Hydrogen peroxide stimulates tetrahydrobiopterin synthesis through the induction of GTP-cyclohydrolase I and increases nitric oxide synthase activity in vascular endothelial cells

Tetrahydrobiopterin (BH4), which is an essential cofactor for nitric oxide synthase (NOS), is generally accepted as an important molecular target for oxidative stress. This study examined whether hydrogen peroxide (H(2)O(2)), one of the reactive oxygen species (ROS), affects the BH4 level in vascular endothelial cells (ECs). Interestingly, the addition of H(2)O(2) to ECs markedly increased the BH4 level, but not its oxidized forms. The H(2)O(2)-induced increase in the BH4 level was blocked by the inhibitor of GTP-cyclohydrolase I (GTPCH), which is the rate-limiting enzyme of BH4 synthesis. Moreover, H(2)O(2) induced the expression of GTPCH mRNA, and the inhibitors of protein synthesis blocked the H(2)O(2)-induced increase in the BH4 level. The expression of the inducible isoform of NOS (iNOS) was slightly induced by the treatment with H(2)O(2). Additionally, the L-citrulline formation from L-arginine, which is the marker for NO synthesis, was stimulated by the treatment with H(2)O(2), and the H(2)O(2)-induced L-citrulline formation was strongly attenuated by NOS or GTPCH inhibitor. These results suggest that H(2)O(2) induces BH4 synthesis via the induction of GTPCH, and the increased BH4 is coupled with NO production by coinduced iNOS. H(2)O(2) appears to be one of the important signaling molecules to regulate the BH4-NOS system.     

Hydrogen peroxide stimulates tyrosine phosphorylation of the insulin receptor and its tyrosine kinase activity in intact cells.

H-35 rat hepatoma cells were labelled with [32P]orthophosphate and their insulin receptors isolated on wheat germ agglutinin (WGA)-agarose and anti-(insulin receptor) serum. The incubation of these cells with 10 mM-H2O2 for 10 min increased the phosphorylation of both the serine and tyrosine residues of the beta subunit of the insulin receptor. Next, insulin receptors were purified on WGA-agarose from control and H2O2-treated H-35 cells and the purified fractions incubated with [gamma-32P]ATP and Mn2+. Phosphorylation of the beta subunit of insulin receptors obtained from H2O2-treated cells was 150% of that of control cells. The kinase activity of the WGA-purified receptor preparation obtained from H2O2-treated cells, as measured by phosphorylation of src-related synthetic peptide, was increased about 4-fold over control cells. These data suggest that in intact cell systems, H2O2 may increase the insulin receptor kinase activity by inducing phosphorylation of the beta subunit of insulin receptor.     

Jak2 tyrosine kinase mediates oxidative stress-induced apoptosis in vascular smooth muscle cells

In vascular smooth muscle cells, Jak2 tyrosine kinase becomes activated in response to oxidative stress in the form of hydrogen peroxide. Although it has been postulated that hydrogen peroxide-induced Jak2 activation promotes cell survival, this has never been tested. We therefore examined the role that Jak2 plays in vascular smooth muscle cell apoptosis following hydrogen peroxide treatment. Here, we report that Jak2 tyrosine kinase activation by hydrogen peroxide is required for apoptosis of vascular smooth muscle cells. Upon treatment of primary rat aortic smooth muscle cells with hydrogen peroxide, we observed laddering of genomic DNA and nuclear condensation, both hallmarks of apoptotic cells. However, apoptosis was prevented by either the expression of a dominant negative Jak2 protein or by the Jak2 pharmacological inhibitor AG490. Moreover, expression of the proapoptotic Bax protein was induced following hydrogen peroxide treatment. Again, expression of a dominant negative Jak2 protein or treatment of cells with AG490 prevented this Bax induction. Following Bax induction by hydrogen peroxide, mitochondrial membrane integrity was compromised, and caspase-9 became activated. In contrast, in cells expressing a Jak2 dominant negative we observed that mitochondrial membrane integrity was preserved, and no caspase-9 activation occurred. These data demonstrate that the activation of Jak2 tyrosine kinase by hydrogen peroxide is essential for apoptosis of vascular smooth muscle cells. Furthermore, this report identifies Jak2 as a potential therapeutic target in vascular diseases in which vascular smooth muscle cell apoptosis contributes to pathological progression.     

Hydrogen peroxide induces GADD153 in Jurkat cells through the protein kinase C-dependent pathway

Growth arrest and DNA damage-inducible gene 153 (GADD153) is a CCAAT/enhancer binding protein (C/EBP) related gene and is induced in response to various stimuli including DNA damaging agents, UV irradiation, and serum starvation. In this study, we investigated which intracellular signals contribute to the expression of GADD153 mRNA in Jurkat cells in response to oxidative stress using several kinds of kinase inhibitors. GADD153 mRNA expression was immediately enhanced following hydrogen peroxide exposure and was significantly inhibited by treatment with H-7, staurosporin, and Ro-31-8220. In particular, rottlerin, a PKCdelta specific inhibitor, markedly attenuated hydrogen peroxide-induced GADD153 mRNA expression even at 1 microM. Treatment with a potent PKC activator, phorbol-12-myristate-13-acetate (PMA), augmented GADD153 mRNA in Jurkat cells in the presence of hydrogen peroxide, although PMA alone induced GADD153 mRNA marginally. Hydrogen peroxide significantly enhanced the AP-1 binding activity of the nuclear extract from Jurkat cells to the GADD153 AP-1 binding site. AP-1 binding activity was suppressed by rottlerin treatment. These findings indicate that PKC, especially PKCdelta, plays an important role in the induction of GADD153 mRNA following oxidative stress.     

Identification of tyrosine 972 as a novel site of Jak2 tyrosine kinase phosphorylation and its role in Jak2 activation

Jak2 is a 130 kDa tyrosine kinase that is important in a number of cellular signaling pathways. Its function is intrinsically regulated by the phosphorylation of a handful of its 49 tyrosines. Here, we report that tyrosine 972 (Y972) is a novel site of Jak2 phosphorylation and, hence, autoregulation. Specifically, we found that Y972 is phosphorylated and confirmed that this residue resides on the surface of the protein. Using expression plasmids that expressed either wild-type Jak2 or a full-length Jak2 cDNA containing a single Y972F substitution mutation, we investigated the consequences of losing Y972 phosphorylation on Jak2 function. We determined that the loss of Y972 phosphorylation significantly reduced the levels of both Jak2 total tyrosine phosphorylation and phosphorylation of Y1007/Y1008. Additionally, Y972 phosphorylation was shown to be important for maximal kinase function. Interestingly, in response to classical cytokine activation, the Jak2 Y972F mutant exhibited a moderately impaired level of activation when compared to the wild-type protein. However, when Jak2 was activated via a GPCR ligand, the ability of the Y972F mutant to be activated was completely lost, therefore suggesting a differential role of Y972 in Jak2 activation. Finally, we found that phosphorylation of Y972 enhances Jak2 kinase function via a mechanism that appears to stabilize the active conformation of the protein. Collectively, our results suggest that Y972 is a novel site of Jak2 phosphorylation and plays an important differential role in ligand-dependent Jak2 activation via a mechanism that involves stabilization of the Jak2 active conformation.     

Direct activation of AMP-activated protein kinase stimulates nitric-oxide synthesis in human aortic endothelial cells

Recent studies have indicated that endothelial nitric-oxide synthase (eNOS) is regulated by reversible phosphorylation in intact endothelial cells. AMP-activated protein kinase (AMPK) has previously been demonstrated to phosphorylate and activate eNOS at Ser-1177 in vitro, yet the function of AMPK in endothelium is poorly characterized. We therefore determined whether activation of AMPK with 5'-aminoimidazole-4-carboxamide ribonucleoside (AICAR) stimulated NO production in human aortic endothelial cells. AICAR caused the time- and dose-dependent stimulation of AMPK activity, with a concomitant increase in eNOS Ser-1177 phosphorylation and NO production. AMPK was associated with immunoprecipitates of eNOS, yet this was unaffected by increasing concentrations of AICAR. AICAR also caused the time- and dose-dependent stimulation of protein kinase B phosphorylation. To confirm that the effects of AICAR were indeed mediated by AMPK, we utilized adenovirus-mediated expression of a dominant negative AMPK mutant. Expression of dominant negative AMPK attenuated AICAR-stimulated AMPK activity, eNOS Ser-1177 phosphorylation and NO production and was without effect on AICAR-stimulated protein kinase B Ser-473 phosphorylation or NO production stimulated by insulin or A23187. These data suggest that AICAR-stimulated NO production is mediated by AMPK as a consequence of increased Ser-1177 phosphorylation of eNOS. We propose that stimuli that result in the acute activation of AMPK activity in endothelial cells stimulate NO production, at least in part due to phosphorylation and activation of eNOS. Regulation of endothelial AMPK therefore provides an additional mechanism by which local vascular tone may be controlled.     

Interferons induce tyrosine phosphorylation of the eIF2alpha kinase PKR through activation of Jak1 and Tyk2

The interferon (IFN)-inducible, double-stranded RNA activated protein kinase (PKR) is a dual-specificity kinase, which has an essential role in the regulation of protein synthesis by phosphorylating the translation eukaryotic initiation factor 2 (eIF2). Here, we show the tyrosine (Tyr) phosphorylation of PKR in response to type I or type II IFNs. We show that PKR physically interacts with either Jak1 or Tyk2 in unstimulated cells and that these interactions are increased in IFN-treated cells. We also show that PKR acts as a substrate of activated Jaks, and is phosphorylated at Tyr 101 and Tyr 293 both in vitro and in vivo. Moreover, we provide strong evidence that both the induction of eIF2alpha phosphorylation and inhibition of protein synthesis by IFN are impaired in cells lacking Jak1 or Tyk2, which corresponds to a lack of induction of PKR tyrosine phosphorylation. We conclude that PKR tyrosine phosphorylation provides an important link between IFN signalling and translational control through the regulation of eIF2alpha phosphorylation.     

Hydrogen peroxide stimulates the epithelial sodium channel through a phosphatidylinositide 3-kinase-dependent pathway

Recent studies indicate that oxidative stress mediates salt-sensitive hypertension. To test the hypothesis that the renal epithelial sodium channel (ENaC) is a target of oxidative stress, patch clamp techniques were used to determine whether ENaC in A6 distal nephron cells is regulated by hydrogen peroxide (H(2)O(2)). In the cell-attached configuration, H(2)O(2) significantly increased ENaC open probability (P(o)) and single-channel current amplitude but not the unit conductance. High concentrations of exogenous H(2)O(2) are required to elevate intracellular H(2)O(2), probably because catalase, the enzyme that promotes the decomposition of H(2)O(2) to H(2)O and O(2), is highly expressed in A6 cells. The effect of H(2)O(2) on ENaC P(o) was enhanced by 3-aminotriazole, a catalase inhibitor, and abolished by overexpression of catalase, indicating that intracellular H(2)O(2) levels are critical to produce the effect. However, H(2)O(2) did not directly activate ENaC in inside-out patches. The effects of H(2)O(2) on ENaC P(o) and amiloride-sensitive Na(+) current were abolished by inhibition of phosphatidylinositide 3-kinase (PI3K). Confocal microscopy data showed that H(2)O(2) elevated phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) in the apical membrane by stimulating PI3K. Because ENaC is stimulated by PI(3,4,5)P(3), these data suggest that H(2)O(2) stimulates ENaC via PI3K-mediated increases in apical PI(3,4,5)P(3).     

Possible involvement of hydroxyl radical on the stimulation of tetrahydrobiopterin synthesis by hydrogen peroxide and peroxynitrite in vascular endothelial cells

We recently described that hydrogen peroxide (H2O2) stimulates the synthesis of tetrahydrobiopterin (BH4) through the induction of the rate-limiting enzyme GTP-cyclohydrolase I (GTPCH), and increases tetrahydrobiopterin content in vascular endothelial cells. Tetrahydrobiopterin is easily oxidized by peroxynitrite (ONOO-), but not by hydrogen peroxide. The aim of this study was to determine the effect of hydroxyl radical and peroxynitrite, which are both toxic biological oxidants, on tetrahydrobiopterin synthesis and the regulation of its content in vascular endothelial cells. In the cell-free assay system, tetrahydrobiopterin was rapidly oxidized by the hydroxyl radical and peroxynitrite, but not by hydrogen peroxide. However, the addition of not only hydrogen peroxide but also the hydroxyl radical and peroxynitrite to vascular endothelial cells transiently decreased tetrahydrobiopterin content, and then markedly increased its content. Interestingly, total biopterin content was also decreased by early treatment with oxidants. Moreover, oxidants induced the expression of GTP-cyclohydrolase I, and the increase of the tetrahydrobiopterin content was blocked by the treatment with GTP-cyclohydrolase I inhibitor. Both the hydrogen peroxide- and peroxynitrite-induced increases in tetrahydrobiopterin content and findings suggest that not only hydrogen peroxide but also the hydroxyl radical and peroxynitrite stimulates tetrahydrobiopterin synthesis through GTP-cyclohydrolase I expression, and that the hydroxyl radical plays a central role in the stimulation of tetrahydrobiopterin synthesis. Moreover, the transient decrease in BH4 to tetrahydrobiopterin.     

Hydrogen peroxide induces apoptosis in HeLa cells through mitochondrial pathway

Cervical cancer is the most common cancer amongst females in India and is associated with high risk HPVs, reactive oxygen species (ROS), and excessive inflammation in most cases. ROS in turn affects the expression of pro- and anti-apoptotic proteins. The objective of the present study was to elucidate the effect of hydrogen peroxide (H(2)O(2)) on apoptotic signaling molecules in vitro. HeLa cell line expresses the Human papilloma virus - 18, E6 oncoprotein which causes the ubiquitin mediated degradation of p53 protein and is thus p53 deficient. p53 is known to act as a cellular stress sensor and triggers apoptosis. p73, a member of the p53 family also induces apoptosis in response to DNA damaging agents but unlike p53, it is infrequently mutated in human tumors. We demonstrate here, that in HeLa cells, apoptosis is triggered by H(2)O(2) via the mitochondrial pathway involving upregulation of p73, and its downstream target Bax. This was accompanied by upregulation of ERK, JNK, c-Myc, Hsp-70 and down regulation of anti-apoptotic Bcl-XL, release of cytochrome c from mitochondria and activation of caspases-9 and -3.     

Globular adiponectin induces adhesion molecule expression through the sphingosine kinase pathway in vascular endothelial cells

The signaling pathways that couple adiponectin receptors to functional, particularly inflammatory, responses have remained elusive. We report here that globular adiponectin induces endothelial cell activation, as measured by the expression of adhesion proteins such as vascular adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), E-selectin and MCP-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with globular adiponectin resulted in NF-kappaB activation and increased mRNA levels of VCAM-1, ICAM-1, E-selectin and MCP-1. Sphingosine 1-phosphate (S1P), but not ceramide or sphingosine, was a potent stimulator of adhesion protein expression. As S1P is generated from sphingosine by SKase, we treated cells with siRNA for SKase to silence the effects of S1P in the endothelial cells. Treatment with SKase siRNA inhibited globular adiponectin-induced NF-kappaB activation and markedly decreased the globular adiponectin-induced mRNA levels of adhesion protein. Thus, we demonstrated that the SKase pathway, through the generation of S1P, is critically involved in mediating globular adiponectin-induced endothelial cell activation.     

Hydrogen peroxide stimulates exocytosis of von Willebrand factor in human umbilical vein endothelial cells

The aim of our research was to study the influence of hydrogen peroxide on the exocytosis of von Willebrand factor (vWF) in human umbilical vein endothelial cells (HUVEC). We have found that H₂O₂ at a non-toxic concentration (100 μM) increases the amount of vWF secreted by HUVEC by 43 ± 14% over control (p < 0.03) and elevates total exposition of vWF on cell surface by 89 ± 5% (p < 0.01). Analysis of immunofluorescent images of HUVEC with CellProfiler program revealed that the average number of antigen positive structures on the single cell surface increases from 11.4 ± 0.16 in control up to 17.5 ± 0.21 after incubation with H₂O₂ (p < 0.01). vWF is exposed on the cell surface as dots with the average sizes around 1–3 μm. H₂O₂ causes an increase in the number of these dots and the appearence of the strings of vWF which are absent in control HUVEC. It is suggested that H₂O₂ may serve as a messenger which stimulates vWF exocytosis.     

Hydrogen peroxide activates the Gas6-Axl pathway in vascular smooth muscle cells

Axl, a receptor tyrosine kinase, is involved in cell survival, proliferation, and migration. We have shown that Axl expression increases in the neointima of balloon-injured rat carotids. Because oxidative stress is known to play a major role in remodeling of injured vessels, we hypothesized that H(2)O(2) might activate Axl by promoting autophosphorylation. H(2)O(2) rapidly stimulated Axl tyrosine phosphorylation in rat vascular smooth muscle cells within 1 min that was maximal at 5 min (6-fold). The response to H(2)O(2) was concentration-dependent with EC(50) of approximately 500 microm. Axl phosphorylation was partly dependent on production of its endogenous ligand, growth arrest gene 6 (Gas6), because Axl-Fc, a fragment of Axl extracellular domain that neutralizes Gas6, inhibited H(2)O(2)-induced Axl phosphorylation by 50%. Axl phosphorylation by H(2)O(2) was also attenuated by warfarin, which inhibits Gas6 activity by preventing post-translational modification. In intact vessels Axl was phosphorylated by H(2)O(2), and Axl phosphorylation was inhibited by warfarin treatment in balloon-injured carotids. Akt, a downstream target of Axl, was phosphorylated by H(2)O(2)in Axl(+/+) mouse aorta but significantly inhibited in Axl(-/-) aorta. Intimal proliferation was decreased significantly in a cuff injury model in Axl(-/-) mice compared with Axl(+/+) mice. In summary, Axl is an important signaling mediator for oxidative stress in cultured vascular smooth muscle cells and intact vessels and may represent an important therapeutic target for vascular remodeling and response to injury.     

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.     

Pyk2/CAKbeta tyrosine kinase activity-mediated angiogenesis of pulmonary vascular endothelial cells.

Endothelial cell spreading, migration, and morphogenesis are essential for angiogenesis, the formation of new blood vessels. In the present study, we explored roles of tyrosine kinase Pyk2 in angiogenesis of pulmonary endothelial cells. We found that tyrosine kinase Pyk2 was particularly enriched in pulmonary vascular endothelial cells and lung, a major organ site for tumor metastasis. By using adenovirus-mediated expression of various Pyk2 mutants, we demonstrated that Pyk2 tyrosine kinase activity was essential for the pulmonary vascular endothelial cell spreading, migration, morphogenesis, as well as pulmonary vein and artery angiogenesis ex vivo. We further showed that Pyk2 kinase activity was required for the expression of focal adhesion kinase, p130Crk-associated substrate, and its homologue human enhancer of filamentation 1, thus regulating formation of focal adhesions and cytoskeletal reorganization. These results indicate that Pyk2 plays a crucial role in the pulmonary endothelial cell motility such as spreading and migration necessary for angiogenesis.     

TNF-alpha increases tyrosine phosphorylation of vascular endothelial cadherin and opens the paracellular pathway through fyn activation in human lung endothelia

Tumor necrosis factor (TNF)-alpha is a key mediator of sepsis-associated multiorgan failure, including the acute respiratory distress syndrome. We examined the role of protein tyrosine phosphorylation in TNF-alpha-induced pulmonary vascular permeability. Postconfluent human lung microvascular and pulmonary artery endothelial cell (EC) monolayers exposed to human recombinant TNF-alpha displayed a dose- and time-dependent increase in transendothelial [(14)C]albumin flux in the absence of EC injury. TNF-alpha also increased tyrosine phosphorylation of EC proteins, and several substrates were identified as the zonula adherens proteins vascular endothelial (VE)-cadherin, and beta-catenin, gamma-catenin, and p120 catenin (p120(ctn)). Prior protein tyrosine kinase (PTK) inhibition protected against the TNF-alpha effect. TNF-alpha activated multiple PTKs, including src family PTKs. Prior PTK inhibition with the src-selective agents PP1 and PP2 each protected against approximately 60% of the TNF-alpha-induced increment in [(14)C]albumin flux. PP2 also blocked TNF-alpha-induced tyrosine phosphorylation of VE-cadherin, gamma-catenin, and p120(ctn). To identify which src family kinase(s) was required for TNF-alpha-induced vascular permeability, small interfering RNA (siRNA) targeting each of the three src family PTKs expressed in human EC, c-src, fyn, and yes, were introduced into the barrier function assay. Only fyn siRNA protected against the TNF-alpha effect, whereas the c-src and yes siRNAs did not. These combined data suggest that TNF-alpha regulates the pulmonary vascular endothelial paracellular pathway, in part, through fyn activation.     

Regulation of tetrahydrobiopterin synthesis and bioavailability in endothelial cells

Tetrahydrobiopterin (BH4) is a member of the pterin family that has a core structure of pyrazino-2,3-d-pyrimidine rings. Because BH4 is an essential cofactor for the biosynthesis of nitric oxide (a major vasodilator), there is growing interest in BH4 biochemistry in endothelial cells (the cells that line blood vessels). BH4 is synthesized via de novo and salvage pathways from guanosine 5′-triphosphate (GTP) and 7,8-dihydrobiopterin, respectively, in animal cells. GTP cyclohydrolase-I (GTP-CH) is the first and rate-controlling enzyme in the de novo pathway. Available evidence shows that endothelial GTP-CH expression and BH4 synthesis are stimulated by a wide array of nutritional (phenylalanine and arginine), hormonal (insulin and estrogen), immunological (inflammatory cytokines including interleukin [IL]-1, interferon-γ, and tumor necrosis factor-α), therapeutic (statins and cyclosporin A), and endothelium-derived (basic fibroblast growth factor and H₂O₂) factors. In contrast, glucocorticoids and anti-inflammatory cytokines (IL-4, IL-10, and transforming growth factor [TGF]-β) inhibit endothelial BH4 synthesis. Because BH4 is oxidized to 7,8-dihydrobiopterin and 7,8-dihydropterin at physiological pH, endothelial BH4 homeostasis is regulated by both BH4 synthesis and its oxidation. Vitamin C, folate, and other antioxidants enhance endothelial BH4 bioavailability through chemical stabilization or scavenging of reactive oxygen species, thereby contributing to the maintenance of physiological homeostasis in the endothelium. New know ledge about the cellular and molecular mechanisms for the regulation of endothelial BH4 synthesis and bioavailability is beneficial for developing effective means to prevent and treat cardiovascular disorders, the leading cause, of death in developed nations.