Regulation Of mTOR Signaling Research Articles

CircRNA-100338 Is Associated With mTOR Signaling Pathway and Poor Prognosis in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the leading cause of cancer-related deaths worldwide. Despite advances in the diagnosis and treatment of HCC, incidence, and mortality continue to rise. For accurate diagnosis and treatment of HCC, there is an urgent need to precisely understand the molecular mechanisms underlying HCC tumorigenesis and progression. Accumulating evidence showed that circRNAs, which are normally produced by scrambling of exons at the splicing process, are recognized as a novel class of endogenous noncoding RNA, which have microRNA sponging properties. In this study, we aim to investigate the circRNA-100338 mediated downstream pathway, and evaluate its association with clinicopathological parameters. Integrated analysis of circRNA-100338, miR-141-3p, and target genes revealed that RHEB, a key regulator in mTOR signaling pathway, was the target of miR-141-3p in hepatitis B-related HCC. CircRNA-100338 regulates the activity of mTOR signaling pathway in vitro. IHC analysis revealed that mTOR signaling pathway was more active in HCC tissues with elevated circRNA-100338 expression. These results indicated that circRNA-100338 could regulate mTOR signaling pathway through circRNA-100338/miR-141-3p/RHEB axis. Finally, correlation analysis of RHEB and EIF5 expression with clinicopathological parameters of HCC patients revealed that the circRNA-100338, RHEB, and EIF5 were indicators of poor prognosis in hepatitis B-related HCC. In conclusion, elevated circRNA-100338 activates mTOR signaling pathway in HCC via circRNA-100338/miR-141-3p/RHEB axis and associates with poor prognosis of hepatitis B-related HCC patients.

Geniposide-mediated protection against amyloid deposition and behavioral impairment correlates with downregulation of mTOR signaling and enhanced autophagy in a mouse model of Alzheimer's disease.

Geniposide, an iridoid glycoside extract from the gardenia fruit, is used in traditional Chinese medicine to alleviate symptoms of liver and inflammatory diseases. Geniposide activates GLP-1 receptors, known to modulate the activity of mechanistic target of rapamycin (mTOR), a key kinase regulating energy balance, proliferation, and survival in cells. mTOR activation inhibits autophagy, which is often disrupted in age-related diseases. Modulation of mTOR function to increase autophagy and inhibit apoptosis is involved in the protective effects of pharmacologic agents targeting diabetes and Alzheimer’s disease (AD). We investigated whether such mechanism could mediate geniposide’s neuroprotective effects in the APP/PS1 mouse model of AD. Eight-week treatment with geniposide improved cognitive scores in behavioral tests, reduced amyloid-β 1-40 plaque deposition, and reduced soluble Aβ1-40 and Aβ1-42 levels in the APP/PS1 mouse brain.This also showed increased p-Akt/Akt, p-mTOR/mTOR and decreased p-4E-BP1/4E-BP1 expression, and these patterns were partially reversed by geniposide. Evidence for enhanced autophagy, denoted by increased expression of LC3-II and Beclin1, was also seen after treatment with geniposide. Our data suggests that down regulation of mTOR signaling, leading to enhanced autophagy and lysosomal clearance of Aβ fibrils, underlies the beneficial effects of geniposide against neuropathological damage and cognitive deficits characteristic of AD.

Hypoxia-induced regulation of mTOR signaling by miR-7 targeting REDD1.

Oxygen is an important factor mediating cell growth and survival under physiological and pathological conditions. Therefore, cells have well-regulated response mechanisms in the face of changes in oxygen levels in their environment. A subset of microRNAs (miRNAs) termed the hypoxamir has been suggested to be a critical mediator of the cellular response to hypoxia. Regulated in development and DNA damage response 1 (REDD1) is a negative regulator of mammalian target of rapamycin (mTOR) signaling in the response to cellular stress, and is elevated in many cell types under hypoxia, with consequent inhibition of mTOR signaling. However, the underlying posttranscriptional regulatory mechanism by miRNAs that contribute to this hypoxia-induced reduction in REDD1 expression remain unknown. Therefore, the aim of the current study was to identify the miRNAs participating in the hypoxic cellular response by scanning the 3'-untranslated region (3'-UTR) of REDD1 for potential miRNA-binding sites using a computer algorithm, TargetScan. miR-7 emerged as a novel hypoxamir that regulates REDD1 expression and is involved in mTOR signaling. miR-7 could repress REDD1 expression posttranscriptionally by directly binding with the 3'-UTR. Upon hypoxia, miR-7 expression was downregulated in HeLa cells to consequently derepress REDD1, resulting in inhibition of mTOR signaling. Moreover, overexpression of miR-7 was sufficient to reverse the hypoxia-induced inhibition of mTOR signaling. Therefore, our findings suggest miR-7 as a key regulator of hypoxia-mediated mTOR signaling through modulation of REDD1 expression. These findings contribute new insight into the miRNA-mediated molecular mechanism of the hypoxic response through mTOR signaling, highlighting potential targets for tumor suppression.

Methionine and valine activate the mammalian target of rapamycin complex 1 pathway through heterodimeric amino acid taste receptor (TAS1R1/TAS1R3) and intracellular Ca2+ in bovine mammary epithelial cells

Amino acids play a key role in regulating milk protein synthesis partly through activation of the mammalian target of rapamycin (mTOR) signaling pathway. However, the involvement of extracellular AA sensing receptors in this process is not well understood. In nonruminants, it is well established that the AA taste 1 receptor member 1/3 (TAS1R1/TAS1R3) heterodimer contributes to the sensing of most l-AA. Whether this receptor is functional in bovine mammary cells is unknown. The objective of this study was to determine essential AA signaling through TAS1R1/TAS1R3 and their roles in regulating mTOR signaling pathway and casein mRNA abundance in primary bovine mammary epithelial cells and the Mac-T cell line. The bovine mammary epithelial cells were stimulated with complete Dulbecco's modified Eagle's medium (+EAA), medium without EAA (-EAA), or medium supplemented with only 1 of the 10 essential AA, respectively. The nonessential AA levels were the same across all treatments. Small interference RNA targeting TAS1R1 were designed and transfected into bovine primary mammary epithelial cells (bPMEC). Supplementation of a complete mixture of essential AA or Arg, Val, Leu, His, Phe, Met, and Ile individually led to greater mTOR phosphorylation. Phosphorylation of ribosomal protein S6 kinase β-1 was greater in the presence of Val, Leu, Trp, Met, and Ile. Valine, Leu, Met, and Ile led to greater eIF4E-binding protein 1 phosphorylation. Although +EAA and a few individual AA tested induced increases in intracellular calcium, Met and Val were the most potent. Knockdown of TAS1R1 decreased intracellular calcium in bPMEC cultured with both Val and Met. Phosphorylation of mTOR, ribosomal protein S6 kinase β-1, and eIF4E-binding protein 1 was lower when TAS1R1 was knocked-down in bPMEC supplemented with Val and Met. In addition, small interference RNA silencing of TAS1R1 resulted in lower β-casein (CSN2) abundance. The TAS1R1/TAS1R3 receptor may sense extracellular AA and activate mTOR signaling in bovine mammary cells, likely by elevating intracellular calcium concentration. This mechanism appears to have a role in Met- and Val-induced changes in CSN2 mRNA abundance. Further in vivo studies will have to be performed to assess the relevance of this mechanism in the mammary gland.

C16‑ceramide and sphingosine1‑phosphate/S1PR2 have opposite effects on cell growth through mTOR signaling pathway regulation.

Recently, sphingolipid derivatives, such as ceramide and sphingosine‑1‑phosphate (S1P), have emerged as key modulators in apoptotic cell death and cell proliferation. This study aimed to clarify the underlying signaling pathways of ceramide and S1P involved in breast cancer cell proliferation. Ceramide acyl chain length is determined by six mammalian ceramide synthases (CerS). We overexpressed CerS1 to6 in MCF‑7 cells to examine whether ceramide signaling propagation varies as a function of acyl chain length. Among the six CerS, only CerS6 overexpression reduced phosphorylation of Akt, S6 kinase (S6K), and extracellular signal‑regulated kinases (ERK) as shown by western blotting. In addition, CerS6 overexpression reduced MCF‑7 cell proliferation. This effect was partially reversed by co‑treatment with MHY1485, an activator of mammalian target of rapamycin (mTOR), demonstrating an important role for the mTOR pathway in the CerS6‑mediated decrease in MCF‑7 cell proliferation. ERK inhibition, but not Akt inhibition, along with mTOR inhibition synergistically reduced MCF‑7 cell proliferation as measured by MTT assay. Notably, the expression of CerS6 and S1P receptor 2 (S1PR2), or CerS6 and sphingosine kinase1 (SphK1), were negatively correlated according to the invasive breast carcinoma patient cohort in The Cancer Genome Atlas database. In addition, both SphK1 overexpression and S1P addition increased mTOR phosphorylation as shown by ELISA, while S1PR2 inhibition had the inverse effect. These data suggest that CerS6 and SphK1 regulate mTOR signaling in breast cancer cell proliferation. Moreover, mTOR activity can be regulated by the balance between S1P and C16‑ceramide, which is generated by CerS6.

Caloric restriction effects on liver mTOR signaling are time-of-day dependent.

The regulation of mechanistic target of rapamycin (mTOR) signaling contributes to the metabolic effects of a calorie restriction (CR) diet. We assayed the effect of CR on the activity of mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) in the liver of mice at six different times across the day. CR effects on mTORC1 and mTORC2 activities were time-of-day dependent. CR induced mTORC1 activity at one time, reduced at two times and has no effect during other times. CR induced mTORC2 activity at one time of the day and has no effects at other times. Circadian clocks are implemented in the regulation of mTOR signaling in mammals and mechanisms of CR. We assayed the effect of CR on mTOR signaling in the liver of mice deficient for circadian transcriptional regulators BMAL1 and CRYs. The CR induced suppression of mTORC1 activity was observed in both clock mutants, while up regulation of mTORC2 was observed in the liver of CRY deficient but not in the liver of BMAL1 deficient mice. Our finding revealed that CR has different time dependent effect on the activity of mTOR complexes 1 and 2 and suggest that circadian clock protein BMAL1 is involved in the up regulation of mTORC2 upon CR in mammals.

Abstract 783: Impact of KRAS and TP53 co-mutations on outcomes following 1st-line therapy among patients with LKB1/STK11 mutated stage IV NSCLC

Abstract * Authors E. M. B. and M. E. M. contributed equally Introduction: LKB1/STK11 is a tumor suppressor and a negative regulator of mTOR signaling. NSCLC patients with STK11 mutations (MT) commonly also have other co-mutations. We evaluated the impact of STK11 MT on outcomes following chemotherapy for metastatic NSCLC, and the role of common co-existing MTs in KRAS and TP53. Methods: We conducted a retrospective review of patients (pts) with NSCLC and STK11 MT treated at the University of Pennsylvania. STK11 MT was identified through next generation sequencing (NGS) in tissue or using the Guardant 360TM platform for plasma. Four treatment groups were analyzed: STK11 alone (A), and 3 co-MT groups: STK11/KRAS (B), STK11/TP53 (C), and STK11/KRAS/TP53 (D). Chi-square analysis was used to assess differences in baseline characteristics between the MT groups. Cox proportional hazard models (HR) were used to determine the relationship of STK11 co-MT to survival. Kaplan-Meier analysis was used to estimate overall survival (OS) and progression-free survival (PFS). Results: Between 2/14/13 and 12/1/16, 77 pts with STK11 MT who received systemic therapy were identified (56 tissue, 21 plasma): median age at diagnosis 66 yrs, 51.5% male, 85% ECOG PS of 0 or 1, 94% of pts received 1st-line chemotherapy. There were no significant differences in baseline characteristics among mutation groups. Pts with STK11/KRAS had a worse median PFS and OS vs. pts with STK11 alone (Table 1). Pts with STK11/TP53 had a significantly better OS compared to STK11/KRAS patients. Conclusion: Among STK11 mutant NSCLC pts treated with chemotherapy, co-MT with KRAS was associated with a significantly worse PFS and OS compared to patients with STK11 alone. By contrast, co-MT with TP53 conferred a better prognosis. These results warrant further validation in a larger study. Table 1: Outcomes by mutation statusA:STK11alone n=16 (24.2%)B:STK11/KRAS n=21 (31.8%)C:STK11/TP53 n=18 (27.3%)D:STK11/KRAS/TP53 n=11 (16.7%)Median PFS5.3 mo2.4 mo*9.9mo4.9 moMedian OS13.1 mo6.9 mo**22.2 mo***13.9 mo*compared to A, HR 2.8, 95% CI 1.1 to 6.9, p = 0.026**compared to A, HR 6.4, 95% CI 2.4 to 17.3, p<0.001***compared to B, HR 0.12, 95% CI 0.04 to 0.31, p<0.001 Citation Format: Erin M. Bange, Melina E. Marmarelis, Wei-Ting Hwang, Yu-Xiao Yang, Jeffrey C. Thompson, Joshua Bauml, Christine A. Ciunci, Evan W. Alley, Tracey L. Evans, Jennifer J. Morrissette, Roger B. Cohen, Corey J. Langer, Erica L. Carpenter, Charu Aggarwal. Impact of KRAS and TP53 co-mutations on outcomes following 1st-line therapy among patients with LKB1/STK11 mutated stage IV NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 783.

H2 O2 induces PP2A demethylation to downregulate mTORC1 signaling in HEK293 cells.

Mammalian target of rapamycin (mTOR) is a Ser/Thr protein kinase that functions as an ATP and amino acid sensor to govern cell growth and proliferation by mediating mitogen- and nutrient-dependent signal transduction. Protein phosphatase 2A (PP2A), a ubiquitously expressed serine/threonine phosphatase, negatively regulates mTOR signaling. Methylation of PP2A is catalyzed by leucine carboxyl methyltransferase-1 (LCMT1) and reversed by protein phosphatase methylesterase 1 (PME-1), which regulates PP2A activity and substrate specificity. However, whether PP2A methylation is related to mTOR signaling is still unknown. In this study, we examined the effect of PP2A methylation on mTOR signaling in HEK293 cells under oxidative stress. Our results show that oxidative stress induces PP2A demethylation and inhibits the mTORC1 signaling pathway. Next, we examined two strategies to block PP2A demethylation under oxidative stress. One strategy was to prevent PP2A demethylation using a PME-1 inhibitor; the other strategy was to activate PP2A methylation via overexpression of LCMT1. The results show that both the PME-1 inhibitor and LCMT1 overexpression prevent the mTORC1 signaling suppression induced by oxidative stress. Additionally, LCMT1 overexpression rescued cell viability and the mitochondrial membrane potential decrease in response to oxidative stress. These results demonstrate that H2 O2 induces PP2A demethylation to downregulate mTORC1 signaling. These findings provide a novel mechanism for the regulation of PP2A demethylation and mTORC1 signaling under oxidative stress.

Long noncoding RNA Hoxb3os is dysregulated in autosomal dominant polycystic kidney disease and regulates mTOR signaling

Autosomal dominant polycystic kidney disease (ADPKD) is a debilitating disease that is characterized by the accumulation of numerous fluid-filled cysts in the kidney. ADPKD is primarily caused by mutations in two genes, PKD1 and PKD2 Long noncoding RNAs (lncRNA), defined by a length >200 nucleotides and absence of a long ORF, have recently emerged as epigenetic regulators of development and disease; however, their involvement in PKD has not been explored previously. Here, we performed deep RNA-Seq to identify lncRNAs that are dysregulated in two orthologous mouse models of ADPKD (kidney-specific Pkd1 and Pkd2 mutant mice). We identified a kidney-specific, evolutionarily conserved lncRNA called Hoxb3os that was down-regulated in cystic kidneys from Pkd1 and Pkd2 mutant mice. The human ortholog HOXB3-AS1 was down-regulated in cystic kidneys from ADPKD patients. Hoxb3os was highly expressed in renal tubules in adult WT mice, whereas its expression was lost in the cyst epithelium of mutant mice. To investigate the function of Hoxb3os, we utilized CRISPR/Cas9 to knock out its expression in mIMCD3 cells. Deletion of Hoxb3os resulted in increased phosphorylation of mTOR and its downstream targets, including p70 S6 kinase, ribosomal protein S6, and the translation repressor 4E-BP1. Consistent with activation of mTORC1 signaling, Hoxb3os mutant cells displayed increased mitochondrial respiration. The Hoxb3os mutant phenotype was partially rescued upon re-expression of Hoxb3os in knockout cells. These findings identify Hoxb3os as a novel lncRNA that is down-regulated in ADPKD and regulates mTOR signaling and mitochondrial respiration.

MiR-33a-5p enhances the sensitivity of lung adenocarcinoma cells to celastrol by regulating mTOR signaling.

MicroRNAs (miRNAs or miRs) have recently become a popular focus of cancer research due to their ability to act as oncogenes or tumor suppressors. In the present study, miR‑33a‑5p expression was identified to be downregulated in lung adenocarcinoma samples compared with normal, which suggested that miR‑33a‑5p may serve as a tumor suppressor gene. Transfection with miR‑33a‑5p mimics inhibited the proliferation and migration of A549 and LTEP‑a‑2 cells and increased cellular apoptosis. A luciferase reporter assay confirmed that miR‑33a‑5p targets the 3'‑untranslated region of the mechanistic target of rapamycin (mTOR) gene. mTOR expression was decreased in A549 and LTEP‑a‑2 cells treated with miR‑33a‑5p mimics, as well as the expression of its downstream effectors phosphorylated (p)‑p70 ribosomal protein S6 kinase (p70S6K) and p‑eukaryotic translation initiation factor4E binding protein1(4EBP1). Following treatment with celastrol, miR‑33a‑5p expression was upregulated, and miR‑33a‑5p could enhance cellular sensitivity to celastrol. Western blot analysis revealed that the expression of mTOR, p‑p70S6K and p‑4EBP1 decreased following celastrol treatment. These results suggested that mTOR was involved in the mechanism by which miR‑33a‑5p enhanced the sensitivity of lung adenocarcinoma cells to celastrol. Furthermore, LTEP‑a‑2 cells were xenografted subcutaneously into nude mice, to examine the effect of celastrol and miR‑33a‑5p on the growth of LTEP‑a‑2 cells invivo. The results demonstrated that tumor growth in the celastrol‑treated or miR‑33a‑5p‑treated group was attenuated compared with the control group. Notably, tumor growth in the combination treatment group was almost arrested after 2weeks. In addition, celastrol upregulated the expression of miR‑33a‑5p, and high expression of miR‑33a‑5p inhibited mTOR and its downstream effectors. In summary, miR‑33a‑5p inhibited the proliferation of lung adenocarcinoma cells, enhanced the antitumor effect of celastrol, and improved sensitivity to celastrol by targeting mTOR in lung adenocarcinoma invitro and invivo.

TSC1 and TSC2 regulate cilia length and canonical Hedgehog signaling via different mechanisms

Primary cilia are sensory organelles that coordinate multiple cellular signaling pathways, including Hedgehog (HH), Wingless/Int (WNT) and Transforming Growth Factor-β (TGF-β) signaling. Similarly, primary cilia have been implicated in regulation of mTOR signaling, in which Tuberous Sclerosis Complex proteins 1 and 2 (TSC1/2) negatively regulate protein synthesis by inactivating the mTOR complex 1 (mTORC1) at energy limiting states. Here we report that TSC1 and TSC2 regulate Smoothened (SMO)-dependent HH signaling in mouse embryonic fibroblasts (MEFs). Reduced SMO-dependent expression of Gli1 was demonstrated in both Tsc1−/− and Tsc2−/− cells, and we found that Tsc1 is required for TGF-β induced phosphorylation of SMAD2/3 and subsequent expression of the HH signaling effector and transcription factor GLI2. Hedgehog signaling was restored in Tsc1−/− cells after exogenous expression of Gli2, whereas rapamycin restored HH signaling in Tsc2−/− cells. Furthermore, we observed that Tsc1−/− MEFs display significantly elongated cilia, whereas cilia in Tsc2−/− MEFs were shorter than normal. The elongated cilium phenotype of Tsc1−/− MEFs is likely due to increased mTORC1-dependent autophagic flux observed in these cells, as both the autophagic flux and the cilia length phenotype was restored by rapamycin. In addition, ciliary length control in Tsc1−/− MEFs was also influenced by reduced expression of Gli2, which compromised expression of Wnt5a that normally promotes cilia disassembly. In summary, our results support distinct functions of Tsc1 and Tsc2 in cellular signaling as the two genes affect ciliary length control and HH signaling via different mechanisms.

Progesterone suppresses Aβ42-induced neuroinflammation by enhancing autophagy in astrocytes

Autophagy is an intracellular catabolic mechanism essential for recycling intracellular unfolding protein and eliminating toxic protein aggregates. Several studies have shown that deficient autophagy is implicated in the development of Alzheimer's disease (AD) progression. To date, rapidly emerging evidence suggests that neurosteroid progesterone (PG) may play an important role in ameliorating AD. However, the role of PG and its neuroprotective mechanism in regulating autophagy still require further investigation. Here, we investigated the protective effects of PG against Aβ-induced inflammatory responses in astrocytes and its underlying mechanism in mediating autophagy. Remarkably, Aβ induced astrocyte dysfunction in autophagic activation and up-regulated inflammatory secretion. However, the autophagy inducer rapamycin (RAPA) significantly suppressed Aβ-induced inflammation in astrocytes. In astrocytes, treatment with Aβ caused autophagy deficiency, whereas PG significantly increased autophagy activation. Finally, PG suppressed Aβ-induced neuroinflammatory production via enhancing autophagy together with regulating mTOR signaling. Taken together, these results show that autophagy is a vital mechanism against Aβ-induced neuroinflammatory responses in astrocytes and demonstrate the potential neuroprotective mechanism of PG in suppressing neuroinflammatory responses by enhancing autophagy. Therefore, uncovering the neuroprotective mechanism of PG may provide new insight into novel therapies for the amelioration of AD.

Liver Reptin/RUVBL2 controls glucose and lipid metabolism with opposite actions on mTORC1 and mTORC2 signalling

ObjectiveThe AAA+ ATPase Reptin is overexpressed in hepatocellular carcinoma and preclinical studies indicate that it could be a relevant therapeutic target. However, its physiological and pathophysiological roles in vivo remain...

Enoyl-CoA hydratase-1 regulates mTOR signaling and apoptosis by sensing nutrients

The oncogenic mechanisms of overnutrition, a confirmed independent cancer risk factor, remain poorly understood. Herein, we report that enoyl-CoA hydratase-1 (ECHS1), the enzyme involved in the oxidation of fatty acids (FAs) and branched-chain amino acids (BCAAs), senses nutrients and promotes mTOR activation and apoptotic resistance. Nutrients-promoted acetylation of lys101 of ECHS1 impedes ECHS1 activity by impairing enoyl-CoA binding, promoting ECHS1 degradation and blocking its mitochondrial translocation through inducing ubiquitination. As a result, nutrients induce the accumulation of BCAAs and FAs that activate mTOR signaling and stimulate apoptosis, respectively. The latter was overcome by selection of BCL-2 overexpressing cells under overnutrition conditions. The oncogenic effects of nutrients were reversed by SIRT3, which deacetylates lys101 acetylation. Severely decreased ECHS1, accumulation of BCAAs and FAs, activation of mTOR and overexpression of BCL-2 were observed in cancer tissues from metabolic organs. Our results identified ECHS1, a nutrients-sensing protein that transforms nutrient signals into oncogenic signals.

Regulated in Development and DNA Damage Response 1 Deficiency Impairs Autophagy and Mitochondrial Biogenesis in Articular Cartilage and Increases the Severity of Experimental Osteoarthritis.

Regulated in development and DNA damage response 1 (REDD1) is an endogenous inhibitor of mechanistic target of rapamycin (mTOR) that regulates cellular stress responses. REDD1 expression is decreased in aged and osteoarthritic (OA) cartilage, and it regulates mTOR signaling and autophagy in articular chondrocytes in vitro. This study was undertaken to investigate the effects of REDD1 deletion in vivo using a mouse model of experimental OA. OA severity was histologically assessed in 4-month-old wild-type and REDD1-/- mice subjected to surgical destabilization of the medial meniscus (DMM). Chondrocyte autophagy, apoptosis, mitochondrial content, and expression of mitochondrial biogenesis markers were determined in cartilage and cultured chondrocytes from wild-type and REDD1-/- mice. REDD1 deficiency increased the severity of changes in cartilage, menisci, subchondral bone, and synovium in the DMM model of OA. Chondrocyte death was increased in the cartilage of REDD1-/- mice and in cultured REDD1-/- mouse chondrocytes under oxidative stress conditions. Expression of key autophagy markers (microtubule-associated protein 1A/1B light chain 3 and autophagy protein 5) was markedly reduced in cartilage from REDD1-/- mice and in cultured human and mouse chondrocytes with REDD1 depletion. Mitochondrial content, ATP levels, and expression of the mitochondrial biogenesis markers peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and transcription factor A, mitochondrial (TFAM) were also decreased in REDD1-deficient chondrocytes. REDD1 was required for AMP-activated protein kinase-induced PGC-1α in chondrocytes. Our findings suggest that REDD1 is a key mediator of cartilage homeostasis through regulation of autophagy and mitochondrial biogenesis and that REDD1 deficiency exacerbates the severity of injury-induced OA.

Effect of Redd1 loss on proliferation and metastasis of pancreatic cancer cells with KrasG12D-LOH by inhibiting glycolysis.

e15741 Background: Aerobic glycolysis, regulated by mammalian target of rapamycin (mTOR) pathway, plays an important role in pancreatic carcinogenesis. Regulated in development and DNA damage response (Redd1) constitutes an important regulator of mTOR signaling. Previously, we observed that the loss of heterozygosity (LOH) status of Kras mutations (e.g. KrasG12D) is associated with an increased Redd1 expression. Here, we investigated the functional relevance of Redd1 in the context of KrasG12D-LOH. Methods: Murine KrasG12D-LOH/KrasG12Dpancreatic cancer cells isolated from genetically engineered mice were used in this study. The glycolysis dependence was detected by CCK8 assays after treated by glycolysis inhibitor 2-Deoxy-D-glucose (2-DG). Redd1 expression was down-regulated using shRNA transfection. Cell proliferation was determined by CCK8 and colony formation assays. Cell invasion and migration was measured by transwell and wound-healing assays. The levels of lactic acid and ATP were tested by ELISA kit. Genes related to glycolysis and mTOR signal were evaluated by western-blot and quantitative RT-PCR. Results: Compared with KRASG12D pancreatic cancer cells, the viability of KRASG12D-LOH cells decreased significantly in the presence of 2-DG. After Redd1 suppression, the proliferative and invasive potentials of KRASG12D-LOH cells decreased significantly when compared with blank group and negative group. The levels of lactic acid and ATP were also reduced by Redd1 down-regulation. Furthermore, the signal activity of mTOR pathway and Pkm2 and Hk2 expression were reduced dramatically, while Tsc1 and Tsc2 expression were unaffected. Conclusions: The LOH status of KRASG12D renders the pancreatic cancer cells additive to glycolysis, which further affect the proliferative and invasive potentials via the function of Redd1/mTOR. These data underscore the importance of KRASG12D-LOH in regulating cancer glucose metabolism.

Identification of mechanically regulated phosphorylation sites on tuberin (TSC2) that control mechanistic target of rapamycin (mTOR) signaling

Mechanistic target of rapamycin (mTOR) signaling is necessary to generate a mechanically induced increase in skeletal muscle mass, but the mechanism(s) through which mechanical stimuli regulate mTOR signaling remain poorly defined. Recent studies have suggested that Ras homologue enriched in brain (Rheb), a direct activator of mTOR, and its inhibitor, the GTPase-activating protein tuberin (TSC2), may play a role in this pathway. To address this possibility, we generated inducible and skeletal muscle-specific knock-out mice for Rheb (iRhebKO) and TSC2 (iTSC2KO) and mechanically stimulated muscles from these mice with eccentric contractions (EC). As expected, the knock-out of TSC2 led to an elevation in the basal level of mTOR signaling. Moreover, we found that the magnitude of the EC-induced activation of mTOR signaling was significantly blunted in muscles from both inducible and skeletal muscle-specific knock-out mice for Rheb and iTSC2KO mice. Using mass spectrometry, we identified six sites on TSC2 whose phosphorylation was significantly altered by the EC treatment. Employing a transient transfection-based approach to rescue TSC2 function in muscles of the iTSC2KO mice, we demonstrated that these phosphorylation sites are required for the role that TSC2 plays in the EC-induced activation of mTOR signaling. Importantly, however, these phosphorylation sites were not required for an insulin-induced activation of mTOR signaling. As such, our results not only establish a critical role for Rheb and TSC2 in the mechanical activation of mTOR signaling, but they also expose the existence of a previously unknown branch of signaling events that can regulate the TSC2/mTOR pathway.

Emerging Role of MicroRNAs in mTOR Signaling.

Mechanistic target of rapamycin (mTOR) is a conserved serine/threonine kinase that plays a critical role in the control of cellular growth and metabolism. Hyperactivation of mTOR pathway is common in human cancers, driving uncontrolled proliferation. MicroRNA (miRNA) is a class of short noncoding RNAs that regulate the expression of a wide variety of genes. Deregulation of miRNAs is a hallmark of cancer. Recent studies have revealed interplays between miRNAs and the mTOR pathway during cancer development. Such interactions appear to provide a fine-tuning of various cellular functions and contribute qualitatively to the behavior of cancer. Here we provide an overview of current knowledge regarding the reciprocal relationship between miRNAs and mTOR pathway: regulation of mTOR signaling by miRNAs and control of miRNA biogenesis by mTOR. Further research in this area may prove important for the diagnosis and therapy of human cancer.

Expression of signaling pathway of mammalian target of rapamycin in articular cartilage cell induced by interleukin 1β

Objective: To observe effect of interleukin(IL)-1β on the expression of signaling pathway of mammalian target of rapamycin(mTOR) of articular cartilage. Methods: Articular cartilage of rats was isolated under sterile technique, cells were digested by type Ⅱ collagenase and trypsin and cultured in vitro, pre-culture the Ⅱ cells for three days, different concentrations of IL-1β were added for 24 hours.The cells were stained with toluidine blue and HE, to observe morphological changes of cells.RT-PCR was used to detect the mRNA expression of typeⅡcollagen gene, aggrecan gene, mTOR gene and P70S6K gene, Western blotting was used to detect the expression of protein related to mTOR. Results: With increasing concentrations of IL-1β, the phenotype of cells appeared polygon into a spindle, the mRNA expression of gene of type Ⅱ collagen (the control group: 0.821±0.014; 1 ng/ml: 0.614±0.014; 10 ng/ml: 0.549±0.009; 100 ng/ml: 0.520±0.008), aggrecan(0.867±0.005; 0.857±0.001; 0.554±0.008; 0.538±0.004) and mTOR(0.845±0.015; 0.785±0.009; 0.569±0.025; 0.518±0.014) reduced, but P70S6K(0.465±0.024; 0.566±0.022; 0.663±0.022; 0.896±0.015) increased by PCR .Expression of protein detected by Western blotting was similar to the trend of PCR. Conclusion: mTOR signaling pathway may play an important role on the degeneration of articular cartilage, regulating mTOR signaling pathway may provides a new idea of delaying the degeneration process of cells.

The effects of reduced dietary protein level on amino acid transporters and mTOR signaling pathway in pigs

Amino acid transporter plays an important role in regulating mTOR signaling pathway. This study investigated the effects of reduced dietary protein levels on amino acid transporters and mTOR signaling pathway. A total of 54 weaning pigs were randomly allocated into a 3 × 3 factorial design, followed by slaughtering the pigs separately after 10-, 25- and 45-day feeding, with 18 pigs from each feeding period divided into three subgroups for treatment with three different protein-level diets: 20% crude protein (CP) diet (normal recommended, high protein, HP), 17% CP diet (medium protein, MP) and 14% CP diet (low protein, LP). The results indicated that reduced dietary protein level decreased the weight of longissimus dorsi. Additionally, quantitative PCR chip analysis showed that mRNA expression of amino acid transporters SLC38A2, SLC1A7, SLC7A1, SLC7A5, SLC16A10 and SLC3A2 in the LP group were significantly (P < 0.05) higher than those in the MP or HP group, and the phosphorylation of mTOR and S6K1 decreased in the LP group after 25-day feeding. Furthermore, the vitro experimental results further confirmed that the mRNA levels for SLC7A1, SLC7A5, SLC3A2, SLC38A2 and SLC36A1 were increased and the phosphorylation of mTOR and S6K1 was decreased when the concentration of amino acids in C2C12 myoblasts was reduced. All these results indicated that the LP diet induced a high expression of amino acid transporters and the inhibition of the mTOR activity, which resulting in restriction on protein synthesis and longissimus dorsi growth.