New Hierarchical Phosphorylation Pathway of the Translational Repressor eIF4E-binding Protein 1 (4E-BP1) in Ischemia-Reperfusion Stress

Eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) is a translational repressor that is characterized by its capacity to bind specifically to eIF4E and inhibit its interaction with eIF4G. Phosphorylation of 4E-BP1 regulates eIF4E availability, and therefore, cap-dependent translation, in cell stress. This study reports a physiological study of 4E-BP1 regulation by phosphorylation using control conditions and a stress-induced translational repression condition, ischemia-reperfusion (IR) stress, in brain tissue. In control conditions, 4E-BP1 was found in four phosphorylation states that were detected by two-dimensional gel electrophoresis and Western blotting, which corresponded to Thr⁶⁹-phosphorylated alone, Thr⁶⁹- and Thr³⁶/Thr⁴⁵-phosphorylated, all these plus Ser⁶⁴ phosphorylation, and dephosphorylation of the sites analyzed. In control or IR conditions, no Thr³⁶/Thr⁴⁵ phosphorylation alone was detected without Thr⁶⁹ phosphorylation, and neither was Ser⁶⁴ phosphorylation without Thr³⁶/Thr⁴⁵/Thr⁶⁹ phosphorylation detected. Ischemic stress induced 4E-BP1 dephosphorylation at Thr⁶⁹, Thr³⁶/Thr⁴⁵, and Ser⁶⁴ residues, with 4E-BP1 remaining phosphorylated at Thr⁶⁹ alone or dephosphorylated. In the subsequent reperfusion, 4E-BP1 phosphorylation was induced at Thr³⁶/Thr⁴⁵ and Ser⁶⁴, in addition to Thr⁶⁹. Changes in 4E-BP1 phosphorylation after IR were according to those found for Akt and mammalian target of rapamycin (mTOR) kinases. These results demonstrate a new hierarchical phosphorylation for 4E-BP1 regulation in which Thr⁶⁹ is phosphorylated first followed by Thr³⁶/Thr⁴⁵ phosphorylation, and Ser⁶⁴ is phosphorylated last. Thr⁶⁹ phosphorylation alone allows binding to eIF4E, and subsequent Thr³⁶/Thr⁴⁵ phosphorylation was sufficient to dissociate 4E-BP1 from eIF4E, which led to eIF4E-4G interaction. These data help to elucidate the physiological role of 4E-BP1 phosphorylation in controlling protein synthesis.     

High-dose rapamycin induces apoptosis in human cancer cells by dissociating mTOR complex 1 and suppressing phosphorylation of 4E-BP1

mTOR, the mammalian target of rapamycin, has been widely implicated in signals that promote cell cycle progression and survival in cancer cells. Rapamycin, which inhibits mTOR with high specificity, has consequently attracted much attention as an anti-cancer therapeutic. Rapamycin suppresses phosphorylation of S6 kinase at nano-molar concentrations, however at higher micro-molar doses, rapamycin induces apoptosis in several human cancer cell lines. While much is known about the effect of low dose rapamycin treatment, the mechanistic basis for the apoptotic effects of high-dose rapamycin treatment is not understood. We report here that the apoptotic effects of high-dose rapamycin treatment correlate with suppressing phosphorylation of the mTOR complex 1 substrate, eukaryotic initiation factor 4E (eIF4E) binding protein-1 (4E-BP1). Consistent with this observation, ablation of eIF4E also resulted in apoptorsis in MDA-MB 231 breast cancer cells. We also provide evidence that the differential dose effects of rapamycin are correlated with partial and complete dissociation of Raptor from mTORC1 at low and high doses, respectively. In contrast with MDA-MB-231 cells, MCF-7 breast cancer cells survived rapamycin-induced suppression of 4E-BP1 phosphorylation. We show that survival correlated with a hyper-phosphorylation of Akt at S473 at high rapamycin doses, the suppression of which conferred rapamycin sensitivity. This study reveals that the apoptotic effect of rapamycin requires doses that completely dissociate Raptor from mTORC1 and suppress that phosphorylation of 4E-BP1 and inhibit eIF4E.     

Progranulin compensates for blocked IGF-1 signaling to promote myotube hypertrophy in C2C12 myoblasts via the PI3K/Akt/mTOR pathway

It is well known that growth hormone (GH)-induced IGF-1 signaling plays a dominant role in postnatal muscle growth. Our previous studies have identified a growth factor, progranulin (PGRN), that is co-induced with IGF-1 upon GH administration. This result prompted us to explore the function of PGRN and its association with IGF-1. In the present study, we demonstrated that, similar to IGF-1, PGRN can promote C2C12 myotube hypertrophy via the PI(3)K/Akt/mTOR pathway. Moreover, PGRN can rescue the muscle atrophy phenotypes in C2C12 myotube when IGF-1 signaling is blocked. This result shows that PGRN can substitute for IGF-1 signaling in the regulation of muscle growth. Our findings provide new insights into IGF-1-modulated complicated networks that regulate muscle growth.     

Ceramide 1-phosphate stimulates proliferation of C2C12 myoblasts

Recent studies have established specific cellular functions for different bioactive sphingolipids in skeletal muscle cells. Ceramide 1-phosphate (C1P) is an important bioactive sphingolipid that has been involved in cell growth and survival. However its possible role in the regulation of muscle cell homeostasis has not been so far investigated. In this study, we show that C1P stimulates myoblast proliferation, as determined by measuring the incorporation of tritiated thymidine into DNA, and progression of the myoblasts through the cell cycle. C1P induced phosphorylation of glycogen synthase kinase-3β and the product of retinoblastoma gene, and enhanced cyclin D1 protein levels. The mitogenic action of C1P also involved activation of phosphatidylinositol 3-kinase/Akt, ERK1/2 and the mammalian target of rapamycin. These effects of C1P were independent of interaction with a putative G(i)-coupled C1P receptor as pertussis toxin, which maintains G(i) protein in the inactive form, did not affect C1P-stimulated myoblast proliferation. By contrast, C1P was unable to inhibit serum starvation- or staurosporine-induced apoptosis in the myoblasts, and did not affect myogenic differentiation. Collectively, these results add up to the current knowledge on cell types targeted by C1P, which so far has been mainly confined to fibroblasts and macrophages, and extend on the mechanisms by which C1P exerts its mitogenic effects. Moreover, the biological activities of C1P described in this report establish that this phosphosphingolipid may be a relevant cue in the regulation of skeletal muscle regeneration, and that C1P-metabolizing enzymes might be important targets for developing cellular therapies for treatment of skeletal muscle degenerative diseases, or tissue injury.     

Protein Phosphatase PPM1G Regulates Protein Translation and Cell Growth by Dephosphorylating 4E Binding Protein 1 (4E-BP1)

Protein translation initiation is a tightly controlled process responding to nutrient availability and mitogen stimulation. Serving as one of the most important negative regulators of protein translation, 4E binding protein 1 (4E-BP1) binds to translation initiation factor 4E and inhibits cap-dependent translation in a phosphorylation-dependent manner. Although it has been demonstrated previously that the phosphorylation of 4E-BP1 is controlled by mammalian target of rapamycin in the mammalian target of rapamycin complex 1, the mechanism underlying the dephosphorylation of 4E-BP1 remains elusive. Here, we report the identification of PPM1G as the phosphatase of 4E-BP1. A coimmunoprecipitation experiment reveals that PPM1G binds to 4E-BP1 in cells and that purified PPM1G dephosphorylates 4E-BP1 in vitro. Knockdown of PPM1G in 293E and colon cancer HCT116 cells results in an increase in the phosphorylation of 4E-BP1 at both the Thr-37/46 and Ser-65 sites. Furthermore, the time course of 4E-BP1 dephosphorylation induced by amino acid starvation or mammalian target of rapamycin inhibition is slowed down significantly in PPM1G knockdown cells. Functionally, the amount of 4E-BP1 bound to the cap-dependent translation initiation complex is decreased when the expression of PPM1G is depleted. As a result, the rate of cap-dependent translation, cell size, and protein content are increased in PPM1G knockdown cells. Taken together, our study has identified protein phosphatase PPM1G as a novel regulator of cap-dependent protein translation by negatively controlling the phosphorylation of 4E-BP1.     

Variation in ACE activity affects myogenic differentiation in C2C12 cells

Variation in ACE activity is related to affect the skeletal muscle function. To elucidate the mechanism by which ACE affects skeletal muscle function, we examined the effects of loss and gain of ACE activity on myogenic differentiation in C2C12 myoblasts. The treatment of captopril, an ACE inhibitor, in differentiating cells significantly induced the up-regulation of myosin heavy chain, and the hypertrophic myotubes. In addition, an AT2 antagonist PD123319, not AT1 antagonist losartan, induced the up-regulation of myosin heavy chain. On the other hand, overexpression of ACE induced the down-regulation of myosin heavy chain. These results suggest that ACE negatively regulate the myogenesis through the mechanism at least in part via production of angiotensin II followed by its binding to AT2 receptor.     

Hepatitis C virus NS5A binds to the mRNA cap-binding eukaryotic translation initiation 4F (eIF4F) complex and up-regulates host translation initiation machinery through eIF4E-binding protein 1 inactivation

Initiation, a major rate-limiting step of host protein translation, is a critical target in many viral infections. Chronic hepatitis C virus (HCV) infection results in hepatocellular carcinoma. Translation initiation, up-regulated in many cancers, plays a critical role in tumorigenesis. mTOR is a major regulator of host protein translation. Even though activation of PI3K-AKT-mTOR by HCV non-structural protein 5A (NS5A) is known, not much is understood about the regulation of host translation initiation by this virus. Here for the first time we show that HCV up-regulates host cap-dependent translation machinery in Huh7.5 cells through simultaneous activation of mTORC1 and eukaryotic translation initiation factor 4E (eIF4E) by NS5A. NS5A, interestingly, overexpressed and subsequently hyperphosphorylated 4EBP1. NS5A phosphorylated eIF4E through the p38 MAPK-MNK pathway. Both HCV infection and NS5A expression augmented eIF4F complex assembly, an indicator of cap-dependent translation efficiency. Global translation, however, was not altered by HCV NS5A. 4EBP1 phosphorylation, but not that of S6K1, was uniquely resistant to rapamycin in NS5A-Huh7.5 cells, indicative of an alternate phosphorylation mechanism of 4EBP1. Resistance of Ser-473, but not Thr-308, phosphorylation of AKT to PI3K inhibitors suggested an activation of mTORC2 by NS5A. NS5A associated with eIF4F complex and polysomes, suggesting its active involvement in host translation. This is the first report that implicates an HCV protein in the up-regulation of host translation initiation apparatus through concomitant regulation of multiple pathways. Because both mTORC1 activation and eIF4E phosphorylation are involved in tumorigenesis, we propose that their simultaneous activation by NS5A might contribute significantly to the development of hepatocellular carcinoma.     

The mechanism of insulin-stimulated 4E-BP protein binding to mammalian target of rapamycin (mTOR) complex 1 and its contribution to mTOR complex 1 signaling

Insulin activation of mTOR complex 1 is accompanied by enhanced binding of substrates. We examined the mechanism and contribution of this enhancement to insulin activation of mTORC1 signaling in 293E and HeLa cells. In 293E, insulin increased the amount of mTORC1 retrieved by the transiently expressed nonphosphorylatable 4E-BP[5A] to an extent that varied inversely with the amount of PRAS40 bound to mTORC1. RNAi depletion of PRAS40 enhanced 4E-BP[5A] binding to ～70% the extent of maximal insulin, and PRAS40 RNAi and insulin together did not increase 4E-BP[5A] binding beyond insulin alone, suggesting that removal of PRAS40 from mTORC1 is the predominant mechanism of an insulin-induced increase in substrate access. As regards the role of increased substrate access in mTORC1 signaling, RNAi depletion of PRAS40, although increasing 4E-BP[5A] binding, did not stimulate phosphorylation of endogenous mTORC1 substrates S6K1(Thr(389)) or 4E-BP (Thr(37)/Thr(46)), the latter already ～70% of maximal in amino acid replete, serum-deprived 293E cells. In HeLa cells, insulin and PRAS40 RNAi also both enhanced the binding of 4E-BP[5A] to raptor but only insulin stimulated S6K1 and 4E-BP phosphorylation. Furthermore, Rheb overexpression in 293E activated mTORC1 signaling completely without causing PRAS40 release. In the presence of Rheb and insulin, PRAS40 release is abolished by Akt inhibition without diminishing mTORC1 signaling. In conclusion, dissociation of PRAS40 from mTORC1 and enhanced mTORC1 substrate binding results from Akt and mTORC1 activation and makes little or no contribution to mTORC1 signaling, which rather is determined by Rheb activation of mTOR catalytic activity, through mechanisms that remain to be fully elucidated.     

Inhibition of chemokine (CXC motif) ligand 12/chemokine (CXC motif) receptor 4 axis (CXCL12/CXCR4)-mediated cell migration by targeting mammalian target of rapamycin (mTOR) pathway in human gastric carcinoma cells

CXCL12/CXCR4 plays an important role in metastasis of gastric carcinoma. Rapamycin has been reported to inhibit migration of gastric cancer cells. However, the role of mTOR pathway in CXCL12/CXCR4-mediated cell migration and the potential of drugs targeting PI3K/mTOR pathway remains unelucidated. We found that CXCL12 activated PI3K/Akt/mTOR pathway in MKN-45 cells. Stimulating CHO-K1 cells expressing pEGFP-C1-Grp1-PH fusion protein with CXCL12 resulted in generation of phosphatidylinositol (3,4,5)-triphosphate, which provided direct evidence of activating PI3K by CXCL12. Down-regulation of p110β by siRNA but not p110α blocked phosphorylation of Akt and S6K1 induced by CXCL12. Consistently, p110β-specific inhibitor blocked the CXCL12-activated PI3K/Akt/mTOR pathway. Moreover, CXCR4 immunoprecipitated by anti-p110β antibody increased after CXCL12 stimulation and G(i) protein inhibitor pertussis toxin abrogated CXCL12-induced activation of PI3K. Further studies demonstrated that inhibitors targeting the PI3K/mTOR pathway significantly blocked the chemotactic responses of MKN-45 cells triggered by CXCL12, which might be attributed primarily to inhibition of mTORC1 and related to prevention of F-actin reorganization as well as down-regulation of active RhoA, Rac1, and Cdc42. Furthermore, rapamycin inhibited the secretion of CXCL12 and the expression of CXCR4, which might form a positive feedback loop to further abolish upstream signaling leading to cell migration. Finally, we found cells expressing high levels of cxcl12 were sensitive to rapamycin in its activity inhibiting migration as well as proliferation. In summary, we found that the mTOR pathway played an important role in CXCL12/CXCR4-mediated cell migration and proposed that drugs targeting the mTOR pathway may be used for the therapy of metastatic gastric cancer expressing high levels of cxcl12.     

Stretch activation of GTP-binding proteins in C2C12 myoblasts

Mechanical stimulation has been proposed as a fundamental determinant of muscle physiology. The mechanotransduction of strain and strain rate in C2C12 myoblasts were investigated utilizing a radiolabeled GTP analogue to detect stretch-induced GTP-binding protein activation. Cyclic uniaxial strains of 10% and 20% at a strain rate of 20% s(-1) rapidly (within 1 min) activated a 25-kDa GTPase (183 +/- 17% and 186 +/- 19%, respectively), while 2% strain failed to elicit a response (109 +/- 11%) relative to controls. One, five, and sixty cycles of 10% strain elicited 187 +/- 20%, 183 +/- 17%, and 276 +/- 38% increases in activation. A single 10% stretch at 20% s(-1), but not 0.3% s(-1), resulted in activation. Insulin activated the same 25-kDa band in a dose-dependent manner. Western blot analysis revealed a panel of GTP-binding proteins in C2C12 myoblasts, and tentatively identified the 25-kDa GTPase as rab5. In separate experiments, a 40-kDa protein tentatively identified as Galpha(i) was activated (240 +/- 16%) by 10% strain at 1 Hz for 15 min. These results demonstrate the rapid activation of GTP-binding proteins by mechanical strain in myoblasts in both a strain magnitude- and strain rate-dependent manner.     

Truncated human LMP-1 triggers differentiation of C2C12 cells to an osteoblastic phenotype in vitro

LIM mineralization protein-1 （LMP-1） is a novel intracellular osteoinductive protein that has been shown to induce bone formation both in vitro and in viva. LMP-1 contains an N-terminal PDZ domain and three C-terminal LIM domains. In this study, we investigated whether a truncated form of human LMP-1 （hLMP-1 [t]）, lacking the three C-terminal LIM domains, triggers the differentiation of pluripotent myoblastic C2C12 cells to the osteoblast lineage. C2C12 cells were transiently transduced with AdS-hLMP-1 （t）-green fluorescent protein or viral vector control. The expression of hLMP-1 （t） RNA and the truncated protein were examined. The results showed that hLMP-1 （t） blocked myotube formation in C2C12 cultures and significantly enhanced the alkaline phosphatase （ALP） activity. In addition, the expressions of ALP, osteocalcin, and bone morphogenetic protein （BMP）-2 and BMP-7 genes were also increased. The induction of these key osteogenic markers suggests that hLMP- 1 （t） can trigger the pluripotent myoblastic C2C12 cells to differentiate into osteoblastic lineage, thus extending our previous observation that LMP-1 and LMP-1 （t） enhances the osteoblastic phenotype in cultures of cells already committed to the osteoblastic lineage. Therefore, C2C12 cells are an appropriate model system for the examination of LMP-1 induction of the osteoblastic phenotype and the study of mechanisms of LMP-1 action.     

Involvement of p38 MAPK-mediated signaling in the calpeptin-mediated suppression of myogenic differentiation and fusion in C2C12 cells

Calpeptin inhibits myoblast fusion by inhibiting the activity of calpain. However, the mechanism by which calpeptin inhibits myogenesis is not completely understood. This study examined how calpeptin affects the expression of the myogenic regulatory factors (MRFs) and the phosphorylation of p38 mitogen-activated protein kinase (MAPK) in differentiating C2C12 myoblasts. Consistent with previous reports, calpeptin inhibited the induction of μ-calpain and the formation of myotubes in these cells. In particular, calpeptin inhibited the expression of the early and mid differentiation markers including MyoD, Myf5, myogenin, and MRF4 as well as the expression of the late markers such as troponin T and myosin heavy chain (MyHC). Calpeptin also suppressed the phosphorylation of p38 MAPK in C2C12 cells. SB203580, a specific p38 inhibitor, prevented the expression of the muscle-specific markers and their fusion into myotubes in these cells, which was further accelerated in the presence of calpeptin. These findings suggest that calpeptin inhibits the myogenesis of skeletal muscle cells by down-regulating the MRFs and involving p38 MAPK signaling.     

TAK1siRNA对C2C12细胞内TAK1基因表达的影响

目的:高效下调TAK1基因表达的小干扰RNA(siRNA)分子的获得.方法:采用脂质体转染方法,将3对(siRNA ID#94455、siRNA ID # 94549、siRNA ID # 189006)人工合成的TAK1基因特异的小干扰RNA(siRNA)分子分别导入小鼠成肌细胞C2C12中,采用实时荧光定量PCR方法分析细胞内TAK1基因的相对表达.结果:和对照组相比,siRNA ID # 94455、siRNA ID # 94549和siRNA ID # 189006分别下调了细胞内TAK1基因的mRNA表达水平33.34%、46.73%和79.97%.结论:实验获得了能够高效下调TAK1基因表达的siRNA.     

cAMP inhibits mammalian target of rapamycin complex-1 and -2 (mTORC1 and 2) by promoting complex dissociation and inhibiting mTOR kinase activity

cAMP and mTOR signalling pathways control a number of critical cellular processes including metabolism, protein synthesis, proliferation and cell survival and therefore understanding the signalling events which integrate these two signalling pathways is of particular interest. In this study, we show that the pharmacological elevation of [cAMP]_i in mouse embryonic fibroblasts (MEFs) and human embryonic kidney 293 (HEK293) cells inhibits mTORC1 activation via a PKA-dependent mechanism. Although the inhibitory effect of cAMP on mTOR could be mediated by impinging on signalling cascades (i.e. PKB, MAPK and AMPK) that inhibit TSC1/2, an upstream negative regulator of mTORC1, we show that cAMP inhibits mTORC1 in TSC2 knockout (TSC2^−/−) MEFs. We also show that cAMP inhibits insulin and amino acid-stimulated mTORC1 activation independently of Rheb, Rag GTPases, TSC2, PKB, MAPK and AMPK, indicating that cAMP may act independently of known regulatory inputs into mTOR. Moreover, we show that the prolonged elevation in [cAMP]_i can also inhibit mTORC2. We provide evidence that this cAMP-dependent inhibition of mTORC1/2 is caused by the dissociation of mTORC1 and 2 and a reduction in mTOR catalytic activity, as determined by its auto-phosphorylation on Ser2481. Taken together, these results provide an important insight into how cAMP signals to mTOR and down-regulates its activity, which may lead to the identification of novel drug targets to inhibit mTOR that could be used for the treatment and prevention of human diseases such as cancer.     

Expression of basic fibroblast growth factor results in the decrease of myostatin mRNA in murine C2C12 myoblasts

During the development and regeneration of skeletal muscle,many growth factors,such asbasic fibroblast growth factor (bFGF,FGF-2) and myostatin,have been shown to play regulating roles.bFGF contributes to promote proliferation and to inhibit differentiation of skeletal muscle,whereas myostatinplays a series of contrasting roles.In order to elucidate whether the expression of bFGF has any relationshipwith the expression of myostatin in skeletal muscle cells,we constructed a eukaryotic expression vector forthe expression of exogenous bFGF in murine C2C12 myoblasts.Quantitative RT-PCR assays indicated thatwith the increase of the expression of exogenous bFGF gene,the expression of endogenous myostatin genewas suppressed at mRNA level and protein level.     

Ischemia-induced phosphorylation of initiation factor 2 in differentiated PC12 cells: role for initiation factor 2 phosphatase

An in vitro model of ischemia was obtained by subjecting PC12 cells differentiated with nerve growth factor to a combination of glucose deprivation plus anoxia. Immediately after the ischemic period, the protein synthesis rate was significantly inhibited (80%) and western blots of cell extracts revealed a significant accumulation of phosphorylated eukaryotic initiation factor 2, alpha subunit, eIF2(alphaP) (42%). Upon recovery, eIF2(alphaP) levels returned to control values after 30 min, whereas protein synthesis was still partially inhibited (33%) and reached almost control values within 2 h. The activities of the mammalian eIF2alpha kinases, double-stranded RNA-activated protein kinase, mammalian GCN2 homologue, and endoplasmic reticulum-resident kinase, were determined. None of the eIF2alpha kinases studied showed increased activity in ischemic cells as compared with controls. Exposure of cells to cell-permeable inhibitors of protein phosphatases 1 and 2A, calyculin A or tautomycin, induced dose- and time-dependent accumulation of eIF2(alphaP), mimicking an ischemic effect. Protein phosphatase activity, as measured with [(32)P]phosphorylase a as a substrate, diminished during ischemia and returned to control levels upon 30-min recovery. In addition, the rate of eIF2(alphaP) dephosphorylation was significantly lower in ischemic cells, paralleling both the greatest translational inhibition and the highest eIF2(alphaP) levels. The endogenous phosphatase activity from control and ischemic extracts showed different sensitivity to inhibitor 2 and fostriecin in in vitro assays, inhibitor-2 effect in ischemic cells being lower than in control cells. Together these results indicate that an eIF2alpha phosphatase, probably protein phosphatase 1, is implicated in the ischemia-induced eIF2(alphaP) accumulation in PC12 cells.     

Sema4C participates in myogenic differentiation in vivo and in vitro through the p38 MAPK pathway

Sema4C is a member of transmembrane semaphorin proteins which regulate axonal guidance in the developing nervous system. The expression of Sema4C was dramatically induced not only during differentiation of C2C12 mouse myoblasts, but also during injury-induced skeletal muscle regeneration. C2C12 cells stably or transiently expressing Sema4C both showed increased myogenic differentiation reflected by accelerated myotube formation and expression of muscle-specific proteins. Overexpression of Sema4C elicited p38 phosphorylation directly, and the effects of Sema4C during myogenic differentiation could be abolished by the p38alpha-specific inhibitor SB203580. Knockdown of Sema4C by siRNA transfection during C2C12 myoblasts differentiation could suppress the phosphorylation of p38 followed by dramatically diminished myotube formation. Sema4C could activate the myogenin promoter during myogenic differentiation. This activation could be abolished by p38 inhibitor SB203580. Taken together, these observations reveal novel functional potentialities of Sema4C which suggest that Sema4C promotes terminal myogenic differentiation in a p38 MAPK-dependent manner.