Regulation of Akt-dependent cell survival by Syk and Rac

Interleukin-2 (IL-2) prevents cell apoptosis and promotes survival, but the involved mechanisms have not been completely defined. Although phosphatidylinositide 3-kinase (PI 3-kinase) has been implicated in IL-2-mediated survival mechanisms, none of the 3 chains of the IL-2 receptor (IL-2R) expresses a binding site for PI 3-kinase. However, IL-2Rbeta does express a Syk-binding motif. By using an IL-2-dependent natural killer (NK) cell line, followed by validation of the results in fresh human NK cells, we identified Syk as a critical effector essential for IL-2-mediated prosurvival signaling in NK cells. Down-regulation of Syk by piceatannol treatment impaired NK cellular viability and induced prominent apoptosis as effectively as suppression of PI 3-kinase function by LY294002. Expression of kinase-deficient Syk or pretreatment with piceatannol markedly suppressed IL-2-stimulated activation of PI 3-kinase and Akt, demonstrating that Syk is upstream of PI 3-kinase and Akt. However, constitutively active PI 3-kinase reversed this loss of Akt function caused by kinase-deficient Syk or piceatannol. Thus, Syk appears to regulate PI 3-kinase, which controls Akt activity during IL-2 stimulation. More important, we observed Rac1 activation by IL-2 and found that it mediated PI 3-kinase activation of Akt. This conclusion came from experiments in which dominant-negative Rac1 significantly decreased IL-2-induced Akt activation, whereas constitutively active Rac1 reelevated Akt activity not only in Syk-impaired but also in PI 3-kinase-impaired NK cells. These results constitute the first report of a Syk --> PI3K --> Rac1 --> Akt signal cascade controlled by IL-2 that mediates NK cell survival.     

Putative role of the phosphatidylinositol 3-kinase-Akt signaling pathway in the survival of granulosa cells

Insulin-like growth factor-1 (IGF-1) is an important differentiation and survival factor for granulosa cells. The purpose of this study was to test the hypothesis that IGF-1 promotes survival of porcine granulosa cells by signaling though the phosphatidylinositol (PI) 3-kinase/Akt signal transduction pathway. Treatment with IGF-1 (100 ng/ml) for 10 min stimulated PI 3-kinase and Akt protein kinase activity. IGF-I stimulated the phosphorylation and activation of Akt in a time- and concentration-dependent manner. The PI 3-kinase inhibitors wortmannin and LY294002 blocked IGF-1 induced increases in PI 3-kinase activity and phosphorylation of Akt. Additionally, IGF-1 treatment prevented apoptosis. The survival response to IGF-I was blocked by treatment with either wortmannin or LY294002. These data suggest that IGF-I-induced phosphorylation of Akt is mediated through PI 3-kinase and that inactivation of this pathway results in granulosa cell apoptosis. We conclude that the P1 3-kinase/Akt signaling serves as a functional survival pathway in the ovary.     

Involvement of protein kinase C and phosphatidylinositol 3-kinase pathways in the survival of B-cell chronic lymphocytic leukemia cells

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived CD5(+) B lymphocytes. TPA (12-O-tetradecanoylphorbol 13- acetate) and interleukin-4 (IL-4) inhibit apoptosis of B-CLL lymphocytes ex vivo. We used specific inhibitors of protein kinase C (PKC), extracellular-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3-kinase) to study their involvement in TPA- and IL-4-induced survival of B-CLL lymphocytes. BisI, a specific inhibitor of PKC, induced apoptosis and inhibited the antiapoptotic activity of TPA and IL-4. B-CLL cells have a basal PKC activity that was increased by TPA but not by IL-4. TPA, but not IL-4, induced ERK activation. However, the inhibition of ERK activation did not affect the viability of B-CLL lymphocytes, demonstrating that this pathway is not involved in their survival. Inhibition of PI3-kinase by LY294002 induced apoptosis of B-CLL cells and inhibited the survival effect of IL-4 and TPA. In addition, Akt, a downstream effector of PI3-kinase activity, was phosphorylated by TPA and IL-4 in B-CLL cells, though PI3-kinase had no effect on PKC-dependent phosphorylation of Akt. Furthermore, the inhibition of PKC or PI3-kinase increased dexamethasone- and fludarabine-induced apoptosis ex vivo in the presence of survival factors. These results demonstrate that PKC and PI3-kinase are involved in the survival of B-CLL cells and suggest that inhibitors of these pathways could be combined with the drugs used in the treatment of B-CLL.     

A member of Forkhead family transcription factor, FKHRL1, is one of the downstream molecules of phosphatidylinositol 3-kinase-Akt activation pathway in erythropoietin signal transduction

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is important for the regulation of a number of cellular responses. Serine/threonine kinase Akt (protein kinase B; PKB) is downstream of PI3K and activated by growth factors. This study found that erythropoietin (EPO) induced tyrosine phosphorylation of Akt in a time- and dose-dependent manner in EPO-dependent human leukemia cell line UT-7/EPO. In vitro kinase assay using histone H2B and glucose synthase kinase as substrates demonstrated that Akt was actually activated by EPO. EPO-induced phosphorylation of Akt was completely blocked by a PI3K-specific inhibitor, LY294002, at 10 micromol/L, indicating that activation of Akt by EPO is dependent on PI3K activity. In addition, overexpression of the constitutively active form of Akt on UT-7/EPO cells partially blocked apoptosis induced by withdrawal of EPO from the culture medium. This finding suggested that the PI3K-Akt activation pathway plays some role in the antiapoptotic effect of EPO. EPO induced phosphorylation of a member of the trancription factor Forkhead family, FKHRL1, at threonine 32 and serine 253 in a dose- and time-dependent manner in UT-7/EPO cells. Moreover, results showed that Akt kinase activated by EPO directly phosphorylated FKHRL1 protein and that FKHRL1 phosphorylation was completely dependent on PI3K activity as is the case for Akt. In conjunction with the evidence that FKHRL1 is expressed in normal human erythroid progenitor cells and erythroblasts, the results suggest that FKHRL1 plays an important role in erythropoiesis as one of the downstream target molecules of PI3K-Akt.     

Transduction of interleukin-2 antiapoptotic and proliferative signals via Akt protein kinase

The interleukin-2 (IL-2) receptor (IL-2R) is composed of three subunits. Of these, IL-2Rα is required for high-affinity IL-2 binding, while IL-2Rβ and IL-2Rγ_c are required for the transduction of IL-2-generated signals. Signals transduced via the S region of the IL-2Rβ (amino acids 267–322) in BAF/3 cells activate the phosphatidylinositol 3-kinase (PI3-kinase) and induce the expression of Bcl-2 and c-myc. Through the induction of Bcl-2, IL-2 inhibits apoptosis and through the combination of Bcl-2 and c-myc it stimulates progression through the cell cycle. Here we show that the protein kinase encoded by the Akt proto-oncogene is activated by IL-2. Akt activation by IL-2 depends on PI3-kinase signals transduced via the S region of the IL-2Rβ and is linked to the translocation of Akt to the cell membrane. Expression of catalytically active Akt mutants in BAF/3 cells expressing IL-2Rβ[A0]ΔS promotes the expression of Bcl-2 and c-myc, inhibits apoptosis induced by IL-3 deprivation or staurosporine, and stimulates cell cycle progression. The same mutants also stimulate cell cycle progression in 2780a, an IL-2-dependent T cell line that undergoes G₁ arrest rather than apoptosis after IL-2 deprivation. The activation of Akt by IL-2 via the PI3-kinase and the rescue of the PI3-kinase-mediated antiapoptotic and proliferative IL-2 signals by catalytically active Akt indicate that these signals are transduced by Akt.     

Modulating Toll-like receptor mediated signaling by (1-->3)-beta-D-glucan rapidly induces cardioprotection

OBJECTIVE: Immune and inflammatory signaling pathways, initiated by the innate response, are involved in myocardial ischemia/reperfusion (I/R) injury. Toll-like receptor (TLR) mediated MyD88-dependent NFkappaB pathways play a role in the induction of innate immunity. We have reported that glucan phosphate (GP) improved survival in experimental sepsis, which correlated with decreased tissue NFkappaB activation. In the present study, we report that GP rapidly induced cardioprotection against I/R injury in vivo. METHODS: Sprague-Dawley rats were pretreated with GP (40 mg/kg, i.p) 1 h before 45 min of ligation of the left anterior descending coronary followed by reperfusion for 4 and 24 h. Infarction size was examined by triphenyltetrazolium chloride (TTC) staining. NFkappaB activation was analyzed by electrophoretic mobility shift assay (EMSA). IkappaB kinase-beta (IKKbeta), IL-1 receptor-associated kinase (IRAK) and Phosphoinositide 3-kinase (PI3K) activities were determined by kinase assay with appropriate substrates. Association of TLR4 with MyD88 or with PI3K p85 was assessed by immunoprecipitation with anti-TLR4 followed by immunoblotting with anti-MyD88 or anti-p85. RESULTS: GP treatment reduced infarct size by 47% in rat hearts subjected to reperfusion for 4 h and by 50% following reperfusion for 24 h. The same protective effect was observed when GP was administrated 5 min after initiation of ischemia. The mechanisms of GP induced cardioprotection involve decreased association of TLR4 with MyD88, inhibition of I/R induced IRAK and IKKbeta activity and decreased NFkappaB activity. In addition, GP increased TLR4 phosphotyrosine, resulting in increasing PI3K/Akt activity in the myocardium, which correlated with decreased cardiac myocyte apoptosis following I/R. CONCLUSION: The results suggest that activation of the TLR mediated MyD88-dependent NFkappaB signaling pathway may play an important role in myocardial I/R injury, while stimulation of the PI3K/Akt signaling could serve a protective role. The data indicates that GP treatment shifts the TLR mediated activation signal in I/R from a predominantly NFkappaB pathway to a predominant PI3K/Akt signaling pathway.     

Anti-apoptotic signaling by hepatocyte growth factor/Met via the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase pathways

Hepatocyte growth factor (HGF) is a ligand of the receptor tyrosine kinase encoded by the c-Met protooncogene. HGF/Met signaling has multifunctional effects on various cell types. We sought to determine the role of HGF/Met in apoptosis and identify signal transducers involved in this process. In experiments with human SK-LMS-1 leiomyosarcoma cells, we show that the Akt kinase is activated by HGF in a time- and dose-dependent manner by phosphatidylinositol 3-kinase (PI3-kinase). Akt is also activated by active tumorigenic forms of Met, i.e., ligand-independent Tpr-Met, a truncated and constitutively dimerized form of Met, and a mutationally activated version of Met corresponding to that found in human hereditary papillary renal carcinoma. In NIH 3T3 cells transfected with wild-type Met, HGF inhibits apoptosis induced by serum starvation and UV irradiation. HGF-induced survival correlates with Akt activity and is inhibited by the specific PI3-kinase inhibitor LY294002, indicating that HGF inhibits cell death through the PI3-kinase/Akt signal transduction pathway. Furthermore, transiently transfected Tpr-Met activates Akt (both Akt1 and Akt2) and protects cells from apoptosis. Mitogen-activated protein kinase (MAPK) also is activated by HGF and rescues cells from apoptosis, although the cytoprotective effect is less marked than for PI3-kinase/Akt. Blocking MAPK with the specific MAPK kinase inhibitor PD098059 impairs the ability of HGF to promote cell survival. Similar results were obtained with NIH 3T3 cells expressing the fusion protein Trk-Met and stimulated with nerve growth factor, the Trk ligand. These results demonstrate that HGF/Met is capable of protecting cells from apoptosis by using both PI3-kinase/Akt and, to a lesser extent, MAPK pathways.     

Lipid rafts are required for Kit survival and proliferation signals

In addition to its physiologic role as central regulator of the hematopoietic and reproductive systems, the Kit receptor tyrosine kinase (RTK) is pathologically overexpressed in some forms of leukemia and constitutively activated by oncogenic mutations in mast-cell proliferations and gastrointestinal stromal tumors. To gain insight into the general activation and signaling mechanisms of RTKs, we investigated the activation-dependent dynamic membrane distributions of wild-type and oncogenic forms of Kit in hematopoietic cells. Ligand-induced recruitment of wild-type Kit to lipid rafts after stimulation by Kit ligand (KL) and the constitutive localization of oncogenic Kit in lipid rafts are necessary for Kit-mediated proliferation and survival signals. KL-dependent and oncogenic Kit kinase activity resulted in recruitment of the regulatory phosphatidylinositol 3-kinase (PI3-K) subunit p85 to rafts where the catalytical PI3-K subunit p110 constitutively resides. Cholesterol depletion by methyl-beta-cyclodextrin prevented Kit-mediated activation of the PI3-K downstream target Akt and inhibited cellular proliferation by KL-activated or oncogenic Kit, including mutants resistant to the Kit inhibitor imatinib-mesylate. Our data are consistent with the notion that Kit recruitment to lipid rafts is required for efficient activation of the PI3-K/Akt pathway and Kit-mediated proliferation.     

Necessary role of phosphatidylinositol 3-kinase in transforming growth factor beta-mediated activation of Akt in normal and rheumatoid arthritis synovial fibroblasts

OBJECTIVE: Rheumatoid arthritis is a disease that, pathologically, is characterized by the progressive growth and invasion of the synovial pannus into the surrounding cartilage and bone. Many cytokines, including transforming growth factor beta1 (TGFbeta1), have been implicated in this process, but their mode of action is incompletely understood. The goal of the present study was to better understand the downstream signaling pathways of TGFbeta in fibroblasts. METHODS: The role of phosphatidylinositol 3-kinase (PI 3-kinase) was determined by chemical inhibition with LY294002 or wortmannin. Activation of protein kinase B (Akt), c-Jun N-terminal kinases (JNKs), and extracellular signal-regulated kinases (ERKs) was evaluated by Western blot analysis using phospho-specific antibodies. RESULTS: Exposure of fibroblasts to TGFbeta rapidly induced activation of a kinase, Akt, that is known to inhibit apoptosis by a variety of pathways. Activation of Akt was blocked by the specific PI 3-kinase inhibitor, LY294002, indicating that TGFbeta-mediated phosphorylation of Akt was dependent on PI 3-kinase activation. This activation pathway was relatively selective for Akt, since inhibition of PI 3-kinase failed to substantially modify activation of ERKs or JNKs in synovial fibroblasts. Inhibition of the PI 3-kinase/Akt pathway resulted in impaired proliferation of synovial fibroblasts and partial attenuation of the protective effect of TGFbeta on Fas-mediated apoptosis. CONCLUSION: TGFbeta exerts its growth and antiapoptotic effects on fibroblasts, at least in part, by activation of the PI 3-kinase/Akt pathway.     

Phosphatidylinositol 3-kinase mutations identified in human cancer are oncogenic

Mutations in genes that encode components of the phosphatidyl-inositol 3-kinase (PI3-kinase) signaling pathway are common in human cancer. The recent discovery of nonrandom somatic mutations in the PIK3CA gene of many human tumors suggests an oncogenic role for the mutated enzyme. We have determined the growth-regulatory and signaling properties of the three most frequently observed PI3-kinase mutations: E542K, E545K, and H1047R. Expressed in chicken embryo fibroblasts, all three mutants induce oncogenic transformation with high efficiency. This transforming ability is correlated with elevated catalytic activity in in vitro kinase assays. The mutant-transformed cells show constitutive phosphorylation of Akt, of p70 S6 kinase, and of the 4E-binding protein 1. Phosphorylation of S6 kinase and of 4E-binding protein 1 is regulated by the target of rapamycin (TOR) kinase and affects rates of protein synthesis. The inhibitor of TOR, rapamycin, strongly interferes with cellular transformation induced by the PI3-kinase mutants, suggesting that the TOR and its downstream targets are essential components of the transformation process. The oncogenic transforming activity makes the mutated PI3-kinase proteins promising targets for small molecule inhibitors that could be developed into effective and highly specific anticancer drugs.     

Antiapoptotic effect of coagulation factor VIIa

Binding of factor VIIa (FVIIa) to its cellular receptor tissue factor (TF) was previously shown to induce various intracellular signaling events, which were thought to be responsible for TF-mediated biologic effects, including angiogenesis, tumor metastasis, and restenosis. To understand the mechanisms behind these processes, we have examined the effect of FVIIa on apoptosis. Serum deprivation-induced apoptosis of BHK(+TF) cells was characterized by apoptotic blebs, nuclei with chromatin-condensed bodies, DNA degradation, and activation of caspase 3. FVIIa markedly decreased the number of cells with apoptotic morphology and prevented the DNA degradation as measured by means of TdT-mediated dUTP nick end labeling (TUNEL). The antiapoptotic effect of FVIIa was confirmed by the observation that FVIIa attenuated caspase 3 activation. FVIIa-induced antiapoptotic effect was dependent on its proteolytic activity and TF but independent of factor Xa and thrombin. FVIIa-induced cell survival correlated with the activation of Akt and was inhibited markedly by the specific PI3-kinase inhibitor, LY294002. Blocking the activation of p44/42 mitogen-activated protein kinase (MAPK) by the specific mitogen-induced extracellular kinase (MEK) inhibitor, U0126, impaired modestly the ability of FVIIa to promote cell survival. In conclusion, FVIIa binding to TF provided protection against apoptosis induced by growth factor deprivation, primarily through activation of PI3-kinase/Akt pathway, and to a lesser extent, p44/42 MAPK pathway.     

Alpha-lipoic acid attenuates LPS-induced inflammatory responses by activating the phosphoinositide 3-kinase/Akt signaling pathway

The phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was recently shown to negatively regulate LPS-induced acute inflammatory responses. We previously observed that the metabolic thiol antioxidant alpha-lipoic acid (LA) inhibits LPS-induced expression of cellular adhesion molecules and adherence of monocytes to human aortic endothelial cells. Here we investigated the mechanism by which LA attenuates LPS-induced monocyte activation in vitro and acute inflammatory responses in vivo. Incubation of human monocytic THP-1 cells with LA induced phosphorylation of Akt in a time- and dose-dependent manner. In cells pretreated with LA followed by LPS, Akt phosphorylation was elevated initially and further increased during incubation with LPS. This LA-dependent increase in Akt phosphorylation was accompanied by inhibition of LPS-induced NF-kappaB DNA binding activity and up-regulation of TNFalpha and monocyte chemoattractant protein 1. Lipoic acid-dependent Akt phosphorylation and inhibition of NF-kappaB activity were abolished by the PI3K inhibitors LY294002 and wortmannin. Furthermore, LA treatment of LPS-exposed C57BL/6N mice strongly enhanced phosphorylation of Akt and glycogen synthase kinase 3beta in blood cells; inhibited the LPS-induced increase in serum concentrations and/or tissue expression of adhesion molecules, monocyte chemoattractant protein 1, and TNFalpha; and attenuated NF-kappaB activation in lung, heart, and aorta. Lipoic acid also improved survival of endotoxemic mice. All of these antiinflammatory effects of LA were abolished by treatment of the animals with wortmannin. We conclude that LA inhibits LPS-induced monocyte activation and acute inflammatory responses in vitro and in vivo by activating the PI3K/Akt pathway. Lipoic acid may be useful in the prevention of sepsis and inflammatory vascular diseases.     

Integrin-dependent cell growth and survival are mediated by different signals in thyroid cells

Cell adhesion to extracellular matrix regulates proliferation and survival of several cell types including epithelial thyroid cells. Activation of integrin receptors by binding to extracellular matrix generates a complex cell type-dependent signaling. Adhesion to extracellular matrix induces proliferation and survival in primary cultures of thyroid cells and induces survival in immortalized human thyrocytes. In this study we demonstrate that in immortalized human thyrocyte cells, adhesion to immobilized fibronectin (FN) stimulates DNA synthesis and proliferation through the p21Ras/MAPK pathway, whereas cell survival is mediated by phosphatidylinositol 3-kinase (PI3K) signal pathway. Integrin activation by immobilized FN induced phosphorylation of pp125 focal adhesion kinase and paxillin and induced the formation of focal adhesion kinase/Grb-2/Sos complex. Western blot and in vitro kinase assay demonstrated the activation of Ras and the p44/p42 MAPK/ERK1/2. Inhibition of p21Ras activity and inhibition of MAPK enzymatic activity completely arrested cell growth but did not induce cell death. Integrin activation by cell adhesion to FN also induced activation of PI3K. Inhibition of PI3K enzymatic activity induced apoptosis demonstrated by annexin V-binding assay and loss of cellular DNA content. These results demonstrate that in thyroid cells adhesion to FN regulates proliferation through the p21Ras/MAPK signal pathway, whereas integrin-mediated cell survival is mediated by PI3K.     

Lesion-targeted thrombopoietin potentiates vasculogenesis by enhancing motility and enlivenment of transplanted endothelial progenitor cells via activation of Akt/mTOR/p70S6kinase signaling pathway

Thrombopoietin (TPO), a physiological regulator of megakaryocyte and platelet development, is a multifunctional positive regulator in early hematopoiesis by hematopoietic stem cells. In this study, we investigated the effect of TPO on endothelial progenitor cells (EPCs) for therapeutic vasculogenesis in vitro and in vivo, and the intracellular signaling mechanism exerting the activity of EPCs. 7-day culture-expanded EPCs derived from human peripheral blood mononuclear cells were applied to each assay. Flow cytometry demonstrated the expression of c-Mpl, the receptor of TPO, in cultured EPCs. In vitro experiments revealed enhanced migration and survival of cultured EPCs by TPO. In vivo, TPO was intramuscularly administered into the foci of ischemic hindlimbs in athymic nude mice, immediately followed by intravenous injection of cultured EPCs, to assess the booster effect of TPO on vascular regeneration. At day 4 post-transplantation, transplanted EPCs were 1.7-fold higher in TPO-treated animals compared to control. At day 28, blood perfusion was recovered in the TPO-treated group, accompanied by an increase in microvascular density. The signaling transduction pathway underlying TPO-mediated activities of cultured EPCs was assessed by Western blotting. TPO induced sequential phosphorylations of Akt to p70S6kinase through mTOR. Inhibition of the PI3-kinase/Akt/mTOR/p70S6kinase signaling pathway negated the biological functions of cultured EPCs, either migration (by LY294002 for PI3-kinase and Rapamycin for mTOR) or survival and tubulogenesis (by Rapamycin). These findings provide evidence that TPO possesses booster potential for therapeutic vasculogenesis, by activating the PI3-kinase/Akt/mTOR/p70S6kinase pathway crucial to the biological activities of EPCs.     

Convergent signal transduction pathways controlling cardiomyocyte survival and function: the role of PI 3-kinase and Akt

Effective cardioprotection in clinical settings requires not only cardiomyocyte survival but also preservation of function. Multiple growth factors protect the heart from ischemic and other injury. While the downstream signaling pathways of these cardioprotective factors are complex, activation of phosphoinositide 3-kinase (PI 3-kinase) and its downstream effector, the serine-threonine kinase Akt (or Protein Kinase B), is a common feature in many cases. Genetic manipulations in cardiomyocytes both in vitro and in vivo suggest that acute activation of this pathway can promote both cardiomyocyte survival and function. Here, we review PI 3-kinase and Akt signaling, with a focus on their role in cardiomyocyte growth, survival, and function. Finally, the clinical implications of these studies will be considered.     

Cell death provoked by loss of interleukin-3 signaling is independent of Bad, Bim, and PI3 kinase, but depends in part on Puma

Growth and survival of hematopoietic cells is regulated by growth factors and cytokines, such as interleukin 3 (IL-3). When cytokine is removed, cells dependent on IL-3 kill themselves by a mechanism that is inhibited by overexpression of Bcl-2 and is likely to be mediated by proapoptotic Bcl-2 family members. Bad and Bim are 2 such BH3-only Bcl-2 family members that have been implicated as key initiators in apoptosis following growth factor withdrawal, particularly in IL-3-dependent cells. To test the role of Bad, Bim, and other proapoptotic Bcl-2 family members in IL-3 withdrawal-induced apoptosis, we generated IL-3-dependent cell lines from mice lacking the genes for Bad, Bim, Puma, both Bad and Bim, and both Bax and Bak. Surprisingly, Bad was not required for cell death following IL-3 withdrawal, suggesting changes to phosphorylation of Bad play only a minor role in apoptosis in this system. Deletion of Bim also had no effect, but cells lacking Puma survived and formed colonies when IL-3 was restored. Inhibition of the PI3 kinase pathway promoted apoptosis in the presence or absence of IL-3 and did not require Bad, Bim, or Puma, suggesting IL-3 receptor survival signals and PI3 kinase survival signals are independent.     

Angiotensin II impairs the insulin signaling pathway promoting production of nitric oxide by inducing phosphorylation of insulin receptor substrate-1 on Ser312 and Ser616 in human umbilical vein endothelial cells

It has been suggested that serine (Ser) phosphorylation of insulin receptor substrate-1 (IRS-1) decreases the ability of IRS-1 to be phosphorylated on tyrosine, thereby attenuating insulin signaling. There is evidence that angiotensin II (AII) may impair insulin signaling to the IRS-1/phosphatydilinositol 3-kinase (PI 3-kinase) pathway by enhancing Ser phosphorylation. Insulin stimulates NO production by a pathway involving IRS-1/PI3-kinase/Akt/endothelial NO synthase (eNOS). We addressed the question of whether AII affects insulin signaling involved in NO production in human umbilical vein endothelial cells and tested the hypothesis that the inhibitory effect of AII on insulin signaling was caused by increased site-specific Ser phosphorylation in IRS-1. Exposure of human umbilical vein endothelial cells to AII resulted in inhibition of insulin-stimulated production of NO. This event was associated with impaired IRS-1 phosphorylation at Tyr612 and Tyr632, two sites essential for engaging the p85 subunit of PI3-kinase, resulting in defective activation of PI 3-kinase, Akt, and eNOS. This inhibitory effect of AII was reversed by the type 1 receptor antagonist losartan. AII increased c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 activity, which was associated with a concomitant increase in IRS-1 phosphorylation at Ser312 and Ser616, respectively. Inhibition of JNK and ERK1/2 activity reversed the negative effects of AII on insulin-stimulated NO production. Our data suggest that AII, acting via the type 1 receptor, increases IRS-1 phosphorylation at Ser312 and Ser616 via JNK and ERK1/2, respectively, thus impairing the vasodilator effects of insulin mediated by the IRS-1/PI 3-kinase/Akt/eNOS pathway.