Differential effects of PDCD4 depletion on protein synthesis in myoblast and myotubes
© Kakade et al.; licensee BioMed Central Ltd. 2014
Received: 1 November 2013
Accepted: 4 January 2014
Published: 9 January 2014
Reduced muscle mass is a hallmark of metabolic diseases like diabetes and cancer. The mammalian (mechanistic) target of rapamycin complex 1/S6 kinase 1 (mTORC1/S6K1) pathway is critical to the regulation of muscle protein synthesis and mass but its mechanism of action is not completely understood.
Using L6 myotubes, we characterized the regulation of programmed cell death 4 (PDCD4), a recently described substrate of S6K1. The abundance, but not Ser67 phosphorylation, of PDCD4 was sensitive to amino acid and serum deprivation: values in starved cells were 4.5X of control (P < 0.001). Refeeding had opposite effects. Growth factors, compared to amino acids, appeared more critical in regulating PDCD4 abundance. Furthermore, inhibition of mTORC1 or the proteasome prevented the refeeding-associated decrease in PDCD4 abundance. Amino acid and serum deprivation significantly increased PDCD4 binding to eIF4A (P < 0.05); this was reversed during refeeding. PDCD4 depletion by RNA interference had no significant effect on phenylalanine incorporation into myotube mixed proteins in control cells but further suppressed (30%) this measure in nutrient-deprived cells (P < 0.0005). This was not observed in myoblasts. In starved myotubes, PDCD4 depletion further reduced the association of eIF4G with eIF4E.
Our data suggest that in myotubes, PDCD4 abundance is sensitive to nutritional manipulation in an mTORC1 and proteasome depended manner. Furthermore, the role of PDCD4 in regulating protein synthesis appears dependent on the developmental state of the cell.
KeywordsPDCD4 mRNA translation S6K1 Protein synthesis Skeletal muscle
The mammalian (mechanistic) target of rapamycin complex 1/ribosomal protein S6 kinase 1 (mTORC1/S6K1) signalling is a critical regulator of skeletal muscle mass and metabolism, and mechanisms that regulate it are studied as possible targets for the treatment/prevention of loss of muscle mass in diverse muscle atrophying conditions [1, 2]. However, the exact mechanism by which S6K1 regulates muscle mass and metabolism remains to be identified. Substrates of S6K1 proposed to mediate its actions are all factors that associate with or regulate mRNA translation initiation. These include the ribosomal protein S6 (S6) and the eukaryotic mRNA translation initiation factor 4B (eIF4B), both of which upon activation induce mRNA translation initiation. S6K1 also phosphorylates eukaryotic mRNA translation elongation factor 2 (eEF2) kinase, an inhibitor of mRNA translation (reviewed in [3, 4]). In skeletal muscle, concurrent increase in phosphorylation of S6K1, S6 and eIF4B are observed in conditions that stimulate muscle protein synthesis, including resistance exercise, provision of amino acid, and stimulation with insulin/IGF-1 [1, 5, 6]. However, the functions/regulation of these substrates do not account for the actions of S6K1 in controlling mRNA translation initiation and muscle mass [6, 7], suggesting a role for other substrates of this kinase.
Programmed cell death 4 (PDCD4), (also known as MA3, TIS (topoisomerase inhibitor-suppressed) , H731 , and interleukin-12 inducible human gene 197/15a  (reviewed in )), is a more recently discovered substrate of S6K1 . In the hypo phosphorylated state, it binds to both eIF4A and eIF4G, leading to both the inhibition of the helicase activity of eIF4A and of the formation of eIF4F complex. These changes will lead to the suppression of translation of mRNA with secondary structures at their 5′-UTR ends [13, 14]. Upon mitogen stimulation, activated S6K1 phosphorylates Ser67 in PDCD4. This targets it for ubiquitination by the ubiquitin protein ligase beta-transducin repeat containing protein (β-TRCP) and subsequent degradation by the proteasome .
Much of what is known about PDCD4 is from cancer studies where PDCD4 is proposed to function as a cell cycle inhibitor/tumor suppressor. Loss of this protein is associated with invasion, progression or increased aggression of numerous, but not all , cancers, including ovarian , lung , breast , liver  and colon cancers . As a substrate of mTORC1/S6K1, PDCD4 may mediate the effect of this kinase pathway on protein synthesis in skeletal muscle. However, not much is known about the role or regulation of PDCD4 in muscle, the tissue that is quantitatively the most important in whole body protein metabolism. It was recently shown that the abundance of PDCD4 in rat skeletal muscle is sensitive to feeding and food deprivation cycle: its abundance increased in skeletal muscle of food-deprived rats, but in fed or refed rats, its abundance decreased along with increase in muscle fractional protein synthesis . These data suggest that interventions that regulate PDCD4 abundance may be explored in the treatment of muscle wasting, a feature of diseases like cancer, AIDS, and trauma. However this study was mainly correlative and did not examine whether or not mTORC1/S6K1 is required for PDCD4 regulation in muscle.
In the present work, using L6 myotubes, our specific objectives were to: 1) examine the requirement for mTORC1/S6K1 and the ubiquitin proteolytic system in regulating PDCD4; 2) examine the contribution of amino acids vs. growth factors in mediating the effect of nutrition on PDCD4; and 3) determine whether nutritional status affects the interaction of PDCD4 with components of eIF4F. Because others have suggested that signalling pathways that regulate protein metabolism may be regulated differently in myotubes versus myoblasts  and because the regulation of PDCD4 may depend on cell type , we also assessed the effect of PDCD4 depletion by RNA interference (RNAi) on myotube total and myofibrillar protein synthesis.
Abundance of PDCD4 in L6 myotubes is sensitive to medium composition and requires mTORC1 and the proteasome
Growth factors, but not amino acids, regulate PDCD4 abundance
Association of PDCD4 with eIF4A in L6 myotubes is sensitive to medium composition
PDCD4 depletion in myotubes had modest effect on protein synthesis
In this study, we demonstrated that in myotubes, the regulation of PDCD4 abundance was reversibly modified by a starvation-refeeding cycle. Collectively, the data presented here are the first evidence to demonstrate a requirement for mTORC1 and the proteasome in regulating the abundance of PDCD4 in muscle cells. We also presented evidence that, at least in myotubes, in the absence of growth factors, amino acids had little effect in regulating the abundance of this protein. Finally, in starved myotubes, and contrary to observations in myoblasts  and non muscle cells , depletion of PDCD4 had minimal effect on the incorporation of phenylalanine into myotube proteins. Rather, in starved myotubes, PDCD4 depletion further reduced eIF4G binding to eIF4E.
In spite of the fact that PDCD4 has been characterized as a substrate of S6K1 and an inhibitor of cap-dependent mRNA translation initiation, there is a paucity of information on the significance of PDCD4 in skeletal muscle. Also, it is unknown if the regulation of PDCD4, like mTORC1/S6K1, is sensitive to nutrients. In the present study, Ser67 and Ser457 phosphorylation of PDCD4 correlates poorly with its abundance. A requirement for mTORC1/S6K1 in regulating PDCD4 abundance suggests that PDCD4 may be phosphorylated on additional residues. However, PDCD4 degradation appears to depend specifically on Ser67 phosphorylation . It is also possible that phosphorylated PDCD4 does not accumulate because degradation by the proteasome is very rapid. However, in refed cells treated with MG132, Ser67 phosphorylated PDCD4 did not accumulate to a greater extent in comparison with cells not treated with the drug (data not shown).
Although amino acids can activate mTORC1 [23, 24], the effects of amino acids require some amount of insulin [25–28]. Our finding that leucine or a medium that contained all the 20 amino acids but lacked growth factors had insignificant effects on PDCD4 abundance is consistent with this view. AKT too may phosphorylate PDCD4 and target it for degradation . In fact, a requirement for serum rather than amino acids might implicate AKT rather than mTORC1/S6K1 in the phosphorylation and degradation of PDCD4 since AKT does not require amino acid for its activation . However, incubation with rapamycin would not only inhibit mTORC1/S6K1 but should lead to a greater activation of PI3K-AKT pathway due to the loss of negative inhibition conveyed by activated S6K1 (reviewed in ). In our study, the fact that inhibition with rapamycin during a 1-h refeeding completely prevented the disappearance of PDCD4 clearly suggests that mTORC1/S6K1 is the main pathway that targets PDCD4 for degradation in myotubes.
Our data showing that PDCD4 knock down suppressed incorporation of phenylalanine into myotube mixed proteins are surprising, given the characterization of the protein as an mRNA translation initiation inhibitor. Furthermore, depletion of PDCD4 in myoblasts ( and Figure 6) and in non muscle cells  increases protein synthesis. A possible explanation might be that the regulation of myofibrillar proteins, the predominant proteins in myotubes, is different from that of total protein. However, we showed that incorporation of phenylalanine into myofibrillar proteins in cells depleted of PDCD4 was 30% lower compared with cells with normal level of PDCD4 (see Additional file 2). We did not measure the rate of synthesis of sarcoplasmic proteins; nevertheless, our data showing a suppression of phenylalanine incorporation into total and myofibrillar proteins suggest that even if depletion of PDCD4 increased the synthesis of sarcoplasmic proteins, such an increase was likely too small to offset the decrease in myofibrillar protein synthesis. It is not clear how PDCD4 depletion would regulate eIF4G abundance and interaction with eIF4E, although there is evidence that PDCD4 can transcriptionally regulate the abundance of some proteins . However, there is no evidence that eIF4G is one of such proteins.
Combined with data from myoblasts and non muscle cells, our data suggest that the effect of PDCD4 on protein synthesis may depend on cell type and/or stage of development, as previously suggested . In this regard, although PDCD4 has been implicated in regulating the abundance of some proteins, including p21 (Waf1/Cip1)  and lysyl oxidase , only c-myb , procaspase-3  and p53  have been demonstrated as natural mRNA translation substrates of PDCD4. These are all factors involved in regulating cell proliferation and migration, and therefore of more relevance in proliferating cells. This is consistent with the notion that the effect of PDCD4 on mRNA translation and protein synthesis might depend on the physiological state of the cell. However, PDCD4 and its targets may still be relevant in regulating muscle protein synthesis and mass during muscle development and regeneration. For example during muscle hypertrophy or repair following injury, satellite cells need to be activated, leading to the proliferation of myoblasts that will subsequently fuse to form myotubes . These can then fuse with existing myofibers or be used to form new fibers [38, 39]. PDCD4 might be involved in this regulation. Consistent with this, abundance of PDCD4 increases during initiation of L6 differentiation into myotubes (Abdullahi A. and Adegoke O.A.J., unpublished observations).
We showed that in L6 myotubes, the regulation of PDCD4 abundance by nutritional factors is sensitive to mTORC1 and ubiquitin dependent proteolytic system. In the absence of growth factors, amino acids, including leucine, appear to play a minor role in regulating PDCD4 abundance. Unlike in proliferating myoblasts and non muscle cells, depletion of PDCD4 had, at best, only modest effect on myotube protein synthesis, indicating that the effect of PDCD4 in muscle cells is dependent on the physiological state of the cell. Additional studies are needed to dissect the mechanisms behind these differential effects of PDCD4.
Fetal Bovine Serum (FBS), Horse Serum (HS), Lipofectamine RNAiMax, Opti-MEM medium, and antibiotic/antimycotic reagents were purchased from Life Technologies (Burlington, Ontario Canada). Amino acid-free medium (RPMI 1640) was obtained from US Biological (Swampscott MA, USA). PDCD4 siRNA oligonucleotides, phosphatase and protease inhibitor cocktails were purchased from Sigma-Aldrich (St. Louis, MO, USA). Alpha Modification of Eagle’s Medium (AMEM) was obtained from Wisent (St-Bruno, Quebec, Canada).
Antibodies to eIF4A, eIF4G, phosphorylated (Thr389) S6K1, and horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit and anti-mouse) were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA). Antibody against PDCD4 was from Cell Signaling Technology or Santa Cruz Biotechnology (Santa Cruz, California, USA). Antibodies against phosphorylated (Ser67, Ser457) PDCD4 were from Sigma-Aldrich or Aviva Systems Biology (San Diego, California, USA).
L6 myoblasts were cultured in 12-well plates in growth medium (GM: AMEM supplemented with FBS and antibiotic-antimycotic agents to final concentrations of 10 and 1%, respectively) until they reached ~80% confluency. Cells were then shifted into differentiation medium (DM: AMEM as above except that 2% HS replaced 10% FBS). Experiments were carried out on day 4–5 of differentiation. For starvation experiments, myotubes were grown in differentiation medium or starved in amino-acid free, serum-free medium (amino acid-free RPMI 1640) for 12 h. They were then refed in DM for 1 or 3 h. To examine the roles of amino acids and growth factors in regulating PDCD4 abundance, in some experiments refeeding was done in incubation media of varied composition. To examine the requirement for mTORC1 or the ubiquitin dependent proteolytic system on the regulation of PDCD4, additional refeeding experiments were carried out in the presence of inhibitors of these pathways (50 nM rapamycin and 10 μM MG132, respectively) or equivalent volumes of DMSO. At the end of the experiments, cells were harvested in a lysis buffer (25 mM Tris, pH 7.5, 1 mM EDTA, 2% (w/v) sodium dodecyl sulphate (SDS), 1 mM DTT, supplemented with protease (10 μl/mL) and phosphatase (10 μl/mL) inhibitor cocktails).
Myotubes on day 4 of differentiation were transfected with 30 nM siRNA oligonucleotides designed against PDCD4 or with a proprietary scrambled oligonucleotide ((Sigma Aldrich, Oakville, Ontario Canada)), using Lipofectamine RNAiMax (Life Technologies) as previously described . We used the following PDCD4 siRNA oligonucleotides: PDCD4 #1 sense (GUCUUCUACUAUUACCAUA [dT] [dT]), PDCD4 #1 antisense (5′UAUGGUAAUAGUAGAAGAC [dT] [dT]), PDCD4 #2 sense (CUACUAUUACCAUAGACCA [dT] [dT]), and PDCD4 #2 antisense (UGGUCUAUGGUAAUAGUAG [dT] [dT]. Thirty eight hours after transfection, cells were cultured in DM or starvation medium. Phenylalanine incorporation into proteins was then measured by assessing the incorporation of radioactive phenylalanine into trichloroacetic acid (TCA) precipitable proteins [29, 40].
Western blotting and immunoprecipitation
Proteins were resolved on 7.5, 10, or 15% SDS-PAGE (depending on the antigens being studied), transferred onto polyvinylidene difluoride (PVDF) membranes, which were then immunoblotted for the indicated antigens, as previously described . Immunoblot signals were quantified using the Carestream Molecular Imaging software (version 188.8.131.52, Carestream Health Inc., Rochester, New York, USA).
To immunoprecipitate eIF4A or PDCD4, myotubes were cultured in 10-cm plates. Following appropriate treatments, cells were rinsed in ice-cold PBS and then lysed in 500 μl of ice-cold lysis buffer (40 mm HEPES (pH 7.5), 120 mM NaCl, 1 mM EDTA, 10 mM pyrophosphate, 10 mM glycerol 2 phosphate, 0.5 mM orthovanadate) supplemented with 0.03% CHAPS, 1 mM DTT, 0.5 mM NaV, 1 mM benzamidine, 6.25 mM N-ethyl-maleimide (NEM) and protease inhibitor cocktail (10 μL/mL). One hundred micrograms of myotube proteins were combined with either anti eIF4A or anti-PDCD4 (Santa Cruz biotechnology) antibodies and the mix rotated overnight at 4°C. The following day and in order to precipitate the antigen-antibody complex, 50 μL of re-suspended BioMag protein G–bound beads (GE Healthcare, Baie d’Urfe, Quebec, Canada) were added to each of the immunoprecipitation tubes and the suspension rocked gently at 4°C for 1 h. The beads were collected on a magnetic stand (Life Technologies) and washed 3 times with 0.1 M sodium phosphate buffer. After the last wash, beads were re-suspended in 1X SDS-PAGE sample buffer and boiled at 95°C for 2 minutes. Following a brief centrifugation, eluates were collected, separated on 10% SDS-PAGE, and blotted for PDCD4 and eIF4A.
Data are presented as means ± SEM. Treatment means were compared using a one-way analysis of variance (ANOVA) and differences among individual means assessed using the Bonferroni multiple comparison test or, as in Figures 5, 6 and 7, by paired Students T-tests. Analyses were done using GraphPAD (Version 6, GraphPAD Software, La Jolla California, USA). The level of significance was set at P < 0.05.
This work was supported by the Natural Sciences and Engineering Research Council of Canada Discovery Grant [#355984] and Faculty of Health York University Minor Research Grants to Olasunkanmi A.J. Adegoke.
- Adegoke OA, Abdullahi A, Tavajohi-Fini P: mTORC1 and the regulation of skeletal muscle anabolism and mass. Appl Physiol Nutr Metab. 2012, 37 (3): 395-406. 10.1139/h2012-009.View ArticlePubMedGoogle Scholar
- Glass D, Roubenoff R: Recent advances in the biology and therapy of muscle wasting. Ann N Y Acad Sci. 2010, 1211 (1): 25-36. 10.1111/j.1749-6632.2010.05809.x.View ArticlePubMedGoogle Scholar
- Ray BN, Kweon HK, Argetsinger LS, Fingar DC, Andrews PC, Carter-Su C: Research resource: identification of novel growth hormone-regulated phosphorylation sites by quantitative phosphoproteomics. Mol Endocrinol. 2012, 26 (6): 1056-1073. 10.1210/me.2011-1258.PubMed CentralView ArticlePubMedGoogle Scholar
- Sengupta S, Peterson TR, Sabatini DM: Regulation of the mTOR complex 1 pathway by nutrients, growth factors, and stress. Mol Cell. 2010, 40 (2): 310-322. 10.1016/j.molcel.2010.09.026.PubMed CentralView ArticlePubMedGoogle Scholar
- Kimball SR, Jefferson LS: Control of translation initiation through integration of signals generated by hormones, nutrients and exercise. J Biol Chem. 2010, 285 (38): 29027-29032. 10.1074/jbc.R110.137208. doi: 10.1074/jbc.R110.137208. Epub 2010 Jun 24. ReviewPubMed CentralView ArticlePubMedGoogle Scholar
- Mieulet V, Roceri M, Espeillac C, Sotiropoulos A, Ohanna M, Oorschot V, Klumperman J, Sandri M, Pende M: S6 kinase inactivation impairs growth and translational target phosphorylation in muscle cells maintaining proper regulation of protein turnover. Am J Physiol Cell Physiol. 2007, 293 (2): C712-C722. 10.1152/ajpcell.00499.2006.View ArticlePubMedGoogle Scholar
- Ruvinsky I, Meyuhas O: Ribosomal protein S6 phosphorylation: from protein synthesis to cell size. Trends Biochem Sci. 2006, 31 (6): 342-348. 10.1016/j.tibs.2006.04.003.View ArticlePubMedGoogle Scholar
- Onishi Y, Hashimoto S, Kizaki H: Cloning of the TIS gene suppressed by topoisomerase inhibitors. Gene. 1998, 215 (2): 453-459. 10.1016/S0378-1119(98)00313-8.View ArticlePubMedGoogle Scholar
- Yoshinaga H, Matsuhashi S, Fujiyama C, Masaki Z: Novel human PDCD4 (H731) gene expressed in proliferative cells is expressed in the small duct epithelial cells of the breast as revealed by an anti-H731 antibody. Pathol Int. 1999, 49 (12): 1067-1077. 10.1046/j.1440-1827.1999.00995.x.View ArticlePubMedGoogle Scholar
- Azzoni L, Zatsepina O, Abebe B, Bennett IM, Kanakaraj P, Perussia B: Differential transcriptional regulation of CD161 and a novel gene, 197/15a, by IL-2, IL-15, and IL-12 in NK and T cells. J Immunol. 1998, 161 (7): 3493-3500.PubMedGoogle Scholar
- Allgayer H: Pdcd4, a colon cancer prognostic that is regulated by a microRNA. Crit Rev Oncol Hematol. 2010, 73 (3): 185-191. 10.1016/j.critrevonc.2009.09.001.View ArticlePubMedGoogle Scholar
- Dorrello NV, Peschiaroli A, Guardavaccaro D, Colburn NH, Sherman NE, Pagano M: S6K1- and betaTRCP-mediated degradation of PDCD4 promotes protein translation and cell growth. Science. 2006, 314 (5798): 467-471. 10.1126/science.1130276.View ArticlePubMedGoogle Scholar
- Yang HS, Cho MH, Zakowicz H, Hegamyer G, Sonenberg N, Colburn NH: A novel function of the MA-3 domains in transformation and translation suppressor Pdcd4 is essential for its binding to eukaryotic translation initiation factor 4A. Mol Cell Biol. 2004, 24 (9): 3894-3906. 10.1128/MCB.24.9.3894-3906.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Yang HS, Jansen AP, Komar AA, Zheng X, Merrick WC, Costes S, Lockett SJ, Sonenberg N, Colburn NH: The transformation suppressor Pdcd4 is a novel eukaryotic translation initiation factor 4A binding protein that inhibits translation. Mol Cell Biol. 2003, 23 (1): 26-37. 10.1128/MCB.23.1.26-37.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Lankat-Buttgereit B, Goke R: The tumour suppressor Pdcd4: recent advances in the elucidation of function and regulation. Biol Cell. 2009, 101 (6): 309-317. 10.1042/BC20080191.View ArticlePubMedGoogle Scholar
- Wei NA, Liu SS, Leung TH, Tam KF, Liao XY, Cheung AN, Chan KK, Ngan HY: Loss of programmed cell death 4 (Pdcd4) associates with the progression of ovarian cancer. Mol Cancer. 2009, 8: 70-10.1186/1476-4598-8-70.PubMed CentralView ArticlePubMedGoogle Scholar
- Chen Y, Knösel T, Kristiansen G, Pietas A, Garber ME, Matsuhashi S, Ozaki I, Petersen I: Loss of PDCD4 expression in human lung cancer correlates with tumour progression and prognosis. J Pathol. 2003, 200 (5): 640-646. 10.1002/path.1378.View ArticlePubMedGoogle Scholar
- Afonja O, Juste D, Das S, Matsuhashi S, Samuels HH: Induction of PDCD4 tumor suppressor gene expression by RAR agonists, antiestrogen and HER-2//neu antagonist in breast cancer cells. Evidence for a role in apoptosis. Oncogene. 2004, 23 (49): 8135-8145. 10.1038/sj.onc.1207983.View ArticlePubMedGoogle Scholar
- Zhang S, Li J, Jiang Y, Xu Y, Qin C: Programmed cell death 4 (PDCD4) suppresses metastastic potential of human hepatocellular carcinoma cells. J Exp Clin Cancer Res. 2009, 28: 71-10.1186/1756-9966-28-71.PubMed CentralView ArticlePubMedGoogle Scholar
- Zargar S, Moreira TS, Samimi-Seisan H, Jeganathan S, Kakade D, Islam N, Campbell J, Adegoke OA: Skeletal muscle protein synthesis and the abundance of the mRNA translation initiation repressor PDCD4 are inversely regulated by fasting and refeeding in rats. Am J Physiol Endocrinol Metab. 2011, 300 (6): E986-E992. 10.1152/ajpendo.00642.2010.View ArticlePubMedGoogle Scholar
- Shi J, Luo L, Eash J, Ibebunjo C, Glass DJ: The SCF-Fbxo40 complex induces IRS1 ubiquitination in skeletal muscle, limiting IGF1 signaling. Dev Cell. 2011, 21 (5): 835-847. 10.1016/j.devcel.2011.09.011.View ArticlePubMedGoogle Scholar
- Lankat-Buttgereit B, Lenschen B, Schmidt H, Goke R: The action of Pdcd4 may be cell type specific: evidence that reduction of dUTPase levels might contribute to its tumor suppressor activity in Bon-1 cells. Apoptosis. 2008, 13 (1): 157-164. 10.1007/s10495-007-0153-x.View ArticlePubMedGoogle Scholar
- Blommaart EF, Luiken JJ, Blommaart PJ, van Woerkom GM, Meijer AJ: Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes. J Biol Chem. 1995, 270 (5): 2320-2326. 10.1074/jbc.270.5.2320.View ArticlePubMedGoogle Scholar
- Hara K, Yonezawa K, Weng QP, Kozlowski MT, Belham C, Avruch J: Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism. J Biol Chem. 1998, 273 (23): 14484-14494. 10.1074/jbc.273.23.14484.View ArticlePubMedGoogle Scholar
- Bolster DR, Jefferson LS, Kimball SR: Regulation of protein synthesis associated with skeletal muscle hypertrophy by insulin-, amino acid- and exercise-induced signalling. Proc Nutr Soc. 2004, 63 (2): 351-356. 10.1079/PNS2004355.View ArticlePubMedGoogle Scholar
- Crozier SJ, Kimball SR, Emmert SW, Anthony JC, Jefferson LS: Oral leucine administration stimulates protein synthesis in rat skeletal muscle. J Nutr. 2005, 135 (3): 376-382.PubMedGoogle Scholar
- Meijer AJ, Dubbelhuis PF: Amino acid signalling and the integration of metabolism. Biochem Biophys Res Commun. 2004, 313 (2): 397-403. 10.1016/j.bbrc.2003.07.012.View ArticlePubMedGoogle Scholar
- Rennie MJ, Bohe J, Smith K, Wackerhage H, Greenhaff P: Branched-chain amino acids as fuels and anabolic signals in human muscle. J Nutr. 2006, 136 (1 Suppl): 264S-268S.PubMedGoogle Scholar
- el-Naggar EA, Kanda F, Okuda S, Maeda N, Nishimoto K, Ishihara H, Chihara K: Direct effects of tumor necrosis factor alpha (TNF-alpha) on L6 myotubes. Kobe J Med Sci. 2004, 50 (1–2): 39-46.PubMedGoogle Scholar
- Nobukuni T, Joaquin M, Roccio M, Dann SG, Kim SY, Gulati P, Byfield MP, Backer JM, Natt F, Bos JL: Amino acids mediate mTOR/raptor signaling through activation of class 3 phosphatidylinositol 3OH-kinase. Proc Natl Acad Sci U S A. 2005, 102 (40): 14238-14243. 10.1073/pnas.0506925102.PubMed CentralView ArticlePubMedGoogle Scholar
- Guertin DA, Sabatini DM: The pharmacology of mTOR inhibition. Sci Signal. 2009, 2 (67): e24-Google Scholar
- Bitomsky N, Wethkamp N, Marikkannu R, Klempnauer KH: siRNA-mediated knockdown of Pdcd4 expression causes upregulation of p21(Waf1/Cip1) expression. Oncogene. 2008, 27 (35): 4820-4829. 10.1038/onc.2008.115.View ArticlePubMedGoogle Scholar
- Santhanam AN, Baker AR, Hegamyer G, Kirschmann DA, Colburn NH: Pdcd4 repression of lysyl oxidase inhibits hypoxia-induced breast cancer cell invasion. Oncogene. 2010, 29 (27): 3921-3932. 10.1038/onc.2010.158.PubMed CentralView ArticlePubMedGoogle Scholar
- Singh P, Wedeken L, Waters LC, Carr MD, Klempnauer KH: Pdcd4 directly binds the coding region of c-myb mRNA and suppresses its translation. Oncogene. 2011, 30 (49): 4864-4873. 10.1038/onc.2011.202.View ArticlePubMedGoogle Scholar
- Eto K, Goto S, Nakashima W, Ura Y, Abe SI: Loss of programmed cell death 4 induces apoptosis by promoting the translation of procaspase-3 mRNA. Cell Death Differ. 2012, 19 (4): 573-581. 10.1038/cdd.2011.126.PubMed CentralView ArticlePubMedGoogle Scholar
- Wedeken L, Singh P, Klempnauer KH: Tumor suppressor protein Pdcd4 inhibits translation of p53 mRNA. J Biol Chem. 2011, 286 (50): 42855-42862. 10.1074/jbc.M111.269456.PubMed CentralView ArticlePubMedGoogle Scholar
- Zammit PS, Partridge TA, Yablonka-Reuveni Z: The skeletal muscle satellite cell: the stem cell that came in from the cold. J Histochem Cytochem. 2006, 54 (11): 1177-1191. 10.1369/jhc.6R6995.2006.View ArticlePubMedGoogle Scholar
- Dhawan J, Rando TA: Stem cells in postnatal myogenesis: molecular mechanisms of satellite cell quiescence, activation and replenishment. Trends Cell Biol. 2005, 15 (12): 666-673. 10.1016/j.tcb.2005.10.007.View ArticlePubMedGoogle Scholar
- Kadi F, Ponsot E: The biology of satellite cells and telomeres in human skeletal muscle: effects of aging and physical activity. Scand J Med Sci Sports. 2010, 20 (1): 39-48. 10.1111/j.1600-0838.2009.00966.x.View ArticlePubMedGoogle Scholar
- Gulve EA, Dice JF: Regulation of protein synthesis and degradation in L8 myotubes. Effects of serum, insulin and insulin-like growth factors. Biochem J. 1989, 260 (2): 377-387.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.