Stretch-induced retinal vascular endothelial growth factor expression is mediated by phosphatidylinositol 3-kinase and protein kinase C (PKC)-zeta but not by stretch-induced ERK1/2, Akt, Ras, or classical/novel PKC pathways.

Stretch-induced expression of vascular endothelial growth factor (VEGF) is thought to be important in mediating the exacerbation of diabetic retinopathy by systemic hypertension. However, the mechanisms underlying stretch-induced VEGF expression are not fully understood. We present novel findings demonstrating that stretch-induced VEGF expression in retinal capillary pericytes is mediated by phosphatidylinositol (PI) 3-kinase and protein kinase C (PKC)-zeta but is not mediated by ERK1/2, classical/novel isoforms of PKC, Akt, or Ras despite their activation by stretch. Cardiac profile cyclic stretch at 60 cpm increased VEGF mRNA expression in a time- and magnitude-dependent manner without altering mRNA stability. Stretch increased ERK1/2 phosphorylation, PI 3-kinase activity, Akt phosphorylation, and PKC-zeta activity. Signaling pathways were explored using inhibitors of PKC, MEK1/2, and PI 3-kinase; adenovirus-mediated overexpression of ERK, PKC-alpha, PKC-delta, PKC-zeta, and Akt; and dominant negative (DN) mutants of ERK, PKC-zeta, Ras, PI 3-kinase and Akt. Although stretch activated ERK1/2 through a Ras- and PKC classical/novel isoform-dependent pathway, these pathways were not responsible for stretch-induced VEGF expression. Overexpression of DN ERK and Ras had no effect on VEGF expression in these cells. In contrast, DN PI 3-kinase as well as pharmacologic inhibitors of PI 3-kinase blocked stretch-induced VEGF expression. Although stretch-induced PI 3-kinase activation increased both Akt phosphorylation and activity of PKC-zeta, VEGF expression was dependent on PKC-zeta but not Akt. In addition, PKC-zeta did not mediate stretch-induced ERK1/2 activation. These results suggest that stretch-induced expression of VEGF involves a novel mechanism dependent upon PI 3-kinase-mediated activation of PKC-zeta that is independent of stretch-induced activation of ERK1/2, classical/novel PKC isoforms, Ras, or Akt. This mechanism may play a role in the well documented association of concomitant hypertension with clinical exacerbation of neovascularization and vascular permeability.     

Translocation of caveolin regulates stretch-induced ERK activity in vascular smooth muscle cells

Mechanical stress contributes to vascular disease related to hypertension. Activation of ERK is key to mediating cellular proliferation and vascular remodeling in response to stretch stress. However, the mechanism by which stretch mediates ERK activation in the vascular tissue is still unclear. Caveolin, a major component of a flasklike invaginated caveolae, acts as an adaptor protein for an integrin-mediated signaling pathway. We found that cyclic stretch transiently induced translocation of caveolin from caveolae to noncaveolar membrane sites in vascular smooth muscle cells (VSMCs). This translocation of caveolin was determined by detergent solubility, sucrose gradient fractionation, and immunocytochemistry. Cyclic stretch induced ERK activation; the activity peaked at 5 min (the early phase), decreased gradually, but persisted up to 120 min (the late phase). Disruption of caveolae by methyl-beta-cyclodextrin, decreasing the caveolar caveolin and accumulating the noncaveolar caveolin, enhanced ERK activation in both the early and late phases. When endogenous caveolins were downregulated, however, the late-phase ERK activation was subsided completely. Caveolin, which was translocated to noncaveolar sites in response to stretch, is associated with beta1-integrins as well as with Fyn and Shc, components required for ERK activation. Taken together, caveolin in caveolae may keep ERK inactive, but when caveolin is translocated to noncaveolar sites in response to stretch stress, caveolin mediates stretch-induced ERK activation through an association with beta1-integrins/Fyn/Shc. We suggest that stretch-induced translocation of caveolin to noncaveolar sites plays an important role in mediating stretch-induced ERK activation in VSMCs.     

Mechanical stretch stimulates growth of vascular smooth muscle cells via epidermal growth factor receptor

We have studied whether activation of epidermal growth factor receptor (EGFR) is involved in stretch-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation and protein synthesis in cultured rat vascular smooth muscle cells (VSMC). Cyclic stretch (1 Hz) induced a rapid (within 5 min) phosphorylation of ERK1/2, an effect that was time and strength dependent and inhibited by an EGFR kinase inhibitor (AG-1478) but not by a platelet-derived growth factor receptor kinase inhibitor (AG-1296). The stretch rapidly (within 2 min) induced tyrosine phosphorylation of several proteins, among which 180-kDa protein was shown to be EGFR as revealed by blockade with AG-1478 as well as immunoprecipitation with anti-EGFR antibody coupled with immunoblotting with anti-phosphotyrosine antibody. The stretch rapidly (within 2 min) induced association of tyrosine-phosphorylated EGFR with adaptor proteins (Shc/Grb2) as revealed by coprecipitation with glutathione-S-transferase-Grb2 fusion protein coupled with immunoblotting with anti-phosphotyrosine, anti-EGFR, and anti-Shc antibodies. Transfection of a dominant-negative mutant of H-Ras also inhibited stretch-induced ERK1/2 activation. Treatment with a stretch-activated ion channel blocker (Gd(3+)) and an intracellular Ca(2+) antagonist (TMB-8) inhibited stretch-induced phosphorylation of EGFR and ERK1/2. Treatment with AG-1478 and a mitogen-activated protein kinase kinase inhibitor (PD-98059), but not AG-1296, blocked [(3)H]leucine uptake stimulated by a high level of stretch. These data suggest that ERK1/2 activation by mechanical stretch requires Ca(2+)-sensitive EGFR activation mainly via stretch-activated ion channels, thereby leading to VSMC growth.     

Static strain stimulates expression of matrix metalloproteinase-2 and VEGF in microvascular endothelium via JNK- and ERK-dependent pathways

VEGF and MMP protein production are both required for exercise-induced capillary growth in skeletal muscle. The underlying process by which muscle activity initiates an angiogenic response is not established, but it is known that mechanical forces such as muscle stretch are involved. We hypothesized that stretch of skeletal muscle microvascular endothelial cells induces production of MMP-2 and VEGF through a common signal pathway. Endothelial cells were grown on Bioflex plates and exposed to 10% static stretch for up to 24 h. MMP-2 protein level was measured by gelatin zymography and VEGF, MMP-2, and MT1-MMP mRNA levels were quantified by real-time quantitative PCR. ERK1/2 and JNK phosphorylation and VEGF protein levels were assessed by Western blotting. Effects of mitogen-activated protein kinases (MAPKs) (ERK1/2, JNK) and reactive oxygen species (ROS) on stretch-induced expression of MMP-2 and VEGF were tested using pharmacological inhibitors. Stretching of endothelial cells for 24 h caused significant increases in MMP-2 protein and mRNA level, but no change in MT1-MMP mRNA. While MMP-2 protein production was enhanced by H(2)O(2) in unstretched cells, ROS inhibition during stretch did not diminish MMP-2 mRNA or protein production. Inhibition of JNK suppressed stretch-induced MMP-2 protein and mRNA, but inhibition of ERK had no effect. In contrast, inhibition of ERK but not JNK attenuated the stretch-induced increase in VEGF mRNA. Our results demonstrate that differential regulation of MMP-2 and VEGF by MAPK signal pathways contribute to stretch-induced activation of microvascular endothelial cells.     

Cyclic mechanical stretch stimulates the proliferation of C2C12 myoblasts and inhibits their differentiation via prolonged activation of p38 MAPK

Mitogen-activated protein kinases (MAPKs) play an indispensable role in activation of the myogenic program, which is responsive to mechanical stimulation. Although there is accumulating evidence of mechanical force-mediated cellular responses, the role of MAPK in regulating the myogenic process in myoblasts exposed to cyclic stretch is unclear. Cyclic stretch induced the proliferation of C2C12 myoblasts and inhibited their differentiation into myotubes. In particular, it induced persistent phosphorylation of p38 kinase, and decreased the level of phosphorylation of extracellular-signal regulated kinase (ERK). Partial inhibition of p38 phosphorylation increased cellular levels of MyoD and p-ERK in stretched C2C12 cells, along with increased myotube formation. Treatment with 10 microM PD98059 prevented myogenin expression in response to a low dose of SB203580 (3 microM) in the stretched cells, suggesting that adequate ERK activation is also needed to allow the cells to differentiate into myotubes. These results suggest that cyclic stretch inhibits the myogenic differentiation of C2C12 cells by activating p38-mediated signaling and inhibiting ERK phosphorylation. We conclude that p38 kinase, not ERK, is the upstream signal transducer regulating cellular responses to mechanical stretch in skeletal muscle cells.     

Targeting of Tumor Necrosis Factor Receptor 1 to Low Density Plasma Membrane Domains in Human Endothelial Cells

TNFR1 (tumor necrosis factor receptor 1) localizes to caveolae of human endothelial-derived EA.hy926 cells. Transduced TNFR1 molecules lacking amino acid residues 229–244 (spanning the transmembrane/intercellular boundary) are expressed on the cell surface equivalently to full-length TNFR1 molecules but incompletely localize to caveolae. A peptide containing this sequence pulls down CAV-1 (caveolin-1) and TNFR1 from cell lysates but fails to do so following disruption of caveolae with methyl-β-cyclodextrin. We previously reported that methyl-β-cyclodextrin eliminates caveolae and blocks tumor necrosis factor (TNF)-induced internalization of TNFR1 but not TNF-induced activation of NF-κB in EA.hy926 cells. Both CAV-1 and FLOT-2 (flotillin-2), organizing proteins of caveolae and lipid rafts, respectively, associate with caveolae in EA.hy926 cells. Small interfering RNA-mediated knockdown of CAV-1 but not FLOT-2 strikingly reduces caveolae number. Both knockdowns reduce total TNFR1 protein expression, but neither prevents TNFR1 localization to low density membrane domains, TNF-induced internalization of TNFR1, or NF-κB activation by TNF. Both CAV-1 and FLOT-2 knockdowns reduce TNF-mediated activation of stress-activated protein kinase (SAPK). However, both knockdowns reduce expression of TRAF2 (TNF receptor-associated factor-2) protein, and small interfering RNA targeting of TRAF2 also selectively inhibits SAPK activation. We conclude that TNFR1 contains a membrane-proximal sequence that targets the receptor to caveolae/lipid rafts. Neither TNFR1 targeting to nor internalization from these low density membrane domains depends upon CAV-1 or FLOT-2. Furthermore, both NF-κB and SAPK activation appear independent of both TNFR1 localization to low density membrane domains and to TNF-induced receptor internalization.     

Activation of ERK by sodium tungstate induces protein synthesis and prevents protein degradation in rat L6 myotubes

The balance between the rates of protein synthesis and degradation in muscle is regulated by PI3K/Akt signaling. Here we addressed the effect of ERK activation by sodium tungstate on protein turnover in rat L6 myotubes. Phosphorylation of ERK by this compound increased protein synthesis by activating MTOR and prevented dexamethasone-induced protein degradation by blocking FoxO3a activity, but it did not alter Akt phosphorylation. Thus, activation of ERK by tungstate improves protein turnover in dexamethasone-treated cells. On the basis of our results, we propose that tungstate be considered an alternative to IGF-I and its analogs in the prevention of skeletal muscle atrophy.     

bFGF and LIF signaling activates STAT3 in proliferating myoblasts

Different mitogens elicit similar effects on growth and differentiation of skeletal muscle, suggesting that potential overlap exists in the signaling cascades activated by such factors. To investigate this possibility, we examined the status of STAT and ERK proteins in C2C12 myoblasts and myotubes following stimulation with bFGF or LIF. Both STAT1 and STAT3 as well as ERK1 and ERK2 proteins were detectable in extracts of myoblasts. LIF stimulation of myoblasts lead to rapid phosphorylation on tyrosine of STAT3 and of ERKs 1 and 2. Similarly, bFGF stimulation of myoblasts resulted in the tyrosine phosphorylation of STAT3. However, unlike LIF, the bFGF induced tyrosine phosphorylation of STAT3 appeared cyclical, with recurrent peaks of phosphorylation even after prolonged exposure. By contrast, STAT1 remained unphosphorylated in myoblasts treated with bFGF or LIF. In differentiated myotubes, LIF treatment resulted in the tyrosine phosphorylation of both STAT3 and STAT1, but ERK phosphorylation was not detectable, and bFGF treatment did not lead to STAT1 or STAT3 tyrosine phosphorylation. Therefore these observations suggest that disparate mitogens can activate similar downstream effectors in proliferating myoblasts. © 1996 Wiley-Liss, Inc.     

Activation and nuclear translocation of PKCdelta,Pyk2 and ERK1/2 by gonadotropin releasing hormone in HEK293 cells

The mechanism of agonist-induced activation of Pyk2 and its relationship with ERK1/2 phosphorylation was analyzed in HEK293 cells stably expressing the gonadotropin releasing hormone (GnRH) receptor. GnRH stimulation caused rapid and sustained phosphorylation of ERK1/2 and Pyk2 that was accompanied by their nuclear translocation. Pyk2 was also localized on cell membranes and at focal adhesions. Dominant negative Pyk2 (PKM) had no effect on GnRH-induced ERK1/2 phosphorylation and c-fos expression. These actions of GnRH on ERK1/2 and Pyk2 were mimicked by activation of protein kinase C (PKC) and were abolished by its inhibition. GnRH caused translocation of PKC and δ, but not of , ι and λ, to the cell membrane, as well as phosphorylation of Raf at Ser338, a major site in the activation of MEK/ERK1/2. Stimulation of HEK293 cells by EGF caused marked ERK1/2 phosphorylation that was attenuated by the selective EGFR receptor (EGF-R) kinase inhibitor, AG1478. However, GnRH-induced ERK1/2 activation was independent of EGF-R activation. These results indicate that activation of PKC is responsible for GnRH-induced phosphorylation of both ERK1/2 and Pyk2, and that Pyk2 activation does not contribute to GnRH signaling. Moreover, GnRH-induced phosphorylation of ERK1/2 and expression of c-fos in HEK293 cells is independent of Src and EGF-R transactivation, and is mediated through the PKC/Raf/MEK cascade.     

Integration of signal pathways for stretch-dependent growth and differentiation in vascular smooth muscle

The vascular smooth muscle phenotype is regulated by environmental factors, such as mechanical forces, that exert effects on signaling to differentiation and growth. We used the mouse portal vein in organ culture to investigate stretch-dependent activation of Akt, ERK, and focal adhesion kinase (FAK), which have been suggested to be involved in the regulation of stretch-dependent protein synthesis. The role of actin polymerization in these signaling events was examined using the actin-stabilizing agent jasplakinolide. Stretch caused a biphasic activation of FAK at 5–15 min and 24–72 h, which may reflect first a direct phosphorylation of preexisting focal adhesions followed by a rearrangement of focal adhesions to accommodate for the increased mechanical load. Phosphorylation of ERK was increased by acute stretch but then decreased, and Akt did not have a distinct peak in stretch-induced phosphorylation. Inhibition of ERK, phosphatidylinositol 3-kinase, or mammalian target of rapamycin reduced global but not contractile protein synthesis with maintained stretch sensitivity. Stabilization of actin filaments with jasplakinolide, in unstretched portal veins, resulted in increased ERK phosphorylation and global protein synthesis as well as the synthesis of contractile proteins. In contrast, stretch during culture with jasplakinolide did not affect FAK phosphorylation or contractility. Therefore, remodeling of smooth muscle cells to adapt to stretch requires a dynamic cytoskeleton. actin polymerization; mitogen-activated protein kinase; phosphatidylinositol 3-kinase; focal adhesion kinase; protein synthesis     

The primary defect in glycogen synthase activity is not based on increased glycogen synthase kinase-3alpha activity in diabetic myotubes

The mechanism responsible for the diminished activation of glycogen synthase (GS) in diabetic myotubes remains unclear, but may involve increased activity and/or expression of glycogen synthase kinase-3 (GSK-3). In myotubes established from type 2 diabetic and healthy control subjects we determined GS activity ratio, protein expression, and activity of GSK-3alpha and beta under basal and insulin-stimulated conditions when precultured in increasing insulin concentrations. In myotubes precultured at low insulin concentrations acute insulin stimulation increased GS activity more in control than in diabetic subjects, whereas the corresponding GSK-3alpha but not GSK-3beta activity was significantly reduced by acute insulin treatment in both groups. However, in myotubes precultured at high insulin concentrations the effect of insulin on GS and GSK-3alpha activity was blunted in both groups. The protein expression of GSK-3alpha or beta was unaffected. In conclusion, myotubes with a primary defect in GS activity express insulin responsive GSK-3alpha, suggesting that failure of insulin to decrease GS phosphorylation involves abnormal activity of another kinase or phosphatase.     

Role of caveolin-1 in EGCG-mediated protection against linoleic-acid-induced endothelial cell activation

Flavonoids can protect against inflammatory diseases such as atherosclerosis by decreasing vascular endothelial cell activation. Plasma microdomains called caveolae may be critical in regulating endothelial activation. Caveolae are particularly abundant in endothelial cells and play a major role in endothelial trafficking and the regulation of signaling pathways associated with the pathology of vascular diseases. We hypothesize that flavonoids can down-regulate endothelial inflammatory parameters by modulating caveolae-regulated cell signaling. We focused on the role of caveolae and its major protein, caveolin-1, in mechanisms of linoleic-acid-induced endothelial cell activation and protection by the catechin epigallocatechin-3-gallate (EGCG). Exposure to linoleic acid for 6 h induced expression of both caveolin-1 and cyclooxygenase (COX)-2. Pretreatment with EGCG blocked fatty-acid-induced caveolin-1 and COX-2 expression in a time- and concentration-dependent manner. Similar results were observed with nuclear factor-kappa B DNA binding activity, which was also reduced by caveolin-1 silencing. Exposure to linoleic acid rapidly increased phosphorylation of several kinases, including p38 MAPK, extracellular signal regulated kinase 1/2 (ERK1/2) and amino kinase terminal (Akt), with maximal induction at about 10 min. Inhibitors of ERK1/2 and Akt down-regulated the linoleic-acid-induced increase in COX-2 protein, which also occurred after pretreatment with EGCG. Caveolin-1 silencing blocked linoleic-acid-induced phosphorylation of ERK1/2 and protein expression of COX-2, suggesting that specific MAPK signaling is caveolae dependent. Our data provide evidence that caveolae may play a critical role in regulating vascular endothelial cell activation and protection by flavonoids such as EGCG.     

PKC mediates cyclic stretch-induced cardiac hypertrophy through Rho family GTPases and mitogen-activated protein kinases in cardiomyocytes

Signaling events, including Rho GTPases and protein kinase C (PKC), are involved in cardiac hypertrophy. However, the mechanisms by which these pathways cooperate during the hypertrophic process remain unclear. Using an in vitro cyclic stretch model with neonatal rat cardiomyocytes, we demonstrated that stretch-induced activation of RhoA, Rac1/Cdc42, and phosphorylation of Rho-guanine nucleotide dissociation inhibitor (GDI) were prevented by inhibition or depletion of PKC, using chelerythrine and phorbol 12-myristate 13-acetate, indicating that phorbol ester-sensitive PKC isozymes may be upstream regulators of Rho GTPases. Using adenoviral-mediated gene transfer of wild-type (WT) and dominant-negative (DN) mutants of PKCalpha and delta, we found that stretch-induced activation of Rho GTPases and phosphorylation of Rho-GDI were mainly regulated by PKCalpha. PKCdelta was involved in regulation of the activation of Rac1. Stretch-induced increases in [(3)H]-leucine incorporation, myofibrillar reorganization and cell size, were blocked by inhibition of Rho GTPases, or overexpression of DN PKCalpha and delta, suggesting that PKCalpha and delta are both required in stretch-induced hypertrophy, through Rho GTPases-mediated signaling pathways. The mechanism, whereby PKC and Rho GTPases regulate hypertrophy, was associated with mitogen-activated protein (MAP) kinases. Stretch-stimulated phosphorylation of MEK1/ERK1/2 and MKK4/JNK was inhibited by overexpression of DN PKCalpha and delta, and that of MKK3/p38 inhibited by DN PKCdelta. The phosphorylation of ERK and JNK induced by overexpression of WT PKCalpha, and the phosphorylation of p38 induced by WT PKCdelta, were regulated by Rho GTPases. This study represents the first evidence that PKCalpha and delta are important regulators in mediating activation of Rho GTPases and MAP kinases, in the cyclic stretch-induced hypertrophic process.     

Differential contribution of insulin receptor substrates 1 versus 2 to insulin signaling and glucose uptake in l6 myotubes

Insulin receptor substrates-1 and 2 (IRS-1 and IRS-2) are pivotal in relaying insulin signaling in insulin-responsive tissues such as muscle. However, the precise contribution of IRS-1 vis-a-vis IRS-2 in insulin-mediated metabolic and mitogenic responses has not been compared directly in differentiated muscle cells. This study aimed to determine the relative contribution of IRS-1 versus IRS-2 in these responses, using small interfering RNA (siRNA)-mediated specific gene silencing. In L6 myotubes, transfection of siRNA targeted specifically against IRS-1 (siIRS-1) or IRS-2 (siIRS-2) reduced the cognate protein expression by 70-75%. Insulin-induced ERK phosphorylation was much more sensitive to IRS-2 than IRS-1 ablation, whereas p38MAPK phosphorylation was reduced by 43 or 62% in myotubes treated with siIRS-1 or siIRS-2, respectively. Insulin-induced Akt1 and Akt2 phosphorylation was reduced in myotubes treated with siIRS-1, but only Akt2 phosphorylation was reduced in myotubes treated with siIRS-2. In contrast, siIRS-1 treatment caused a marked reduction in insulin-induced actin remodeling, glucose uptake, and GLUT4 translocation, and siIRS-2 was without effect on these responses. Notably, combined siIRS-1 and siIRS-2, although reducing each IRS by around 75%, caused no further drop in glucose uptake than that achieved with siIRS-1 alone, but abolished p38MAPK phosphorylation. We conclude that insulin-stimulated Akt1 phosphorylation, actin remodeling, GLUT4 translocation, and glucose uptake are regulated mainly by IRS-1, whereas IRS-2 contributes selectively to ERK signaling, and Akt2 and p38MAPK lie downstream of both IRS in muscle cells.     

Mitogen-activated protein kinases mediate stretch-induced c-fos mRNA expression in myometrial smooth muscle cells

Evidence indicates that stretch of theuterus imposed by the growing fetus contributes to the onset of labor.Previously we have shown that mechanically stretching rat myometrialsmooth muscle cells (SMCs) induces c-fos expression. Toinvestigate this stretch-induced signaling, we examined the involvementof the mitogen-activated protein kinase (MAPK) family. We show thatstretching rat myometrial SMCs induces a rapid and transientphosphorylation (activation) of MAPKs: extracellular signal-regulatedprotein kinase (ERK), c-Jun NH₂-terminal kinase (JNK), andp38. The use of selective inhibitors for the ERK pathway (PD-98059 andU-0126), p38 (SB-203580), and JNK pathway (curcumin) demonstrated that activation of all three MAPK signaling pathways was necessary foroptimal stretch-induced c-fos expression. We alsodemonstrate that upstream tyrosine kinase activity is involved in themechanotransduction pathway leading to stretch-induced MAPK activationand c-fos mRNA expression. To further examine the role ofMAPKs in vivo, we used a unilaterally pregnant rat model. MAPKs (ERKand p38) are expressed in the pregnant rat myometrium with maximal ERKand p38 phosphorylation occurring in the 24 h immediatelypreceding labor. Importantly, the rise in MAPK phosphorylation wasconfined to the gravid horn and was absent in the empty uterine horn,suggesting that mechanical strain imposed by the growing fetus controlsMAPK activation in the myometrium. Collectively, this data indicatethat mechanical stretch modulates MAPK activity in the myometriumleading to c-fos expression.

     

TNF-alpha increases protein content in C2C12 and primary myotubes by enhancing protein translation via the TNF-R1, PI3K, and MEK

Recent evidence supports that TNF-alpha, long considered a catabolic factor, may also have a physiological function in skeletal muscle. The catabolic view, mainly based on correlative studies in human and in vivo animal models, was challenged by experiments with myoblasts, in which TNF-alpha induced differentiation. The biological effects of TNF-alpha in differentiated muscle, however, remain poorly understood. In the present study, we tested whether TNF-alpha has growth-promoting effects in myotubes, and we characterized the mechanisms leading to these effects. Treatment of C(2)C(12) myotubes with TNF-alpha for 24 h increased protein synthesis (PS) and enhanced cellular dehydrogenase activity by 22 and 26%, respectively, without changing cell numbers. These effects were confirmed in myotubes differentiated from primary rat myoblasts. TNF-alpha activated two signaling cascades: 1) ERK1/2 and its target eIF4E and 2) Akt and its downstream effectors GSK-3, p70(S6K), and 4E-BP1. TNF-alpha-induced phosphorylation of Akt, and ERK1/2 was inhibited by an antibody against TNF-alpha receptor 1 (TNF-R1). PD-98059 pretreatment abolished TNF-alpha-induced phosphorylation of ERK1/2 and eIF4E, whereas PS was only partially inhibited. LY-294002 completely abolished TNF-alpha-induced stimulation of PS as well as phosphorylation of Akt and its downstream targets GSK-3, p70(S6K), and 4E-BP1. Rapamycin inhibited TNF-alpha-induced phosphorylation of the mTOR C1 target p70(S6K) without altering TNF-alpha-induced PS and 4E-BP1 phosphorylation. In conclusion, our results provide evidence that TNF-alpha enhances PS in myotubes and that this is based on enhanced protein translation mediated by the TNF-R1 and PI3K-Akt and MEK-ERK signaling cascades.