Regulation of Cytokine Signal Transduction in Hematopoietic Stem Cells by Mammalian Target of Rapamycin

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1239-1239
Author(s):  
Emily A Partridge ◽  
Jesse Vrecenak ◽  
Miroslaw Kozlowski ◽  
Alan W Flake
Keyword(s):  
Signal Transduction ◽  
Growth Factor ◽  
Mammalian Target Of Rapamycin ◽  
Tgf Beta ◽  
Mtor Inhibition ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Rapamycin Treatment ◽  
Receptor Endocytosis ◽  
Smad Proteins

Abstract Abstract 1239 Background: Hematopoiesis is a coordinated process in which hematopoietic stem cells (HSCs) undergo self-renewal and differentiation to produce multilineage blood cells. Maintenance of the HSC compartment has been shown to require regulation by a diverse profile of cytokines including members of the receptor tyrosine kinase (RTK) superfamily, with thrombopoietin (TPO) identified as a central regulator of HSC fate. HSC regulation is also mediated by serine-threonine kinases, such as transforming growth factor-beta (TGF-beta). Improved understanding of the mechanisms regulating HSC proliferation, self-renewal, and quiescence is essential for improving transplantation, ex vivo expansion, and other therapeutic applications of these cells. Mammalian target of rapamycin (mTOR) is a central regulator of cellular metabolism, nutrient sensing and autophagy. mTOR has also emerged as a central regulator of HSC proliferation and self-renewal. Mouse models of mTOR hyperactivation demonstrate an imbalance of HSC proliferation and self-renewal leading to exhaustion of repopulating cells in a rapamycin-dependent manner, and pretreatment of human cord blood CD34+ cells with rapamycin has been shown to increase engraftment in immunodeficient mice. However, the mechanisms responsible for mTOR regulation of HSCs remain poorly understood. Here we show that inhibition of mTOR by rapamycin significantly enhances signal transduction through both the TPO and TGF-beta pathways by inhibiting receptor endocytosis, resulting in increased cell surface levels of growth factor receptors and enhanced engraftment in a mouse model of prenatal HSC transplantation. Methods: We employed primary and immortalized HSCs, as well as 293T cells stably overexpressing the thrombopoietin receptor, to study the effects of mTOR inhibition. Cells were treated with rapamycin for 72 hours prior to growth factor stimulation, and activation of signaling proteins were measured by immunoblotting and immunofluorescence studies. Surface receptor levels were determined by biotinylation and streptavidin immunoprecipitation. Clathrin- and caveolin-dependent endocytosis was inhibited by potassium depletion and nystatin treatment, respectively. Bone marrow transplantation studies were preformed in a well-characterized mouse model of in utero hematopoietic cell transplantation at 14 days gestational age, with engraftment quantified at 96 hours and 2 weeks postnatally. Results: Rapamycin treatment resulted in enhanced signal transduction through both the TPO and TGF-beta pathways, with significantly increased levels of phosphorylation and nuclear translocation of MAPK and Smad proteins respectively (Figure 1A). Biotinylation studies revealed increased levels of cell surface receptors following mTOR inhibition. Pharmacologic disruption of endocytosis was found to ablate the effects of rapamycin treatment, with equivalent and robust activation of MAPK and Smad proteins in rapamycin-treated and untreated cells (Figure 1B). To test the hypothesis that enhanced cytokine signaling would result in an optimal profile of HSC function, in utero transplantation of rapamycin-treated primary bone marrow cells was performed. Rapamycin treatment resulted in enhanced HSC engraftment levels at 96 hours post-transplantation (4.2 +/− 0.6% vs 23.8 +/− 2.2%, p<0.005), and postnatal chimerism levels assessed at 2 weeks of age were similarly found to be significantly higher in the rapamycin-treatment group compared to controls (2.2 +/− 0.6% vs 29.8 +/− 10.7%, p<0.005). Conclusions: These results support a role for mTOR in modulating HSC signal transduction by regulation of cytokine receptor endocytosis, resulting in enhanced levels of engraftment following transplantation. The observed effects of mTOR inhibition on multiple diverse cytokine profiles suggests a central mechanism of regulation of intracellular trafficking, which may be mediated at the level of formation of early endosomal and autophagosome complexes. Further studies will delineate receptor fate following internalization by these pathways. These studies will ultimately improve understanding of how the manipulation of multiple signaling circuits may be optimized to control self-renewal and quiescence compatible for in vitro expansion and transplantation of HSCs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Maintenance of Hematopoietic Stem Cells Through Regulation of Wnt and mTOR Pathways.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2309-2309
Author(s):  
Jian Huang ◽  
Peter S. Klein
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Signaling Pathways ◽  
Glycogen Synthase ◽  
Dependent Manner ◽  
Mtor Inhibition ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 2309 Hematopoietic stem cells (HSCs) maintain the ability to self-renew and to differentiate into all lineages of the blood. The signaling pathways regulating hematopoietic stem cell (HSCs) self-renewal and differentiation are not well understood. We are very interested in understanding the roles of glycogen synthase kinase-3 (Gsk3) and the signaling pathways regulated by Gsk3 in HSCs. In our previous study (Journal of Clinical Investigation, December 2009) using loss of function approaches (inhibitors, RNAi, and knockout) in mice, we found that Gsk3 plays a pivotal role in controlling the decision between self-renewal and differentiation of HSCs. Disruption of Gsk3 in bone marrow transiently expands HSCs in a b-catenin dependent manner, consistent with a role for Wnt signaling. However, in long-term repopulation assays, disruption of Gsk3 progressively depletes HSCs through activation of mTOR. This long-term HSC depletion is prevented by mTOR inhibition and exacerbated by b-catenin knockout. Thus GSK3 regulates both Wnt and mTOR signaling in HSCs, with opposing effects on HSC self-renewal such that inhibition of Gsk3 in the presence of rapamycin expands the HSC pool in vivo. In the current study, we found that suppression of the mammalian target of rapamycin (mTOR) pathway, an established nutrient sensor, combined with activation of canonical Wnt/ß-catenin signaling, allows the ex vivo maintenance of human and mouse long-term HSCs under cytokine-free conditions. We also show that combining two clinically approved medications that activate Wnt/ß-catenin signaling and inhibit mTOR increases the number of long-term HSCs in vivo. Disclosures: No relevant conflicts of interest to declare.

scholarly journals mTOR inhibitors lower an intrinsic barrier to virus infection mediated by IFITM3

2018 ◽  
Vol 115 (43) ◽  
pp. E10069-E10078 ◽  
Author(s):  
Guoli Shi ◽  
Stosh Ozog ◽  
Bruce E. Torbett ◽  
Alex A. Compton
Keyword(s):  
Influenza A ◽  
Lentiviral Vector ◽  
Sendai Virus ◽  
Mammalian Target Of Rapamycin ◽  
Mtor Inhibitors ◽  
Endosomal Trafficking ◽  
Mtor Inhibition ◽  
Rapamycin Treatment ◽  
Endosomal Sorting ◽  
Basal Expression

Rapamycin and its derivatives are specific inhibitors of mammalian target of rapamycin (mTOR) kinase and, as a result, are well-established immunosuppressants and antitumorigenic agents. Additionally, this class of drug promotes gene delivery by facilitating lentiviral vector entry into cells, revealing its potential to improve gene therapy efforts. However, the precise mechanism was unknown. Here, we report that mTOR inhibitor treatment results in down-regulation of the IFN-induced transmembrane (IFITM) proteins. IFITM proteins, especially IFITM3, are potent inhibitors of virus–cell fusion and are broadly active against a range of pathogenic viruses. We found that the effect of rapamycin treatment on lentiviral transduction is diminished upon IFITM silencing or knockout in primary and transformed cells, and the extent of transduction enhancement depends on basal expression of IFITM proteins, with a major contribution from IFITM3. The effect of rapamycin treatment on IFITM3 manifests at the level of protein, but not mRNA, and is selective, as many other endosome-associated transmembrane proteins are unaffected. Rapamycin-mediated degradation of IFITM3 requires endosomal trafficking, ubiquitination, endosomal sorting complex required for transport (ESCRT) machinery, and lysosomal acidification. Since IFITM proteins exhibit broad antiviral activity, we show that mTOR inhibition also promotes infection by another IFITM-sensitive virus, Influenza A virus, but not infection by Sendai virus, which is IFITM-resistant. Our results identify the molecular basis by which mTOR inhibitors enhance virus entry into cells and reveal a previously unrecognized immunosuppressive feature of these clinically important drugs. In addition, this study uncovers a functional convergence between the mTOR pathway and IFITM proteins at endolysosomal membranes.

scholarly journals The effect of rapamycin treatment on cerebral ischemia: A systematic review and meta-analysis of animal model studies

2018 ◽  
Vol 14 (2) ◽  
pp. 137-145 ◽  
Author(s):  
Daniel J Beard ◽  
Gina Hadley ◽  
Neal Thurley ◽  
David W Howells ◽  
Brad A Sutherland ◽  
...  
Keyword(s):  
Systematic Review ◽  
Cerebral Ischemia ◽  
Infarct Volume ◽  
Therapeutic Option ◽  
Meta Analysis ◽  
Mammalian Target Of Rapamycin ◽  
Point Estimate ◽  
Study Quality ◽  
Mtor Inhibition ◽  
Rapamycin Treatment

Background Amplifying endogenous neuroprotective mechanisms is a promising avenue for stroke therapy. One target is mammalian target of rapamycin (mTOR), a serine/threonine kinase regulating cell proliferation, cell survival, protein synthesis, and autophagy. Animal studies investigating the effect of rapamycin on mTOR inhibition following cerebral ischemia have shown conflicting results. Aim To conduct a systematic review and meta-analysis evaluating the effectiveness of rapamycin in reducing infarct volume in animal models of ischemic stroke. Summary of review Our search identified 328 publications. Seventeen publications met inclusion criteria (52 comparisons: 30 reported infarct size and 22 reported neurobehavioral score). Study quality was modest (median 4 of 9) with no evidence of publication bias. The point estimate for the effect of rapamycin was a 21.6% (95% CI, 7.6%–35.7% p < 0.01) improvement in infarct volume and 30.5% (95% CI 17.2%–43.8%, p < 0.0001) improvement in neuroscores. Effect sizes were greatest in studies using lower doses of rapamycin. Conclusion Low-dose rapamycin treatment may be an effective therapeutic option for stroke. Modest study quality means there is a potential risk of bias. We recommend further high-quality preclinical studies on rapamycin in stroke before progressing to clinical trials.

PI3-Kinase Inhibition Decreases the Proliferation of ALL Cells, Potentiates mTOR Inhibition, and Abrogates Growth Factor-Mediated Reversal of mTOR Inhibition.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1383-1383
Author(s):  
Jonathan Fish ◽  
Jessica Hulitt ◽  
Marlo Bruno ◽  
Stephan A. Grupp ◽  
Valerie I. Brown
Keyword(s):  
Signal Transduction ◽  
Cell Proliferation ◽  
Growth Factor ◽  
Cell Lines ◽  
Low Dose ◽  
Great Promise ◽  
Dependent Manner ◽  
Inhibitory Effects ◽  
Mtor Inhibition ◽  
The Impact

Abstract Biologically targeted cancer agents, including signal transduction inhibitors, have shown great promise in treating hematologic malignancies. However, used as single agents, these drugs may not be curative secondary to innate or acquired cellular resistance. Thus, acute lymphoblastic leukemia (ALL) and other cancer cells may become resistant to rapamycin, an mTOR inhibitor (MTI), following extended exposure to the drug. A strategy to overcome such resistance is to combine targeted agents, and thereby inhibit multiple targets simultaneously. Previously, we have shown activity of MTI in models of both human and murine ALL. In mouse models, treatment of ALL with MTI prolongs survival but may not cure disease. IL-7, a lymphoid growth factor important in the regulation of progenitor B cell development and proliferation, can reverse the inhibitory effects of MTI on human and murine pre-B ALL cells. We wished to further explore the mechanisms by which IL-7-mediated signaling protects ALL cells from the inhibitory effects of MTI, through the investigation of modulators of growth factor signaling in ALL. Thus, we have evaluated the impact of LY294002, an inhibitor of phosphatidyl inositol-3 kinase (PI3K). PI3K is a critical signaling molecule in cell survival and proliferation, with one of its central roles being signal transduction from growth factor receptors to the activation of AKT (an upstream regulator of mTOR). PI3K/AKT pathway over-activation has been implicated in many different cancers. Treatment of ALL cell lines with the PI3K inhibitor LY294002 markedly decreased cell proliferation in a dose-dependent manner. More importantly, the inhibitory effects of LY294002 were additive or synergistic with the inhibitory effects of MTI, and prevented the ability of IL-7 to reverse the inhibitory effects of rapamycin. Treatment of pre-B ALL cell lines with 2.5 μM LY294002 resulted in decreased proliferation to 20–45% of baseline as compared to untreated cells, whereas treatment with a higher dose (5 μM) reduced cell proliferation to 10–20%. Combinations of LY294002 and rapamycin, even at low doses, inhibited cell proliferation to a greater degree than each drug individually. Co-treatment with 2.5 μM LY294002 and low dose rapamycin (1 ng/ml) resulted in profound inhibition of proliferation to &lt;=5%, compared to 20–30% with rapamycin alone. Furthermore, co-treatment with low-dose LY294002 and low-dose rapamycin resulted in greater inhibition than even higher doses of each of these agents individually. While the addition of IL-7 (1 U/ml) to rapamycin-treated cells resulted in the reversal of rapamycin-mediated cell inhibition, the further addition of 2.5 μM LY294002 significantly antagonized this growth factor rescue of MTI-treated ALL cells. The blockade by LY294002 of the IL-7 effect was most apparent in ALL cell lines that were IL-7 dependent, with cell proliferation reduced to &lt;20%. However, the effects were still significant in IL-7 independent cell lines, with proliferation reduced to 20–60%. Similar results were seen using human ALL cell lines. These data suggest that the PI3K signaling pathway serves as a potential rescue pathway from mTOR inhibition, mediating the ability of growth factors to rescue cells from rapamycin;PI3K itself is a therapeutic target for ALL; andcombination therapy with MTI and PI3K inhibitors may be more active than either agent alone.

scholarly journals Activation of ULK1 Kinase Mediates Clearance of Free Alpha-Globin in Human Beta-Thalassemic Erythroblasts

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 411-411
Author(s):  
Christophe Lechauve ◽  
Julia Keith ◽  
Eugene Khandros ◽  
Stephanie Fowler ◽  
Kalin Mayberry ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Protein Quality ◽  
Therapeutic Potential ◽  
Dependent Manner ◽  
Ineffective Erythropoiesis ◽  
Advisory Committees ◽  
Mtor Inhibition ◽  
Hematopoietic Stem ◽  
Rapamycin Treatment ◽  
Α Chain

Abstract β-Thalassemia is a common, frequently debilitating, inherited anemia caused by HBB gene mutations that reduce or eliminate the expression of the β-globin subunit of adult hemoglobin (HbA, α2β2). Consequently, excess free α-globin forms toxic precipitates in red blood cells (RBCs) and their precursors, leading to ineffective erythropoiesis and hemolytic anemia. Previously, we showed that free α-globin is eliminated by protein quality-control pathways, including the ubiquitin-proteasome system and autophagy (Khandros et al., Blood 2012;119:5265). In β-thalassemic mice, disruption of the Unc-51-like autophagy activating kinase gene (Ulk1) increased α-globin precipitates and worsened the pathologies of β-thalassemia. Treatment of β-thalassemic mice with rapamycin to inhibit mTOR (an ULK1 inhibitor) reduced α-globin precipitates, lessened ineffective erythropoiesis, and increased the lifespan of circulating RBCs in an Ulk1-dependent fashion. To investigate the therapeutic potential of rapamycin in human β-thalassemia, we treated erythroid precursors generated by in vitro differentiation of patient-derived CD34+ hematopoietic stem and progenitor cells. Reverse-phase high-performance liquid chromatography (HPLC) analysis of hemoglobinized erythroblasts generated from transfusion-dependent (TD, n = 5) or non-transfusion-dependent (NTD, n = 5) β-thalassemia patients revealed α-chain excesses (α-chain/β-like [β + γ + δ] chain) of approximately 40% and 15%, respectively (compared to 7 normal donors; P < 0.001). Rapamycin (10µM or 20µM) or the proteasome inhibitor MG132 (2.5µM) was added to day 13 cultures, which contained mid- to late-stage erythroblasts, and α-globin accumulation was determined by HPLC 2 days later. As expected, proteasome inhibition by MG132 raised free α-globin levels in thalassemic erythroblasts (P < 0.01) and induced cell death (P < 0.01). In contrast, rapamycin reduced free α-globin in a dose-dependent manner by 40% and 85% in TD (P < 0.0001) and NTD β-thalassemia (P < 0.001), respectively, but had no effect on erythroblasts derived from normal CD34+ cells (figure). We also observed decreases in the accumulation of autophagic markers, such as SQSTM1/p62 protein, by Western blotting. We observed no negative effects of rapamycin on the survival of patient-derived erythroblasts. Also of note, under our experimental conditions, rapamycin treatment of erythroblasts did not induce fetal hemoglobin production, as has been previously reported, thereby excluding this potential mechanism for reducing globin chain imbalances. Overall, rapamycin treatment significantly reduced the accumulation of free α-globin in TD β-thalassemia and almost fully corrected the imbalance in NTD β-thalassemia cells. Our findings identify a new drug-regulatable pathway for ameliorating β-thalassemia. Rapamycin is approved and well studied, and it has a generally manageable toxicity profile. Moreover, there are additional pharmacologic approaches to activating ULK via mTOR inhibition or other pathways. These approaches may lead to effective drug therapies for β-thalassemia, particularly NTD or intermittently TD forms of the disease. Disclosures Cappellini: Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Vifor: Membership on an entity's Board of Directors or advisory committees; Sanofi/Genzyme: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria.

scholarly journals Growth Factor Receptor-Bound Protein 10 (Grb10) Regulates Hematopoietic Stem Cell (HSC) Self-Renewal Via Control of mTOR Signaling

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1160-1160
Author(s):  
Xiao Yan ◽  
Heather A Himburg ◽  
Phuong L Doan ◽  
Mamle Quarmyne ◽  
Evelyn Tran ◽  
...  
Keyword(s):  
Stem Cell ◽  
Growth Factor ◽  
Hematopoietic Stem Cell ◽  
Growth Factor Receptor ◽  
Post Transplantation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
Duke University

Abstract Elucidation of the mechanisms governing HSC regeneration has been impeded by difficulty in isolating HSCs early following genotoxic injury, such as total body irradiation (TBI). Using multiparametric flow cytometric cell sorting of BM ckit+sca-1+lin- cells coupled with gene expression analysis, we identified growth factor receptor-bound protein 10 (Grb10), a co-receptor which regulates Insulin Receptor/IGF-1 signaling, to be significantly overexpressed by BM KSL cells at the earliest detectable point of regeneration (day +10) following TBI (3.3-fold, p<0.0001). Grb10 is a member of the imprinted gene family which is predominately expressed in the stem cells of a variety of tissues, including embryonic stem cells, bone marrow, skin and muscle. Viral shRNA-mediated knockdown of Grb10 in BM KSL cells caused a significant decrease in KSL cells and colony forming cells (CFCs) detected in 7-day culture (p=0.03 and p=0.002, respectively). Furthermore, mice competitively transplanted with Grb10-deficient HSCs displayed 10-fold lower donor, multilineage hematopoietic cell engraftment than mice transplanted with Grb10-expressing HSCs (p=0.007 for %CD45.1+ donor cells). Secondary competitive repopulation assays confirmed a greater than 10-fold deficit in long-term repopulating capacity in Grb10-deficient KSL cells compared to Grb10-expressing KSL cells (p=0.006 for %CD45.1+ donor cells). In order to determine if Grb10 was necessary for HSC maintenance and normal hematopoiesis in vivo, we generated maternally-derived Grb10-deficient mice. Heterozygous 8 week old Grb10m/+ (1 mutant allele, 1 wild type allele) had 10-fold decreased Grb10 expression in BM lin-cells. BM CFCs and SLAM+ KSL cells were significantly decreased in Grb10m/+ mice compared to Grb10+/+ mice (p=0.006 and p=0.04, respectively). Competitive repopulation assays demonstrated significantly decreased donor hematopoietic cell repopulation in recipient mice transplanted with Grb10m/+ BM cells versus mice transplanted with Grb10+/+ BM cells (p=0.003 for %CD45.1+ donor cells). Mice transplanted with BM cells from homozygous Grb10-/- mice showed a similar decrease in donor-derived hematopoietic repopulation compared to mice transplanted with BM cells from Grb10+/+ mice (p=0.02 at 20 weeks post-transplantation). These results confirmed that Grb10 regulates HSC self-renewal capacity in vivo. To determine whether Grb10 regulates HSC regeneration after myelotoxic injury, we irradiated Grb10m/+ mice with 550cGy TBI, and monitored hematopoietic recovery over time in comparison to Grb10+/+ controls. Interestingly, Grb10m/+ mice displayed accelerated hematopoietic regeneration early following TBI. At day+10 after 550cGy, Grb10m/+ mice contained significantly increased numbers of BM SLAM+ KSL cells (p=0.04) and CFCs (p=0.009), compared to Grb10+/+ littermates. Similarly, mice transplanted with BM cells from irradiated, Grb10m/+ mice displayed 5-fold increased donor hematopoietic repopulation at 20 weeks post-transplantation compared to mice transplanted with BM cells from irradiated, Grb10+/+ mice (p=0.006). These data suggest that Grb10 deficiency accelerates hematopoietic recovery in the early period following myelosuppressive radiation injury. Mechanistically, Grb10-deficiency caused an increase in the percentage of BM KSL cells in G1 and G2/S/M phase of cell cycle compared to Grb10+/+ KSL cells (p=0.003). We also observed significantly increased levels of mTOR activation in Grb10m/+ BM KSL cells compared to Grb10+/+ BM KSL cells (p=0.001 for pS6, p=0.001 for pS6k and p=0.02 for p4EBP1). Furthermore, mTOR inhibition via siRNA-mTOR targeting rescued the defect in BM hematopoietic progenitor content (colony forming cells) in Grb10-deficient BM cells (p<0.0001). Taken together, our results suggest that Grb10 is necessary for HSC maintenance in steady state, while, paradoxically, Grb10 inhibition accelerates HSC regeneration early following injury. Furthermore, our data suggest that Grb10 mediates these effects via regulation of mTOR signaling. Selective modulation of Grb10 signaling has the potential to augment HSC self-renewal in steady state and to accelerate HSC regeneration following myelotoxic injury. Disclosures Himburg: Duke University: Patents & Royalties: Patent Application for use of Pleiotrophin as a hematopoietic stem cell growth factor. Chute:C2 Regenerate: Equity Ownership; Duke University: Patents & Royalties: Application to use PTN as growth factor as hematopoietic stem cell growth factor.

Signal transduction pathways that regulate muscle growth

2008 ◽  
Vol 44 ◽  
pp. 99-108 ◽  
Author(s):  
Henning Wackerhage ◽  
Aivaras Ratkevicius
Keyword(s):  
Signal Transduction ◽  
Protein Synthesis ◽  
Growth Factor ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Muscle Growth ◽  
Mammalian Target Of Rapamycin ◽  
High Growth ◽  
Signal Transduction Pathways ◽  
Cellular Functions

Progressive high-resistance exercise with 8–12 repetitions per set to near failure for beginners and 1–12 repetitions for athletes will increase muscle protein synthesis for up to 72 h; approx. 20 g of protein, especially when ingested directly after exercise, will promote high growth by elevating protein synthesis above breakdown. Muscle growth is regulated by signal transduction pathways that sense and compute local and systemic signals and regulate various cellular functions. The main signalling mechanisms are the phosphorylation of serine, threonine and tyrosine residues by kinases and their dephosphorylation by phosphatases. Muscle growth is stimulated by the mTOR (mammalian target of rapamycin) system, which senses (i) IGF-1 (insulin-like growth factor 1)/MGF (mechano-growth factor)/insulin and/or (ii) mechanical signals, (iii) amino acids and (iv) the energetic state of the muscle, and regulates protein synthesis accordingly. The action of the mTOR system is opposed by myostatin-Smad signalling which inhibits muscle growth via gene transcription.

Enhanced jun gene expression is an early genomic response to transforming growth factor beta stimulation

1989 ◽  
Vol 9 (3) ◽  
pp. 1255-1262
Author(s):  
L Pertovaara ◽  
L Sistonen ◽  
T J Bos ◽  
P K Vogt ◽  
J Keski-Oja ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Transcription Factors ◽  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
A549 Cells ◽  
Base Line ◽  
Tgf Beta ◽  
Early Effect ◽  
Growth Factor Beta

Transforming growth factor beta (TGF beta) is a multifunctional polypeptide that regulates proliferation, differentiation, and other functions of many cell types. The pathway of TGF beta signal transduction in cells is unknown. We report here that an early effect of TGF beta is an enhancement of the expression of two genes encoding serum- and phorbol ester tumor promoter-regulated transcription factors: the junB gene and the c-jun proto-oncogene, respectively. This stimulation was observed in human lung adenocarcinoma A549 cells which were growth inhibited by TGF beta, AKR-2B mouse embryo fibroblasts which were growth stimulated by TGF beta, and K562 human erythroleukemia cells, which were not appreciably affected in their growth by TGF beta. The increase in jun mRNA occurred with picomolar TGF beta concentrations within 1 h of TGF beta stimulation, reached a peak between 1 and 5 h in different cells, and declined gradually to base-line levels. This mRNA response was followed by a large increase in the biosynthesis of the c-jun protein (AP-1), as shown by metabolic labeling and immunoprecipitation analysis. However, differential and cell type-specific regulation appeared to determine the timing and magnitude of the response of each jun gene in a given cell. In AKR-2B and NIH 3T3 cells, only junB was induced by TGF beta, evidently in a protein synthesis-independent fashion. The junB response to TGF beta was maintained in c-Ha-ras and neu oncogene-transformed cells. Thus, one of the earliest genomic responses to TGF beta may involve nuclear signal transduction and amplification by the junB and c-jun transcription factors in concert with c-fos, which is also induced. The differential activation of the jun genes may explain some of the pleiotropic effects of TGF beta.

RLIP76, an effector of the GTPase Ral, interacts with the AP2 complex: involvement of the Ral pathway in receptor endocytosis

2000 ◽  
Vol 113 (16) ◽  
pp. 2837-2844 ◽  
Author(s):  
V. Jullien-Flores ◽  
Y. Mahe ◽  
G. Mirey ◽  
C. Leprince ◽  
B. Meunier-Bisceuil ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Growth Factor ◽  
Egf Receptor ◽  
The Other ◽  
Ras Signaling ◽  
Receptor Endocytosis ◽  
Epidermal Growth ◽  
Two Hybrid

RLIP76 is a modular protein that was identified as a putative effector of Ral, a GTPase activated during Ras signaling. To explore further the contribution of the Ral-RLIP76 pathway to Ras signaling, we have looked for partners of RLIP76. Mu2, the medium chain of the AP2 complex is shown to interact with RLIP76. We show also that in vivo endogenous AP2 and RLIP76 form a complex and that this in vivo interaction is independent of cells being stimulated by a growth factor. Furthermore, RLIP76 differentiates AP2 from AP1 in vivo as RLIP76 differentiates mu2 from mu1 in vitro and in two hybrid assays. We show that activated Ral interferes with both tranferrin receptor endocytosis and epidermal growth factor (EGF) receptor endocytosis in HeLa cells. We propose a model where the Ral-RLIP76 pathway connects signal transduction and endocytosis through interaction on one hand between the Ras-Ral pathway and RLIP, on the other hand between RLIP and proteins belonging to the endocytotic machinery.

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