Differential role of hydrogen peroxide in UV-induced signal transduction

The present study investigated the differential requirement of ROS in UV-induced activation of these pathways. Exposure of the mouse epidermal Cl41 cells to UV radiation led to generation of ROS as measured by electron spin resonance (ESR) and by H₂O₂ and O₂ ^–; fluorescence staining assay. Treatment of cells with UV radiation or H₂O₂ also markedly activated Erks, JNKs, p38 kinase and led to increases in phosphorylation of Akt and p70^S6k in mouse epidermal JB6 cells. The scavenging of UV-generated H₂O₂ by N-acety-L-cyteine (NAC, a general antioxidant) or catalase (a specific H₂O₂ inhibitor) inhibited UV-induced activation of JNKs, p38 kinase, Akt and p70^S6k, while it did not show any inhibitory effects on Erks activation. Further, pretreatment of cells with sodium formate (an OH radical scavenger) or superoxide dismutase (O₂ ^– radical scavenger) did not inhibit any of these pathways. These results demonstrate that H₂O₂ generation is required for UV-induced phosphorylation of Akt and p70^S6k, and involved in activation of JNKs and p38 kinase, but not Erks.     

Translocation of protein kinase Cepsilon and protein kinase Cdelta to membrane is required for ultraviolet B-induced activation of mitogen-activated protein kinases and apoptosis.

UV-induced signal transduction may be involved in tumor promotion and induction of apoptosis. The role of protein kinase C (PKC) in UVB-induced signal transduction is not well understood. This study showed that UVB markedly induced translocation of membrane-associated PKCepsilon and PKCdelta, but not PKCalpha, from cytosol to membrane. Dominant negative mutant (DNM) PKCepsilon or PKCdelta inhibited UVB-induced translocation of PKCepsilon and PKCdelta, respectively. UVB-induced activation of extracellular signal-regulated protein kinases (Erks) and c-Jun NH2-terminal kinases (JNKs) was strongly inhibited by DNM PKCepsilon and PKCdelta, whereas the DNM of PKCalpha was less effective on the UVB-induced phosphorylation of Erks and JNKs. Among the PKC inhibitors used only rottlerin, a selective inhibitor of PKCdelta, markedly inhibited the UVB-induced activation of Erks and JNKs, but not p38 kinases. Safingol, a selective inhibitor for PKCalpha, did not show any inhibitory effect on UVB-induced mitogen-activated protein kinase activation. GF109203X is a stronger inhibitor of classical PKC than novel PKC. Lower concentrations of GF109203X (<10 microM) had no effect on UVB-induced activation of Erks or JNKs. However, at higher concentrations (over 20 microM), GF109203X inhibited UVB-induced activation of JNKs, Erks, and even p38 kinases. Meanwhile, rottlerin and GF109203X markedly inhibited UVB-induced apoptosis of JB6 cells, whereas safingol had little inhibitory effect. DNM-Erk2 cells and PD98059, a selective inhibitor for mitogen-activated protein kinase/extracellular signal-regulated kinase 1 that directly activates Erks, inhibited UVB-induced apoptosis. DNM-JNK1 cells also blocked UVB-induced apoptosis, whereas SB202190, a specific inhibitor for p38 kinases, did not produce the inhibitory effect. These data demonstrate that PKCdelta and PKCepsilon, but not PKCalpha, mediate UVB-induced signal transduction and apoptosis in JB6 cells through activation of Erks and JNKs.     

Inhibitory mechanisms of tea polyphenols on the ultraviolet B-activated phosphatidylinositol 3-kinase-dependent pathway

In this study, we investigated the effect of tea polyphenols, (-)-epigallocatechin-3-gallate or theaflavins, on UVB-induced phosphatidylinositol 3-kinase (PI3K) activation in mouse epidermal JB6 Cl 41 cells. Pretreatment of cells with these polyphenols inhibited UVB-induced PI3K activation. Furthermore, UVB-induced activation of Akt and ribosomal p70 S6 kinase (p70 S6-K), PI3K downstream effectors, were also attenuated by the polyphenols. In addition to LY294002, a PI3K inhibitor, pretreatment with a specific mitogen-activated protein/extracellular signal-regulated protein kinases (Erks) kinase 1 inhibitor, U0126, or a specific p38 kinase inhibitor, SB202190, blocked UVB-induced activation of both Akt and p70 S6-K. Pretreatment with LY294002 restrained UVB-induced phosphorylation of Erks, suggesting that in UVB signaling, the Erk pathway is mediated by PI3K. Moreover, pretreatment with rapamycin, an inhibitor of p70 S6-K, inhibited UVB-induced activation of p70 S6-K, but UVB-induced activation of Akt did not change. Interestingly, UVB-induced p70 S6-K activation was directly blocked by the addition of (-)-epigallocatechin-3-gallate or theaflavins, whereas these polyphenols showed only a weak inhibition on UVB-induced Akt activation. Because PI3K is an important factor in carcinogenesis, the inhibitory effect of these polyphenols on activation of PI3K and its downstream effects may further explain the anti-tumor promotion action of these tea constituents.     

Induction of EGFR-dependent and EGFR-independent signaling pathways by ultraviolet A irradiation

Most of the signal pathways involved in ultraviolet (UV)-induced skin carcinogenesis are thought to originate at plasma membrane receptors. However, UVA-induced signal transduction to downstream ribosomal protein S6 kinases, p70(S6K) and p90(RSK), is not well understood. In this report, we show that UVA stimulation of the epidermal growth factor receptor (EGFR) may lead to activation of p70(S6K)/p90(RSK) through phosphatidyl isositol (PI)-3 kinase and extracellular receptor-activated kinases (ERKs). Evidence is provided that phosphorylation and activation of p70(S6K)/p90(RSK) induced by UVA were prevented in Egfr(-/-) cells and were also markedly inhibited by the EGFR-specific tyrosine kinase inhibitors AG1478 and PD153035. Furthermore, EGFR tyrosine kinase inhibitors and EGFR deficiency significantly suppressed activation of PI-3 kinase and ERKs in regulating activation of p90(RSK)/p70(S6K) but had no effect on activation of c-Jun NH(2)-terminal kinases (JNKs) and p38 kinase in response to UVA. Thus, our results suggest that UVA-induced EGFR signaling may be required for activation of p90(RSK)/p70(S6K), PI-3 kinase, and ERKs but not JNKs or p38 kinase.     

K(+) channel activity and redox status are differentially required for JNK activation by UV and reactive oxygen species

Upon exposure to ultraviolet (UV) radiation, osmotic changes or the presence of reactive oxygen species (ROS) c-Jun N-terminal kinases (JNKs) are rapidly activated. Extensive studies have elucidated molecular components that mediate the activation of JNKs. However, it remains unclear whether activation of JNKs by various stress signals involves different pathways. Here we show that K(+) channel activity is involved in mediating apoptosis induced by UV but not by H(2)O(2) in myelocytic leukemic ML-1 cells. Specifically, JNKs were rapidly phosphorylated upon treatment of ML-1 cells with UV and H(2)O(2). UV-induced, but not H(2)O(2)-induced, JNK-1 phosphorylation was inhibited by pretreatment with 4-aminopyridine (4-AP), a K(+) channel blocker. 4-AP also blocked UV-induced increase in JNK activity as well as p38 phosphorylation. Immunofluorescent microscopy revealed that phosphorylated JNKs were concentrated at centrosomes in ML-1 cells and that these proteins underwent rapid subcellular translocation upon UV treatment. Consistently, the subcellular translocation of JNKs induced by UV was largely blocked by 4-AP. Furthermore, UV-induced JNK activation was blocked by NEM, a sulfhydryl alkylating agent also affecting K(+) current. Both UV- and H(2)O(2)-induced JNK activities were inhibited by glutathione, suggesting that the redox status does play an important role in the activation of JNKs. Taken together, our findings suggest that JNK activation by UV and H(2)O(2) is mediated by distinct yet overlapping pathways and that K(+) channel activity and redox status are differentially required for UV- and H(2)O(2)-induced activation of JNKs.     

Oxidative signaling in renal epithelium: Critical role of cytosolic phospholipase A2 and p38(SAPK)

Previous studies from this laboratory have demonstrated a critical role of cytosolic phospholipase A2 (cPLA2) and arachidonic acid in angiotensin II (Ang II) AT2 receptor-mediated signal transduction in renal epithelium. In primary proximal tubular epithelial cells exposed to hydrogen peroxide (H2O2), both the selective cPLA2 inhibitors and the cPLA2 antisense oligonucleotides significantly attenuated H2O2-induced arachidonic acid liberation and activation of p38(SAPK), ERK1/2, and Akt1. This H2O2-induced kinase activation was significantly attenuated by a Src kinase inhibitor PP2, or by transient transfection of carboxyl-terminal Src kinase (CSK) that maintained Src in the dormant form. Under basal conditions, Src coimmunoprecipitated with epidermal growth factor receptor (EGFR), while H2O2 increased EGFR phosphorylation in the complex. We observed that inhibition of EGFR kinase activity with AG1478 significantly attenuated H2O2-induced p38(SAPK) and ERK1/2 activation, but did not inhibit Akt1 activation. Furthermore, it seems that p38(SAPK) is upstream of ERK1/2 and Akt1, since a p38(SAPK) inhibitor SB203580 significantly blocked H2O2-induced activation of ERK1/2 and Akt1. Interestingly, overexpression of the dominant-negative p38(SAPK) isoform alpha inhibited ERK1/2 but not Akt1 activation. Our observations demonstrate that in these nontransformed cells, activation of cPLA2 is a converging point for oxidative stress and Ang II, which share common downstream signaling mechanisms including Src and EGFR. In addition, p38(SAPK) provides a positive input to both growth and antiapoptotic signaling pathways induced by acute oxidative stress.     

Induction of activator protein-1 through reactive oxygen species by crystalline silica in JB6 cells

We reported previously that freshly fractured silica (FFSi) induces activator protein-1 (AP-1) activation through extracellular signal-regulated protein kinases (ERKs) and p38 kinase pathways. In the present study, the biologic activities of FFSi and aged silica (ASi) were compared by measuring their effects on the AP-1 activation and phosphorylation of ERKs and p38 kinase. The roles of reactive oxygen species (ROS) in this silica-induced AP-1 activation were also investigated. We found that FFSi-induced AP-1 activation was four times higher than that of ASi in JB6 cells. FFSi also caused greater phosphorylation of ERKs and p38 kinase than ASi. FFSi generated more ROS than ASi when incubated with the cells as measured by electron spin resonance (ESR). Studies using ROS-sensitive dyes and oxygen consumption support the conclusion that ROS are generated by silica-treated cells. N-Acetylcysteine (an antioxidant) and polyvinyl pyridine-N-oxide (an agent that binds to Si-OH groups on silica surfaces) decreased AP-1 activation and phosphorylation of ERKs and p38 kinase. Catalase inhibited phosphorylation of ERKs and p38 kinase, as well as AP-1 activation induced by FFSi, suggesting the involvement of H(2)O(2) in the mechanism of silica-induced AP-1 activation. Sodium formate (an ( small middle dot)OH scavenger) had no influence on silica-induced MAPKs or AP-1 activation. Superoxide dismutase enhanced both AP-1 and MAPKs activation, indicating that H(2)O(2), but not O(2), may play a critical role in silica-induced AP-1 activation. These studies indicate that freshly ground silica is more biologically active than aged silica and that ROS, in particular H(2)O(2), play a significant role in silica-induced AP-1 activation.     

Hydrogen peroxide mediates arsenite activation of p70(s6k) and extracellular signal-regulated kinase

To define the mechanism of arsenite-induced tumor promotion, we examined the role of reactive oxygen species (ROS) in the signaling pathways of cells exposed to arsenite. Arsenite treatment resulted in the persistent activation of p70(s6k) and extracellular signal-regulated kinase 1/2 (ERK1/2) which was accompanied by an increase in intracellular ROS production. The predominant produced appeared to be H(2)O(2), because the arsenite-induced increase in dichlorofluorescein (DCF) fluorescence was completely abolished by pretreatment with catalase but not with heat-inactivated catalase. Elimination of H(2)O(2) by catalase or N-acetyl-L-cysteine inhibited the arsenite-induced activation of p70(s6k) and ERK1/2, indicating the possible role of H(2)O(2) in the arsenite activation of the p70(s6k) and the ERK1/2 signaling pathways. A specific inhibitor of p70(s6k), rapamycin, and calcium chelators significantly blocked the activation of p70(s6k) induced by arsenite. While the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 completely abrogated arsenite activation of p70(s6k), ERK1/2 activation by arsenite was not affected by these inhibitors, indicating that H(2)O(2) might act as an upstream molecule of PI3K as well as ERK1/2. Consistent with these results, none of the inhibitors impaired H(2)O(2) production by arsenite. DNA binding activity of AP-1, downstream of ERK1/2, was also inhibited by catalase, N-acetyl-L-cysteine, and the MEK inhibitor PD98059, which significantly blocked arsenite activation of ERK1/2. Taken together, these studies provide insight into mechanisms of arsenite-induced tumor promotion and suggest that H(2)O(2) plays a critical role in tumor promotion by arsenite through activation of the ERK1/2 and p70(s6k) signaling pathways.     

Signal transduction pathways involved in phosphorylation and activation of p70S6K following exposure to UVA irradiation

Ultraviolet light A (UVA) plays an important role in the etiology of human skin cancer, and UVA-induced signal transduction has a critical role in UVA-induced skin carcinogenesis. The upstream signaling pathways leading to p70(S6K) phosphorylation and activation are not well understood. Here, we observed that UVA induces phosphorylation and activation of p70(S6K). Further, UVA-stimulated p70(S6K) activity and phosphorylation at Thr(389) were blocked by wortmannin, rapamycin, PD98059, SB202190, and dominant negative mutants of phosphatidylinositol (PI) 3-kinase p85 subunit (DNM-Deltap85), ERK2 (DNM-ERK2), p38 kinase (DNM-p38), and JNK1 (DNM-JNK1) and were absent in Jnk1-/- or Jnk2-/- knockout cells. The p70(S6K) phosphorylation at Ser(411) and Thr(421)/Ser(424) was inhibited by rapamycin, PD98059, or DNM-ERK2 but not by wortmannin, SB202190, DNM-Deltap85, or DNM-p38. However, Ser(411), but not Thr(421)/Ser(424) phosphorylation, was suppressed in DNM-JNK1 and abrogated in Jnk1-/- or Jnk2-/- cells. In vitro assays indicated that Ser(411) on immunoprecipitated p70(S6K) proteins is phosphorylated by active JNKs and ERKs, but not p38 kinase, and Thr(421)/Ser(424) is phosphorylated by ERK1, but not ERK2, JNKs, or p38 kinase. Moreover, p70(S6K) co-immunoprecipitated with PI 3-kinase and possibly PDK1. The complex possibly possessed a partial basal level of phosphorylation, but not at MAPK sites, which was available for its activation by MAPKs in vitro. Thus, these results suggest that activation of MAPKs, like PI 3-kinase/mTOR, may be involved in UVA-induced phosphorylation and activation of p70(S6K).     

Differential regulation of the phosphatidylinositol 3-kinase/Akt and p70 S6 kinase pathways by the alpha(1A)-adrenergic receptor in rat-1 fibroblasts

Phosphatidylinositol (PI) 3-kinase and its downstream effector Akt are thought to be signaling intermediates that link cell surface receptors to p70 S6 kinase. We examined the effect of a G(q)-coupled receptor on PI 3-kinase/Akt signaling and p70 S6 kinase activation using Rat-1 fibroblasts stably expressing the human alpha(1A)-adrenergic receptor. Treatment of the cells with phenylephrine, a specific alpha(1)-adrenergic receptor agonist, activated p70 S6 kinase but did not activate PI 3-kinase or any of the three known isoforms of Akt. Furthermore, phenylephrine blocked the insulin-like growth factor-I (IGF-I)-induced activation of PI 3-kinase and the phosphorylation and activation of Akt-1. The effect of phenylephrine was not confined to signaling pathways that include insulin receptor substrate-1, as the alpha(1)-adrenergic receptor agonist also inhibited the platelet-derived growth factor-induced activation of PI 3-kinase and Akt-1. Although increasing the intracellular Ca(2+) concentration with the ionophore A23187 inhibited the activation of Akt-1 by IGF-I, Ca(2+) does not appear to play a role in the phenylephrine-mediated inhibition of the PI 3-kinase/Akt pathway. The differential ability of phenylephrine and IGF-I to activate Akt-1 resulted in a differential ability to protect cells from UV-induced apoptosis. These results demonstrate that activation of p70 S6 kinase by the alpha(1A)-adrenergic receptor in Rat-1 fibroblasts occurs in the absence of PI 3-kinase/Akt signaling. Furthermore, this receptor negatively regulates the PI 3-kinase/Akt pathway, resulting in enhanced cell death following apoptotic insult.     

Activation of a PKC is required for vanadate-induced phosphorylation of protein kinase B (Akt), but not p70S6k in mouse epidermal JB6 cells

Vanadium is a metal widely distributed in the environment. Although vanadate-containing compounds exert potent toxic effects on a wide variety of biological systems, the mechanisms by which vanadate mediates adverse effects are not well understood. The present study investigated the vanadate-induced phosphorylation of Akt and p70S6K, two kinases known to be vital for cell survival, growth, transformation, and transition of the cell cycle in mammals. Exposure of mouse epidermal JB6 cells to vanadium led to phosphorylation of Akt and p70S6K in a time- and dose-dependent manner. Vanadium exposure also caused translocation of atypical isoforms of PKC (lambda, zeta) from the cytosol to the membrane, but had no effect on PKCalpha translocation, suggesting that the atypical PKCs (aPKC) were specifically involved in vanadium-induced cellular response. Importantly, overexpression of a dominant negative mutant PKClambda blocked Akt phosphorylation at Ser473 and Thr308, whereas it did not inhibit p70S6k phosphorylation at Thr389 and Thr421/Ser424, suggesting that aPKC activation is specifically involved in vanadium-induced activation of Akt, but not in activation of p70S6k. Furthermore, vanadium-induced p70S6k phosphorylation at Thr389 and Thr421/Ser424 and Akt phosphorylation at Thr308 occurred through a PI-3K-dependent pathway because a PI-3K dominant negative mutant inhibited induction as compared with vector control cells. These results indicate that there was a differential role of aPKC in vanadate-induced phosphorylation of Akt and p70S6k, suggesting that signal transduction pathways leading to the activation of Akt and p70S6k were different.     

Extracellular zinc activates p70 S6 kinase through the phosphatidylinositol 3-kinase signaling pathway

We have studied a possible role of extracellular zinc ion in the activation of p70S6k, which plays an important role in the progression of cells from the G(1) to S phase of the cell cycle. Treatment of Swiss 3T3 cells with zinc sulfate led to the activation and phosphorylation of p70S6k in a dose-dependent manner. The activation of p70S6k by zinc treatment was biphasic, the early phase being at 30 min followed by the late phase at 120 min. The zinc-induced activation of p70S6k was partially inhibited by down-regulation of phorbol 12-myristate 13-acetate-responsive protein kinase C (PKC) by chronic treatment with phorbol 12-myristate 13-acetate, but this was not significant. Moreover, Go6976, a specific calcium-dependent PKC inhibitor, did not significantly inhibit the activation of p70S6k by zinc. These results demonstrate that the zinc-induced activation of p70S6k is not related to PKC. Also, extracellular calcium was not involved in the activation of p70S6k by zinc. Further characterization of the zinc-induced activation of p70S6k using specific inhibitors of the p70S6k signaling pathway, namely rapamycin, wortmannin, and LY294002, showed that zinc acted upstream of mTOR/FRAP/RAFT and phosphatidylinositol 3-kinase (PI3K), because these inhibitors caused the inhibition of zinc-induced p70S6k activity. In addition, Akt, the upstream component of p70S6k, was activated by zinc in a biphasic manner, as was p70S6k. Moreover, dominant interfering alleles of Akt and PDK1 blocked the zinc-induced activation of p70S6k, whereas the lipid kinase activity of PI3K was potently activated by zinc. Taken together, our data suggest that zinc activates p70S6k through the PI3K signaling pathway.     

Grape seed proanthocyanidins inhibit UV-radiation-induced oxidative stress and activation of MAPK and NF-kappaB signaling in human epidermal keratinocytes

Solar ultraviolet (UV) radiation-induced oxidative stress has been implicated in various skin diseases. Here, we report the photoprotective effect of grape seed proanthocyanidins (GSPs) on UV-induced oxidative stress and activation of mitogen-activated protein kinase (MAPK) and NF-kappaB signaling pathways using normal human epidermal keratinocytes (NHEK). Treatment of NHEK with GSPs inhibited UVB-induced hydrogen peroxide (H2O2), lipid peroxidation, protein oxidation, and DNA damage in NHEK and scavenged hydroxyl radicals and superoxide anions in a cell-free system. GSPs also inhibited UVB-induced depletion of antioxidant defense components, such as glutathione peroxidase, catalase, superoxide dismutase, and glutathione. As UV-induced oxidative stress mediates activation of MAPK and NF-kappaB signaling pathways, we determined the effects of GSPs on these pathways. Treatment of NHEK with GSPs inhibited UVB-induced phosphorylation of ERK1/2, JNK, and p38 proteins of the MAPK family at the various time points studied. As UV-induced H2O2 plays a major role in activation of MAPK proteins, NHEK were treated with H2O2 with or without GSPs and other known antioxidants, viz. (-)-epigallocatechin-3-gallate, silymarin, ascorbic acid, and N-acetylcysteine. It was observed that H2O2-induced phosphorylation of ERK1/2, JNK, and p38 was decreased by these antioxidants. Under identical conditions, GSPs also inhibited UVB-induced activation of NF-kappaB/p65, which was mediated through inhibition of degradation and activation of IkappaBalpha and IKKalpha, respectively. Together, these results suggest that GSPs could be useful in the attenuation of UV-radiation-induced oxidative stress-mediated skin diseases in human skin.     

PI-3K and Akt are mediators of AP-1 induction by 5-MCDE in mouse epidermal Cl41 cells

5-Methylchrysene has been found to be a complete carcinogen in laboratory animals. However, the tumor promotion effects of (+/-)-anti-5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE) remain unclear. In the present work, we found that 5-MCDE induced marked activator protein-1 (AP-1) activation in Cl41 cells. 5-MCDE also induced a marked activation of phosphatidylinositol 3-kinase (PI-3K). Inhibition of PI-3K impaired 5-MCDE-induced AP-1 transactivation, suggesting that PI-3K is an upstream kinase involved in AP-1 activation by 5-MCDE. Furthermore, we found that Akt is a PI-3K downstream mediator for 5-MCDE-induced AP-1 transactivation, whereas another PI-3K downstream kinase, p70(S6K), was not involved in AP-1 activation by 5-MCDE. Moreover, inhibition of Akt activation blocked 5-MCDE-induced activation of extracellular signal-regulated protein kinases (ERKs) and c-Jun NH(2)-terminal kinases (JNKs), whereas it did not affect p38K activation. Consistently, overexpression of a dominant-negative mutant of ERK2 or JNK1 blocked the AP-1 activation by 5-MCDE. These results demonstrate that 5-MCDE is able to induce AP-1 activation, and the AP-1 induction is specifically through a PI-3K/Akt-dependent and p70(S6K)-independent pathway.     

The expression of the NADPH oxidase subunit p22phox is regulated by a redox-sensitive pathway in endothelial cells

Endothelial dysfunction is characterized by increased levels of reactive oxygen species (ROS) and a prothrombotic state. The mechanisms linking thrombosis to ROS production in the endothelium are not well understood. We investigated the role of thrombin in regulating NADPH oxidase-dependent ROS production and expression of its subunit p22phox in the endothelial cell line EaHy926. Thrombin elicited a biphasic increase in ROS generation peaking within 15 min, but also at 3 h. The delayed response was accompanied by increased p22phox mRNA and protein expression. Two-photon confocal laser microscopy showed colocalization between p22phox and ROS production. Antioxidant treatment with vitamin C or diphenyleneiodonium abrogated thrombin-induced ROS production and p22phox expression, whereas H2O2 elevated ROS production and p22phox levels. Both responses were dependent on p38 MAP kinase and phosphatidylinositol-3-kinase (PI3 kinase)/Akt. Finally, p22phox was required for thrombin- or H2O2-stimulated proliferation. These data show that thrombin rapidly increases ROS production in endothelial cells, resulting, via activation of p38 MAP kinase and PI3 kinase/Akt, in upregulation of p22phox accompanied by a delayed increase in ROS generation and enhanced proliferation. These findings suggest a positive feedback mechanism whereby ROS, possibly generated by the NADPH oxidase, lead to elevated levels of p22phox and, thus, sustained ROS generation as is observed in endothelial dysfunction.     

Regulation of Akt by arachidonic acid and phosphoinositide 3-kinase in angiotensin II-stimulated vascular smooth muscle cells

Angiotensin (Ang) II stimulates cytosolic phospholipase A2(cPLA(2))-dependent release of arachidonic acid (ArAc) in vascular smooth muscle cells (VSMC). ArAc release and production of reactive oxygen species (ROS) lead to the activation of downstream kinases resulting in VSMC growth. To determine the role of Akt in this pathway, we used VSMC to link Ang II-induced ArAc release and ROS production to the activation of Akt and VSMC growth. We observed that Ang II, ArAc, or H(2)O(2) increased Akt activation. The Akt inhibitor SH6 blocked Ang II-, ArAc-, or H(2)O(2)-induced Akt activation, as did inhibition of phosphoinositide 3-kinase (PI(3)K). Inhibition of cPLA(2) blocked Ang II effects, while the ROS scavenger NaC decreased Ang II- and ArAc-induced Akt activation. Inhibition of Akt blocked the (3)H-thymidine incorporation induced by all three agonists. Thus, Ang II-induced ArAc release and ROS production leads to the PI(3)K-dependant activation of Akt and VSMC growth.