA-binding activity. We found that REDD1 acted upstream of IκB by enhancing both K63-ubiquitination and auto-phosphorylation of IκB kinase complex. In contrast with STZ-diabetic REDD1+/+ mice, IκB kinase complex autophosphorylation and macrophage infiltration were not observed in the retina of STZ-diabetic REDD1-/- mice. The findings provide new insight into how diabetes promotes retinal inflammation and support a model wherein REDD1 sustains activation of canonical NF-κB signaling.

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.

Blocking REDD1/TXNIP Complex Ameliorates HG-Induced Renal Tubular Epithelial Cell Apoptosis and EMT through Repressing Oxidative Stress.

Diabetic nephropathy (DN) has become the most common secondary kidney disease causing end-stage renal disease (ESRD). Nevertheless, the underlying mechanisms responsible for DN remain largely unknown. Regulated in development and DNA damage response 1 (REDD1) is a prooxidative molecule known to contribute to diabetes mellitus and its complications. However, it has not been previously examined whether and how REDD1 can further drive renal tubular epithelial cell (RTEC) apoptosis and epithelial-to-mesenchymal transition in DN. The expression of REDD1 was elevated in the kidneys of DN patients and diabetic mice in this study. By generating the DN model in REDD1 knockout mice, we demonstrated that REDD1 deficiency significantly improved apoptosis and EMT in diabetic mice. In vitro experiments showed that REDD1 generation was induced by high glucose (HG) in HK-2 cells. Similarly, the transfection of REDD1 siRNA plasmid also suppressed HG-induced apoptosis and EMT. Furthermore, we discovered that the inhibition of REDD1 suppressed the expression of Nox4-induced HG and reactive oxygen species (ROS) synthesis in HK-2 cells. In addition, HG could induce endogenous REDD1 and TXNIP to form a powerful complex. In summary, our findings demonstrate that blocking the REDD1/TXNIP complex can prevent HG-induced apoptosis and EMT by inhibiting ROS production, highlighting REDD1 as a valuable therapeutic priority site for DN.

NEDD8-Activating Enzyme Inhibitor MLN4924 Inhibits Both the Tumor Stroma and Angiogenesis in Pancreatic Cancer via Gli1 and REDD1.

Pancreatic cancer is characterized by a dense desmoplasia stroma, which hinders efficient drug delivery and plays a critical role in tumor progression and metastasis. MLN4924 is a first-in-class NEDD8-activating enzyme inhibitor that exhibits anti-tumor activities toward pancreatic cancer, and given the comprehensive effects that MLN4924 could have, we ask what impact MLN4924 would have on the stroma of pancreatic cancer and its underlying mechanisms. Primary pancreatic stellate cells (PSCs) and human HMEC-1 cells were treated with MLN4924 in vitro. The proliferation and extracellular matrix protein levels of PSCs were tested, and their relationship with transcription factor Gli1 in PSCs was investigated. The angiogenic phenotypes of HMEC-1 cells were evaluated using capillary-like tube formation assay, and their relationship with REDD1 in HMEC-1 cells was investigated. In this study, we found that MLN4924 inhibited the proliferation of pancreatic stellate cells and their secretion of collagen and CXCL-1, and the collagen secretion inhibiting effect of MLN4924 was related with transcription factor Gli1. MLN4924 inhibited multiple angiogenic phenotypes of HMEC-1 cells, and mTOR agonist partially relieved the inhibition of MLN4924 on HEMCs. MLN4924 increased the expression of REDD1 and REDD1 knockdown promoted the angiogenic phenotypes of HMEC-1 cells. Our study suggests that MLN4924 inhibits both the tumor stroma and angiogenesis in pancreatic cancer, and the inhibition effect is related with Gli1 in pancreatic stellate cells and REDD1 in vascular endothelial cells, respectively.

REDD1 Ablation Attenuates the Development of Renal Complications in Diabetic Mice.

Chronic hyperglycemia contributes to development of diabetic kidney disease by promoting glomerular injury. In this study, we evaluated the hypothesis that hyperglycemic conditions promote expression of the stress response protein regulated in development and DNA damage response 1 (REDD1) in the kidney in a manner that contributes to the development of oxidative stress and renal injury. After 16 weeks of streptozotocin-induced diabetes, albuminuria and renal hypertrophy were observed in wild-type (WT) mice coincident with increased renal REDD1 expression. In contrast, diabetic REDD1 knockout (KO) mice did not exhibit impaired renal physiology. Histopathologic examination revealed that glomerular damage including mesangial expansion, matrix deposition, and podocytopenia in the kidneys of diabetic WT mice was reduced or absent in diabetic REDD1 KO mice. In cultured human podocytes, exposure to hyperglycemic conditions enhanced REDD1 expression, increased reactive oxygen species (ROS) levels, and promoted cell death. In both the kidney of diabetic mice and in podocyte cultures exposed to hyperglycemic conditions, REDD1 deletion reduced ROS and prevented podocyte loss. Benefits of REDD1 deletion were recapitulated by pharmacological GSK3β suppression, supporting a role for REDD1-dependent GSK3β activation in diabetes-induced oxidative stress and renal defects. The results support a role for REDD1 in diabetes-induced renal complications.

Abstract EC103: A Novel Nuclear Role For REDD1 In Cardiac Insulin Sensitivity And Resistance

Insulin resistance (IR) is a hallmark of type II diabetes (TIID) and causes cardiac dysfunction independent of hypertension and coronary artery disease, or diabetic cardiomyopathy (DC). Impaired metabolism and energetics are a major cause of DC, however the molecular mechanisms underlying these defects remain ill-defined. Our unbiased RNA sequencing (RNA-Seq) studies identified ‘regulated in development and DNA damage (REDD)1’ as a potential critical regulator of cardiac insulin sensitivity, as a 1.5-fold increase in REDD1 expression was observed in the hearts of mice treated with acute insulin (1 hour (h)) versus vehicle (n=3). REDD1 is an insulin-sensitive, negative regulator of mTORC1 and global REDD1 deletion leads to whole body IR. Interestingly, cardiac REDD1 is also upregulated in several models of IR. Thus, we hypothesized that while REDD1 is critical for cardiac insulin sensitivity, sustained elevated levels contribute to IR via chronic inhibition of mTORC1. Our findings confirm that acute insulin stimulation or chronic high fat diet (HFD) (2 to 16 weeks) induce cardiac REDD1 expression in vivo (1.7-fold±0.1, n=6). In neonatal rat ventricular myocytes (NRVM) or Hap1 cells, insulin (1 h) or palmitate (24 h) also induce REDD1 expression (1.3-fold±0.1, n=3 or 1.6-fold±0.1, n=9, respectively), which indeed inhibits mTORC1. Notably, insulin treatment following HFD or palmitate, does not further enhance REDD1. In addition, subcellular fractionation shows that REDD1 is uniquely detected in nuclear and chromatin fractions and increases with insulin (1.5-fold±0.1, n=6). We confirmed REDD1 nuclear localization via microscopy and chromatin binding via chromatin immunoprecipitation-deep sequencing (ChIP-Seq) (n=5, pooled). In addition, we determined that REDD1 primarily binds the transcription start sites of metabolic genes and increases with insulin, corresponding to reduced gene expression. Overall, our findings suggest that insulin-inducible cardiac REDD1 exists as part of negative feedback loops that prevent the overactivation of mTORC1 and metabolic gene expression, contributing to insulin sensitivity. Further, sustained fat-induced REDD1 expression, may drive IR via chronic inhibition of mTORC1 and metabolic gene expression.

Berberine-mediated REDD1 down-regulation ameliorates senescence of retinal pigment epithelium by interrupting the ROS-DDR positive feedback loop

BackgroundAccumulation of age-associated senescent cells accompanied with increased reactive oxygen species (ROS) and inflammatory factors contributes to the progression of age-related macular degeneration (AMD), the main cause of blindness in the elderly. Berberine (BBR) has shown efficacy in the treatment of age-related diseases including diabetes and obesity by decreasing ROS. However, the pharmacological effect of BBR on alleviating retinal aging remains largely unknown. PurposeOur study aimed to investigate the pharmacological effect of BBR as an anti-aging agent in retinal aging and its further molecular mechanisms. MethodsD-galactose (DG)-induced ARPE-19 cell senescence and retinal aging were employed to evaluate the anti-aging effect of BBR in vivo and in vitro. The siRNA transfection, Western-Blot analyses, SA-β-Gal assay and immunofluorescence were performed to investigate the potential mechanisms of BBR on anti-aging of RPE. ResultsIn RPE-choroid of both natural aged and DG-induced accelerated aged mice, oxidative stress was increased along with the up-regulation of p21 expression, which was ameliorated by BBR treatment. BBR down-regulated the expression of REDD1 to decrease intracellular ROS content, attenuating DG-induced senescence in vitro and in vivo. Furthermore, p53 instead of HIF-1α was identified as the transcriptional regulator of REDD1 in DG-induced premature senescence. Importantly, NAC and BBR reversed the expression of p53 and the content of 8-OHdG, indicating that the positive feedback loop of ROS-DNA damage response (DDR) was formed, and BBR interrupted this feedback loop to alleviate DG-induced premature senescence by reducing REDD1 expression. In addition, BBR restored DG-damaged autophagy flux by up-regulating TFEB-mediated lysosomal biosynthesis by inhibiting REDD1 expression, thereby attenuating cellular senescence. ConclusionBBR down-regulates REDD1 expression to interrupt the ROS-DDR positive feedback loop and restore autophagic flux, thereby reducing premature senescence of RPE. Our findings elucidate the promising effects of REDD1 on cellular senescence and the great potential of BBR as a therapeutic approach.

ZFP36L2 suppresses mTORc1 through a P53-dependent pathway to prevent peripartum cardiomyopathy in mice.

Pregnancy is associated with substantial physiological changes of the heart, and disruptions in these processes can lead to peripartum cardiomyopathy (PPCM). The molecular processes that cause physiological and pathological changes in the heart during pregnancy are not well characterized. Here, we show that mTORc1 was activated in pregnancy to facilitate cardiac enlargement that was reversed after delivery in mice. mTORc1 activation in pregnancy was negatively regulated by the mRNA-destabilizing protein ZFP36L2 through its degradation of Mdm2 mRNA and P53 stabilization, leading to increased SESN2 and REDD1 expression. This pathway impeded uncontrolled cardiomyocyte hypertrophy during pregnancy, and mice with cardiac-specific Zfp36l2 deletion developed rapid cardiac dysfunction after delivery, while prenatal treatment of these mice with rapamycin improved postpartum cardiac function. Collectively, these data provide what we believe to be a novel pathway for the regulation of mTORc1 through mRNA stabilization of a P53 ubiquitin ligase. This pathway was critical for normal cardiac growth during pregnancy, and its reduction led to PPCM-like adverse remodeling in mice.