Activation of Rac1, RhoA, and mitogen-activated protein kinases is required for Ras transformation.

Although substantial evidence supports a critical role for the activation of Raf-1 and mitogen-activated protein kinases (MAPKs) in oncogenic Ras-mediated transformation, recent evidence suggests that Ras may activate a second signaling pathway which involves the Ras-related proteins Rac1 and RhoA. Consequently, we used three complementary approaches to determine the contribution of Rac1 and RhoA function to oncogenic Ras-mediated transformation. First, whereas constitutively activated mutants of Rac1 and RhoA showed very weak transforming activity when transfected alone, their coexpression with a weakly transforming Raf-1 mutant caused a greater than 35-fold enhancement of transforming activity. Second, we observed that coexpression of dominant negative mutants of Rac1 and RhoA reduced oncogenic Ras transforming activity. Third, activated Rac1 and RhoA further enhanced oncogenic Ras-triggered morphologic transformation, as well as growth in soft agar and cell motility. Finally, we also observed that kinase-deficient MAPKs inhibited Ras transformation. Taken together, these data support the possibility that oncogenic Ras activation of Rac1 and RhoA, coupled with activation of the Raf/MAPK pathway, is required to trigger the full morphogenic and mitogenic consequences of oncogenic Ras transformation.     

Activation of mitogen-activated protein kinases is essential for hydrogen peroxide -induced apoptosis in retinal pigment epithelial cells

Retinal pigment epithelial (RPE) cells are constantly exposed to oxidative injury while clearing byproducts of photoreceptor turnover, a circumstance thought to be responsible for degenerative retinal diseases. The mechanisms of hydrogen peroxide (H₂O₂)-induced apoptosis in RPE cells are not fully understood. We studied signal transduction mechanisms of H₂O₂-induced apoptosis in the human RPE cell line ARPE-19. Activation of two stress kinases (JNK and p38) occurs during H₂O₂ stimulation, and H₂O₂-mediated cell death was significantly reduced by their specific inhibition. Exposure to a lethal dose of H₂O₂ elicited Bax translocation to the mitochondria and release of apoptosis-inducing factor (AIF) from the mitochondria, both of which were abolished by either JNK- or p38-specific inhibitors. Both H₂O₂-induced cell death and JNK/p38 phosphorylation were partially inhibited by C. difficile toxin B, inhibitor of Rho, Rac, and cdc42. Use of pull-down assays revealed that the small GTPase activated by H₂O₂ is Rac1. This study is the first to demonstrate that H₂O₂ induces a Rac1/JNK1/p38 signaling cascade, and that JNK and p38 activation is important for H₂O₂-induced apoptosis as well as AIF/Bax translocation of RPE cells. Y.-C. Yang and T.-C. Ho contributed equally to the work described herein.     

Activation of protein kinase C alpha is required for TPA-triggered ERK (MAPK) signaling and growth inhibition of human hepatoma cell HepG2

The signaling mechanisms for most of the antiproliferative processes are not fully understood. We have demonstrated that ERK(MAPK) signaling was involved in the induction of both p15^INK4band p16^INK4a CDK inhibitors and growth inhibition of hepatoma cell HepG2 triggered by the tumor promoter tetradecanoyl phorbol acetate (TPA). In this study, the upstream signal mechanism for TPA-induced ERK(MAPK) activation was investigated. In HepG2 cells only one of the cPKC isozymes, PKC, but not cPKCII, nPKC or aPKC was activated by TPA as demonstrated by its membrane translocation within 10–30 min and down-regulation at 24 h after TPA treatment. Pretreatment of 0.2–2.0 M Bisindolylmaleimides, an inhibitor of PKC, attenuated the TPA-induced phosphorylation of ERK, gene expressions of p15^INK4band p16^INK4a, and growth inhibition of HepG2 cell in a dose-dependent manner. Consistently, transfection of HepG2 with 1.0–3.0 M antisense (AS) PKC, but not (AS) PKCII, or nPKC oligonucleotides (ODN), for 36 h prior to TPA treatment also prevented the TPA-induced molecular and cellular effects described above. Taken together, we concluded that PKC is specifically required for TPA-induced ERK(MAPK) signaling to trigger gene expressions of p15^INK4band p16^INK4a leading to HepG2 growth inhibition.     

Activation of mitogen-activated protein kinases (Erk1 and Erk2) cascade results in phosphorylation of NF-M tail domains in transfected NIH 3T3 cells.

Neurofilaments (NFs) are neuron-specific intermediate filaments, and are the major cytoskeletal component in large myelinated axons. Lysine-serine-proline (KSP) repeats in the tail domains of high molecular weight NF proteins (NF-M and NF-H) are extensively phosphorylated in vivo in the axon. This phosphorylation in the tail domain has been postulated to play an important role in mediating neuron-specific properties, including axonal caliber and conduction velocity. Recent studies have shown that the mitogen-activated protein kinases (extracellular signal-regulated kinases, Erk1 and Erk2) phosphorylate KSP motifs in peptide substrates derived from the NF-M and NF-H tail domains in vitro. However, it is not clear whether activation of the mitogen activated protein (MAP) kinase pathway is able to phosphorylate these domains in vivo. To answer this question, a constitutively active form of mitogen-activated Erk activating kinase (MEK1) was cotransfected with an NF-M expression construct into NIH 3T3 cells. The activated mutant, but not the dominant negative mutant, induced phosphorylation of NF-M. In addition, it was shown that epidermal growth factor, which induces the MAP kinase cascade in NIH 3T3 cells, also activated endogenous Erk1 and Erk2 and NF-M tail domain phosphorylation in the transfected cells. These results present direct evidence that in-vivo activation of Erk1 and Erk 2 is sufficient for NF-M tail domain phosphorylation in transfected cells.     

Delayed transactivation of the receptor for nerve growth factor is required for sustained signaling and differentiation by alpha2-adrenergic receptors in transfected PC12 cells

Alpha2-adrenergic receptors have been reported to induce subtype-specific neuronal differentiation in vitro, but the signaling mechanisms that mediate this effect have not been characterized. In the present study we found that stimulated alpha2-ARs induce delayed transactivation of TrkA in PC12 cells. The transactivation of TrkA was sensitive to the PP1 inhibitor of the Src family kinases and required prior transactivation of the EGF receptor. Moreover, alpha2-adrenergic receptors induced sustained activation of MAPK and Akt. The sustained activation of Akt, but not of MAPK, was subtype-specific and correlated with the neuronal differentiation of PC12 cells, with the order alpha2A相似文献   

Subtype-specific neuronal differentiation of PC12 cells transfected with alpha2-adrenergic receptors

Cells of the PC12 rat pheochromocytoma cell line acquire characteristics of sympathetic neurons under appropriate treatment. Stably transfected PC12 cells expressing individual alpha2-adrenergic receptor (alpha2-AR) subtypes were used to assess the role of alpha2-ARs in neuronal differentiation and to characterise the signalling pathways activated by the alpha2-AR agonist epinephrine in these cells. The effects of alpha2-AR activation were compared with the differentiating action and the signalling mechanisms of nerve growth factor (NGF). Epinephrine induced neuronal differentiation of PC12alpha2 cells through alpha2-AR activation in a subtype-dependent manner, internalization of all human alpha2-AR subtypes, and activation of mitogen-activated protein kinase (MAPK) and the serine-threonine protein kinase Akt. Epinephrine and NGF showed synergism in their differentiating effects. The MAPK kinase (MEK-1) inhibitor PD 98059 abolished the differentiating effect of epinephrine indicating that the differentiation is dependent on MAPK activation. Activating protein-1 (AP-1) DNA-binding activity was increased after epinephrine treatment in all three PC12alpha2 subtype clones. Evaluation of the potential physiological consequences of these findings requires further studies on endogenously expressed alpha2-ARs in neuronal cells.     

Centaurin-alpha1 is a phosphatidylinositol 3-kinase-dependent activator of ERK1/2 mitogen-activated protein kinases

Centaurin-alpha1 is known to be a phosphatidylinositol 3,4,5-triphosphate (PIP3)-binding protein that has two pleckstrin homology domains and a putative ADP ribosylation factor GTPase-activating protein domain. However, the physiological function of centaurin-alpha1 is still not understood. Here we have shown that transient expression of centaurin-alpha1 in COS-7 cells results in specific activation of ERK, and the activation is inhibited by co-expression of a dominant negative form of Ras. We have also found that a mutant form of centaurin-alpha1 that is unable to bind PIP3 fails to induce ERK activation and that a phosphatidylinositol 3-kinase inhibitor LY294002 inhibits centaurin-alpha1-dependent ERK activation. Furthermore, transient knockdown of centaurin-alpha1 by small interfering RNAs results in reduced ERK activation after epidermal growth factor stimulation in T-REx 293 cells. These results suggest that centaurin-alpha1 contributes to ERK activation in growth factor signaling, linking the PI3K pathway to the ERK mitogen-activated protein kinase pathway through its ability to interact with PIP3.     

Activation and targeting of mitogen-activated protein kinases by G-protein-coupled receptors

Over the past decade, it has become apparent that many G-protein-coupled receptors (GPCRs) generate signals that control cellular differentiation and growth, including stimulation of Ras family GTPases and activation of mitogen-activated protein (MAP) kinase pathways. The mechanisms that GPCRs use to control the activity of MAP kinases vary between receptor and cell type but fall broadly into one of three categories: signals initiated by classical G protein effectors, e.g., protein kinase (PK)A and PKC, signals initiated by cross-talk between GPCRs and classical receptor tyrosine kinases, e.g., "transactivation" of epidermal growth factor (EGF) receptors, and signals initiated by direct interaction between beta-arrestins and components of the MAP kinase cascade, e.g., beta-arrestin "scaffolds". While each of these pathways results in increased cellular MAP kinase activity, emerging data suggest that they are not functionally redundant. MAP kinase activation occurring via PKC-dependent pathways and EGF receptor transactivation leads to nuclear translocation of the kinase and stimulates cell proliferation, while MAP kinase activation via beta-arrestin scaffolds primarily increases cytosolic kinase activity. By controlling the spatial and temporal distribution of MAP kinase activity within the cell, the consequences of GPCR-stimulated MAP kinase activation may be determined by the mechanism by which they are activated.     

AMP-activated protein kinase is required for the lipid-lowering effect of metformin in insulin-resistant human HepG2 cells

The antidiabetic drug metformin stimulates AMP-activated protein kinase (AMPK) activity in the liver and in skeletal muscle. To better understand the role of AMPK in the regulation of hepatic lipids, we studied the effect of metformin on AMPK and its downstream effector, acetyl-CoA carboxylase (ACC), as well as on lipid content in cultured human hepatoma HepG2 cells. Metformin increased Thr-172 phosphorylation of the alpha subunit of AMPK in a dose- and time-dependent manner. In parallel, phosphorylation of ACC at Ser-79 was increased, which was consistent with decreasing ACC activity. Intracellular triacylglycerol and cholesterol contents were also decreased. These effects of metformin were mimicked or completely abrogated by adenoviral-mediated expression of a constitutively active AMPKalpha or a kinase-inactive AMPKalpha, respectively. An insulin-resistant state was induced by exposing cells to 30 mm glucose as indicated by decreased phosphorylation of Akt and its downstream effector, glycogen synthase kinase 3alpha/beta. Under these conditions, the phosphorylation of AMPK and ACC was also decreased, and the level of hepatocellular triacylglycerols increased. The inhibition of AMPK and the accumulation of lipids caused by high glucose concentrations were prevented either by metformin or by expressing the constitutively active AMPKalpha. The kinase-inactive AMPKalpha increased lipid content and blocked the ability of metformin to decrease lipid accumulation caused by high glucose concentrations. Taken together, these results indicate that AMPKalpha negatively regulates ACC activity and hepatic lipid content. Inhibition of AMPK may contribute to lipid accumulation induced by high concentrations of glucose associated with insulin resistance. Metformin lowers hepatic lipid content by activating AMPK, thereby mediating beneficial effects in hyperglycemia and insulin resistance.     

Oocyte-dependent activation of mitogen-activated protein kinase (ERK1/2) in cumulus cells is required for the maturation of the mouse oocyte-cumulus cell complex

Luteinizing hormone (LH) induces maturational processes in oocyte-cumulus cell complexes (OCC) of preovulatory follicles that include both resumption of meiosis in the oocyte and expansion (mucification) of the cumulus oophorus. Both processes require activation of mitogen-activated protein kinase (MAPK) in granulosa cells. Here, it is reported that inhibition of MAPK activation prevented gonadotropin-stimulated resumption of meiosis as well as the rise in expression of two genes whose products are necessary for normal cumulus expansion, Has2 and Ptgs2. However, inhibition of MAPK did not block gonadotropin-induced elevation of granulosa cell cAMP, indicating that the activation of MAPK required for inducing GVB and cumulus expansion is downstream of cAMP. Moreover, activation of MAPK in cumulus cells requires one or more paracrine factors from the oocyte to induce GVB and cumulus expansion; MAPK activation alone is not sufficient to initiate these maturational processes. This study demonstrates a remarkable interaction between the oocyte and cumulus cells that is essential for gonadotropin-induced maturational processes in OCC. By enabling gonadotropin-dependent MAPK activation in granulosa cells, oocytes promote the generation of a return signal from these cells that induces the resumption of meiosis. It also appears that an oocyte-dependent pathway downstream from oocyte-enabled activation of MAPK, and distinct from that promoting the resumption of meiosis, governs cumulus expansion.     

Ethanol inhibits L1 cell adhesion molecule activation of mitogen-activated protein kinases

Inhibition of the functions of L1 cell adhesion molecule (L1) by ethanol has been implicated in the pathogenesis of the neurodevelopmental aspects of the fetal alcohol syndrome (FAS). Ethanol at pharmacological concentrations has been shown to inhibit L1-mediated neurite outgrowth of rat post-natal day 6 cerebellar granule cells (CGN). Extracellular signal-related kinases (ERK) 1/2 activation occurs following L1 clustering. Reduction in phosphoERK1/2 by inhibition of mitogen-activated protein kinase kinase (MEK) reduces neurite outgrowth of cerebellar neurons. Here, we examine the effects of ethanol on L1 activation of ERK1/2, and whether this activation occurs via activation of fibroblast growth factor receptor 1 (FGFR1). Ethanol at 25 mm markedly inhibited ERK1/2 activation by both clustering L1 with cross-linked monoclonal antibodies, or by L1-Fc chimeric proteins. Clustering L1 with subsequent ERK1/2 activation did not result in tyrosine phosphorylation of the FGFR1. In addition, inhibition of FGFR1 tyrosine kinase blocked basic fibroblast growth factor (bFGF) activation of ERK1/2, but did not affect activation of ERK1/2 by clustered L1. We conclude that ethanol disrupts the signaling pathway between L1 clustering and ERK1/2 activation, and that this occurs independently of the FGFR1 pathway in cerebellar granule cells.     

Phospholipase C-gamma is required for agonist-induced Ca2+ entry

We report here that PLC-gamma isoforms are required for agonist-induced Ca2+ entry (ACE). Overexpressed wild-type PLC-gamma1 or a lipase-inactive mutant PLC-gamma1 each augmented ACE in PC12 cells, while a deletion mutant lacking the region containing the SH3 domain of PLC-gamma1 was ineffective. RNA interference to deplete either PLC-gamma1 or PLC-gamma2 in PC12 and A7r5 cells inhibited ACE. In DT40 B lymphocytes expressing only PLC-gamma2, overexpressed muscarinic M5 receptors (M5R) activated ACE. Using DT40 PLC-gamma2 knockout cells, M5R stimulation of ER Ca2+ store release was unaffected, but ACE was abolished. Normal ACE was restored by transient expression of PLC-gamma2 or a lipase-inactive PLC-gamma2 mutant. The results indicate a lipase-independent role of PLC-gamma in the physiological agonist-induced activation of Ca2+ entry.     

Activation of P2Y2 receptor induces c-FOS protein through a pathway involving mitogen-activated protein kinases and phosphoinositide 3-kinases in HeLa cells

The effects of P2Y2 purinoceptor activation on c-Fos expression and the signaling pathways evoked by extracellular ATP/UTP in HeLa cells were investigated. We found that P2Y2 activation induced c-Fos protein and phosphorylated the extracellular signal-regulated kinases 1 and 2 (ERK1/2). The P2Y2-stimulated c-Fos induction was partly blocked (a) by U73122, a phospholipase C inhibitor, (b) by G?6976, a conventional PKC inhibitor, (c) by PD098059, a mitogen-activated protein kinase kinase inhibitor, and, moreover, (d) by the inhibitors of phosphoinositide 3-kinases (PI3K), LY294002 and wortmannin. When G?6976 and PD098059, or G?6976 and wortmannin, were combined there was a totally inhibition of P2Y2-induced c-Fos increase. Either U73122 or G?6976 did not inhibit ERK1/2 phosphorylation induced by ATP/UTP, while it was inhibited by LY294002 (or wortmannin) and by staurosporine. Additionally, wortmannin inhibited the cytosol-to-membrane translocation of PKC- epsilon induced by ATP/UTP. These data indicated that agonist-induced PI3K and downstream PKC- epsilon activation mediated the effect of ATP/UTP on ERK1/2 activation. To test the biological consequences of ERK1/2 activation, the effect of P2Y2 on cell functions were examined. P2Y2 stimulation increased cell proliferation and this effect was attenuated by PD098059 in a dose-dependent manner, thereby indicating that the ERK pathway mediates mitogenic signaling by P2Y2. In conclusion, the activation of conventional PKCs through P2Y2 receptor acts in concert with ERK and PI3K/PKC- epsilon pathways to induce c-Fos protein and HeLa cell proliferation.