Regulation of mTOR and cell growth in response to energy stress by REDD1

The tuberous sclerosis tumor suppressors TSC1 and TSC2 regulate the mTOR pathway to control translation and cell growth in response to nutrient and growth factor stimuli. We have recently identified the stress response REDD1 gene as a mediator of tuberous sclerosis complex (TSC)-dependent mTOR regulation by hypoxia. Here, we demonstrate that REDD1 inhibits mTOR function to control cell growth in response to energy stress. Endogenous REDD1 is induced following energy stress, and REDD1-/- cells are highly defective in dephosphorylation of the key mTOR substrates S6K and 4E-BP1 following either ATP depletion or direct activation of the AMP-activated protein kinase (AMPK). REDD1 likely acts on the TSC1/2 complex, as regulation of mTOR substrate phosphorylation by REDD1 requires TSC2 and is blocked by overexpression of the TSC1/2 downstream target Rheb but is not blocked by inhibition of AMPK. Tetracycline-inducible expression of REDD1 triggers rapid dephosphorylation of S6K and 4E-BP1 and significantly decreases cellular size. Conversely, inhibition of endogenous REDD1 by short interfering RNA increases cell size in a rapamycin-sensitive manner, and REDD1-/- cells are defective in cell growth regulation following ATP depletion. These results define REDD1 as a critical transducer of the cellular response to energy depletion through the TSC-mTOR pathway.     

Role of hypoxia‐induced fibronectin‐integrin β1 expression in embryonic stem cell proliferation and migration: Involvement of PI3K/Akt and FAK

Cell migration is largely dependent on integrin (IN) binding to the extracellular matrix, and several signaling pathways involved in these processes have been shown to be modified by hypoxia. Therefore, the aim of this study was to determine the influence of hypoxia on fibronectin (FN) and IN β1 expression in mouse embryonic stem cells (mESCs) and their signaling pathways to modulate proliferation. FN and IN β1 expression were significantly increased in hypoxic mESCs by 24 h. Hypoxia also increased cell attachment, which was accompanied by concomitant increases in the binding level of FN and IN β1. Hypoxia‐induced FN expression was mediated by increased phosphatidylinositol 3 kinase (PI3K)/Akt and mammalian target of rapamycin (mTOR) phosphorylation, and hypoxia‐inducible factor‐1α (HIF‐1α) expression. Moreover, under hypoxic conditions, focal adhesion kinase (FAK) and Src phosphorylation were increased in a time‐dependent fashion; these increases were blocked by IN β1 antibody. In addition, the hypoxia induced increase of F‐actin distribution and cell migration (activation of matrix metalloproteinase‐2 and ‐9) was inhibited by IN β1 antibody. Indeed, hypoxia increased the level of cell‐cycle regulatory protein and DNA synthesis. In conclusion, hypoxia increases the proliferation and migration of mESCs via FN‐IN β1 production through the PI3K/Akt, mTOR, and HIF‐1α pathways, followed by FAK activation. J. Cell. Physiol. 226: 484–493, 2011. © 2010 Wiley‐Liss, Inc.     

Intracellular parasitism with Toxoplasma gondii stimulates mammalian‐target‐of‐rapamycin‐dependent host cell growth despite impaired signalling to S6K1 and 4E‐BP1

The Ser/Thr kinase mammalian‐target‐of‐rapamycin (mTOR) is a central regulator of anabolism, growth and proliferation. We investigated the effects of Toxoplasma gondii on host mTOR signalling. Toxoplasma invasion of multiple cell types rapidly induced sustained mTOR activation that was restricted to infected cells, as determined by rapamycin‐sensitive phosphorylation of ribosomal protein S6; however, phosphorylation of the growth‐associated mTOR substrates 4E‐BP1 and S6K1 was not detected. Infected cells still phosphorylated S6K1 and 4E‐BP1 in response to insulin, although the S6K1 response was blunted. Parasite‐induced S6 phosphorylation was independent of S6K1 and did not require activation of canonical mTOR‐inducing pathways mediated by phosphatidylinositol 3‐kinase–Akt and ERK. Host mTOR was localized in a vesicular pattern surrounding the parasitophorous vacuole, suggesting potential activation by phosphatidic acid in the vacuolar membrane. In spite of a failure to phosphorylate 4E‐BP1 and S6K1, intracellular T. gondii triggered host cell cycle progression in an mTOR‐dependent manner and progression of infected cells displayed increased sensitivity to rapamycin. Moreover, normal cell growth was maintained during parasite‐induced cell cycle progression, as indicated by total cellular S6 levels. The Toxoplasma‐infected cell provides a unique example of non‐canonical mTOR activation supporting growth that is independent of signalling through either S6K1 or 4E‐BP1.     

Hypoxia induces BMP‐2 expression via ILK,Akt, mTOR,and HIF‐1 pathways in osteoblasts

It has been shown that hypoxia stimulation regulates bone formation, maintenance, and repair. Bone morphogenetic protein (BMP) plays important roles in osteoblastic differentiation and bone formation. However, the effects of hypoxia exposure on BMP‐2 expression in cultured osteoblasts are largely unknown. Here we found that hypoxia stimulation increased mRNA and protein levels of BMP‐2 by qPCR, Western blot and ELISA assay in osteoblastic cells MG‐63, hFOB and bone marrow stromal cells M2‐10B4. Integrin‐linked kinase (ILK) inhibitor (KP‐392), Akt inhibitor (1L‐6‐hydroxymethyl‐chiro‐inositol‐2‐[(R)‐2‐O‐methyl‐3‐O‐octadecylcarbonate]) or mammalian target of rapamycin (mTOR) inhibitor (rapamycin) inhibited the potentiating action of hypoxia. Exposure to hypoxia increased the kinase activity of ILK and phosphorylation of Akt and mTOR. Furthermore, hypoxia also increased the stability and activity of HIF‐1 protein. The binding of HIF‐1α to the HRE elements after exposure to hypoxia was measured by EMSA assay. Moreover, the use of pharmacological inhibitors or genetic inhibition revealed that both ILK/Akt and mTOR signaling pathway were potentially required for hypoxia‐induced HIF‐1α activation and subsequent BMP‐2 up‐regulation. Taken together, our results provide evidence that hypoxia enhances BMP‐2 expression in osteoblasts by an HIF‐1α‐dependent mechanism involving the activation of ILK/Akt and mTOR pathways. J. Cell. Physiol. 223:810–818, 2010. © 2010 Wiley‐Liss, Inc.     

Growth Control Under Stress: mTOR Regulation through the REDD1-TSC Pathway

Dysregulated signaling by the checkpoint kinase TOR (target of rapamycin) has been linked to numerous human cancers. The tuberous sclerosis tumor suppressors TSC1 and TSC2 form a protein complex that integrates and transmits cellular growth factor and stress signals to negatively regulate TOR activity. Several recent reports have identified the stress response gene REDD1 as an essential regulator of TOR activity through the TSC1/2 complex in both Drosophila and mammalian cells. REDD1 is induced in response both to hypoxia and energy stress, and cells that lack REDD1 exhibit highly defective TOR regulation in response to either of these stress signals. While the precise mechanism of REDD1 function remains to be determined, the finding that REDD1-dependent TOR regulation contributes to cell growth/cell size control in flies and mammals suggests that abnormalities of REDD1-mediated signaling might disrupt energy homeostasis and/or promote tumorigenesis.     

S6K1 is a multifaceted regulator of Mdm2 that connects nutrient status and DNA damage response

p53 mediates DNA damage‐induced cell‐cycle arrest, apoptosis, or senescence, and it is controlled by Mdm2, which mainly ubiquitinates p53 in the nucleus and promotes p53 nuclear export and degradation. By searching for the kinases responsible for Mdm2 S163 phosphorylation under genotoxic stress, we identified S6K1 as a multifaceted regulator of Mdm2. DNA damage activates mTOR‐S6K1 through p38α MAPK. The activated S6K1 forms a tighter complex with Mdm2, inhibits Mdm2‐mediated p53 ubiquitination, and promotes p53 induction, in addition to phosphorylating Mdm2 on S163. Deactivation of mTOR‐S6K1 signalling leads to Mdm2 nuclear translocation, which is facilitated by S163 phosphorylation, a reduction in p53 induction, and an alteration in p53‐dependent cell death. These findings thus establish mTOR‐S6K1 as a novel regulator of p53 in DNA damage response and likely in tumorigenesis. S6K1–Mdm2 interaction presents a route for cells to incorporate the metabolic/energy cues into DNA damage response and links the aging‐controlling Mdm2–p53 and mTOR‐S6K pathways.     

OTUB1 modulates c‐IAP1 stability to regulate signalling pathways

The cellular inhibitor of apoptosis (c‐IAP) proteins are E3 ubiquitin ligases that are critical regulators of tumour necrosis factor (TNF) receptor (TNFR)‐mediated signalling. Through their E3 ligase activity c‐IAP proteins promote ubiquitination of receptor‐interaction protein 1 (RIP1), NF‐κB‐inducing kinase (NIK) and themselves, and regulate the assembly of TNFR signalling complexes. Consequently, in the absence of c‐IAP proteins, TNFR‐mediated activation of NF‐κB and MAPK pathways and the induction of gene expression are severely reduced. Here, we describe the identification of OTUB1 as a c‐IAP‐associated deubiquitinating enzyme that regulates c‐IAP1 stability. OTUB1 disassembles K48‐linked polyubiquitin chains from c‐IAP1 in vitro and in vivo within the TWEAK receptor‐signalling complex. Downregulation of OTUB1 promotes TWEAK‐ and IAP antagonist‐stimulated caspase activation and cell death, and enhances c‐IAP1 degradation. Furthermore, knockdown of OTUB1 reduces TWEAK‐induced activation of canonical NF‐κB and MAPK signalling pathways and modulates TWEAK‐induced gene expression. Finally, suppression of OTUB1 expression in zebrafish destabilizes c‐IAP (Birc2) protein levels and disrupts fish vasculature. These results suggest that OTUB1 regulates NF‐κB and MAPK signalling pathways and TNF‐dependent cell death by modulating c‐IAP1 stability.     

Regulated in Development and DNA Damage Responses -1 (REDD1) Protein Contributes to Insulin Signaling Pathway in Adipocytes

REDD1 (Regulated in development and DNA damage response 1) is a hypoxia and stress response gene and is a negative regulator of mTORC1. Since mTORC1 is involved in the negative feedback loop of insulin signaling, we have studied the role of REDD1 on insulin signaling pathway and its regulation by insulin. In human and murine adipocytes, insulin transiently stimulates REDD1 expression through a MEK dependent pathway. In HEK-293 cells, expression of a constitutive active form of MEK stabilizes REDD1 and protects REDD1 from proteasomal degradation mediated by CUL4A-DDB1 ubiquitin ligase complex. In 3T3-L1 adipocytes, silencing of REDD1 with siRNA induces an increase of mTORC1 activity as well as an inhibition of insulin signaling pathway and lipogenesis. Rapamycin, a mTORC1 inhibitor, restores the insulin signaling after downregulation of REDD1 expression. This observation suggests that REDD1 positively regulates insulin signaling through the inhibition of mTORC1 activity. In conclusion, our results demonstrate that insulin increases REDD1 expression, and that REDD1 participates in the biological response to insulin.     

Suppressing mTORC1 on the lysosome

The mechanistic target of rapamycin, mTOR, is a protein kinase that integrates environmental and nutritional inputs into regulation of cell growth and metabolism. Key outputs of mTOR signalling occur from the lysosome membrane in the form of the multi‐subunit mTOR complex 1 (mTORC1), which phosphorylates multiple targets. While class I phosphoinositide kinase (PI3K‐I) is a well‐known activator of mTORC1, a recent paper (Marat et al, 2017) shows that a class II PI3K with a different substrate specificity, PI3K‐C2β, serves to inhibit mTORC1 on lysosomes under conditions of growth factor deprivation.     

Signalling pathways involved in hypoxia‐induced renal fibrosis

Renal fibrosis is the common pathological hallmark of progressive chronic kidney disease (CKD) with diverse aetiologies. Recent researches have highlighted the critical role of hypoxia during the development of renal fibrosis as a final common pathway in end‐stage kidney disease (ESKD), which joints the scientist's attention recently to exploit the molecular mechanism underlying hypoxia‐induced renal fibrogenesis. The scaring formation is a multilayered cellular response and involves the regulation of multiple hypoxia‐inducible signalling pathways and complex interactive networks. Therefore, this review will focus on the signalling pathways involved in hypoxia‐induced pathogenesis of interstitial fibrosis, including pathways mediated by HIF, TGF‐β, Notch, PKC/ERK, PI3K/Akt, NF‐κB, Ang II/ROS and microRNAs. Roles of molecules such as IL‐6, IL‐18, KIM‐1 and ADO are also reviewed. A comprehensive understanding of the roles that these hypoxia‐responsive signalling pathways and molecules play in the context of renal fibrosis will provide a foundation towards revealing the underlying mechanisms of progression of CKD and identifying novel therapeutic targets. In the future, promising new effective therapy against hypoxic effects may be successfully translated into the clinic to alleviate renal fibrosis and inhibit the progression of CKD.     

Qiliqiangxin protects against anoxic injury in cardiac microvascular endothelial cells via NRG‐1/ErbB‐PI3K/Akt/mTOR pathway

Cardiac microvascular endothelial cells (CMECs) are important angiogenic components and are injured rapidly after cardiac ischaemia and anoxia. Cardioprotective effects of Qiliqiangxin (QL), a traditional Chinese medicine, have been displayed recently. This study aims to investigate whether QL could protect CMECs against anoxic injury and to explore related signalling mechanisms. CMECs were successfully cultured from Sprague‐Dawley rats and exposed to anoxia for 12 hrs in the absence and presence of QL. Cell migration assay and capillary‐like tube formation assay on Matrigel were performed, and cell apoptosis was determined by TUNEL assay and caspase‐3 activity. Neuregulin‐1 (NRG‐1) siRNA and LY294002 were administrated to block NRG‐1/ErbB and PI3K/Akt signalling, respectively. As a result, anoxia inhibited cell migration, capillary‐like tube formation and angiogenesis, and increased cell apoptosis. QL significantly reversed these anoxia‐induced injuries and up‐regulated expressions of NRG‐1, phospho‐ErbB2, phospho‐ErbB4, phospho‐Akt, phospho‐mammalian target of rapamycin (mTOR), hypoxia‐inducible factor‐1α (HIF‐1α) and vascular endothelial growth factor (VEGF) in CMECs, while NRG‐1 knockdown abolished the protective effects of QL with suppressed NRG‐1, phospho‐ErbB2, phospho‐ErbB4, phospho‐Akt, phospho‐mTOR, HIF‐1α and VEGF expressions. Similarly, LY294002 interrupted the beneficial effects of QL with down‐regulated phospho‐Akt, phospho‐mTOR, HIF‐1α and VEGF expressions. However, it had no impact on NRG‐1/ErbB signalling. Our data indicated that QL could attenuate anoxia‐induced injuries in CMECs via NRG‐1/ErbB signalling which was most probably dependent on PI3K/Akt/mTOR pathway.     

Polycystin‐1 affects cancer cell behaviour and interacts with mTOR and Jak signalling pathways in cancer cell lines

Polycystic Kidney Disease (PKD), which is attributable to mutations in the PKD1 and PKD2 genes encoding polycystin‐1 (PC1) and polycystin‐2 (PC2) respectively, shares common cellular defects with cancer, such as uncontrolled cell proliferation, abnormal differentiation and increased apoptosis. Interestingly, PC1 regulates many signalling pathways including Jak/STAT, mTOR, Wnt, AP‐1 and calcineurin‐NFAT which are also used by cancer cells for sending signals that will allow them to acquire and maintain malignant phenotypes. Nevertheless, the molecular relationship between polycystins and cancer is unknown. In this study, we investigated the role of PC1 in cancer biology using glioblastoma (GOS3), prostate (PC3), breast (MCF7), lung (A549) and colorectal (HT29) cancer cell lines. Our in vitro results propose that PC1 promotes cell migration in GOS3 cells and suppresses cell migration in A549 cells. In addition, PC1 enhances cell proliferation in GOS3 cells but inhibits it in MCF7, A549 and HT29 cells. We also found that PC1 up‐regulates mTOR signalling and down‐regulates Jak signalling in GOS3 cells, while it up‐regulates mTOR signalling in PC3 and HT29 cells. Together, our study suggests that PC1 modulates cell proliferation and migration and interacts with mTOR and Jak signalling pathways in different cancer cell lines. Understanding the molecular details of how polycystins are associated with cancer may lead to the identification of new players in this devastating disease.