Mechanism of angiotensin II-induced superoxide production in cells reconstituted with angiotensin type 1 receptor and the components of NADPH oxidase

The mechanism of angiotensin II (Ang II)-induced superoxide production was investigated with HEK293 or Chinese hamster ovary cells reconstituted with the angiotensin type 1 receptor (AT(1)R) and NADPH oxidase (either Nox1 or Nox2) along with a pair of adaptor subunits (either NOXO1 with NOXA1 or p47(phox) with p67(phox)). Ang II enhanced the activity of both Nox1 and Nox2 supported by either adaptor pair, with more effective activation of Nox1 in the presence of NOXO1 and NOXA1 and of Nox2 in the presence of p47(phox) and p67(phox). Expression of several AT(1)R mutants showed that interaction of the receptor with G proteins but not that with beta-arrestin or with other proteins (Jak2, phospholipase C-gamma1, SH2 domain-containing phosphatase 2) that bind to the COOH-terminal region of AT(1)R, was necessary for Ang II-induced superoxide production. The effects of constitutively active alpha subunits of G proteins and of various pharmacological agents implicated signaling by a pathway comprising AT(1)R, Galpha(q/11), phospholipase C-beta, and protein kinase C as largely, but not exclusively, responsible for Ang II-induced activation of Nox1 and Nox2 in the reconstituted cells. A contribution of Galpha(12/13), phospholipase D, and phosphatidyl-inositol 3-kinase to Ang II-induced superoxide generation was also suggested, whereas Src and the epidermal growth factor receptor did not appear to participate in this effect of Ang II. In reconstituted cells stimulated with Ang II, Nox2 exhibited a more sensitive response than Nox1 to the perturbation of protein kinase C, phosphatidylinositol 3-kinase, or the small GTPase Rac1.     

Functional and biochemical consequences of abrogating the activation of multiple diverse early signaling pathways in Kit. Role for Src kinase pathway in Kit-induced cooperation with erythropoietin receptor

Kit receptor tyrosine kinase and erythropoietin receptor (Epo-R) cooperate in regulating blood cell development. Mice that lack the expression of Kit or Epo-R die in utero of severe anemia. Stimulation of Kit by its ligand, stem cell factor activates several distinct early signaling pathways, including phospholipase C gamma, phosphatidylinositol 3-kinase, Src kinase, Grb2, and Grb7. The role of these pathways in Kit-induced growth, proliferation, or cooperation with Epo-R is not known. We demonstrate that inactivation of any one of these early signaling pathways in Kit significantly impairs growth and proliferation. However, inactivation of the Src pathway demonstrated the most profound defect. Combined stimulation with Epo also resulted in impaired cooperation between Src-defective Kit mutant and Epo-R and, to a lesser extent, with Kit mutants defective in the activation of phosphatidylinositol 3-kinase or Grb2. The impaired cooperation between the Src-defective Kit mutant and Epo-R was associated with reduced transphosphorylation of Epo-R and expression of c-Myc. Remarkably, restoration of only the Src pathway in a Kit receptor defective in the activation of all early signaling pathways demonstrated a 50% correction in proliferation in response to Kit stimulation and completely restored the cooperation with Epo-R. These data demonstrate an essential role for Src pathway in regulating growth, proliferation, and cooperation with Epo-R downstream from Kit.     

Inhibiting Src family tyrosine kinase activity blocks glutamate signalling to ERK1/2 and Akt/PKB but not JNK in cultured striatal neurones

Glutamate receptor activation of mitogen-activated protein (MAP) kinase signalling cascades has been implicated in diverse neuronal functions such as synaptic plasticity, development and excitotoxicity. We have previously shown that Ca2+-influx through NMDA receptors in cultured striatal neurones mediates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt/protein kinase B (PKB) through a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathway. Exposing neurones to the Src family tyrosine kinase inhibitor PP2, but not the inactive analogue PP3, inhibited NMDA receptor-induced phosphorylation of ERK1/2 and Akt/PKB in a concentration-dependent manner, and reduced cAMP response element-binding protein (CREB) phosphorylation. To establish a link between Src family tyrosine kinase-mediated phosphorylation and PI 3-kinase signalling, affinity precipitation experiments were performed with the SH2 domains of the PI 3-kinase regulatory subunit p85. This revealed a Src-dependent phosphorylation of a focal adhesion kinase (FAK)-p85 complex on glutamate stimulation. Demonstrating that PI3-kinase is not ubiquitously involved in NMDA receptor signal transduction, the PI 3-kinase inhibitors wortmannin and LY294002 did not prevent NMDA receptor Ca2+-dependent phosphorylation of c-Jun N-terminal kinase 1/2 (JNK1/2). Further, inhibiting Src family kinases increased NMDA receptor-dependent JNK1/2 phosphorylation, suggesting that Src family kinase-dependent cascades may physiologically limit signalling to JNK. These results demonstrate that Src family tyrosine kinases and PI3-kinase are pivotal regulators of NMDA receptor signalling to ERK/Akt and JNK in striatal neurones.     

pp60Src mediates insulin-stimulated sequestration of the beta(2)-adrenergic receptor: insulin stimulates pp60Src phosphorylation and activation

Insulin stimulates a rapid phosphorylation and sequestration of the beta(2)-adrenergic receptor. Analysis of the signaling downstream of the insulin receptor with enzyme inhibitors revealed roles for both phosphatidylinositol 3-kinase and pp60Src. Inhibition of Src with PP2, like the inhibition of phosphatidylinositol 3-kinase with LY294002 [2-(4-morpholynyl)-8-phenyl-4H-1-benzopyran-4-one], blocked the activation of Src as well as insulin-stimulated sequestration of the beta(2)-adrenergic receptor. Depletion of Src with antisense morpholinos also suppressed insulin-stimulated receptor sequestration. Src is shown to be phosphorylated/activated in response to insulin in human epidermoid carcinoma A431 cells as well as in mouse 3T3-L1 adipocytes and their derivative 3T3-F422A cells, well-known models of insulin signaling. Inhibition of Src with PP2 blocks the ability of insulin to sequester beta(2)-adrenergic receptors and the translocation of the GLUT4 glucose transporters. Insulin stimulates Src to associate with the beta(2)-adrenergic receptor/AKAP250/protein kinase A/protein kinase C signaling complex. We report a novel positioning of Src, mediating signals from insulin to phosphatidylinositol 3-kinase and to beta(2)-adrenergic receptor trafficking.     

Osteopontin induces AP-1-mediated secretion of urokinase-type plasminogen activator through c-Src-dependent epidermal growth factor receptor transactivation in breast cancer cells

We have recently reported that osteopontin (OPN) stimulates cell motility and nuclear factor kappaB-mediated secretion of urokinase-type plasminogen activator (uPA) through phosphatidylinositol 3-kinase/Akt signaling pathways in breast cancer cells (Das, R., Mahabeleshwar, G. H., and Kundu, G. C. (2003) J. Biol. Chem. 278, 28593-28606). However, the role(s) of OPN on AP-1-mediated uPA secretion and cell motility and the involvement of c-Src/epidermal growth factor receptor (EGFR) in these processes in breast cancer cells are not well defined. In this study we report that OPN induces alpha(v)beta(3) integrin-mediated c-Src kinase activity in both highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. Ligation of OPN with alpha(v)beta(3) integrin induces kinase activity and tyrosine phosphorylation of EGFR in MDA-MB-231 and wild type EGFR-transfected MCF-7 cells, and this was inhibited by the dominant negative form of c-Src (dn c-Src) indicating that c-Src kinase plays a crucial role in this process. OPN induces association between alpha(v)beta(3) integrin and EGFR on the cell membrane in a macromolecular form with c-Src. Furthermore, OPN induces alpha(v)beta(3) integrin/EGFR-mediated ERK1/2 phosphorylation and AP-1 activation. Moreover, dn c-Src also suppressed the OPN-induced phosphatidylinositol (PI) 3-kinase activity in these cells indicating that c-Src acts as master switch in regulating MEK/ERK1/2 and phosphatidylinositol 3-kinase/Akt signaling pathways. OPN-induced ERK phosphorylation, AP-1 activation, uPA secretion, and cell motility were suppressed when cells were transfected with dn c-Src or pretreated with alpha(v)beta(3) integrin antibody, c-Src kinase inhibitor (pp2), EGFR tyrosine kinase inhibitor (PD153035), and MEK-1 inhibitor (PD98059). To our knowledge, this is the first report that OPN induces alpha(v)beta(3) integrin-mediated AP-1 activity and uPA secretion by activating c-Src/EGFR/ERK signaling pathways and further demonstrates a functional molecular link between OPN-induced integrin/c-Src-dependent EGFR phosphorylation and ERK/AP-1-mediated uPA secretion, and all of these ultimately control the motility of breast cancer cells.     

Involvement of the Src kinase Lyn in phospholipase C-gamma 2 phosphorylation and phosphatidylinositol 3-kinase activation in Epo signalling

We examined the role of the Src kinase Lyn in phospholipase C-gamma 2 (PLC-gamma 2) and phosphatidylinositol (PI) 3-kinase activation in erythropoietin (Epo)-stimulated FDC-P1 cells transfected with a wild type (WT) Epo-receptor (Epo-R). We showed that two inhibitors of Src kinases, PP1 and PP2, abolish both PLC-gamma 2 tyrosine phosphorylation and PI 3-kinase activity in WT Epo-R FDC-P1 cells. We also demonstrated that Epo-phosphorylated Lyn is associated with tyrosine phosphorylated PLC-gamma 2 and PI 3-kinase in WT Epo-R FDC-P1-stimulated cells. Moreover Epo-activated Lyn phosphorylates in vitro PLC-gamma 2 immunoprecipitated from unstimulated cells. Our results suggest that the Src kinase Lyn is involved in PLC-gamma 2 phosphorylation and PI 3-kinase activation induced by Epo.     

Lysophosphatidic acid promotes survival and differentiation of rat Schwann cells

Lysophosphatidic acid (LPA; 1-acyl-sn-glycerol-3-phosphate), an abundant constituent of serum, mediates multiple biological responses via G protein-coupled serpentine receptors. Schwann cells express the LPA receptors (Edg receptors), which, once activated, have the potential to signal through G(alphai) to activate p21(ras) and phosphatidylinositol 3-kinase, through G(alphaq) to activate phospholipase C, or through G(q12/13) to activate the Rho pathway. We found that the addition of serum or LPA to serum-starved Schwann cells rapidly (10 min) induced the appearance of actin stress fibers via a Rho-mediated pathway. Furthermore, LPA was able to rescue Schwann cells from apoptosis in a G(alphai)/phosphatidylinositol 3-kinase/MEK/MAPK-dependent manner. In addition, LPA increased the expression of myelin protein P(0) in Schwann cells in a Galpha(i)-independent manner but dependent on protein kinase C. By means of pharmacological and overexpression approaches, we found that the novel isozyme protein kinase Cdelta was required for myelin P(0) expression. Thus, the multiple effects of LPA in Schwann cells (actin reorganization, survival, and myelin gene expression) appear to be mediated through the different G protein-dependent pathways activated by the LPA receptor.     

Src family protein-tyrosine kinases alter the function of PTEN to regulate phosphatidylinositol 3-kinase/AKT cascades

Src family protein-tyrosine kinases, which play an important role in signal integration, have been implicated in tumorigenesis in multiple lineages, including breast cancer. We demonstrate, herein, that Src kinases regulate the phosphatidylinositol 3-kinase (PI3K) signaling cascade via altering the function of the PTEN tumor suppressor. Overexpression of activated Src protein-tyrosine kinases in PTEN-deficient breast cancer cells does not alter AKT phosphorylation, an indicator of signal transduction through the PI3K pathway. However, in the presence of functional PTEN, Src reverses the activity of PTEN, resulting in an increase in AKT phosphorylation. Activated Src reduces the ability of PTEN to dephosphorylate phosphatidylinositols in micelles and promotes AKT translocation to cellular plasma membranes but does not alter PTEN activity toward water-soluble phosphatidylinositols. Thus, Src may alter the capacity of the PTEN C2 domain to bind cellular membranes rather than directly interfering with PTEN enzymatic activity. Tyrosine phosphorylation of PTEN is increased in breast cancer cells treated with pervanadate, suggesting that PTEN contains sites for tyrosine phosphorylation. Src kinase inhibitors markedly decreased pervanadate-mediated tyrosine phosphorylation of PTEN. Further, expression of activated Src results in marked tyrosine phosphorylation of PTEN. SHP-1, a SH2 domain-containing protein-tyrosine phosphatase, selectively binds and dephosphorylates PTEN in Src transfected cells. Both Src inhibitors and SHP-1 overexpression reverse Src-induced loss of PTEN function. Coexpression of PTEN with activated Src reduces the stability of PTEN. Taken together, the data indicate that activated Src inhibits PTEN function leading to alterations in signaling through the PI3K/AKT pathway.     

The protein-tyrosine phosphatase SHP-2 is required during angiotensin II-mediated activation of cyclin D1 promoter in CHO-AT1A cells

Angiotensin II (Ang II) binds to specific G protein-coupled receptors and is mitogenic in Chinese hamster ovary (CHO) cells stably expressing a rat vascular angiotensin II type 1A receptor (CHO-AT(1A)). Cyclin D1 protein expression is regulated by mitogens, and its assembly with the cyclin-dependent kinases induces phosphorylation of the retinoblastoma protein pRb, a critical step in G(1) to S phase cell cycle progression contributing to the proliferative responses. In the present study, we found that in CHO-AT(1A) cells, Ang II induced a rapid and reversible tyrosine phosphorylation of various intracellular proteins including the protein-tyrosine phosphatase SHP-2. Ang II also induced cyclin D1 protein expression in a phosphatidylinositol 3-kinase and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)-dependent manner. Using a pharmacological and a co-transfection approach, we found that p21(ras), Raf-1, phosphatidylinositol 3-kinase and also the catalytic activity of SHP-2 and its Src homology 2 domains are required for cyclin D1 promoter/reporter gene activation by Ang II through the regulation of MAPK/ERK activity. Our findings suggest for the first time that SHP-2 could play an important role in the regulation of a gene involved in the control of cell cycle progression resulting from stimulation of a G protein-coupled receptor independently of epidermal growth factor receptor transactivation.     

Estradiol regulates the insulin-like growth factor-I (IGF-I) signalling pathway: a crucial role of phosphatidylinositol 3-kinase (PI 3-kinase) in estrogens requirement for growth of MCF-7 human breast carcinoma cells

Estrogens can stimulate the proliferation of estrogen-responsive breast cancer cells by increasing their proliferative response to insulin-like growth factors. With a view to investigating the molecular mechanisms implicated, we studied the effect of estradiol on the expression of proteins implicated in the insulin-like growth factor signalling pathway. Estradiol dose- and time-dependently increased the expression of insulin receptor substrate-1 and the p85/p110 subunits of phosphatidylinositol 3-kinase but did not change those of ERK2 and Akt/PKB. ICI 182,780 did not inhibit estradiol-induced IRS-1 and p85 expression. Moreover, two distinct estradiol-BSA conjugate compounds were as effective as estradiol in inducing IRS-1 and p85/p110 expression indicating the possible implication of an estradiol membrane receptor. Comparative analysis of steroids-depleted and steroids-treated cells showed that IGF-I only stimulates cell growth in the latter condition. Nevertheless, expression of a constitutively active form of PI 3-kinase in steroid-depleted cells triggers proliferation. These results demonstrate that estradiol positively regulates essential proteins of the IGF signalling pathway and put in evidence that phosphatidylinositol 3-kinase plays a central role in the synergistic pro-proliferative action of estradiol and IGF-I.     

Epidermal growth factor receptor-dependent control of keratinocyte survival and Bcl-xL expression through a MEK-dependent pathway

Previous work has shown that the epidermal growth factor receptor (EGFR) tyrosine kinase moiety provides protection to normal human keratinocytes against apoptosis. This protection is, at least in part, due to EGFR-dependent expression of the antiapoptotic Bcl-2 family member, Bcl-x(L). Here we focused on intracellular signaling pathways relevant to keratinocyte survival and/or Bcl-x(L) expression. By using pharmacological inhibitors and dominant negative expression constructs, we observed that phosphatidylinositol 3-kinase/AKT and phospholipase C gamma/protein kinase C alpha activation were required for keratinocyte survival independently of EGFR activation or Bcl-x(L) expression. By contrast, MEK activity required EGFR activation and, as shown by use of the MEK inhibitor PD98059 and a dominant negative MEK construct, was necessary for Bcl-x(L) expression and survival. Consistent with an earlier study, blocking SRC kinase activities similarly led to down-regulation of Bcl-x(L) protein expression and impaired keratinocyte survival. In conclusion, our results demonstrate that EGFR-dependent MEK activity contributes to both Bcl-x(L) expression and survival of normal keratinocytes. Other signaling pathways (i.e. phosphatidylinositol 3-kinase/AKT and phospholipase C gamma/protein kinase C alpha) are obligatory to keratinocyte survival but not to Bcl-x(L) expression, and control of these pathways by EGFR activation is not rate-limiting to normal keratinocyte survival.     

Saturated fatty acid-induced apoptosis in MDA-MB-231 breast cancer cells. A role for cardiolipin

Little is known about the biochemical basis of the action of free fatty acids (FFA) on breast cancer cell proliferation and apoptosis. Here we report that unsaturated FFAs stimulated the proliferation of human MDA-MB-231 breast cancer cells, whereas saturated FFAs inhibited it and caused apoptosis. Saturated FFA palmitate decreased the mitochondrial membrane potential and caused cytochrome c release. Palmitate-induced apoptosis was enhanced by the fat oxidation inhibitor etomoxir, whereas it was reduced by fatty-acyl CoA synthase inhibitor triacsin C. The non-metabolizable analog 2-bromopalmitate was not cytotoxic. This indicates that palmitate must be metabolized to exert its toxic effect but that its action does not involve fat oxidation. Pharmacological studies showed that the action of palmitate is not mediated via ceramides, reactive oxygen species, or changes in phosphatidylinositol 3-kinase activity. Palmitate caused early enhancement of cardiolipin turnover and decreased the levels of this mitochondrial phospholipid, which is necessary for cytochrome c retention. Cosupplementation of oleate, or increasing beta-oxidation with the AMP-activated protein kinase activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside, both restored cardiolipin levels and blocked palmitate-induced apoptosis. Oleate was preferentially metabolized to triglycerides, and oleate cosupplementation channeled palmitate esterification processes to triglycerides. Overexpression of Bcl-2 family members blocked palmitate-induced apoptosis. The results provide evidence that a decrease in cardiolipin levels and altered mitochondrial function are involved in palmitate-induced breast cancer cell death. They also suggest that the antiapoptotic action of oleate on palmitate-induced cell death involves both restoration of cardiolipin levels and redirection of palmitate esterification processes to triglycerides.     

ACh and adenosine activate PI3-kinase in rabbit hearts through transactivation of receptor tyrosine kinases

Adenosine and acetylcholine (ACh) trigger preconditioning through different signaling pathways. We tested whether either could activate myocardial phosphatidylinositol 3-kinase (PI3-kinase), a putative signaling protein in ischemic preconditioning. We used phosphorylation of Akt, a downstream target of PI3-kinase, as a reporter. Exposure of isolated rabbit hearts to ACh increased Akt phosphorylation 2.62 +/- 0.33 fold (P = 0.001), whereas adenosine caused a significantly smaller increase (1.52 +/- 0.08 fold). ACh-induced activation of Akt was abolished by the tyrosine kinase blocker genistein indicating at least one tyrosine kinase between the muscarinic receptor and Akt. ACh-induced Akt activation was blocked by the Src tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and by 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG-1478), an epidermal growth factor receptor (EGFR) inhibitor, suggesting phosphorylation of a receptor tyrosine kinase in an Src tyrosine kinase-dependent manner. ACh caused tyrosine phosphorylation of the EGFR, which could be blocked by PP2, thus supporting this receptor hypothesis. AG-1478 failed to block the cardioprotection of ACh, however, suggesting that other receptor tyrosine kinases might be involved. Therefore, G(i) protein-coupled receptors can activate PI3-kinase/Akt through transactivation of receptor tyrosine kinases in an Src tyrosine kinase-dependent manner.     

Tyrosine kinase-independent activation of extracellular-regulated kinase (ERK) 1/2 by the insulin-like growth factor-1 receptor

The extracellular-regulated kinase (ERK1/2) is a key conduit for transduction of signals from growth factor receptors to the nucleus. Previous work has shown that ERK1/2 activation in response to IGF-1 may require the participation of G proteins, but the role of the receptor tyrosine kinase in this process has not been clearly resolved. This investigation of IGF-1 receptor function was therefore designed to examine the contribution of the receptor tyrosine kinase to ERK1/2 activation. Phosphorylation of ERK1/2 in smooth muscle cells following treatment with IGF-1 was not blocked by pretreatment with AG1024 or picropodophylin, inhibitors of the IGF-1 receptor tyrosine kinase. Likewise, IGF-1 activated ERK1/2 in cells expressing a kinase-dead mutant of the IGF-1 receptor. ERK1/2 activation was unaffected by the phosphatidylinositol 3-kinase inhibitor LY-294002, but was sensitive to inhibitors of Src kinase, phospholipase C and Gβγ subunit signalling. Treatment with αIR-3, a neutralizing monoclonal antibody, also stimulated ERK1/2 phosphorylation without concomitant activation of the receptor tyrosine kinase. Phosphoprotein mapping of IGF-1 and αIR-3 treated cells confirmed that antibody-induced ERK1/2 phosphorylation occurred in the absence of tyrosine kinase phosphorylation, and enabled extension of these findings to p38 MAPK. These results suggest that stimulation of ERK1/2 phosphorylation by IGF-1 does not require activation of the receptor tyrosine kinase.     

Signal transduction by human-restricted Fc gamma RIIa involves three distinct cytoplasmic kinase families leading to phagocytosis.

Recent experiments indicate an important role for Src family and Syk protein tyrosine kinases and phosphatidylinositol 3-kinase in the signal transduction process initiated by mouse receptors for IgG and leading to phagocytosis. Considerably less is known regarding signal transduction by the human-restricted IgG receptor, FcgammaRIIa. Furthermore, the relationship among the Src family, Syk, and phosphatidylinositol 3-kinase in phagocytosis is not understood. Here, we show that FcgammaRIIa is phosphorylated by an Src family member, which results in recruitment and concomitant activation of the distal enzymes Syk and phosphatidylinositol 3-kinase. Using a FcgammaRI-p85 receptor chimera cotransfected with kinase-inactive mutants of Syk or application of a pharmacological inhibitor of Syk, we show that Syk acts in parallel with phosphatidylinositol 3-kinase. Our results indicate that FcgammaRIIa-initiated monocyte or neutrophil phagocytosis proceeds from the clustered IgG receptor to Src to phosphatidylinositol 3-kinase and Syk.     

Angiotensin II activation of focal adhesion kinase and pp60c-Src in relation to mitogen-activated protein kinases in hepatocytes

We have investigated signaling pathways leading to angiotensin II (Ang II) activation of mitogen-activated protein kinase (MAPK) in hepatocytes. MAPK activation by Ang II was abolished by the Ang II type 1 (AT1) receptor antagonist losartan, but not by the Ang II type 2 (AT2) receptor antagonist PD123319. Ang II (100 nM) induced a rapid phosphorylation of Src (peak approximately 2 min) and focal adhesion kinase (FAK, peak approximately 5 min) followed by a decrease to basal levels in 30 min. An increased association between FAK and Src in response to Ang II was detected after 1 min, which declined to basal levels after 30 min. Treatment with the Src kinase inhibitor PP-1 inhibited FAK phosphorylation. Downregulation of PKC, intracellular Ca2+ chelator BAPTA or inhibitors of PKC, Src kinase, MAPK kinase (MEK), Ca2+/calmodulin dependent protein kinase, phosphatidylinositol 3-kinase all blocked Ang II-induced MAPK phosphorylation. In contrast to other cells, there was no evidence for the role of EGF receptor transactivation in the activation of MAPK by Ang II. However, PDGF receptor phosphorylation is involved in the Ang II stimulated MAPK activation. Furthermore, Src/FAK and Ca/CaM kinase activation serve as potential links between the Ang II receptor and MAPK activation. These studies offer insight into the signaling network upstream of MAPK activation by AT1 receptor in hepatocytes.     

Eph B4 receptor signaling mediates endothelial cell migration and proliferation via the phosphatidylinositol 3-kinase pathway

The goals of this study were 2-fold: 1) to determine whether stimulation of Eph B4 receptors promotes microvascular endothelial cell migration and/or proliferation, and 2) to elucidate signaling pathways involved in these responses. The human endothelial cells used possessed abundant Eph B4 receptors with no endogenous ephrin B2 expression. Stimulation of these receptors with ephrin B2/Fc chimera resulted in dose- and time-dependent phosphorylation of Akt. These responses were inhibited by LY294002 and ML-9, blockers of phosphatidylinositol 3-kinase (PI3K) and Akt, respectively. Eph B4 receptor activation increased proliferation by 38%, which was prevented by prior blockade with LY294002, ML-9, and inhibitors of protein kinase G (KT5823) and MEK (PD98059). Nitrite levels increased over 170% after Eph B4 stimulation, indicating increased nitric oxide production. Signaling of endothelial cell proliferation appears to be mediated by a PI3K/Akt/endothelial nitric-oxide synthase/protein kinase G/mitogen-activated protein kinase cascade. Stimulation with ephrin B2 also increased migration by 63% versus controls. This effect was inhibited by blockade with PP2 (Src inhibitor), LY294002 or ML-9 but was unaffected by the PKG and MEK blockers. Eph B4 receptor stimulation increased activation of both matrix metalloproteinase-2 and -9. The results from these studies indicate that Eph B4 stimulates migration and proliferation and may play a role in angiogenesis.