ATF4-Mediated Upregulation of REDD1 and Sestrin2 Suppresses mTORC1 Activity during Prolonged Leucine Deprivation.

BACKGROUND: The protein kinase target of rapamycin (mTOR) in complex 1 (mTORC1) is activated by amino acids and in turn upregulates anabolic processes. Under nutrient-deficient conditions, e.g., amino acid insufficiency, mTORC1 activity is suppressed and autophagy is activated. Intralysosomal amino acids generated by autophagy reactivate mTORC1. However, sustained mTORC1 activation during periods of nutrient insufficiency would likely be detrimental to cellular homeostasis. Thus, mechanisms must exist to prevent amino acids released by autophagy from reactivating the kinase. OBJECTIVE: The objective of the present study was to test whether mTORC1 activity is inhibited during prolonged leucine deprivation through ATF4-dependent upregulation of the mTORC1 suppressors regulated in development and DNA damage response 1 (REDD1) and Sestrin2. METHODS: Mice (8 wk old; C57Bl/6 × 129SvEV) were food deprived (FD) overnight and one-half were refed the next morning. Mouse embryo fibroblasts (MEFs) deficient in ATF4, REDD1, and/or Sestrin2 were deprived of leucine for 0-16 h. mTORC1 activity and ATF4, REDD1, and Sestrin2 expression were assessed in liver and cell lysates. RESULTS: Refeeding FD mice resulted in activation of mTORC1 in association with suppressed expression of both REDD1 and Sestrin2 in the liver. In cells in culture, mTORC1 exhibited a triphasic response to leucine deprivation, with an initial suppression followed by a transient reactivation from 2 to 4 h and a subsequent resuppression after 8 h. Resuppression occurred concomitantly with upregulated expression of ATF4, REDD1, and Sestrin2. However, in cells lacking ATF4, neither REDD1 nor Sestrin2 expression was upregulated by leucine deprivation, and resuppression of mTORC1 was absent. Moreover, in cells lacking either REDD1 or Sestrin2, mTORC1 resuppression was attenuated, and in cells lacking both proteins resuppression was further blunted. CONCLUSIONS: The results suggest that leucine deprivation upregulates expression of both REDD1 and Sestrin2 in an ATF4-dependent manner, and that upregulated expression of both proteins is involved in resuppression of mTORC1 during prolonged leucine deprivation.

Evidence for a role for Sestrin1 in mediating leucine-induced activation of mTORC1 in skeletal muscle.

Previous studies established that leucine stimulates protein synthesis in skeletal muscle to the same extent as a complete mixture of amino acids, and the effect occurs through activation of the mechanistic target of rapamycin in complex 1 (mTORC1). Recent studies using cells in culture showed that the Sestrins bind leucine and are required for leucine-dependent activation of mTORC1. However, the role they play in mediating leucine-dependent activation of the kinase in vivo has been questioned because the dissociation constant of Sestrin2 for leucine is well below circulating and intramuscular levels of the amino acid. The goal of the present study was to compare expression of the Sestrins in skeletal muscle to other tissues and to assess their relative role in mediating activation of mTORC1 by leucine. The results show that the relative expression of the Sestrin proteins varies widely among tissues and that in skeletal muscle Sestrin1 expression is higher than Sestrin3, whereas Sestrin2 expression is markedly lower. Analysis of the dissociation constants of the Sestrins for leucine as assessed by leucine-induced dissociation of the Sestrin·GAP activity toward Rags 2 (GATOR2) complex revealed that Sestrin1 has the highest affinity for leucine and that Sestrin3 has the lowest affinity. In agreement with the dissociation constants calculated using cells in culture, oral leucine administration promotes disassembly of the Sestrin1·GATOR2 complex but not the Sestrin2 or Sestrin3·GATOR2 complex. Overall, the results presented herein are consistent with a model in which leucine-induced activation of mTORC1 in skeletal muscle in vivo occurs primarily through release of Sestrin1 from GATOR2.

Suppression of p16 Induces mTORC1-Mediated Nucleotide Metabolic Reprogramming.

Reprogrammed metabolism and cell cycle dysregulation are two cancer hallmarks. p16 is a cell cycle inhibitor and tumor suppressor that is upregulated during oncogene-induced senescence (OIS). Loss of p16 allows for uninhibited cell cycle progression, bypass of OIS, and tumorigenesis. Whether p16 loss affects pro-tumorigenic metabolism is unclear. We report that suppression of p16 plays a central role in reprogramming metabolism by increasing nucleotide synthesis. This occurs by activation of mTORC1 signaling, which directly mediates increased translation of the mRNA encoding ribose-5-phosphate isomerase A (RPIA), a pentose phosphate pathway enzyme. p16 loss correlates with activation of the mTORC1-RPIA axis in multiple cancer types. Suppression of RPIA inhibits proliferation only in p16-low cells by inducing senescence both in vitro and in vivo. These data reveal the molecular basis whereby p16 loss modulates pro-tumorigenic metabolism through mTORC1-mediated upregulation of nucleotide synthesis and reveals a metabolic vulnerability of p16-null cancer cells.

Validation of a commercially available anti-REDD1 antibody using RNA interference and REDD1-/- mouse embryonic fibroblasts.

REDD1 is a transcriptional target gene of p53 and HIF-1, and an inhibitor of mTOR (mechanistic target of rapamycin) complex 1 (mTORC1)-signaling through PP2A-dependent interaction, making it an important convergence point of both tumor suppression and cell growth pathways. In accordance with this positioning, REDD1 levels are transcriptionally upregulated in response to a variety of cellular stress factors such as nutrient deprivation, hypoxia and DNA damage. In the absence of such conditions, and in particular where growth factor signaling is activated, REDD1 expression is typically negligible; therefore, it is necessary to induce REDD1 prior to experimentation or detection in model systems. Here, we evaluated the performance of a commercially available polyclonal antibody recognizing REDD1 by Western blotting in the presence of thapsigargin, a pharmacological inducer of ER stress well known to upregulate REDD1 protein expression. Further, REDD1 antibody specificity was challenged in HEK-293 cells in the presence of RNA interference and with a REDD1 (-/-) mouse embryonic fibroblast knockout cell line. Results showed reproducibility and specificity of the antibody, which was upheld in the presence of thapsigargin treatment. We conclude that this antibody can be used to reliably detect REDD1 endogenous expression in samples of both human and mouse origin.

Disruption of genes encoding eIF4E binding proteins-1 and -2 does not alter basal or sepsis-induced changes in skeletal muscle protein synthesis in male or female mice.

Sepsis decreases skeletal muscle protein synthesis in part by impairing mTOR activity and the subsequent phosphorylation of 4E-BP1 and S6K1 thereby controlling translation initiation; however, the relative importance of changes in these two downstream substrates is unknown. The role of 4E-BP1 (and -BP2) in regulating muscle protein synthesis was assessed in wild-type (WT) and 4E-BP1/BP2 double knockout (DKO) male mice under basal conditions and in response to sepsis. At 12 months of age, body weight, lean body mass and energy expenditure did not differ between WT and DKO mice. Moreover, in vivo rates of protein synthesis in gastrocnemius, heart and liver did not differ between DKO and WT mice. Sepsis decreased skeletal muscle protein synthesis and S6K1 phosphorylation in WT and DKO male mice to a similar extent. Sepsis only decreased 4E-BP1 phosphorylation in WT mice as no 4E-BP1/BP2 protein was detected in muscle from DKO mice. Sepsis decreased the binding of eIF4G to eIF4E in WT mice; however, eIF4E•eIF4G binding was not altered in DKO mice under either basal or septic conditions. A comparable sepsis-induced increase in eIF4B phosphorylation was seen in both WT and DKO mice. eEF2 phosphorylation was similarly increased in muscle from WT septic mice and both control and septic DKO mice, compared to WT control values. The sepsis-induced increase in muscle MuRF1 and atrogin-1 (markers of proteolysis) as well as TNFα and IL-6 (inflammatory cytokines) mRNA was greater in DKO than WT mice. The sepsis-induced decrease in myocardial and hepatic protein synthesis did not differ between WT and DKO mice. These data suggest overall basal protein balance and synthesis is maintained in muscle of mice lacking both 4E-BP1/BP2 and that sepsis-induced changes in mTOR signaling may be mediated by a down-stream mechanism independent of 4E-BP1 phosphorylation and eIF4E•eIF4G binding.

Toll-like receptor activation suppresses ER stress factor CHOP and translation inhibition through activation of eIF2B.

Activation of Toll-like receptors (TLRs) induces the endoplasmic reticulum (ER) unfolded protein response (UPR) to accommodate essential protein translation. However, despite increased levels of phosphorylated eIF2α (p-eIF2α), a TLR-TRIF-dependent pathway assures that the cells avoid CHOP induction, apoptosis and translational suppression of critical proteins. As p-eIF2α decreases the functional interaction of eIF2 with eIF2B, a guanine nucleotide exchange factor (GEF), we explored the hypothesis that TLR-TRIF signalling activates eIF2B GEF activity to counteract the effects of p-eIF2α. We now show that TLR-TRIF signalling activates eIF2B GEF through PP2A-mediated serine dephosphorylation of the eIF2B ɛ-subunit. PP2A itself is activated by decreased Src-family-kinase-induced tyrosine phosphorylation of its catalytic subunit. Each of these processes is required for TLR-TRIF-mediated CHOP suppression in ER-stressed cells in vitro and in vivo. Thus, in the setting of prolonged, physiologic ER stress, a unique TLR-TRIF-dependent translational control pathway enables cells to carry out essential protein synthesis and avoid CHOP-induced apoptosis while still benefiting from the protective arms of the UPR.

Multi-modal proteomic analysis of retinal protein expression alterations in a rat model of diabetic retinopathy.

BACKGROUND: As a leading cause of adult blindness, diabetic retinopathy is a prevalent and profound complication of diabetes. We have previously reported duration-dependent changes in retinal vascular permeability, apoptosis, and mRNA expression with diabetes in a rat model system. The aim of this study was to identify retinal proteomic alterations associated with functional dysregulation of the diabetic retina to better understand diabetic retinopathy pathogenesis and that could be used as surrogate endpoints in preclinical drug testing studies. METHODOLOGY/PRINCIPAL FINDINGS: A multi-modal proteomic approach of antibody (Luminex)-, electrophoresis (DIGE)-, and LC-MS (iTRAQ)-based quantitation methods was used to maximize coverage of the retinal proteome. Transcriptomic profiling through microarray analysis was included to identify additional targets and assess potential regulation of protein expression changes at the mRNA level. The proteomic approaches proved complementary, with limited overlap in proteomic coverage. Alterations in pro-inflammatory, signaling and crystallin family proteins were confirmed by orthogonal methods in multiple independent animal cohorts. In an independent experiment, insulin replacement therapy normalized the expression of some proteins (Dbi, Anxa5) while other proteins (Cp, Cryba3, Lgals3, Stat3) were only partially normalized and Fgf2 and Crybb2 expression remained elevated. CONCLUSIONS/SIGNIFICANCE: These results expand the understanding of the changes in retinal protein expression occurring with diabetes and their responsiveness to normalization of blood glucose through insulin therapy. These proteins, especially those not normalized by insulin therapy, may also be useful in preclinical drug development studies.

Rapid turnover of the mTOR complex 1 (mTORC1) repressor REDD1 and activation of mTORC1 signaling following inhibition of protein synthesis.

mTORC1 is a complex of proteins that includes the mammalian target of rapamycin (mTOR) and several regulatory proteins. It is activated by a variety of hormones (e.g. insulin) and nutrients (e.g. amino acids) that act to stimulate cell growth and proliferation and repressed by hormones (e.g. glucocorticoids) that act to reduce cell growth. Curiously, mTORC1 signaling is reported to be rapidly (e.g. within 1-2 h) activated by inhibitors of protein synthesis that act on either mRNA translation elongation or gene transcription. However, the basis for the mTORC1 activation has not been satisfactorily delineated. In the present study, mTORC1 signaling was found to be activated in response to inhibition of either the initiation or elongation phases of mRNA translation. Changes in mTORC1 signaling were inversely proportional to alterations in the expression of the mTORC1 repressor, REDD1, but not the expression of TRB3 or TSC2. Moreover the cycloheximide-induced increase in mTORC1 signaling was significantly attenuated in cells lacking REDD1, showing that REDD1 plays an integral role in the response. Finally, the half-life of REDD1 was estimated to be 5 min or less. Overall, the results are consistent with a model in which inhibition of protein synthesis leads to a loss of REDD1 protein because of its rapid degradation, and in part reduced REDD1 expression subsequently leads to de-repression of mTORC1 activity.