Nox4 NAD(P)H oxidase mediates Src-dependent tyrosine phosphorylation of PDK-1 in response to angiotensin II: role in mesangial cell hypertrophy and fibronectin expression

Activation of glomerular mesangial cells (MCs) by angiotensin II (Ang II) leads to hypertrophy and extracellular matrix accumulation. Here, we demonstrate that, in MCs, Ang II induces an increase in PDK-1 (3-phosphoinositide-dependent protein kinase-1) kinase activity that required its phosphorylation on tyrosine 9 and 373/376. Introduction into the cells of PDK-1, mutated on these tyrosine residues or kinase-inactive, attenuates Ang II-induced hypertrophy and fibronectin accumulation. Ang II-mediated PDK-1 activation and tyrosine phosphorylation (total and on residues 9 and 373/376) are inhibited in cells transfected with small interfering RNA for Src, indicating that Src is upstream of PDK-1. In cells expressing oxidation-resistant Src mutant C487A, Ang II-induced hypertrophy and fibronectin expression are prevented, suggesting that the pathway is redox-sensitive. Ang II also up-regulates Nox4 protein, and siNox4 abrogates the Ang II-induced increase in intracellular reactive oxygen species (ROS) generation. Small interfering RNA for Nox4 also inhibits Ang II-induced activation of Src and PDK-1 tyrosine phosphorylation (total and on residues 9 and 373/376), demonstrating that Nox4 functions upstream of Src and PDK-1. Importantly, inhibition of Nox4, Src, or PDK-1 prevents the stimulatory effect of Ang II on fibronectin accumulation and cell hypertrophy. This work provides the first evidence that Nox4-derived ROS are responsible for Ang II-induced PDK-1 tyrosine phosphorylation and activation through stimulation of Src. Importantly, this pathway contributes to Ang II-induced MC hypertrophy and fibronectin accumulation. These data shed light on molecular processes underlying the oxidative signaling cascade engaged by Ang II and identify potential targets for intervention to prevent renal hypertrophy and fibrosis.     

Src and Pyk2 mediate angiotensin II effects in cultured rat astrocytes

Angiotensin II (Ang II)-induced proliferation of rat astrocytes is mediated by multiple signaling pathways. In the present study, we investigated the role of non-receptor tyrosine kinases on Ang II-signaling and proliferation of astrocytes cultured from neonatal rat pups. Ang II stimulated astrocyte growth, ERK1/2 phosphorylation and the phosphorylation of Src and proline-rich tyrosine kinase-2 (Pyk2), in astrocytes obtained from brainstem and cerebellum. Pretreatment with 10 microM PP2, a selective Src inhibitor, inhibited Ang II stimulated ERK1/2 phosphorylation by 59% to 91% both in brainstem and cerebellum astrocytes. PP2 also inhibited Ang II induction of brainstem (76% inhibition) and cerebellar (64% inhibition) astrocyte growth. Similarly, pretreatment with 25 microM dantrolene, the Pyk2 inhibitor, attenuated ERK1/2 activity in brainstem (62% inhibition) and in cerebellum astrocytes (44% inhibition). Interestingly, inhibition of Pyk2 inhibited Ang II-induced Src activation suggesting that these two non-receptor tyrosine kinases may be acting in concert to mediate Ang II effects in astrocytes. In summary, we found that Ang II stimulates the non-receptor tyrosine kinases Src and Pyk2 which mediate Ang II-induced ERK1/2 activation leading to stimulation of astrocyte growth. In addition, these two tyrosine kinases may be interacting to regulate effects of the peptide in these cells.     

Flow shear stress stimulates Gab1 tyrosine phosphorylation to mediate protein kinase B and endothelial nitric-oxide synthase activation in endothelial cells

Fluid shear stress generated by blood flow modulates endothelial cell function via specific intracellular signaling events. We showed previously that flow activated the phosphatidylinositol 3-kinase (PI3K), Akt, and endothelial nitric-oxide synthase (eNOS) via Src kinase-dependent transactivation of vascular endothelial growth factor receptor 2 (VEGFR2). The scaffold protein Gab1 plays an important role in receptor tyrosine kinase-mediated signal transduction. We found here that laminar flow (shear stress = 12 dynes/cm2) rapidly stimulated Gab1 tyrosine phosphorylation in both bovine aortic endothelial cells and human umbilical vein endothelial cells, which correlated with activation of Akt and eNOS. Gab1 phosphorylation as well as activation of Akt and eNOS by flow was inhibited by the Src kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and VEGFR2 kinase inhibitors SU1498 and VTI, suggesting that flow-mediated Gab1 phosphorylation is Src kinase-dependent and VEGFR2-dependent. Tyrosine phosphorylation of Gab1 by flow was functionally important, because flow stimulated the association of Gab1 with the PI3K subunit p85 in a time-dependent manner. Furthermore, transfection of a Gab1 mutant lacking p85 binding sites inhibited flow-induced activation of Akt and eNOS. Finally, knockdown of endogenous Gab1 by small interference RNA abrogated flow activation of Akt and eNOS. These data demonstrate a critical role of Gab1 in flow-stimulated PI3K/Akt/eNOS signal pathway in endothelial cells.     

A unique autophosphorylation site on Tie2/Tek mediates Dok-R phosphotyrosine binding domain binding and function

Tie2/Tek is an endothelial cell receptor tyrosine kinase that induces signal transduction pathways involved in cell migration upon angiopoietin-1 (Ang1) stimulation. To address the importance of the various tyrosine residues of Tie2 in signal transduction, we generated a series of Tie2 mutants and examined their signaling properties. Using this approach in conjunction with a phosphorylation state-specific antibody, we identified tyrosine residue 1106 on Tie2 as an Ang1-dependent autophosphorylation site that mediates binding and phosphorylation of the downstream-of-kinase-related (Dok-R) docking protein. This tyrosine residue is contained within a unique interaction motif for the phosphotyrosine binding domain of Dok-R, and the pleckstrin homology domain of Dok-R further contributes to Tie2 binding in a phosphatidylinositol 3'-kinase-dependent manner. Introduction of a Tie2 mutant lacking tyrosine residue 1106 into endothelial cells interferes with Dok-R phosphorylation in response to Ang1. Furthermore, this mutant is unable to restore the migration potential of endothelial cells derived from mice lacking Tie2. Together, these findings demonstrate that tyrosine residue 1106 on Tie2 is critical for coupling downstream cell migration signal transduction pathways with Ang1 stimulation in endothelial cells.     

Molecular control of angiopoietin signalling

The angiopoietins act through the endothelial receptor tyrosine kinase Tie2 to regulate vessel maturation in angiogenesis and control quiescence and stability of established vessels. The activating ligand, Ang1 (angiopoietin-1), is constitutively expressed by perivascular cells, and the ability of endothelial cells to respond to the ligand is controlled at the level of the Ang1 receptor. This receptor interacts with the related protein Tie1 on the cell surface, and Tie1 inhibits Ang1 signalling through Tie2. The responsiveness of endothelium to Ang1 is determined by the relative levels of Tie2 and the inhibitory co-receptor Tie1 in the cells. Tie1 undergoes regulated ectodomain cleavage which is stimulated by a range of factors including VEGF (vascular endothelial growth factor), inflammatory cytokines and changes in shear stress. Ectodomain cleavage of Tie1 relieves inhibition of Tie2 and enhances Ang1 signalling. This mechanism regulates Ang1 signalling without requiring changes in the level of the ligand and allows Ang1 signalling to be co-ordinated with other signals in the cellular environment. Regulation of signalling at the level of receptor responsiveness may be an important adaptation in systems in which an activating ligand is normally present in excess or where the ligand provides a constitutive maintenance signal.     

Angiotensin II induces focal adhesion kinase/paxillin phosphorylation and cell migration in human umbilical vein endothelial cells

In the present study, we demonstrated that Ang II provokes a transitory enhancement of focal adhesion kinase (FAK) and paxillin phosphorylation in human umbilical endothelial cells (HUVEC). Moreover, Ang II induces a time- and dose-dependent augmentation in cell migration, but does not affect HUVEC proliferation. The effect of Ang II on FAK and paxillin phosphorylation was markedly attenuated in cells pretreated with wortmannin and LY294002, indicating that phosphoinositide 3-kinase (PI3K) plays an important role in regulating FAK activation. Similar results were observed when HUVEC were pretreated with genistein, a non-selective tyrosine kinases inhibitor, or with the specific inhibitor PP2 for Src family kinases, demonstrating the involvement of protein tyrosine kinases, and particularly Src family of tyrosine kinases, in the downstream signalling pathway of Ang II receptors. Furthermore, FAK and paxillin phosphorylation was markedly blocked after treatment of HUVEC with AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) phosphorylation. Pretreatment of cells with inhibitors of PI3K, Src family tyrosine kinases, and EGFR also decreased HUVEC migration. In conclusion, these results suggest that Ang II mediates an increase in FAK and paxillin phosphorylation and induces HUVEC migration through signal transduction pathways dependent on PI3K and Src tyrosine kinase activation and EGFR transactivation.     

Sphingosine 1-phosphate induces membrane ruffling and increases motility of human umbilical vein endothelial cells via vascular endothelial growth factor receptor and CrkII

Sphingosine 1-phosphate (S1P), a ligand for endothelial differentiation gene family proteins, is one of the most potent signal mediators released from activated platelets. Here, we report that S1P induces membrane ruffling of human umbilical vein endothelial cells (HUVECs) via the vascular endothelial growth factor receptor (VEGFR), Src family tyrosine kinase(s), and the CrkII adaptor protein. S1P induced prominent phosphorylation of CrkII in HUVECs, indicating that CrkII was involved in the S1P-induced signaling pathway. S1P-induced CrkII phosphorylation was blocked by pertussis toxin and overexpression of the carboxyl terminus of beta-adrenergic receptor kinase, indicating that the betagamma subunit of G(i) was required for the phosphorylation. Notably, the S1P-induced CrkII phosphorylation was also abolished by inhibitors of VEGFR or Src family tyrosine kinases. By using Picchu, a real time monitoring protein for CrkII phosphorylation, we found that S1P induced rapid CrkII phosphorylation at membrane ruffles. Finally, we observed that expression of a dominant negative mutant of CrkII inhibited the S1P-induced membrane ruffling and cell migration. These results delineated a novel S1P signaling pathway that involves sequential activation of G(i)-coupled receptor(s), VEGFR, Src family tyrosine kinase(s), and the CrkII adaptor protein, and which is responsible for both the induction of membrane ruffling and the increase in cell motility.     

Lipid rafts serve as signaling platforms for Tie2 receptor tyrosine kinase in vascular endothelial cells

The Tie2 receptor tyrosine kinase plays a pivotal role in vascular and hematopoietic development. The major intracellular signaling systems activated by Tie2 in response to Angiopoietin-1 (Ang1) include the Akt and Erk1/2 pathways. Here, we investigated the role of cholesterol-rich plasma membrane microdomains (lipid rafts) in Tie2 regulation. Tie2 could not be detected in the lipid raft fraction of human umbilical vein endothelial cells (HUVECs) unless they were first stimulated with Ang1. After stimulation, a minor fraction of Tie2 associated tightly with the lipid rafts. Treatment of HUVECs with the lipid raft disrupting agent methyl-β-cyclodextrin selectively inhibited Ang1-induced Akt phosphorylation, but not Erk1/2 phosphorylation. It has been reported that inhibition of FoxO activity is an important mechanism for Ang1-stimulated Tie2-mediated endothelial function. Consistent with this, we found that phosphorylation of FoxO mediated by Tie2 activation was attenuated by lipid raft disruption. Therefore, we propose that lipid rafts serve as signaling platforms for Tie2 receptor tyrosine kinase in vascular endothelial cells, especially for the Akt pathway.     

Reactive oxygen species induce reversible PECAM-1 tyrosine phosphorylation and SHP-2 binding

Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) functions to control the activation and survival of the cells on which it is expressed. Many of the regulatory functions of PECAM-1 are dependent on its tyrosine phosphorylation and subsequent recruitment of the Src homology (SH2) domain containing protein tyrosine phosphatase SHP-2. The recent demonstration that PECAM-1 tyrosine phosphorylation occurs in cells exposed to the reactive oxygen species hydrogen peroxide (H2O2) suggested that this form of oxidative stress may also support PECAM-1/SHP-2 complex formation. In the present study, we show that PECAM-1 tyrosine phosphorylation in response to exposure of cells to H2O2 is reversible, involves a shift in the balance between kinase and phosphatase activities, and supports binding of SHP-2 and recruitment of this phosphatase to cell-cell borders. We speculate, however, that the unique ability of H2O2 to reversibly oxidize the reactive site cysteine residues of protein tyrosine phosphatases may result in transient inactivation of the SHP-2 that is bound to PECAM-1 under these conditions. Finally, we provide evidence that PECAM-1 tyrosine phosphorylation and SHP-2 binding in endothelial cells requires exposure to an "oxidative burst" of H2O2, but that exposure of these cells to sufficiently high concentrations of H2O2 for a sufficiently long period of time abrogates binding of SHP-2 to tyrosine-phosphorylated PECAM-1. These findings support a role for PECAM-1 as a sensor of oxidative stress, perhaps most importantly during the process of inflammation.     

Bile acids alter the subcellular localization of CNT2 (concentrative nucleoside cotransporter) and increase CNT2-related transport activity in liver parenchymal cells

PI3K (phosphoinositide 3-kinase) activity is involved in Ang (angiotensin) II-stimulated VSMC (vascular smooth muscle cell) growth and hypertrophy. In the present study, we demonstrate that the inhibition of PI3K in VSMCs by expression of a dominant-negative p85alpha mutant lacking the p110-binding domain (Deltap85), or by treatment of cells with LY294002, inhibited Ang II-stimulated PAI-1 (plasminogen activator inhibitor-1) mRNA expression. Using a GST (glutathione S-transferase) fusion protein containing the p85 N-terminal SH2 (Src homology 2) domain as 'bait' followed by MS/MS (tandem MS), we identified a 70 kDa fragment of the p70 PDGFR-beta (platelet-derived growth factor receptor-beta) as a signalling adapter that is phosphorylated and recruits the p85 subunit of PI3K after Ang II stimulation of AT1 (Ang II subtype 1) receptors on VSMCs. This fragment of the PDGFR-beta, which has a truncation of its extracellular domain, accounted for approx. 15% of the total PDGFR-beta detected in VSMCs with an antibody against its cytoplasmic domain. Stimulation of VSMCs with Ang II increased tyrosine-phosphorylation of p70 PDGFR-beta at Tyr751 and Tyr1021 and increased its binding to p85. PDGF also induced phosphorylation of p70 PDGFR-beta, a response inhibited by the PDGF tyrosine kinase selective inhibitor, AG1296. By contrast, Ang II-induced phosphorylation of the 70 kDa receptor was not affected by AG1296. Ang II-stimulated phosphorylation of the p70 PDGFR-beta was blocked by the AT1 receptor antagonist, candesartan (CV 11974) and was partially inhibited by PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine}, an Src family kinase inhibitor. Our result suggests that the p70 PDGFR-beta functions as an adapter that recruits PI3K to the membrane upon AT1 receptor stimulation.     

Regulation of calcium-sensitive tyrosine kinase Pyk2 by angiotensin II in endothelial cells. Roles of Yes tyrosine kinase and tyrosine phosphatase SHP-2

Calcium-sensitive tyrosine kinase Pyk2 has been implicated in the regulation of ion channels, cellular adhesion, and mitogenic and hypertrophic reactions. In this study, we have investigated the regulation of Pyk2 by angiotensin II (Ang II) in pulmonary vein endothelial cells. We found that the Ang II-induced tyrosine phosphorylation of Pyk2, which requires the activity of Src family kinase, was specifically regulated by the Src family kinase member, Yes kinase. Moreover, we identified for the first time the constitutive association of Pyk2 with an Src homology 2 (SH2) domain-containing tyrosine phosphatase SHP-2. SHP-2 interacts with Pyk2 through a region other than its SH2 domains. Pyk2 can be dephosphorylated in vitro in SHP-2 immunoprecipitates and in intact cells expressing an NH(2) terminus-truncated form of SHP-2, which lacks the two SH2 domains but has an enhanced phosphatase activity. Ang II activates the endogenous SHP-2. Finally, the SHP-2-mediated dephosphorylation of Pyk2 correlates with the negative effect of SHP-2 on the Ang II-induced activation of extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase. Thus, the balance of Pyk2 tyrosine phosphorylation in response to Ang II is controlled by Yes kinase and by a tyrosine phosphatase SHP-2 in endothelial cells.     

Tyrosine phosphorylation of cortactin is required for H2O2-mediated injury of human endothelial cells

Injury of endothelial cells induced by reactive oxygen species plays an important role in the development of early stages of vascular diseases such as hypertension and atherosclerosis. Exposure of human umbilical vein endothelial cells to hydrogen peroxide (H(2)O(2)), a common form of reaction oxygen species, triggers a series of intracellular events, including actin cytoskeletal reorganization, cytoplasm shrinkage, membrane blebbing and protein-tyrosine phosphorylation. The effect of H(2)O(2) on endothelial cells is dramatically enhanced when a survival pathway involving extracellular signal-regulated kinase is blocked by PD098059. In contrast, the injury of endothelial cells mediated by H(2)O(2) is inhibited by PP2, a selective specific inhibitor for protein-tyrosine kinase Src. Cortactin, a filamentous actin (F-actin)-associated protein, becomes phosphorylated at tyrosine residues upon stimulation by H(2)O(2) in a manner dependent on the activity of Src. The level of tyrosine phosphorylation of cortactin is correlated with the formation of membrane blebs. Overexpression of wild-type cortactin tagged with green fluorescent protein in endothelial cells via a retroviral vector substantiates the H(2)O(2)-induced morphological changes, whereas overexpression of a green fluorescent protein-cortactin mutant deficient in tyrosine phosphorylation renders endothelial cells resistant to H(2)O(2). The functional role of cortactin in H(2)O(2)-mediated shape changes was also evaluated in NIH 3T3 cells. Stable 3T3 transfectants expressing wild-type cortactin in the presence of either H(2)O(2)/PD098059 or H(2)O(2) alone at 200 microm exhibited a dramatic shape change characterized by rounding up or aggregation. However, the similar changes were not detected with cells overexpressing a cortactin mutant deficient in tyrosine phosphorylation. These data demonstrate an important role of the Src/cortactin-dependent actin reorganization in the injury of endothelial cells mediated by reactive oxygen species.     

Characterization and expression of a novel alternatively spliced human angiopoietin-2

Angiopoietin-2 (Ang2) is a naturally occurring antagonist of angiopoietin-1 (Ang1) that competes for binding to the Tie2 receptor and blocks Ang1-induced Tie2 autophosphorylation during vasculogenesis. Using the polymerase chain reaction, we isolated a cDNA encoding a novel shorter form of Ang2 from human umbilical vein endothelial cell cDNA and have designated it angiopoietin-2(443) (Ang2(443)), because it contains 443 amino acids. Part of the coiled-coil domain (amino acids 96-148) is absent in Ang2(443) because of alternative splicing of the gene. Like Ang2, recombinant Ang2(443) expressed in COS-7 cells is secreted as a glycosylated homodimeric protein. Recombinant Ang2(443) binds to the Tie2 receptor but does not induce Tie2 phosphorylation. Pre-occupation of Ang2(443) on Tie2 inhibits Ang1 or Ang2 binding and inhibits Ang1-induced phosphorylation. Expression of Ang2(443) mRNA is detectable in primary endothelial cells, several nonendothelial tumor cell lines, and primary tumor tissues. Interestingly, two cervical carcinoma cell lines express relatively moderate levels of Ang2(443) mRNA and protein. Macrophages express mainly Ang2 mRNA, but the expression of Ang2(443) mRNA is temporarily up-regulated during macrophage differentiation. These results suggest that Ang2(443) is a functional antagonist of Ang1 and could be an important regulator of angiogenesis during some tumorigenic and inflammatory processes.     

Thrombin-stimulated Pyk2 phosphorylation in human endothelium is dependent on intracellular calcium and independent of protein kinase C and Src kinases

G-protein-coupled receptor agonists (GPCAs) cause functional responses in endothelial cells including secretion, proliferation, and altering monolayer permeability. These events are mediated in part by activation of the p42/44 mitogen-activated protein kinase (MAPK) cascade. The cytosolic tyrosine kinase Pyk2 is postulated to link GPCA-induced changes in intracellular calcium to activation of the MAP kinase cascade. We have investigated the regulation of Pyk2 in human umbilical vein endothelial cells in response to GPCAs and show that (1) thrombin, a PAR-1 peptide, and histamine cause rapid concentration- and time-dependent phosphorylation on tyrosines 402 (Src kinase binding site), 881 (Grb2 binding site), and 580 (an autophosphorylation site), (2) thrombin-stimulated phosphorylation is dependent on intracellular calcium and independent of PKC and PI-3 kinase, and (3) inhibition of Src kinases has no significant effect on thrombin-stimulated phosphorylation, implying that tyrosine phosphorylation of Pyk2 is independent of Src binding.