GM1 ganglioside induces phosphorylation and activation of Trk and Erk in brain

We investigated the ability of GM1 to induce phosphorylation of the tyrosine kinase receptor for neurotrophins, Trk, in rat brain, and activation of possible down-stream signaling cascades. GM1 increased phosphorylated Trk (pTrk) in slices of striatum, hippocampus and frontal cortex in a concentration- and time-dependent manner, and enhanced the activity of Trk kinase resulting in receptor autophosphorylation. The ability of GM1 to induce pTrk was shared by other gangliosides, and was blocked by the selective Trk kinase inhibitors K252a and AG879. GM1 induced phosphorylation of TrkA > TrkC > TrkB in a region-specific distribution. Adding GM1 to brain slices activated extracellular-regulated protein kinases (Erks) in all three brain regions studied. In striatum, GM1 elicited activation of Erk2 > Erk1 in a time-and concentration-dependent manner. The GM1 effect on Erk2 was mimicked by other gangliosides, and was blocked by the Trk kinase inhibitors K252a and AG879. Pertussis toxin, as well as Src protein tyrosine kinase and protein kinase C inhibitors, did not prevent the GM1-induced activation of Erk2, apparently excluding the participation of Gi and Gq/11 protein-coupled receptors. Intracerebroventricular administration of GM1 induced a transient phosphorylation of TrkA and Erk1/2 in the striatum and hippocampus complementing the in situ studies. These observations support a role for GM1 in modulating Trk and Erk phosphorylation and activity in brain.     

The Protein Phosphatase 2A Regulatory Subunits B′β and B′δ Mediate Sustained TrkA Neurotrophin Receptor Autophosphorylation and Neuronal Differentiation

Nerve growth factor (NGF) is critical for the differentiation and maintenance of neurons in the peripheral and central nervous system. Sustained autophosphorylation of the TrkA receptor tyrosine kinase and long-lasting activation of downstream kinase cascades are hallmarks of NGF signaling, yet our knowledge of the molecular mechanisms underlying prolonged TrkA activity is incomplete. Protein phosphatase 2A (PP2A) is a heterotrimeric Ser/Thr phosphatase composed of a scaffolding, catalytic, and regulatory subunit (B, B′, and B" gene families). Here, we employ a combination of pharmacological inhibitors, regulatory subunit overexpression, PP2A scaffold subunit exchange, and RNA interference to show that PP2A containing B′ family regulatory subunits participates in sustained NGF signaling in PC12 cells. Specifically, two neuron-enriched regulatory subunits, B′β and B′δ, recruit PP2A into a complex with TrkA to dephosphorylate the NGF receptor on Ser/Thr residues and to potentiate its intrinsic Tyr kinase activity. Acting at the receptor level, PP2A/ B′β and B′δ enhance NGF (but not epidermal growth factor or fibroblast growth factor) signaling through the Akt and Ras-mitogen-activated protein kinase cascades and promote neuritogenesis and differentiation of PC12 cells. Thus, select PP2A heterotrimers oppose desensitization of the TrkA receptor tyrosine kinase, perhaps through dephosphorylation of inhibitory Ser/Thr phosphorylation sites on the receptor itself, to maintain neurotrophin-mediated developmental and survival signaling.     

A Functional Role of Fibroblast Growth Factor Receptor 1 (FGFR1) in the Suppression of Influenza A Virus Replication

Influenza A virus causes annual epidemics and occasional pandemics in humans. Here, we investigated four members of the fibroblast growth factor receptor (FGFR) family; FGFR1 to 4, and examined their expression patterns in human lung epithelial cells A549 with influenza A virus infection. We identified a functional role of FGFR1 in influenza A/Puerto Rico/8/1934 (PR8) and A/Anhui/01/2005 (H5N1) virus replication. Our results showed that FGFR1 silencing by siRNA interference promoted influenza A/PR8 and H5N1 virus replication in A549 cells, while lentivirus-mediated exogenous FGFR1 expression significantly suppressed influenza A virus replication; however, FGFR4 did not have the same effects. Moreover, FGFR1 phosphorylation levels were downregulated in A549 cells by influenza A virus infection, while the repression of FGFR1 kinase using PD173074, a potent and selective FGFR1 inhibitor, could enhance virus replication. Furthermore, we found that FGFR1 inhibits influenza virus internalization, but not binding, during viral entry. These results suggested that FGFR1 specifically antagonizes influenza A virus replication, probably by blocking viral entry.     

Host cellular signaling induced by influenza virus

A wide range of host cellular signal transduction pathways can be stimulated by influenza virus infection. Some of these signal transduction pathways induce the host cell’s innate immune response against influenza virus, while others are essential for efficient influenza virus replication. This review examines the cellular signaling induced by influenza virus infection in host cells, including host pattern recognition receptor (PRR)-related signaling, protein kinase C (PKC), Raf/MEK/ERK and phosphatidylinositol- 3-kinase (PI3K)/Akt signaling, and the corresponding effects on the host cell and/or virus, such as recognition of virus by the host cell, viral absorption and entry, viral ribonucleoprotein (vRNP) export, translation control of cellular and viral proteins, and virus-induced cell apoptosis. Research into influenza virus-induced cell signaling promotes a clearer understanding of influenza virus-host interactions and assists in the identification of novel antiviral targets and antiviral strategies.     

Pleiotropic coupling of G protein-coupled receptors to the mitogen-activated protein kinase cascade. Role of focal adhesions and receptor tyrosine kinases.

G protein-coupled receptors (GPCRs) initiate Ras-dependent activation of the Erk 1/2 mitogen-activated protein kinase cascade by stimulating recruitment of Ras guanine nucleotide exchange factors to the plasma membrane. Both integrin-based focal adhesion complexes and receptor tyrosine kinases have been proposed as scaffolds upon which the GPCR-induced Ras activation complex may assemble. Using specific inhibitors of focal adhesion complex assembly and receptor tyrosine kinase activation, we have determined the relative contribution of each to activation of the Erk 1/2 cascade following stimulation of endogenous GPCRs in three different cell types. The tetrapeptide RGDS, which inhibits integrin dimerization, and cytochalasin D, which depolymerizes the actin cytoskeleton, disrupt the assembly of focal adhesions. In PC12 rat pheochromocytoma cells, both agents block lysophosphatidic acid (LPA)- and bradykinin-stimulated Erk 1/2 phosphorylation, suggesting that intact focal adhesion complexes are required for GPCR-induced mitogen-activated protein kinase activation in these cells. In Rat 1 fibroblasts, Erk 1/2 activation via LPA and thrombin receptors is completely insensitive to both agents. Conversely, the epidermal growth factor receptor-specific tyrphostin AG1478 inhibits GPCR-mediated Erk 1/2 activation in Rat 1 cells but has no effect in PC12 cells. In HEK-293 human embryonic kidney cells, LPA and thrombin receptor-mediated Erk 1/2 activation is partially sensitive to both the RGDS peptide and tyrphostin AG1478, suggesting that both focal adhesion and receptor tyrosine kinase scaffolds are employed in these cells. The dependence of GPCR-mediated Erk 1/2 activation on intact focal adhesions correlates with expression of the calcium-regulated focal adhesion kinase, Pyk2. In all three cell types, GPCR-stimulated Erk 1/2 activation is significantly inhibited by the Src kinase inhibitors, herbimycin A and 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-D-3,4-pyrimidine (PP1), suggesting that Src family nonreceptor tyrosine kinases represent a point of convergence for signals originating from either scaffold.     

Inhibition of platelet-derived growth factor-induced mitogenesis and tyrosine kinase activity in cultured bone marrow fibroblasts by tyrphostins.

Tyrphostins, which block protein tyrosine kinase activity, were studied for their inhibitory action on platelet-derived growth factor (PDGF)-induced proliferation of human bone marrow fibroblasts. Of the seven tryphostins examined, tyrphostin AG370 was found to be the most potent blocker against PDGF-induced mitogenesis (IC50 = 20 microM). This PTK blocker also blocks mitogenesis induced by epidermal growth factor (IC50 = 50 microM) and human serum (IC50 = 50 microM), but with lower efficacy. In digitonin-permeabilized fibroblasts as well as in intact fibroblasts, tyrphostin AG370 inhibits PDGF receptor autophosphorylation and the tyrosine phosphorylation of intracellular protein substrates (pp120, pp85, and pp75) which coprecipitate with the PDGF receptor. In comparison to AG370, AG18, a potent EGF receptor blocker, was less efficient in inhibiting PDGF-induced proliferation of fibroblasts and phosphorylation of the intracellular protein substrates. Under the conditions in which AG370 inhibits PDGF-induced mitogenesis and phosphorylation, it does not affect [125I]PDGF internalization and enhance [125I]PDGF binding. These findings suggest that AG370, which is an indole tyrphostin, may serve as a model for developing analogues with a therapeutic potential for treatment of diseases which involve abnormal cellular proliferation induced by PDGF.     

Epidermal growth factor stimulates 3-hydroxy-3-methylglutaryl-coenzyme A reductase expression via the ErbB-2 pathway in human breast adenocarcinoma cells.

HMG-CoA reductase is the key enzyme for the biosynthesis of isoprenoid compounds essential for cell growth and differentiation. Its tyrosine kinase-dependent modulation has recently been suggested and described in the ErbB-2 overexpressing cell line SKBR-3 [Asslan et al. (1998) Biochem. J. 330, 241-246]. Epidermal growth factor (EGF) increased the HMG-CoA reductase activity, protein, and mRNA levels only in ErbB-2-expressing cells (SKBR-3 and MCF-7) but not in MDA-MB-468 cells that do not express ErbB-2 even though their EGF receptor was efficiently phosphorylated. Tyrphostin AG 879, a specific inhibitor of ErbB-2 tyrosine kinase activity, decreased HMG-CoA reductase activity only in cells that expressed ErbB-2. A functional EGF receptor appeared to be necessary since its inhibition by the specific tyrphostin AG 1478 abolished the EGF effects. Phosphatidylinositol 3-kinase (PI 3-kinase) might be a crucial enzyme in the signaling pathway since the specific inhibitor, LY 294002, was shown to inhibit HMG-CoA reductase activity and to completely abolish the stimulation by EGF in SKBR-3 cells.     

A cell-based high-throughput screen identifies tyrphostin AG 879 as an inhibitor of animal cell phospholipid and fatty acid biosynthesis

Inhibition of animal cell phospholipid biosynthesis has been proposed for anticancer and antiviral therapies. Using CHOK1 derived cell lines, we have developed and used a cell-based high-throughput procedure to screen a 1280 compound, small molecule library for inhibitors of phospholipid biosynthesis. We identified tyrphostin AG 879 (AG879), which inhibited phospholipid biosynthesis by 85–90% at a concentration of 10 μM, displaying an IC₅₀ of 1–3 μM. The synthesis of all phospholipid head group classes was heavily affected. Fatty acid biosynthesis was also dramatically inhibited (90%). AG879 inhibited phospholipid biosynthesis in all additional cell lines tested, including MDCK, HUH7, Vero, and HeLa cell lines. In CHO cells, AG879 was cytostatic; cells survived for at least four days during exposure and were able to divide following its removal. AG879 is an inhibitor of receptor tyrosine kinases (RTK) and inhibitors of signaling pathways known to be activated by RTK's also inhibited phospholipid biosynthesis. We speculate that inhibition of RTK by AG879 results in an inhibition of fatty acid biosynthesis with a resulting decrease in phospholipid biosynthesis and that AG879's effect on fatty acid synthesis and/or phospholipid biosynthesis may contribute to its known capacity as an effective antiviral/anticancer agent.     

The Interactomes of Influenza Virus NS1 and NS2 Proteins Identify New Host Factors and Provide Insights for ADAR1 Playing a Supportive Role in Virus Replication

Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.     

Crosstalks between the receptors tyrosine kinase EGFR and TrkA and the GPCR,FPR, in human monocytes are essential for receptors-mediated cell activation

The G-protein coupled receptor (GPCR) fMLP receptor (FPR) and the two receptors tyrosine kinase (RTK), the nerve growth factor (NGF) receptor TrkA and the epidermal growth factor (EGF) receptor (EGFR) are involved in reactive oxygen species (ROS), matrix metalloproteinase-9 (MMP-9) production and CD11b membrane integrin upregulation. We show that in monocytes the three receptors crosstalk each other to modulate these pro-inflammatory mediators. Tyrphostin AG1478, the EGFR inhibitor, inhibits fMLP and NGF-associated ROS production, fMLP-associated CD11b upregulation and NGF-induced TrkA phosphorylation; K252a, the NGF receptor inhibitor, inhibits fMLP or EGF-associated ROS production, CD11b expression and EGF-induced EGFR phosphorylation; cyclosporine H, the FPR inhibitor inhibits EGF or NGF-associated ROS production, EGF-associated CD11b upregulation and prevents EGFR and TrkA phosphorylation by their respective ligand EGF and NGF. In response to fMLP, TrkA phosphorylation is inhibited by the EGFR inhibitor while EGFR phosphorylation is inhibited by the TrkA inhibitor. Receptor crosstalks are Src and ERK dependent. Down-regulation of each receptor by specific siRNA suppresses the ability of the two other receptors to promote ligand-mediated ERK phosphorylation and pro-inflammatory activities including ROS, MMP-9 production and CD11b upregulation. Thus, in monocytes GPCR ligands' activity involves activation of RTK while RTK-ligands activity engages GPCR-signalling molecules.     

Growth factor/growth factor receptor loops in autocrine growth regulation of human prostate cancer DU145 cells

Autocrine growth factors produced by epithelial cells mediate the development and proliferation of neoplastic human prostate tissue. Various approaches have been used to down-regulate neoplastic growth of prostate cancer using natural flavonoids, soluble receptors, pseudo-ligands, monoclonal antibodies and tyrosine kinase inhibitors (tyrphostins). Selected growth factor/growth factor receptor loops (mainly TGFα/EGFR and IGFs/IGFIR) have been proposed as regulators of prostate cancer cell growth. We have previously determined that blockade of IGFIR or VEGF2R signaling pathways by tyrphostin AG1024 and SU1498 inhibits autocrine growth and viability of DU145 cells in vitro. Recently, we compared the activity of AG1024 and SU1498 with the inhibiting effect of tyrphostin A23 (a selective inhibitor of EGFR). The results described in this paper confirm that DU145 cells do not produce IGFI or EGF. In contrast, DU145 cells produce a great amount of VEGF, much more than TGFα (about 60-fold), and VEGF may be the real autocrine growth factor of the investigated cells. The results indicate that the growth of DU145 may be regulated by at least three autocrine loops: TGFα/EGFR, IGFII/IGFIR and VEGF/VEGFR2. Neither AG1024 nor SU1498 affected the production of TGFα substantially, which excludes the possibility that IGFRs or VEGFR2 inhibitors arrest the growth of these cells by inhibition of synthesis and/or secretion of TGFα. The obtained data indicate that all tree investigated tyrphostins (AG1024, SU1498 and A23) inhibit signal transmission by Akt (PKB), ERK(1/2), Src and STAT in a similar manner. A comparison of the effects of the investigated tyrphostins indicates that TGFα, IGFII and VEGF stimulate cell growth by affecting the same signaling pathway. The hypothesis was confirmed by the effect of the investigated tyrphostins on activation of EGFR. All these inhibitors decreased phosphorylation of EGFR to the same extent, and after the same time of incubation with cell culture. These results strongly suggest that stimulation of EGFR kinase is the main step in the initiation of mitogen signaling in DU145 cells, regardless of the type of ligand (TGFα, IGFs or VEGF) and their specific receptors.     

Tyrosine kinase inhibitors block calcium channel currents in vascular smooth muscle cells.

Selective inhibitors of tyrosine kinases, tyrphostin 23 and genistein, produced concentration-dependent inhibition of voltage-operated calcium channel currents in vascular smooth muscle cells isolated from rabbit ear artery. The potency of these two structurally dissimilar inhibitors was similar to that reported for their action as inhibitors of tyrosine kinases. Daidzein, an inactive analogue of genistein, had little inhibitory effect on calcium channel currents at concentrations below 300 microM consistent with an action of these agents at a tyrosine kinase. However, tyrphostin 1, a reportedly less active tyrphostin derivative, also inhibited calcium channel currents with a potency similar to tyrphostin 23. These findings suggest that voltage-operated calcium channels in vascular smooth muscle may be modulated by endogenous tyrosine kinase(s) which display different sensitivities to inhibitors compared with the epidermal growth factor (EGF) receptor. Alternatively the possibility of direct blocking actions of these inhibitors at voltage-operated calcium channels cannot be excluded.     

Influenza virus replication in lung epithelial cells depends on redox‐sensitive pathways activated by NOX4‐derived ROS

An overproduction of reactive oxygen species (ROS) mediated by NADPH oxidase 2 (NOX2) has been related to airway inflammation typical of influenza infection. Virus‐induced oxidative stress may also control viral replication, but the mechanisms underlying ROS production, as well as their role in activating intracellular pathways and specific steps of viral life cycle under redox control have to be fully elucidated. In this study, we demonstrate that influenza A virus infection of lung epithelial cells causes a significant ROS increase that depends mainly on NOX4, which is upregulated at both mRNA and protein levels, while the expression of NOX2, the primary source of ROS in inflammatory cells, is downregulated. Inhibition of NOX4 activity through chemical inhibitors or RNA silencing blocks the ROS increase, prevents MAPK phosphorylation, and inhibits viral ribonucleoprotein (vRNP) nuclear export and viral release. Overall these data, obtained in cell lines and primary culture, describe a so far unrecognized role for NOX4‐derived ROS in activating redox‐regulated intracellular pathways during influenza virus infection and highlight their relevance in controlling specific steps of viral replication in epithelial cells. Pharmacological modulation of NOX4‐mediated ROS production may open the way for new therapeutic approaches to fighting influenza by targeting cell and not the virus.     

Effect of protein tyrosine kinase inhibitors on the current through the Ca(V)3.1 channel

In the present study, we have investigated the effects of protein tyrosine kinase (PTK) inhibitors on the Ca(V)3.1 calcium channel stably transfected in HEK293 cells using the whole-cell configuration of the patch-clamp technique. We have tested two different tyrosine kinase inhibitors, genistein and tyrphostin AG213, and their inactive analogs, genistin and tyrphostin AG9. Bath application of genistein, but not genistin, decreased the T-type calcium current amplitude in a concentration-dependent manner with an IC(50) of 24.7+/-2.0 microM. This effect of genistein was accompanied by deceleration of channel activation and acceleration of channel inactivation. Intracellular application of neither genistein nor genistin had a significant effect on the calcium current. Extracellular application of 50 microM tyrphostin AG213 and its inactive analogue, tyrphostin AG9, did not affect the current through the Ca(V)3.1 channel. The effect of genistein on the channel was also not affected by the presence of catalytically active PTK, p60(c-src) inside the cell. We have concluded that genistein directly inhibited the channel. This mechanism does not involve a PTK-dependent pathway. The alteration of the channel kinetics by genistein suggests an interaction with the voltage sensor of the channel together with the channel pore occlusion.     

Transforming growth factor beta1 (TGF-beta1) promotes endothelial cell survival during in vitro angiogenesis via an autocrine mechanism implicating TGF-alpha signaling

Mouse capillary endothelial cells (1G11 cell line) embedded in type I collagen gels undergo in vitro angiogenesis. Cells rapidly reorganize and form capillary-like structures when stimulated with serum. Transforming growth factor beta1 (TGF-beta1) alone can substitute for serum and induce cell survival and tubular network formation. This TGF-beta1-mediated angiogenic activity depends on phosphatidylinositol 3-kinase (PI3K) and p42/p44 mitogen-activated protein kinase (MAPK) signaling. We showed that specific inhibitors of either pathway (wortmannin, LY-294002, and PD-98059) all suppressed TGF-beta1-induced angiogenesis mainly by compromising cell survival. We established that TGF-beta1 stimulated the expression of TGF-alpha mRNA and protein, the tyrosine phosphorylation of a 170-kDa membrane protein representing the epidermal growth factor (EGF) receptor, and the delayed activation of PI3K/Akt and p42/p44 MAPK. Moreover, we showed that all these TGF-beta1-mediated signaling events, including tubular network formation, were suppressed by incubating TGF-beta1-stimulated endothelial cells with a soluble form of an EGF receptor (ErbB-1) or tyrphostin AG1478, a specific blocker of EGF receptor tyrosine kinase. Finally, addition of TGF-alpha alone poorly stimulated angiogenesis; however, by reducing cell death, it strongly potentiated the action of TGF-beta1. We therefore propose that TGF-beta1 promotes angiogenesis at least in part via the autocrine secretion of TGF-alpha, a cell survival growth factor, activating PI3K/Akt and p42/p44 MAPK.