Dobesilate diminishes activation of the mitogen-activated protein kinase ERK1/2 in glioma cells

Fibroblast growth factors (FGFs) and their receptors, regularly expressed at high levels in gliomas, are further upregulated during the transition of the tumor from low- to high-grade malignancy, and are essential for glioma progression. FGFs induce upregulation of the mitogen-activated protein kinase (MAPK) signaling cascade in cultured glioma cells, which suggests that MAPK pathway participates in the FGF-dependent glioma development. Recently, it has been shown that dobesilate, an inhibitor of FGF mitogenic activity, shows antiproliferative and proapoptotic activities in glioma cell cultures. Accordingly, it should be expected this new synthetic FGF inhibitor to affect the activation levels of MAPK. Here we report that immunocytochemical and Western blot data unequivocally show that treatment of cell cultures with dobesilate causes a significant decrease of the intracellular levels of ERK1/2 activation, one of the components of the MAPK signalling cascade. This finding supports an important role for dobesilate in glioma growth, suggesting that dobesilate should be a treatment to be born in mind for glioma management.     

STAT1 and STAT3 activation by oxidative stress in A431 cells involves Src-dependent EGF receptor transactivation

Different cellular signal transduction cascades are affected by environmental stressors (UV-radiation, gamma-irradiation, hyperosmotic conditions, oxidants). In this study, we examined oxidative stress-evoked signal transduction pathways leading to activation of STATs in A431 carcinoma cells. Oxidative stress, initiated by addition of H2O2 (1-2 mM) to A431 cells, activates STAT3 and, to a lesser extent, STAT1 in dose- and time-dependent manner. Maximum phosphorylation levels were observed after a 2 minutes stimulation at 1-2 mM H2O2. Phosphorylation was blocked by AG1478, a pharmacological inhibitor of the epidermal growth factor receptor tyrosine kinase, implicating intrinsic EGF receptor tyrosine kinase in this process. Consistent with this observation, H2O2-stimulated EGFR tyrosine phosphorylation was abolished by specific Src kinase family inhibitor CGP77675, implicating Src in H2O2-induced EGFR activation. An essential role for Src and JAK2 in STATs activation was suggested by three findings. 1. Src kinase family inhibitor CGP77675 blocked STAT3 and STAT1 activation by H2O2 in a concentration-dependent manner. 2. In Src-/-fibroblasts, activation of both STAT3 and STAT1 by H2O2 was significantly attenuated. 3. Inhibiting JAK2 activity with the specific inhibitor AG490 reduced the level of H2O2-induced STAT3 phosphorylation, but not STAT1 in A431 cells. These data show essential roles for Src and JAK2 inactivation of STAT3. In contrast, H2O2-mediated activation of STAT1 requires only Src kinase activity. Herein, we postulate also that H2O2-induced STAT activation in carcinoma cells involves Src-dependent EGFR transactivation.     

Rapid activation of ERK1/2 mitogen-activated protein kinase by corticosterone in PC12 cells

Although the nongenomic effects of glucocorticoids have been well acknowledged, its precise intracellular signal transduction pathway remains to be elucidated. The present study using Western immunoblot and protein kinase activity assay, for the first time, showed that corticosterone (B) can induce a rapid activation of Erk1/2 mitogen-activated protein kinase (MAPK) in PC12 cells. The dose-response curve was bell shaped, with the maximal activation at 10(-9) M in 15 min. The results from immunofluorescence staining also revealed that the activated Erk1/2 MAPK was translocated from cytoplasm to nucleus of PC12 cells in 15 min. Activation of Erk1/2 MAPK by B was apparently not mediated by the classical cytosolic steroid receptors, for B-BSA can induce the phosphorylation of Erk1/2 MAPK, but the antagonist (RU38486) cannot block the phosphorylation of Erk1/2 MAPK induced by B. Phosphorylation of Erk1/2 MAPK induced by B was not affected by a tyrosine kinase inhibitor (genistein), suggesting that the pathway did not involve the tyrosine kinase activity. On the other hand, protein kinase C activator (PMA) can activate and protein kinase C inhibitor (G?6976) can block the activation of Erk1/2 MAPK induced by B. Taken together, these data clearly demonstrated that B might act via putative membrane receptor and rapidly activate Erk1/2 MAPK through protein kinase C alpha in PC12 cells.     

Dependence of EGF receptor and STAT factor activation on redox of A431 cells

Reactive oxygen species (ROS) were established to play an important role in cellular signaling as second messengers by integrating different pathways. Recently, we showed that EGF initiated a rapid tyrosine phosphorylation of both EGF-receptor and STAT factors with simultaneous increase in the intracellular ROS level. Now, we have investigated the effect of intracellular red-ox state on EGF- and H2O2-induced activation of EGF receptor, STAT1 and STAT3. We demonstrated that the pretreatment of A431 cells with antioxidant N-acetyl-L-cysteine (NAC) partly reduced the level of EGF-induced phosphorylation of proteins under investigation. Besides, H2O2-induced activation of EGF receptor, and STAT factors was fully prevented by NAC pretreatment. The inhibition of ROS generation by DPI declined EGF-dependent activation of EGF receptor and STAT factors to basal level. Our results demonstrate the essential role of cellular red-ox status in the modulation of EGF-mediated activation of receptor and STAT factors. We have postulated that EGF-induced ROS generation is a very important initial event promoting physiological activation of EGF receptor and subsequent STAT factor activation.     

Epidermal growth factor induces activation of protein kinase FA and ATP.Mg-dependent protein phosphatase in A431 cells

The cytosolic fractions from epidermal growth factor (EGF)-treated A431 cells exhibit a marked increase in activities of ATP.Mg-dependent protein phosphatase and its activating factor (protein kinase FA) when compared to controls in the absence of EGF. By contrast, the Triton X-100-solubilized membrane fractions from the same EGF-treated cells exhibit a corresponding decrease in protein kinase FA activity. The EGF-dependent activation of protein kinase FA and ATP.Mg-dependent protein phosphatase occurred within physiological concentrations of EGF (ED50 = 5 x 10(-10) M). The changes of kinase and phosphatase activities which were measured concomitantly exhibit very similar characteristics as to EGF sensitivity and time dependence. The EGF-induced kinase and phosphatase activation occurred very rapidly, reaching the maximal activity levels within 3 min. Moreover, the EGF effect is transient; both EGF-stimulated phosphatase and kinase activities returned to control levels within 30 min. Taken together, the results suggest that EGF may induce the activation of kinase FA in the membrane and thereby promotes the activation of ATP.Mg-dependent phosphatase in the cytosol. Exposure of A431 cells to exogenous phospholipase C also resulted in the activation of endogenous kinase FA and ATP.Mg-dependent phosphatase in a similar pattern produced by EGF. This further suggests that phospholipase C can mimic EGF to mediate the activation of kinase FA and ATP.Mg-dependent phosphatase in A431 cells. By its dual role as a multisubstrate protein kinase and as an activating factor of multisubstrate protein phosphatase, protein kinase FA may represent a transmembrane signal of EGF.     

Mechanism of mitogen-activated protein kinase phosphatase-3 activation by ERK2

The mitogen-activated protein kinase phosphatase 3 (MKP3)-catalyzed hydrolysis of aryl phosphates in the absence and presence of extracellular signal-regulated kinase 2 (ERK2) was investigated in order to provide insights into the molecular basis of the ERK2-induced MKP3 activation. In the absence of ERK2, the MKP3-catalyzed hydrolysis of simple aryl phosphates does not display any dependence on pH, viscosity, and the nature of the leaving group. Increased catalytic activity and enhanced affinity for oxyanions are observed for MKP3 in the presence of ERK2. In addition, normal bell-shaped pH dependence on the reaction catalyzed by MKP3 is restored in the presence of ERK2. Collectively, these results suggest that the rate-limiting step in the absence of ERK2 for the MKP3 reaction corresponds to a substrate-induced conformational change in MKP3 involving active site rearrangement and general acid loop closure. The binding of ERK2 to the N-terminal domain of MKP3 facilitates the repositioning of active site residues and speeds up the loop closure in MKP3 such that chemistry becomes rate-limiting in the presence of ERK2. Remarkably, it is found that the extent of ERK2-induced MKP3 activation is substrate dependent, with smaller activation observed for bulkier substrates. Unlike simple aryl phosphates, the MKP3-catalyzed hydrolysis of bulky polycyclic substrates exhibits bell-shaped pH rate profiles in the absence of ERK2. Furthermore, it is found that glycerol can also activate the MKP3-catalyzed reaction, increase the affinity of MKP3 for oxyanion, and restore the bell-shaped pH rate profile for the MKP3-catalyzed reaction. Thus, the rate of repositioning of catalytic groups and the reorienting of the electrostatic environment in the MKP3 active site can be enhanced not only by ERK2 but also by high affinity substrates or by glycerol.     

H2O2-induced activation of transcription factors STAT1 and STAT3: the role of EGF receptor and tyrosine kinase JAK2

Reactive oxygen species initiate multiple signal transduction pathways including tyrosine kinase signaling. Here, we demonstrate tyrosine phosphorylation of EGF receptor, STAT3, and, to a lesser extent, STAT1 upon H2O2 treatment of HER14 cells (NIH3T3 fibroblasts transfected with full-length EGF receptor). Maximum phosphorylation levels were observed in 5 min of stimulation at 1-2 mM H2O2. It has been shown that the intrinsic EGF-receptor tyrosine kinase is responsible for the receptor phosphorylation upon H2O2 stimulation. STAT3 and STAT1 activation in HER14 cells was demonstrated to depend on EGF receptor kinase activity, rather than JAK2 activity, while in both K721A and CD126 cells (NIH3T3 transfected with kinase-dead EGF receptor, and EGF receptor lacking major autophosphorylation sites, respectively) STAT1 and STAT3 tyrosine phosphorylation requires JAK2 kinase activity. Furthermore, STAT3 is constitutively phosphorylated in K721A and CD126 cells, and STAT1 H2O2-stimulated activation in these cells is much more prominent than in HER14. In all the cell lines used, Src-kinase activity was demonstrated to be unnecessary for ROS-initiated phosphorylation of STATs. Herein, we postulate that EGF receptor plays a role in H2O2-induced STAT activation in HER14 cells. Our data also prompted a hypothesis of constitutive inhibition of JAK2-dependent STAT activation in this cell line.     

Spred-1 negatively regulates interleukin-3-mediated ERK/mitogen-activated protein (MAP) kinase activation in hematopoietic cells

Sprouty/Spred family proteins have been identified as negative regulators of growth factor-induced ERK/mitogen-activated protein (MAP) kinase activation. However, it has not been clarified whether these proteins regulate cytokine-induced ERK activity. We found that Spred-1 is highly expressed in interleukin-3 (IL-3)-dependent hematopoietic cell lines and bone marrow-derived mast cells. To investigate the roles of Spred-1 in hematopoiesis, we expressed wild-type Spred-1 and a dominant negative form of Spred-1, DeltaC-Spred, in IL-3- and stem cell factor (SCF)-dependent cell lines as well as hematopoietic progenitor cells from mouse bone marrow by retrovirus gene transfer. In IL-3-dependent Ba/F3 cells expressing c-kit, forced expression of Spred-1 resulted in a reduced proliferation rate and ERK activation in response to not only SCF but also IL-3. In contrast, DeltaC-Spred augmented IL-3-induced cell proliferation and ERK activation. Wild-type Spred-1 inhibited colony formation of bone marrow cells in the presence of cytokines, whereas DeltaC-Spred-1 expression enhanced colony formation. Augmentation of ERK activation and proliferation in response to IL-3 was also observed in Spred-1-deficient bone marrow-derived mast cells. These data suggest that Spred-1 negatively regulates hematopoiesis by suppressing not only SCF-induced but also IL-3-induced ERK activation.     

Epidermal growth factor stimulates phosphatidylinositol turnover for ten hours in A431 cells without activation of protein kinase C

Epidermal Growth Factor stimulated phosphatidylinositol turnover in A431 cells for up to ten hours. There was an increase in phosphatidylinositol, phosphatidylinositol-4-phosphate, phosphatidylinositol-4,5-bisphosphate and phosphatidic acid at all time points. The effects on the inositol phosphates were variable. Despite the activation of the phosphatidylinositol cycle, we were unable to demonstrate activation of protein kinase C.     

Extracellular S100A1 protein inhibits apoptosis in ventricular cardiomyocytes via activation of the extracellular signal-regulated protein kinase 1/2 (ERK1/2)

S100A1 is a Ca2+-binding protein of the EF-hand type that belongs to the S100 protein family. It is specifically expressed in the myocardium at high levels and is considered to be an important regulator of cardiac contractility. Because the S100A1 protein is released into the extracellular space during ischemic myocardial injury, we examined the cardioprotective potential of the extracellular S100A1 protein on ventricular cardiomyocytes in vitro. In this report we show that extracellularly added S100A1 protein is endocytosed into the endosomal compartment of neonatal ventricular cardiomyocytes via a Ca2+-dependent clathrin-mediated process. S100A1 uptake protects neonatal ventricular cardiomyocytes from 2-deoxyglucose and oxidative stress-induced apoptosis in vitro. S100A1-mediated anti-apoptotic effects involve specific activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) pro-survival pathway, including activation of phospholipase C, protein kinase C, mitogen-activated protein kinase kinase 1, and ERK1/2. In contrast, neither transsarcolemmal Ca2+ influx via the L-type channel nor protein kinase A activity seems to take part in the S100A1-mediated signaling pathway. In conclusion, this study provides evidence for the S100A1 protein serving as a novel cardioprotective factor in vitro. These findings warrant speculation that injury-dependent release of the S100A1 protein from cardiomyocytes may serve as an intrinsic mechanism to promote survival of the myocardium in vivo.     

Angiotensin-(1-7)-induced activation of ERK1/2 is cAMP/protein kinase A-dependent in glomerular mesangial cells

The renin-angiotensin system (RAS) plays an important role in renal physiology and kidney injury. Although the cellular effects of the RAS activation are generally attributed to angiotensin II (ANG II), the recent identification of angiotensin-converting enzyme 2 has shifted the focus to other peptides including Ang-(1-7). The G protein-coupled receptor for Ang-(1-7), mas, is expressed by mesangial cells (MC) but the signal transduction pathways activated by Ang-(1-7) in MC have not been fully elucidated. Accordingly, we studied the effect of Ang-(1-7) on extracellular signal-related kinase (ERK)1/2 activation in rat MC. Ang-(1-7)-induced ERK1/2 phosphorylation in MC is time- and concentration-dependent. Pretreatment of MC with the mas receptor antagonist A-779 but not the AT(1) antagonist losartan or the AT(2) antagonist PD123319 abrogated ERK1/2 activation. Neither pretreatment with the NADPH oxidase inhibitors diphenyleneiodonium and apocynin nor pretreatment with the epidermal growth factor (EGF) receptor antagonists AG1478 and PD158780 attenuated Ang-(1-7)-induced activation of ERK1/2. Even though each of these compounds abolished ANG II-induced activation of ERK1/2. Ang-(1-7) increased intracellular cAMP levels and activated protein kinase A (PKA) and inhibition of either adenylyl cyclase or PKA activity attenuated Ang-(1-7)-induced ERK1/2 activation. In conclusion, Ang-(1-7)-induced activation of ERK1/2 is cAMP/PKA-dependent in MC, but independent of NADPH oxidase and the EGF receptor.