Sphingosine 1-phosphate stimulation of NADPH oxidase activity: relationship with platelet-derived growth factor receptor and c-Src kinase

This study demonstrates for the first time that sphingosine 1-phosphate (S1P) increases H2O2 production in NIH3T3 fibroblasts through NADPH oxidase activation, confirming the involvement of phosphoinositide-3-kinase and protein kinase C in the activation of this enzyme in non-phagocyte mammalian cells. The results demonstrate also that both platelet-derived growth factor (PDGF) and S1P-mediated NADPH oxidase activation and H2O2 production by Gi-protein coupled receptors (GPCRs) and c-Src kinase. Moreover, both PDGF and S1P activate c-Src kinase through GPCRs, indicating that this kinase can constitute a connection factor between PDGF and S1P signaling, confirming the cross-talk previously found between their receptors. Thus, Gi-protein-mediated NADPH oxidase activation with the consequent H2O2 increase constitutes an early event in the PDGF and S1P pathways. However, a different time course of H2O2 production in S1P-stimulated cells compared to that obtained in PDGF-stimulated cells has been observed, and this seems to be related to the different activation behavior of c-Src kinase induced after S1P or PDGF stimulation. Finally, these data demonstrate that S1P-induced H2O2 production is necessary to maximize c-Src kinase activation, confirming that this is a redox regulated kinase. After which, c-Src plays an important role both upstream and downstream from NADPH oxidase activation.     

GSH role on platelet-derived growth factor receptor tyrosine phosphorylation induced by H2O2

This study, conducted on NIH3T3 cells, demonstrates that GSH depletion obtained by buthionine sulfoximine (BSO) treatment does not affect platelet-derived growth-factor receptor (PDGFr) autophosphorylation or cell protein phosphorylation induced by exogenous addition of H2O2, while it does decrease tyrosine phosphorylation obtained by PDGF stimulation. This last effect seems due to the lack of H2O2 generation; for the first time a relation between intracellular GSH content and H2O2 production induced by PDGF has been demonstrated. Therefore, changes of GSH levels can affect the early events of the PDGFr signal pathways by redox regulation. It has also demonstrated that in NIH3T3 cells, H2O2 can directly activate tyrosine phosphorylation by a reversible effect with the involvement of SH-group. This H2O2 effect is increased by vanadate and by GSH depleting agent, diethylmaleate, which unlike BSO is able to produce H2O2 as the current study shows.     

Phosphoinositide 3-kinase regulates the phosphorylation of NADPH oxidase component p47(phox) by controlling cPKC/PKCdelta but not Akt

Superoxide production by NADPH oxidase is essential for the bactericidal properties of phagocytes. Phosphorylation of p47(phox), one of the cytosolic components of NADPH oxidase, is a crucial step of the oxidase activation. Some evidences suggest that phosphoinositide 3-kinase (PI3K) is involved in p47(phox) phosphorylation, but it has not been fully understood how PI3K regulates it. The aim of this study was to examine the mechanism underlying the PI3K regulation of p47(phox) phosphorylation. Pharmacological inhibition of PI3K attenuated both fMLP-stimulated p47(phox) phosphorylation and NADPH oxidase activity in HL-60 cells differentiated to a neutrophil-like phenotype. Although fMLP elicited Akt activation in a PI3K-dependent manner, an Akt inhibitor had no effect on the oxidase activity triggered by fMLP. In vitro kinase assay revealed that Akt was unable to catalyze p47(phox) phosphorylation. Interestingly, the activation of cPKC and PKCdelta after fMLP stimulation was dependent on PI3K. Furthermore, PI3K inhibitors reduced the activation of phospholipase Cgamma2 without affecting tyrosine phosphorylation on it. These results suggest that PI3K regulates the phosphorylation of NADPH oxidase component p47(phox) by controlling diacylglycerol-dependent PKCs but not Akt.     

Involvement of PI3-kinase and its association with c-Src in PTH-stimulated rat enterocytes

Phosphoinositide-3-kinase (PI3K) is a lipid kinase, which phosphorylates the D3 position of phosphoinositides, and is known to be activated by a host of protein tyrosine kinases. PI3K plays an important role in mitogenesis in several cell systems. However, whether parathyroid hormone (PTH) affects the activity and functional roles of PI3K in intestinal cells remain to be determined. The objective of this study was to identify and characterize the PI3K pathway, and its relation to other non-receptor tyrosine kinases in mediating PTH signal transduction in rat enterocytes. PTH dose- and time-dependently increased PI3K activity with a peak occurring at 2 min. The tyrosine kinase inhibitor genistein, c-Src inhibitor PP1 and two structurally different inhibitors of PI3K, LY294002 and wortmannin, suppressed PI3K activity dependent on PTH. Co-immunoprecipitation analysis showed a constitutive association between c-Src and PI3K, which was enhanced by PTH treatment, suggesting that the cytosolic tyrosine kinase forms an immunocomplex with PI3K probably via the N-SH2 domain of the p85alpha regulatory subunit. In response to PTH, tyrosine phosphorylation of p85alpha was enhanced, effect that was abolished by PP1, the inhibitor of c-Src kinase. PTH causes a rapid (0.5-5 min) phosphorylation of Akt/PKB, effect that was abrogated by PI3K inhibitors, indicating that in rat enterocytes, PI3K is an upstream mediator of Akt/PKB activation by PTH. We report here that PI3K is also required for PTH activation of the mitogen-activated protein kinases ERK1 and ERK2. Taken together, the present study demonstrate, for the first time, that PTH rapidly and transiently stimulates PI3K activity and its down effector Akt/PKB in rat enterocytes playing c-Src kinase a central role in PTH-dependent PI3K activation and that PI3K signaling pathway contributes to PTH-mediated MAPK activation.     

Roles of p38 MAPK, PKC and PI3-K in the signaling pathways of NADPH oxidase activation and phagocytosis in bovine polymorphonuclear leukocytes

Stimulation of bovine polymorphonuclear leukocytes (PMN) with serum-opsonized zymosan (sOZ) induced the activation of p38 mitogen-activated protein kinase (MAPK), protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3-K) and sOZ-induced O(2)(-) production was significantly attenuated by their inhibitors (SB203580 for p38 MAPK, GF109203X for PKC and wortmannin for PI3-K). They caused significant attenuation of sOZ-induced phosphorylation of p47phox as well. Flow cytometric analysis, however, revealed that SB203580 and wortmannin attenuated phagocytosis, but GF109203X facilitated it. The results suggest that p38 MAPK and PI3-K participated in both signaling pathways of NADPH oxidase activation (O(2)(-) production) and phagocytosis, and PKC participated in the signaling pathway of NADPH oxidase activation alone.     

Mechanisms of inhibition by heparin of PDGF stimulated MAP kinase activation in vascular smooth muscle cells

Heparin and heparan are potent inhibitors of vascular smooth muscle cell (VSMC) proliferation. To investigate the mechanisms by which heparin suppresses growth factor stimulated mitogenesis, the present experiments investigated the effects of heparin on platelet-derived growth factor (PDGF) stimulated signal transduction pathways. Heparin treatment substantially inhibited PDGF-BB stimulated rat VSMC growth. Western analysis showed a 30 min PDGF-BB treatment of VSMC induced the tyrosine phosphorylation of multiple protein bands; cotreatment with heparin inhibited mitogen-activated protein (MAP) kinase tyrosine phosphorylation but had little effect on PDGF receptor tyrosine phosphorylation. In-gel kinase assays demonstrated that heparin inhibited PDGF-BB stimulated MAP kinase activity at late (25 min) but not early (10 min) time points. These data indicate that heparin does not inhibit the initial signalling events after PDGF-BB binding but instead acts through an alternate mechanism to inhibit MAP kinase. To investigate if heparin directly stimulates tyrosine phosphatase-mediated suppression of MAP kinase, we treated VSMC with orthovanadate, a tyrosine phosphatase inhibitor. Heparin inhibited MAP kinase tyrosine phosphorylation after orthovanadate treatment, indicating that heparin does not suppress MAP kinase by enlistment of a tyrosine phosphatase. Experiments were performed to investigate signalling pathways upstream of MAP kinase. To determine if protein kinase C (PKC) mediates PDGF-BB, serum, and EGF stimulation of MAP kinase, we treated VSMC overnight with phorbol ester (PMA) to downregulate PKC. Abolition of conventional and novel PKC activity significantly suppressed both serum and PDGF-BB induced MAP kinase activation, indicating protein kinase C is an important mediator for these mitogens. In contrast, downregulation of these PKC isoforms had little effect on EGF stimulation of MAP kinase. As heparin inhibits PDGF and serum but not EGF stimulation of MAP kinase, these data precisely correlate heparin inhibition of MAP kinase with activation through PKC-dependent pathways. Immunoprecipitation analysis found that heparin inhibited serum, PMA, and PDGF but not EGF induced raf-1 phosphorylation. These studies demonstrate that heparin did not block PDGF-BB receptor activation, which initiates the mitogenic signalling cascade. Heparin did inhibit specific postreceptor second messenger signals, such as the late phase activation of MAP kinase, which may be mediated by suppression of PKC-dependent pathways. J. Cell. Physiol. 172:69–78, 1997. © 1997 Wiley-Liss, Inc.     

Overexpression of c-Src enhances cell-matrix adhesion and cell migration in PDGF-stimulated NIH3T3 fibroblasts

c-Src is normally associated with the plasma membrane, but upon activation by tyrosine kinase receptors it translocates to the cytoskeleton. Activation of c-Src alters its conformation and induces the association of c-Src with cytoskeletal proteins. c-Src is implicated in tyrosine phosphorylation of cytoskeletal proteins, which might affect the cytoskeletal architecture. Rearrangements of the cytoskeleton affect cell-matrix adhesion and cell migration. In this study NIH3T3 fibroblasts, that overexpress c-Src, were used to analyze the effect of c-Src on both cell-matrix adhesion and cell migration. Upon PDGF stimulation translocation of c-Src to the cytoskeleton was detected. PDGF treatment also increased cell-matrix adhesion and cell migration. The cell line with the highest c-Src expression showed the largest increases in both phenomena. These findings suggest that translocation of c-Src to the cytoskeleton results in enhanced cell-matrix adhesion and cell migration.     

Platelet-derived growth factor-induced disruption of gap junctional communication and phosphorylation of connexin43 involves protein kinase C and mitogen-activated protein kinase

Previously we showed a rapid and transient inhibition of gap junctional communication (GJC) by platelet-derived growth factor (PDGF) in T51B rat liver epithelial cells expressing wild-type platelet-derived growth factor β receptors (PDGFrβ). This action of PDGF correlated with the hyperphosphorylation of the gap junction protein connexin43 (Cx43) and required PDGFrβ tyrosine kinase activity, suggesting the participation of protein kinases and phosphatases many of which are activated by PDGF treatment. In the present study, two such kinases, namely protein kinase C (PKC) and mitogen-activated protein kinase (MAPK), are investigated for their possible involvement in PDGF-induced closure of junctional channels and Cx43-phosphorylation. Down-regulation of PKC-isoforms by 12-O-tetradecanoylphorbol-13-acetate or pretreatment with the PKC inhibitor calphostin C, completely blocked PDGF action on GJC and Cx43. Activation of MAPK correlated with PDGF-induced Cx43 phosphorylation, and prevention of MAPK activation by PD98059 eliminated the PDGF effects. Interestingly, elimination of GJC recovery by cycloheximide was associated with a sustained activated-MAPK level. Based on these results we postulate that the activation of PKC and MAPK are required in PDGF-mediated Cx43 phosphorylation and junctional closure. J. Cell. Physiol. 176:332–341, 1998. © 1998 Wiley-Liss, Inc.     

Effect of pertussis toxin on insulin-induced signal transduction in rat adipocytes and soleus muscles

It has been reported that pertussis toxin (PTX) suppresses the function of trimeric guanine nucleotide binding protein (G-protein). We examined the effect of PTX on insulin-induced glucose uptake, diacylglycerol (DG)-protein kinase C (PKC) signalling, phosphatidylinositol (PI) 3-kinase and PKC zeta activation and insulin-induced tyrosine phosphorylation of Gialpha to clarify the role of G-protein for insulin-mediated signal transduction mechanism in rat adipocytes and soleus muscles. Isolated adipocytes and soleus muscles were preincubated with 0.01 approximately 1 ng/ml PTX for 2 hours, followed by stimulation with 10-100 nM insulin or 1 microM tetradecanoyl phorbol-13-acetate (TPA). Pretreatment with PTX resulted in dose-responsive decreases in insulin-stimulated [3H]2-deoxyglucose (DOG) uptake, and unchanged TPA-stimulated [3H]2-DOG uptake, without affecting basal [3H]2-DOG uptake. In adipocytes, insulin-induced DG-PKC signalling, PI 3-kinase activation and PKC zeta translocation from cytosol to the membrane were suppressed when treated with PTX, despite no changes in [125I]insulin-specific binding and insulin receptor tyrosine kinase activity. Moreover, to elucidate insulin-stimulated tyrosine phosphorylation of 40 kDa alpha-subunit of G-protein (Gialpha-2), adipocytes were stimulated with 10 nM insulin for 10 minutes, homogenized, immunoprecipitated with anti-phosphotyrosine antibody, and immunoblotted with anti-Gialpha-2 antibody. Insulin-induced tyrosine phosphorylation of Gialpha-2 was found by immunoblot analysis with anti-Gialpha-2 antibody. These results suggest that G-protein regulates DG-PKC signalling by binding of Gialpha-2 with GTP and PI 3-kinase-PKC zeta signalling by releasing of Gbetagamma via dissociation of trimeric G-protein after insulin receptor tyrosine phosphorylation in insulin-sensitive tissues.     

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.     

PKCalpha reduces the lipid kinase activity of the p110alpha/p85alpha PI3K through the phosphorylation of the catalytic subunit

The modulation of phosphoinositide 3-kinase (PI3K) activity influences the quality of cellular responses triggered by various receptor tyrosine kinases. Protein kinase C (PKC) has been reported to phosphorylate signalling molecules upstream of PI3K and thereby it may affect the activation of PI3K. Here, we provide the first evidence for a direct effect of a PKC isoenzyme on the activity of PI3K. PKCalpha but not PKCepsilon phosphorylated the catalytic subunit of the p110alpha/p85alpha PI3K in vitro in a manner inhibited by the PKC inhibitor bisindolylmaleimide I (BIM I). The incubation of PI3K with active PKCalpha resulted in a significant decrease in its lipid kinase activity and this effect was also attenuated by BIM I. We conclude that PKCalpha is able to modulate negatively the lipid kinase activity of the p110alpha/p85alpha PI3K through the phosphorylation of the catalytic subunit.     

A-Raf associates with and regulates platelet-derived growth factor receptor signalling

Raf kinases are important intermediates in epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) mediated activation of the mitogen-activated protein kinase (MAPK) pathway. In this report, we show that the A-Raf kinase is associated with activated EGF receptor complexes and with PDGF receptor (PDGFR) complexes independent of prior PDGF treatment. The ability of A-Raf to associate with receptor tyrosine kinases could provide a Ras-GTP-independent mechanism for the membrane localization of A-Raf. Expression of a partially activated A-Raf mutant resulted in decreased tyrosine phosphorylation of the PDGFR, specifically on Y857 (autophosphorylation site) and Y1021 (phospholipase Cgamma1 (PLCgamma1) binding site), but not the binding sites for other signalling proteins (Nck, phosphatidylinositol 3'-kinase (PI3K), RasGAP, Grb2, SHP). Activated A-Raf expression also altered the activation of PLCgamma1, and p85-associated PI3K. Thus, A-Raf can regulate PLCgamma1 signalling via a PDGFR-dependent mechanism and may also regulate PI3K signalling via a PDGFR-independent mechanism.     

Hydrogen peroxide generated during cellular insulin stimulation is integral to activation of the distal insulin signaling cascade in 3T3-L1 adipocytes

In a variety of cell types, insulin stimulation elicits the rapid production of H(2)O(2), which causes the oxidative inhibition of protein-tyrosine phosphatases and enhances the tyrosine phosphorylation of proteins in the early insulin action cascade (Mahadev, K., Zilbering, A., Zhu, L., and Goldstein, B. J. (2001) J. Biol. Chem. 276, 21938-21942). In the present work, we explored the potential role of insulin-induced H(2)O(2) generation on downstream insulin signaling using diphenyleneiodonium (DPI), an inhibitor of cellular NADPH oxidase that blocks insulin-stimulated cellular H(2)O(2) production. DPI completely inhibited the activation of phosphatidylinositol (PI) 3'-kinase activity by insulin and reduced the insulin-induced activation of the serine kinase Akt by up to 49%; these activities were restored when H(2)O(2) was added back to cells that had been pretreated with DPI. Interestingly, the H(2)O(2)-induced activation of Akt was entirely mediated by upstream stimulation of PI 3'-kinase activity, since treatment of 3T3-L1 adipocytes with the PI 3'-kinase inhibitors wortmannin or LY294002 completely blocked the subsequent activation of Akt by exogenous H(2)O(2). Preventing oxidant generation with DPI also blocked insulin-stimulated glucose uptake and GLUT4 translocation to the plasma membrane, providing further evidence for an oxidant signal in the regulation of the distal insulin-signaling cascade. Finally, in contrast to the cellular mechanism of H(2)O(2) generation by other growth factors, such as platelet-derived growth factor, we also found that insulin-stimulated cellular production of H(2)O(2) may occur through a unique pathway, independent of cellular PI 3'-kinase activity. Overall, these data provide insight into the physiological role of insulin-dependent H(2)O(2) generation, which is not only involved in the regulation of tyrosine phosphorylation events in the early insulin signaling cascade but also has important effects on the regulation of downstream insulin signaling, involving the activation of PI 3'-kinase, Akt, and ultimately cellular glucose transport in response to insulin.     

Convergence of multiple signaling cascades at glycogen synthase kinase 3: Edg receptor-mediated phosphorylation and inactivation by lysophosphatidic acid through a protein kinase C-dependent intracellular pathway

Lysophosphatidic acid (LPA) is a natural phospholipid with multiple biological functions. We show here that LPA induces phosphorylation and inactivation of glycogen synthase kinase 3 (GSK-3), a multifunctional serine/threonine kinase. The effect of LPA can be reconstituted by expression of Edg-4 or Edg-7 in cells lacking LPA responses. Compared to insulin, LPA stimulates only modest phosphatidylinositol 3-kinase (PI3K)-dependent activation of protein kinase B (PKB/Akt) that does not correlate with the magnitude of GSK-3 phosphorylation induced by LPA. PI3K inhibitors block insulin- but not LPA-induced GSK-3 phosphorylation. In contrast, the effect of LPA, but not that of insulin or platelet-derived growth factor (PDGF), is sensitive to protein kinase C (PKC) inhibitors. Downregulation of endogenous PKC activity selectively reduces LPA-mediated GSK-3 phosphorylation. Furthermore, several PKC isotypes phosphorylate GSK-3 in vitro and in vivo. To confirm a specific role for PKC in regulation of GSK-3, we further studied signaling properties of PDGF receptor beta subunit (PDGFRbeta) in HEK293 cells lacking endogenous PDGF receptors. In clones expressing a PDGFRbeta mutant wherein the residues that couple to PI3K and other signaling functions are mutated with the link to phospholipase Cgamma (PLCgamma) left intact, PDGF is fully capable of stimulating GSK-3 phosphorylation. The process is sensitive to PKC inhibitors in contrast to the response through the wild-type PDGFRbeta. Therefore, growth factors, such as PDGF, which control GSK-3 mainly through the PI3K-PKB/Akt module, possess the ability to regulate GSK-3 through an alternative, redundant PLCgamma-PKC pathway. LPA and potentially other natural ligands primarily utilize a PKC-dependent pathway to modulate GSK-3.     

Differential signalling pathways for EGF versus PDGF activation of Erk1/2 MAP kinase and cell proliferation in brown pre-adipocytes

Stimulation by both adrenergic and non-adrenergic pathways can induce proliferation of brown pre-adipocytes. To understand the signalling pathways involved in non-adrenergic stimulation of cell proliferation, we examined Erk1/2 activation. In primary cultures of mouse brown pre-adipocytes, both EGF (epidermal growth factor) and PDGF (platelet-derived growth factor) induced Erk1/2 activation. EGF-stimulated Erk1/2 activation involved Src tyrosine kinases, but not PKC or PI3K, whereas in PDGF-induced Erk1/2 activation, PI3K, PKC (probably the atypical ζ isoform) and Src were involved sequentially. Both EGF and PDGF induced PI3K-dependent Akt activation that was not involved in Erk1/2 activation. By comparing effects of signalling inhibitors (wortmannin, SH-6, TPA, Gö6983, PP2, PD98059) on EGF- and PDGF-induced Erk1/2 activation and cell proliferation (³H-thymidine incorporation), we conclude that while the signal transduction pathways initiated by these growth factors are clearly markedly different, their effects on cell proliferation can be fully explained through their stimulation of Erk1/2 activation; thus Erk1/2 is a common, essential step for stimulation of proliferation in these cells.     

胞浆Ca~(2+)和某些激酶对NADPH氧化酶激活和肌动蛋白聚合的作用

 为澄清中性粒细胞胞浆 Ca2 +和某些 O-·2 产生相关激酶对 NADPH氧化酶激活和肌动蛋白聚合的作用 ,利用分化为中性粒细胞样的 HL- 60细胞研究了胞浆 Ca2 +螯合剂 BAPTA- AM和激酶抑制剂对这些激酶激活、NADPH氧化酶激活和肌动蛋白聚合的影响 .使用 1 0 μmol/L的 Ca2 +螯合剂 BAPTA- AM去除胞浆 Ca2 +后 ,趋化肽 f MLP诱导的 O-·2 产生明显减少 ,但不影响 f MLP诱导的肌动蛋白聚合 ;8μmol/L的 PKC激酶抑制物 GF1 0 92 0 3x几乎完全抑制 O-·2 产生 ;50 μmol/L的p38激酶抑制物 SB2 0 3580、50 μmol/L的 ERK激酶抑制物 PD0 980 59和 0 .1 μmol/L的 PI3激酶抑制物渥曼青霉素 (Wortmannin)使 f MLP诱导的 O-·2 产生大约减少一半 ;其中 Wortmannin还抑制 f MLP诱导的肌动蛋白聚合 ;f MLP刺激细胞后 ,PI3- K、p38和 ERK激酶迅速激活 ,但这些激酶的激活对 Ca2 +是非必需的 .这些结果说明 Ca2 +依赖途径 (PKC)和 Ca2 +非依赖途径 (PI3- K、p38和ERK)对 NADPH氧化酶激活都起着重要作用 ,而 Ca2 +非依赖途径中的 PI3- K激酶还参与中性粒细胞样 HL- 60细胞的肌动蛋白聚合 .     

Mitogen-activated protein kinase and phosphorylation of connexin43 are not sufficient for the disruption of gap junctional communication by platelet-derived growth factor and tetradecanoylphorbol acetate

Disruption of gap junctional communication (GJC) by various compounds, including growth factors and tumor promoters, is believed to be modulated by the phosphorylation of a gap junctional protein, connexin43 (Cx43). We have previously demonstrated a platelet-derived growth factor (PDGF)-induced blockade of GJC and phosphorylation of Cx43 in T51B rat liver epithelial cells expressing wild-type PDGF receptor beta (PDGFr beta). Both of these actions of PDGF required participation of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). Similar requirements of MAPK were suggested in the modulation of GJC by other agents, including epidermal growth factor (EGF) and lysophosphatidic acid (LPA). Since many of these agents activate additional protein kinases, our present study examined whether activation of MAPK was sufficient for Cx43 phosphorylation and GJC blockade. By utilizing a variety of MAPK activators, we now show that activation of MAPK is not always associated with either Cx43 phosphorylation or disruption of GJC, which suggests a requirement for additional factors. Furthermore, pretreatment with hydrogen peroxide (H2O2), a potent MAPK activator but inefficient GJC/Cx43 modulator, abrogated PDGF- or TPA-induced disruption of GJC. While a 5 min H2O2 pretreatment abolished both PDGF- and TPA-induced Cx43 phosphorylation and GJC blockade, a simultaneous H2O2 treatment interfered only with GJC closure but not with the phosphorylation of Cx43 induced by PDGF and TPA. This finding indicates that, in addition to the Cx43 phosphorylation step, inhibition of GJC requires interaction with other components. H2O2-mediated abrogation of PDGF/TPA signaling can be neutralized by the antioxidant N-acetylcysteine (NAC) or by the tyrosine kinase inhibitor genistein. Taken together, our results suggest that disruption of GJC is not solely mediated by either activated MAPK or Cx43 phosphorylation but requires the participation of additional kinases and regulatory components. This complex mode of regulation is perhaps essential for the proposed functional role of GJC.     

Eosinophil major basic protein stimulates neutrophil superoxide production by a class IA phosphoinositide 3-kinase and protein kinase C-zeta-dependent pathway

Eosinophil major basic protein (MBP) is an effective stimulus for neutrophil superoxide (O(2)(-)) production, degranulation, and IL-8 production. In this study we evaluated the participation of phosphoinositide 3-kinase (PI3K) and PI3K-associated signaling events in neutrophil activation by MBP. Inhibition of PI3K activity blocked MBP-stimulated O(2)(-) production, but not degranulation or IL-8 production. Measurement of Akt phosphorylation at Ser(473) and Thr(308) confirmed that MBP stimulated PI3K activity and also demonstrated indirectly activation of phosphoinositide-dependent kinase-1 by MBP. Genistein and the Src kinase family inhibitor, 4-amino-5-(4-methyphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, inhibited MBP-stimulated phosphorylation of Akt. 4-Amino-5-(4-methyphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine also inhibited MBP-stimulated O(2)(-) production. MBP stimulated phosphorylation and translocation of the p85 subunit of class I(A) PI3K, but not translocation of the p110gamma subunit of class I(B) PI3K, to the neutrophil membrane. Inhibition of protein kinase Czeta (PKCzeta) inhibited MBP-stimulated O(2)(-) production. Measurement of phosphorylated PKCzeta (Thr(410)) and PKCdelta (Thr(505)) confirmed that PKCzeta, but not PKCdelta, is activated in MBP-stimulated neutrophils. The time courses for phosphorylation and translocation of the p85 subunit of class I(A) PI3K, activation of Akt, and activation of PKCzeta were similar. Moreover, inhibition of PI3K activity inhibited MBP-induced activation of PKCzeta. We conclude that MBP stimulates a Src kinase-dependent activation of class I(A) PI3K and, in turn, activation of PKCzeta in neutrophils, which contributes to the activation of NADPH oxidase and the resultant O(2)(-) production in response to MBP stimulation.