Mechanistic Target Of Rapamycin Activity Research Articles

A role of dentate gyrus mechanistic target of rapamycin activation in epileptogenesis in a mouse model of posttraumatic epilepsy.

The mechanistic target of rapamycin (mTOR) pathway has been implicated in promoting epileptogenesis in animal models of acquired epilepsy, such as posttraumatic epilepsy (PTE) following traumatic brain injury (TBI). However, the specific anatomical regions and neuronal populations mediating mTOR's role in epileptogenesis are not well defined. In this study, we tested the hypothesis that mTOR activation in dentate gyrus granule cells promotes neuronal death, mossy fiber sprouting, and PTE in the controlled cortical impact (CCI) model of TBI. An adeno-associated virus (AAV)-Cre viral vector was injected into the hippocampus of Rptorflox/flox (regulatory-associated protein of mTOR) mutant mice to inhibit mTOR activation in dentate gyrus granule cells. Four weeks after AAV-Cre or AAV-vehicle injection, mice underwent CCI injury and were subsequently assessed for mTOR pathway activation by Western blotting, neuronal death, and mossy fiber sprouting by immunopathological analysis, and posttraumatic seizures by video-electroencephalographic monitoring. AAV-Cre injection primarily affected the dentate gyrus and inhibited hippocampal mTOR activation following CCI injury. AAV-Cre-injected mice had reduced neuronal death in dentate gyrus detected by Fluoro-Jade B staining and decreased mossy fiber sprouting by ZnT3 immunostaining. Finally, AAV-Cre-injected mice exhibited a decrease in incidence of PTE. mTOR pathway activation in dentate gyrus granule cells may at least partly mediate pathological abnormalities and epileptogenesis in models of TBI and PTE. Targeted modulation of mTOR activity in this hippocampal network may represent a focused therapeutic approach for antiepileptogenesis and prevention of PTE.

Endothelial Mechanistic Target of Rapamycin Activation with Different Strains of R. rickettsii: Possible Role in Rickettsial Pathogenesis.

Rickettsia rickettsii is an obligate intracellular pathogen that primarily targets endothelial cells (ECs), leading to vascular inflammation and dysfunction. Mechanistic target of rapamycin (mTOR) regulates several cellular processes that directly affect host immune responses to bacterial pathogens. Here, we infected ECs with two R. rickettsii strains, avirulent (Iowa) and highly virulent Sheila Smith (SS) to identify differences in the kinetics and/or intensity of mTOR activation to establish a correlation between mTOR response and bacterial virulence. Endothelial mTOR activation with the highly virulent SS strain was significantly higher than with the avirulent Iowa strain. Similarly, there was increased LC3-II lipidation with the virulent SS strain compared with the avirulent Iowa strain of R. rickettsii. mTOR inhibitors rapamycin and Torin2 significantly increased bacterial growth and replication in the ECs, as evidenced by a more than six-fold increase in rickettsia copy numbers at 48 h post-infection. Further, the knockdown of mTOR with Raptor and Rictor siRNA resulted in a higher rickettsial copy number and the altered expression of the pro-inflammatory cytokines interleukin (IL)-1α, IL-6, and IL-8. These results are the first to reveal that endothelial mTOR activation and the early induction of autophagy might be governed by bacterial virulence and have established the mTOR pathway as an important regulator of endothelial inflammation, host immunity, and microbial replication.

Autophagy preserves hematopoietic stem cells by restraining MTORC1-mediated cellular anabolism

ABSTRACT Adult stem cells are long-lived and quiescent with unique metabolic requirements. Macroautophagy/autophagy is a fundamental survival mechanism that allows cells to adapt to metabolic changes by degrading and recycling intracellular components. Here we address why autophagy depletion leads to a drastic loss of the stem cell compartment. Using inducible deletion of autophagy specifically in adult hematopoietic stem cells (HSCs) and in mice chimeric for autophagy-deficient and normal HSCs, we demonstrate that the stem cell loss is cell-intrinsic. Mechanistically, autophagy-deficient HSCs showed higher expression of several amino acid transporters (AAT) when compared to autophagy-competent cells, resulting in increased amino acid (AA) uptake. This was followed by sustained MTOR (mechanistic target of rapamycin) activation, with enlarged cell size, glucose uptake and translation, which is detrimental to the quiescent HSCs. MTOR inhibition by rapamycin treatment in vivo was able to rescue autophagy-deficient HSC loss and bone marrow failure and resulted in better reconstitution after transplantation. Our results suggest that targeting MTOR may improve aged stem cell function, promote reprogramming and stem cell transplantation. List of abbreviations: 5FU: fluoracil; AA: amino acids; AKT/PKB: thymoma viral proto-oncogene 1; ATF4: activating transcription factor 4; BafA: bafilomycin A1; BM: bone marrow; EIF2: eukaryotic initiation factor 2; EIF4EBP1/4EBP1: eukaryotic translation initiation factor 4E binding protein 1; KIT/CD117/c-Kit: KIT proto-oncogene receptor tyrosine kinase; HSCs: hematopoietic stem cells; HSPCs: hematopoietic stem and progenitor cells; Kyn: kynurenine; LSK: lineage− (Lin−), LY6A/Sca-1+, KIT/c-Kit/CD117+; LY6A/Sca-1: lymphocyte antigen 6 family member A; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; MTORC2: MTOR complex 2; OPP: O-propargyl-puromycin; PI3K: phosphoinositide 3-kinase; poly(I:C): polyinosinic:polycytidylic acid; RPS6/S6: ribosomal protein S6; tam: tamoxifen; TCA: tricarboxylic acid; TFEB: transcription factor EB; PTPRC/CD45: Protein Tyrosine Phosphatase Receptor Type C, CD45 antigen.

Inhibition of autophagy and induction of glioblastoma cell death by NEO214, a perillyl alcohol-rolipram conjugate

ABSTRACT Glioblastoma (GBM) is the most aggressive primary brain tumor, exhibiting a high rate of recurrence and poor prognosis. Surgery and chemoradiation with temozolomide (TMZ) represent the standard of care, but, in most cases, the tumor develops resistance to further treatment and the patients succumb to disease. Therefore, there is a great need for the development of well-tolerated, effective drugs that specifically target chemoresistant gliomas. NEO214 was generated by covalently conjugating rolipram, a PDE4 (phosphodiesterase 4) inhibitor, to perillyl alcohol, a naturally occurring monoterpene related to limonene. Our previous studies in preclinical models showed that NEO214 harbors anticancer activity, is able to cross the blood-brain barrier (BBB), and is remarkably well tolerated. In the present study, we investigated its mechanism of action and discovered inhibition of macroautophagy/autophagy as a key component of its anticancer effect in glioblastoma cells. We show that NEO214 prevents autophagy-lysosome fusion, thereby blocking autophagic flux and triggering glioma cell death. This process involves activation of MTOR (mechanistic target of rapamycin kinase) activity, which leads to cytoplasmic accumulation of TFEB (transcription factor EB), a critical regulator of genes involved in the autophagy-lysosomal pathway, and consequently reduced expression of autophagy-lysosome genes. When combined with chloroquine and TMZ, the anticancer impact of NEO214 is further potentiated and unfolds against TMZ-resistant cells as well. Taken together, our findings characterize NEO214 as a novel autophagy inhibitor that could become useful for overcoming chemoresistance in glioblastoma. Abbreviations: ATG: autophagy related; BAFA1: bafilomycin A1; BBB: blood brain barrier; CQ: chloroquine; GBM: glioblastoma; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MGMT: O-6-methylguanine-DNA methyltransferase; MTOR: mechanistic target of rapamycin kinase; MTORC: MTOR complex; POH: perillyl alcohol; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TMZ: temozolomide

Protein Kinase A and 5' AMP-Activated Protein Kinase Signaling Pathways Exert Opposite Effects on Induction of Autophagy in Luteal Cells.

In the absence of pregnancy the ovarian corpus luteum undergoes regression, a process characterized by decreased production of progesterone and structural luteolysis involving apoptosis. Autophagy has been observed in the corpus luteum during luteal regression. Autophagy is a self-degradative process important for balancing sources of cellular energy at critical times in development and in response to nutrient stress, but it can also lead to apoptosis. Mechanistic target of rapamycin (MTOR) and 5′ AMP-activated protein kinase (AMPK), key players in autophagy, are known to inhibit or activate autophagy, respectively. Here, we analyzed the signaling pathways regulating the initiation of autophagy in bovine luteal cells. In vivo studies showed increased activating phosphorylation of AMPKα (Thr172) and elevated content of LC3B, a known marker of autophagy, in luteal tissue during PGF2α-induced luteolysis. In vitro, AMPK activators 1) stimulated phosphorylation of regulatory associated protein of MTOR (RPTOR) leading to decreased activity of MTOR, 2) increased phosphorylation of Unc-51-Like Kinase 1 (ULK1) and Beclin 1 (BECN1), at sites specific for AMPK and required for autophagy initiation, 3) increased levels of LC3B, and 4) enhanced colocalization of autophagosomes with lysosomes indicating elevated autophagy. In contrast, LH/PKA signaling in luteal cells 1) reduced activation of AMPKα and phosphorylation of RPTOR, 2) elevated MTOR activity, 3) stimulated phosphorylation of ULK1 at site required for ULK1 inactivation, and 4) inhibited autophagosome formation as reflected by reduced content of LC3B-II. Pretreatment with AICAR, a pharmacological activator of AMPK, inhibited LH-mediated effects on RPTOR, ULK1 and BECN1. Our results indicate that luteotrophic signaling via LH/PKA/MTOR inhibits, while luteolytic signaling via PGF2α/Ca2+/AMPK activates key signaling pathways involved in luteal cell autophagy.

The active component of ginseng, ginsenoside Rb1, improves erythropoiesis in models of Diamond–Blackfan anemia by targeting Nemo-like kinase

Nemo-like kinase (NLK) is a member of the mitogen-activated protein kinase family of kinases and shares a highly conserved kinase domain with other mitogen-activated protein kinase family members. The activation of NLK contributes to the pathogenesis of Diamond–Blackfan anemia (DBA), reducing c-myb expression and mechanistic target of rapamycin activity, and is therefore a potential therapeutic target. Unlike other anemias, the hematopoietic effects of DBA are largely restricted to the erythroid lineage. Mutations in ribosomal genes induce ribosomal insufficiency and reduced protein translation, dramatically impacting early erythropoiesis in the bone marrow of patients with DBA. We sought to identify compounds that suppress NLK and increases erythropoiesis in ribosomal insufficiency. We report that the active component of ginseng, ginsenoside Rb1, suppresses NLK expression and improves erythropoiesis in in vitro models of DBA. Ginsenoside Rb1–mediated suppression of NLK occurs through the upregulation of miR-208, which binds to the 3′-UTR of NLK mRNA and targets it for degradation. We also compare ginsenoside Rb1–mediated upregulation of miR-208 with metformin-mediated upregulation of miR-26. We conclude that targeting NLK expression through miRNA binding of the unique 3′-UTR is a viable alternative to the challenges of developing small-molecule inhibitors to target the highly conserved kinase domain of this specific kinase.

Nucleotide-binding domain and leucine-rich-repeat-containing protein X1 deficiency induces nicotinamide adenine dinucleotide decline, mechanistic target of rapamycin activation, and cellular senescence and accelerates aging lung-like changes.

Mitochondrial dysfunction has long been implicated to have a causative role in organismal aging. A mitochondrial molecule, nucleotide‐binding domain and leucine‐rich‐repeat‐containing protein X1 (NLRX1), represents the only NLR family member that targets this cellular location, implying that NLRX1 probably establishes a fundamental link between mitochondrial functions and cellular physiology. However, the significance of NLRX1 function in cellular senescence, a key conceptual constituent in aging biology, is yet to be defined. Here, we demonstrate that molecular hallmarks involved in aging biology including NAD+ decline, and activation of mTOR, p53, and p16INK4A are significantly enhanced in NLRX1 deficiency in vitro. Mechanistic studies of replicative cellular senescence in the presence or absence of NLRX1 in vitro reveal that NLRX1‐deficient fibroblasts fail to maintain optimal NAD+/NADH ratio, which instigates the decline of SIRT1 and the activation of mTOR, p16INK4A, and p53, leading to the increase in senescence‐associated beta‐galactosidase (SA‐β‐gal)‐positive cells. Importantly, the enhanced cellular senescence response in NLRX1 deficiency is significantly attenuated by pharmacological inhibition of mTOR signaling in vitro. Finally, our in vivo murine studies reveal that NLRX1 decreases with age in murine lungs and NLRX1 deficiency in vivo accelerates pulmonary functional and structural changes that recapitulate the findings observed in human aging lungs. In conclusion, the current study provides evidence for NLRX1 as a crucial regulator of cellular senescence and in vivo lung aging.

THU317 - Cross-linking of liver metabolism - hedgehog as a key regulator of energy metabolism, regulating mechanistic target of rapamycin activity

THU317 - Cross-linking of liver metabolism - hedgehog as a key regulator of energy metabolism, regulating mechanistic target of rapamycin activity

69 Phosphorylation of mechanistic target of rapamycin in porcine blastocyst-stage embryos is dependent on the concentration of glutamine in the medium

Supplementation of glutamine to porcine embryo culture medium improves blastocyst development, increases leucine consumption, and enhances mitochondrial activity. In cancer cells, glutamine has been implicated in the phosphorylation and activation of mechanistic target of rapamycin (MTOR) to support rapid cellular proliferation. The objective of this study was to determine if phosphorylation of MTOR in porcine blastocysts was dependent upon the concentration of glutamine in the medium and presence of leucine, another activator of MTOR. Presumptive zygotes (n=732 per treatment across 6 replicates) were split into four groups and cultured in 0, 1, 3.75, or 10mM GlutaMAX, an L-glutamine alternative, with or without 0.2mM leucine. On Day 6, percentages of embryos developed to the blastocyst stage were recorded. Blastocyst-stage embryos (n=100 per treatment) were collected for immunoblotting to detect total MTOR and phosphorylated (Ser2448) MTOR (pMTOR) with 3 replicates per target. To assess MTOR and lysosomal colocalization as another indicator of MTOR activation, blastocyst-stage embryos (n=15 per treatment) were fixed, probed with antibodies against MTOR and lysosomal associated membrane protein 1 (LAMP1), and imaged on a confocal microscope. Data were analysed by using the GLM procedure in SAS 9.4 (SAS Institute Inc.), and differences between means were detected by using one-way ANOVA followed by a least significant difference test with P<0.05 declared significant. Compared with the other treatments, culture in 0mM GlutaMAX resulted in decreased blastocyst development with (26.2±4.8%) or without (23.2±3.0%) leucine. Embryos cultured in 3.75mM GlutaMAX had increased development to the blastocyst stage (+Leu: 49.7±4.2%; −Leu: 46.2±2.6%) compared with culture in 1mM (+Leu: 39.5±3.5%; −Leu: 37.8±2.1%). In the presence of leucine, culture in 0mM GlutaMAX resulted in decreased pMTOR abundance compared with 3.75 or 10mM GlutaMAX. In the absence of leucine, embryos cultured in 0mM GlutaMAX had decreased abundance of total MTOR compared with all other groups, and pMTOR abundance was decreased in embryos cultured in 0 or 1mM compared with 3.75 or 10mM GlutaMAX. Furthermore, embryos cultured in 0mM GlutaMAX had decreased colocalization of MTOR and LAMP1 compared with those cultured in 3.75 or 10mM GlutaMAX. Therefore, glutamine supplementation is sufficient to prompt MTOR phosphorylation in porcine embryos. Further analyses are examining the phosphorylation status of MTOR downstream targets and effects of inhibiting enzymes involved in glutaminolysis. Funding was provided by the United States Department of Agriculture, National Institute of Food and Agriculture (2019-67011-29543) and Food for the 21st Century at the University of Missouri.

Hypothalamic orexin and mechanistic target of rapamycin activation mediate sleep dysfunction in a mouse model of tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is a genetic disease related to hyperactivation of the mechanistic target of rapamycin (mTOR) pathway and manifested by neurological symptoms, such as epilepsy and sleep disorders. The pathophysiology of sleep dysfunction is poorly understood and is likely multifactorial, but may involve intrinsic biological regulators in the brain. Here, we characterized a mouse model of sleep disorders in TSC and investigated mechanisms of sleep dysfunction in this conditional knockout model involving inactivation of the Tsc1 gene in neurons and astrocytes (Tsc1GFAPCKO mice). Sleep studies utilizing EEG, EMG, and behavioral analysis found that Tsc1GFAPCKO mice have decreased REM sleep and impaired sleep-wake differentiation between light and dark phases. mTOR activity and orexin expression were increased in hypothalamic sections and cultured hypothalamic neurons from Tsc1GFAPCKO mice. Both the sleep abnormalities and increased orexin expression in Tsc1GFAPCKO mice were reversed by rapamycin treatment, indicating their dependence on mTOR activation. An orexin antagonist, suvorexant, also restored normal REM levels in Tsc1GFAPCKO mice. These results identify a novel mechanistic link between mTOR and orexin in the hypothalamus related to sleep dysfunction and suggest a targeted therapeutic approach to sleep disorders in TSC.

Inactivation of MTOR promotes autophagy-mediated epithelial injury in particulate matter-induced airway inflammation

ABSTRACT Particulate matter (PM) is able to induce airway epithelial injury, while the detailed mechanisms remain unclear. Here we demonstrated that PM exposure inactivated MTOR (mechanistic target of rapamycin kinase), enhanced macroautophagy/autophagy, and impaired lysosomal activity in HBE (human bronchial epithelial) cells and in mouse airway epithelium. Genetic or pharmaceutical inhibition of MTOR significantly enhanced, while inhibition of autophagy attenuated, PM-induced IL6 expression in HBE cells. Consistently, club-cell-specific deletion of Mtor aggravated, whereas loss of Atg5 in bronchial epithelium reduced, PM-induced airway inflammation. Interestingly, the augmented inflammatory responses caused by MTOR deficiency were markedly attenuated by blockage of downstream autophagy both in vitro and in vivo. Mechanistically, the dysregulation of MTOR-autophagy signaling was partially dependent on activation of upstream TSC2, and interacted with the TLR4-MYD88 to orchestrate the downstream NFKB activity and to regulate the production of inflammatory cytokines in airway epithelium. Moreover, inhibition of autophagy reduced the expression of EPS15 and the subsequent endocytosis of PM. Taken together, the present study provides a mechanistic explanation for how airway epithelium localized MTOR-autophagy axis regulates PM-induced airway injury, suggesting that activation of MTOR and/or suppression of autophagy in local airway might be effective therapeutic strategies for PM-related airway disorders. Abbreviations ACTB: actin beta; AKT: AKT serine/threonine kinase; ALI: air liquid interface; AP2: adaptor related protein complex 2; ATG: autophagy related; BALF: bronchoalveolar lavage fluid; COPD: chronic obstructive pulmonary disease; CXCL: C-X-C motif chemokine ligand; DOX: doxycycline; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; EPS15: epidermal growth factor receptor pathway substrate 15; HBE: human bronchial epithelial; H&E: hematoxylin & eosin; IKK: IKB kinase; IL: interleukin; LAMP2: lysosomal-associated membrane protein 2; LPS: lipopolysaccharide; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MTEC: mouse tracheal epithelial cells; MTOR: mechanistic target of rapamycin kinase; MYD88: MYD88 innate immune signal transduction adaptor; NFKB: nuclear factor of kappa B; NFKBIA: NFKB inhibitor alpha; PM: particulate matter; PtdIns3K: phosphatidylinositol 3-kinase; Rapa: rapamycin; RELA: RELA proto-oncogene, NFKB subunit; SCGB1A1: secretoglobin family 1A member 1; siRNA: small interfering RNAs; SQSTM1: sequestosome 1; TEM: transmission electronic microscopy; TLR4: toll like receptor 4; TSC2: TSC complex subunit 2.

Spliced X-box Binding Protein 1 Stimulates Adaptive Growth Through Activation of mTOR

The unfolded protein response plays versatile roles in physiology and pathophysiology. Its connection to cell growth, however, remains elusive. Here, we sought to define the role of unfolded protein response in the regulation of cardiomyocyte growth in the heart. We used both gain- and loss-of-function approaches to genetically manipulate XBP1s (spliced X-box binding protein 1), the most conserved signaling branch of the unfolded protein response, in the heart. In addition, primary cardiomyocyte culture was used to address the role of XBP1s in cell growth in a cell-autonomous manner. We found that XBP1s expression is reduced in both human and rodent cardiac tissues under heart failure. Furthermore, deficiency of XBP1s leads to decompensation and exacerbation of heart failure progression under pressure overload. On the other hand, cardiac-restricted overexpression of XBP1s prevents the development of cardiac dysfunction. Mechanistically, we found that XBP1s stimulates adaptive cardiac growth through activation of the mechanistic target of rapamycin signaling, which is mediated via FKBP11 (FK506-binding protein 11), a novel transcriptional target of XBP1s. Moreover, silencing of FKBP11 significantly diminishes XBP1s-induced mechanistic target of rapamycin activation and adaptive cell growth. Our results reveal a critical role of the XBP1s-FKBP11-mechanistic target of rapamycin axis in coupling of the unfolded protein response and cardiac cell growth regulation.

P53 induces miR-199a-3p to suppress mechanistic target of rapamycin activation in cisplatin-induced acute kidney injury.

How p53 participates in acute kidney injury (AKI) progress and what are the underlying mechanisms remain illusive. For this issue, it is important to probe into the role of p53 in cisplatin-induced AKI. We find that p53 was upregulated in cisplatin-induced AKI, yet, pifithrin-α inhibites the p53 expression to attenuated renal injury and cell apoptosis both in vivo cisplatin-induced AKI mice and in vitro HK-2 human renal tubular epithelial cells. To knock down p53 by siRNA significantly decreased the miRNA, miR-199a-3p, expression in HK-2 cells. Blockade of miR-199a-3p significantly reduced cisplatin-induced cell apoptosis and inhibited caspase-3 activity. Mechanistically, we identified that miR-199a-3p directly bound to mechanistic target of rapamycin (mTOR) 3'-untranslated region and overexpressed miR-199a-3p reduce the expression and phosphorylation of mTOR. Furthermore, we demonstrated that p53 inhibited mTOR activation through activating miR-199a-3p. In conclusion, our findings reveal that p53, upregulating the expression of miR-199a-3p affects the progress of cisplatin-induced AKI, which might provide a promising therapeutic target of AKI.

MPB, a novel berberine derivative, enhances lysosomal and bactericidal properties via TGF-β-activated kinase 1-dependent activation of the transcription factor EB.

Lysosome has a crucial role in clearance of endocytosed pathogens from the cell. Small molecules that can boost lysosome function and bactericidal ability to cope with subsequent infection are urgently needed. Here, we report that MPB, a novel berberine derivative, induced lysosome-based degradation and clearance of methicillin-resistant Staphylococcus aureus and enteroinvasive Escherichia coli in macrophages. MPB caused nuclear translocation of transcription factor EB (TFEB), which boosted expression of lysosome genes. TFEB silencing repressed the MPB-mediated enhancements in degradation and bacterial eradication. MPB switched on TFEB nuclear translocation by coupling 2 parallel signaling pathways. MPB-triggered JNK activation led to 14-3-3δ being released from TFEB, which, in turn, caused TFEB nuclear translocation. In addition, MPB induced AMPK activation and subsequent inhibition of mechanistic target of rapamycin activity, which also contributed to TFEB nuclear translocation. Importantly, genetical or pharmaceutical inhibition of TGF-β-activated kinase 1 (TAK1) reduced MPB action remarkably. MPB acted through TAK1 at lysine 158 to activate JNK and AMPK and, thus, induced TFEB-dependent bactericidal activity in macrophages. Therefore, our study reveals a novel mechanism by which MPB controls JNK and AMPK phosphorylation cascades to activate lysosomal function and bactericidal activity via TAK1 K158-dependent manner, which may offer insight into novel therapeutic strategies to control bacterial infection.-Liu, X., Zhang, N., Liu, Y., Liu, L., Zeng, Q., Yin, M., Wang, Y., Song, D., Deng, H. MPB, a novel berberine derivative, enhances lysosomal and bactericidal properties via TGF-β-activated kinase 1-dependent activation of the transcription factor EB.

1087 Age- and UV-induced overstimulation of mechanistic target of rapamycin activity in keratinocytes and dermal fibroblasts

1087 Age- and UV-induced overstimulation of mechanistic target of rapamycin activity in keratinocytes and dermal fibroblasts

PPP2R2D, a regulatory subunit of protein phosphatase2A, promotes gastric cancer growth and metastasis via mechanistic target of rapamycin activation.

Protein phosphatase2A (PP2A) is an essential serine/threonine protein phosphatase that regulates the basic activities of eukaryotes by dephosphorylating its substrates. The function and substrate specificity of PP2A are generally determined by its regulatory subunits. In the present study, the clinical significance and roles of PPP2R2D, one of the regulatory subunits of PP2A, were demonstrated in gastric cancer(GC) carcinogenesis. Through a tissue microarray and quantitative polymerase chain reaction analysis, it was demonstrated that PPP2R2D was commonly upregulated in GC samples. This upregulation was positively correlated with the patients' tumor stage (P<0.01), Tclassification (P<0.01) and N classification (P=0.01). Furthermore, a high expression of PPP2R2D was closely associated with poor prognosis of patients. Knockdown of PPP2R2D significantly inhibited the proliferation and migration of GC cells invitro, as well as the tumorigenicity and metastasis invivo in an animal GC model. By contrast, overexpression of PPP2R2D promoted GC cell proliferation and migration invitro. The analysis of underlying mechanisms indicated that PPP2R2D silencing decreased the phosphorylation level of mechanistic target of rapamycin (mTOR), thereby implicating that PPP2R2D is involved in the regulation of mTOR activity during tumorigenesis. Thus, the findings of the present study suggested that PPP2R2D may serve as a potential oncogene in GC and as a novel target for therapeutic strategies against this disease.

MTOR signaling is essential for the development of thymic epithelial cells and the induction of central immune tolerance

ABSTRACTThymic epithelial cells (TECs) are critical for the establishment and maintenance of appropriate microenvironment for the positive and negative selection of thymocytes and the induction of central immune tolerance. Yet, little about the molecular regulatory network on TEC development and function is understood. Here, we demonstrate that MTOR (mechanistic target of rapamycin [serine/threonine kinase]) is essential for proper development and functional maturation of TECs. Pharmacological inhibition of MTOR activity by rapamycin (RPM) causes severe thymic atrophy and reduction of TECs. TEC-specific deletion of Mtor causes the severe reduction of mTECs, the blockage of thymocyte differentiation and output, the reduced generation of thymic regulatory T (Treg) cells and the impaired expression of tissue-restricted antigens (TRAs) including Fabp2, Ins1, Tff3 and Chrna1 molecules. Importantly, specific deletion of Mtor in TECs causes autoimmune diseases characterized by enhanced tissue immune cell infiltration and the presence of autoreactive antibodies. Mechanistically, Mtor deletion causes overdegradation of CTNNB1/Beta-Catenin due to excessive autophagy and the attenuation of WNT (wingless-type MMTV integration site family) signaling in TECs. Selective inhibition of autophagy significantly rescued the poor mTEC development caused by Mtor deficiency. Altogether, MTOR is essential for TEC development and maturation by regulating proliferation and WNT signaling activity through autophagy. The present study also implies that long-term usage of RPM might increase the risk of autoimmunity by impairing TEC maturation and function.

Cathepsin K in Lymphangioleiomyomatosis: LAM Cell–Fibroblast Interactions Enhance Protease Activity by Extracellular Acidification

Lymphangioleiomyomatosis (LAM) is a rare disease in which LAM cells and fibroblasts form lung nodules and it is hypothesized that LAM nodule-derived proteases cause cyst formation and tissue damage. Onprotease gene expression profiling in whole lung tissue, cathepsin K gene expression was 40-fold overexpressed in LAM compared with control lung tissue (P ≤ 0.0001). Immunohistochemistry confirmed cathepsin K protein was expressed in LAM but not control lungs. Cathepsin K gene expression and protein and protease activity were detected in LAM-associated fibroblasts but not the LAM cell line 621-101. In lung nodules, cathepsin K immunoreactivity predominantly co-localized with LAM-associated fibroblasts. In vitro, fibroblast extracellular cathepsin K activity was minimal at pH 7.5 but significantly enhanced at pH 7 and 6. 621-101 cells reduced extracellular pH with acidification dependent on 621-101 mechanistic target of rapamycin activity and net hydrogen ion exporters, particularly sodium bicarbonate co-transporters and carbonic anhydrases, which were also expressed inLAM lung tissue. In LAM cell-fibroblast co-cultures, acidification paralleled cathepsin K activity, andboth were reduced by sodium bicarbonate co-transporter (P ≤ 0.0001) and carbonic anhydrase inhibitors (P = 0.0021). Our findings suggest that cathepsin K activity is dependent on LAM cell-fibroblast interactions, and inhibitors of extracellular acidification may be potential therapies for LAM.

Cathepsin B modulates lysosomal biogenesis and host defense against Francisella novicida infection.

Lysosomal cathepsins regulate an exquisite range of biological functions, and their deregulation is associated with inflammatory, metabolic, and degenerative diseases in humans. In this study, we identified a key cell-intrinsic role for cathepsin B as a negative feedback regulator of lysosomal biogenesis and autophagy. Mice and macrophages lacking cathepsin B activity had increased resistance to the cytosolic bacterial pathogen Francisella novicida Genetic deletion or pharmacological inhibition of cathepsin B down-regulated mechanistic target of rapamycin activity and prevented cleavage of the lysosomal calcium channel TRPML1. These events drove transcription of lysosomal and autophagy genes via transcription factor EB, which increased lysosomal biogenesis and activation of autophagy initiation kinase ULK1 for clearance of the bacteria. Our results identified a fundamental biological function of cathepsin B in providing a checkpoint for homeostatic maintenance of lysosome populations and basic recycling functions in the cell.

A novel curcumin analog binds to and activates TFEB in vitro and in vivo independent of MTOR inhibition

ABSTRACTAutophagy dysfunction is a common feature in neurodegenerative disorders characterized by accumulation of toxic protein aggregates. Increasing evidence has demonstrated that activation of TFEB (transcription factor EB), a master regulator of autophagy and lysosomal biogenesis, can ameliorate neurotoxicity and rescue neurodegeneration in animal models. Currently known TFEB activators are mainly inhibitors of MTOR (mechanistic target of rapamycin [serine/threonine kinase]), which, as a master regulator of cell growth and metabolism, is involved in a wide range of biological functions. Thus, the identification of TFEB modulators acting without inhibiting the MTOR pathway would be preferred and probably less deleterious to cells. In this study, a synthesized curcumin derivative termed C1 is identified as a novel MTOR-independent activator of TFEB. Compound C1 specifically binds to TFEB at the N terminus and promotes TFEB nuclear translocation without inhibiting MTOR activity. By activating TFEB, C1 enhances autophagy and lysosome biogenesis in vitro and in vivo. Collectively, compound C1 is an orally effective activator of TFEB and is a potential therapeutic agent for the treatment of neurodegenerative diseases.