Inositol polyphosphate multikinase is a physiologic PI3-kinase that activates Akt/PKB

The second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)), formed by the p110 family of PI3-kinases, promotes cellular growth, proliferation, and survival, in large part by activating the protein kinase Akt/PKB. We show that inositol polyphosphate multikinase (IPMK) physiologically generates PIP(3) as well as water soluble inositol phosphates. IPMK deletion reduces growth factor-elicited Akt signaling and cell proliferation caused uniquely by loss of its PI3-kinase activity. Inhibition of p110 PI3-kinases by wortmannin prevents IPMK phosphorylation and activation. Thus, growth factor stimulation of Akt signaling involves PIP(3) generation through the sequential activations of the p110 PI3-kinases and IPMK. As inositol phosphates inhibit Akt signaling, IPMK appears to act as a molecular switch, inhibiting or stimulating Akt via its inositol phosphate kinase or PI3-kinase activities, respectively. Drugs regulating IPMK may have therapeutic relevance in influencing cell proliferation.     

Glimepiride induces proliferation and differentiation of rat osteoblasts via the PI3-kinase/Akt pathway

Glimepiride is a third-generation sulfonylurea agent and is widely used in the treatment of type 2 diabetes mellitus. In addition to the stimulatory effects on pancreatic insulin secretion, glimepiride has also been reported to have extrapancreatic functions including activation of PI3 kinase (PI3K) and Akt in rat adipocytes and skeletal muscle. PI3-kinase and Akt are important signaling molecules in the regulation of proliferation and differentiation in various cells. This study investigated the actions of glimepiride in rat osteoblasts and the role of PI3K/Akt pathway. Cell proliferation was determined by measuring absorbance at 550 nm. Supernatant assay was used for measuring alkaline phosphatase activity. Western blot analysis was used for determining collagen I, insulin receptor substrate-1/2, PI3K/Akt, and endothelial nitric oxide synthase expression. We found that glimepiride significantly enhanced proliferation and differentiation of osteoblasts and led to activation of several key signaling molecules including insulin receptor substrate-1/2, PI3K/Akt, and endothelial nitric oxide synthase. Furthermore, a specific inhibitor of PI3K abolished the stimulatory effects of glimepiride on proliferation and differentiation. Taken together, these observations provide concrete evidence that glimepiride activates the PI3K/Akt pathway; and this activation is likely required for glimepiride to stimulate proliferation and differentiation of rat osteoblasts.     

Phosphatidylinositol 3-kinase/Akt regulates angiotensin II-induced inhibition of apoptosis in microvascular endothelial cells by governing survivin expression and suppression of caspase-3 activity

Angiotensin II (Ang II) plays essential roles in vascular homeostasis, neointimal formation, and postinfarct remodeling. Although Ang II has been shown to regulate apoptosis in cardiomyocytes and vascular smooth muscle cells, its role in vascular endothelial cells (ECs) remains elusive. To address this issue, we first performed TUNEL and caspase-3 activity assays with porcine microvascular ECs challenged by serum deprivation. Ang II significantly reduced the ratio of apoptotic cells and caspase-3 activity. The Ang II type 1 receptor (AT1) was responsible for these effects. Among the signaling molecules downstream of AT1, we revealed that PI3-kinase/Akt pathway plays a predominant role in the antiapoptotic effect of Ang II. Interestingly, the expression of survivin, a central molecule of cell survival, increased after Ang II stimulation. Overexpression of a dominant-negative form of Akt abolished both Ang II-induced antiapoptosis and survivin protein expression. In a murine model of hyperoxygen-induced retinal vascular regression, AT1a knockout mice showed a significant increase in retinal avascular areas. Our data indicate that Ang II plays a critical antiapoptotic role in vascular ECs by a mechanism involving PI3-kinase/Akt activation, subsequent upregulation of survivin, and suppression of caspase-3 activity.     

Phosphoinositol lipids bind to phosphatidylinositol 3 (PI3)-kinase enhancer GTPase and mediate its stimulatory effect on PI3-kinase and Akt signalings

Phosphatidylinositol 3 (PI3)-kinase enhancer (PIKE) is a nuclear GTPase that enhances PI3-kinase activity in a GTP-dependent manner. Both PIKE-L and -A isoforms contain GTPase, pleckstrin homology (PH), ADP ribosylation factor-GTPase-activating protein, and two ankyrin repeats domains, and C-terminal ADP ribosylation factor-GTPase-activating protein activates its internal GTPase activity. However, whether PH domain modulates the intramolecular action and subsequently influences its downstream signalings remains elusive. Here we show that PH domain from PIKE-L robustly binds PI(3,4,5)P(3) and exclusively resides in the nucleus. By contrast, the mutant (K679,687N), unable to bind phosphoinositol lipids, translocates to the cytoplasm. This mutation substantially compromises the stimulatory effects on PI3-kinase by PIKE-L. Surprisingly, PH domain from PIKE-A distributes in the cytoplasm. Similar mutation in PH domain of PIKE-A abolishes its binding to PI(3,4,5)P(3) and significantly decreases its activation of Akt. Moreover, amplified PIKE-A from human cancers contains mutations and highly stimulates Akt kinase activity, correlating with its GTPase activity. Thus, phosphatidylinositols regulate PIKE GTPase activity, mediating its downstream PI3-kinase/Akt signaling through a feedback mechanism by binding to its PH domain.     

Mechanism of angiotensin II-induced proliferation in bovine adrenocortical cells.

The peptide hormone angiotensin-II (AII) is a potent vasoconstrictor and major regulator of aldosterone synthesis. In addition, AII also has growth-promoting effects. We have recently shown that the lipoxygenase (LO) pathway of arachidonic acid plays a major role in AII-induced aldosterone synthesis in adrenal glomerulosa cells. The LO pathway is also involved in the vasopressor and renin-inhibitory effects of AII. However, the role of LO products in AII-induced mitogenic effects have not yet been investigated. In the present studies we have evaluated the role of the LO pathway in AII-induced proliferative responses in a bovine adrenocortical cell clone termed AC1 cells. In addition, the potential receptor type and mechanism of AII-induced proliferation was studied by evaluating the effect of specific nonpeptide type 1 and type 2 AII receptor antagonists and the role of protein kinase-C (PKC). AII-induced DNA synthesis was significantly attenuated by two structurally dissimilar LO inhibitors, baicalein and phenidone. In addition, the LO product 12-hydroxyeicosatetraenoic acid (12-HETE) itself caused a significant increase in DNA synthesis, suggesting that the 12-LO pathway in part plays a role in AII-mediated mitogenesis. AII-induced proliferative responses were blocked by the type 1 AII receptor antagonist. Both AII- and 12-HETE-induced increases in DNA synthesis were markedly inhibited by two PKC blockers, staurosporine and sangivamycin. Further, both AII and 12-HETE could activate PKC by translocating it from the cytosol to the membrane fraction, as determined by Western immunoblotting. These results suggest that both 12-LO activation and protein kinase-C have an important role in AII-induced adrenal cell proliferation.     

Contribution of PI 3-kinase isoforms to angiotensin II- and alpha-adrenoceptor-mediated signalling pathways in cardiomyocytes

OBJECTIVE: Angiotensin II stimulation increases the formation of reactive oxygen species (ROS), the phosphorylation of p38 mitogen-activated protein kinase (MAPK), and the expression of transforming growth factor beta (TGFbeta) in adult cardiomyocytes. The aim of this study was to determine the involvement of PI 3-kinase and to specify the participation of different isoforms in the angiotensin II-induced formation of ROS in comparison to the hypertrophic pathway triggered by alpha-adrenoceptor stimulation. METHODS: Freshly isolated myocytes were used to examine formation of ROS via H(2)DCF fluorescence. p38 MAPK phosphorylation, p70(S6)-kinase phosphorylation, PI 3-kinase, and TGFbeta expression were measured by Western blotting. Sense and antisense oligonucleotides were used to down-regulate diverse PI 3-kinase isoforms. Hypertrophy was measured by (14)C-phenylalanine incorporation and cell volume. RESULTS: Inhibition of PI 3-kinase by Ly294002 or wortmannin, two inhibitors, decreased formation of ROS, phosphorylation of p38 MAPK, and TGFbeta expression. Down-regulation of the p110beta isoform by antisense oligonucleotides inhibited the angiotensin II-induced signalling pathway but not the alpha-adrenoceptor-mediated hypertrophic growth of cardiomyocytes. In contrast, down-regulation of the p110alpha isoform decreased the alpha-adrenoceptor-mediated hypertrophic growth of cardiomyocytes but did not affect the angiotensin II-mediated signalling pathway. CONCLUSION: Thus, our study identifies an involvement of PI 3-kinase in the angiotensin II-induced formation of ROS and provides a biochemical basis for ligand-specific responses for angiotensin II and alpha-adrenoceptor stimulation as relates to hypertrophy.     

Endothelial nitric oxide synthase lies downstream from angiotensin II-induced angiogenesis in ischemic hindlimb

We assessed the role of angiotensin (Ang) II in ischemia-induced angiogenesis and analyzed the molecular pathways involved in such an effect. Ischemia was produced by unilateral artery femoral occlusion in control, in valsartan-treated (Ang II receptor type I antagonist, 20 mg/kg per day), in Ang II-treated (5 ng/kg per min), and in Ang II and valsartan-treated rats. After 28 days, angiogenesis was assessed by microangiography and capillary density measurement in hindlimbs. The ischemic/nonischemic leg ratio for angiographic score and capillary number increased by 2.6- and 2-fold, respectively, in Ang II-treated rats compared with controls (P<0.01). This was associated with an increase in vascular endothelial growth factor (VEGF; 1.6-fold) and endothelial NO synthase (eNOS; 1.8-fold) protein content within the ischemic leg, assessed by Western blot. Angiotensin type 1 receptor blockade and administration of VEGF neutralizing antibody (2.5 microg IP, twice a week) in Ang II-treated rats completely prevented such Ang II angiogenic effects. The key role of eNOS was then emphasized by using mice deficient in gene encoding for eNOS. In wild-type mice, Ang II (0.3 mg/kg per min) treatment increased by 1.7- and 1.6-fold the ischemic/nonischemic leg for angiographic score and blood perfusion (assessed by laser Doppler perfusion imaging) ratios, respectively (P<0.01). Conversely, no significant changes were observed in Ang II-treated mice deficient in gene encoding for eNOS. Subhypertensive dose of Ang II enhanced angiogenesis associated with tissue ischemia through angiotensin type 1 receptor activation that involved the VEGF/eNOS-dependent pathway.     

Critical role for PI 3-kinase in the control of erythropoietin-induced erythroid progenitor proliferation

The production of red blood cells is tightly regulated by erythropoietin (Epo). The phosphoinositide 3-kinase (PI 3-kinase) pathway was previously shown to be activated in response to Epo. We studied the role of this pathway in the control of Epo-induced survival and proliferation of primary human erythroid progenitors. We show that phosphoinositide 3 (PI 3)-kinase associates with 4 tyrosine-phosphorylated proteins in primary human erythroid progenitors, namely insulin receptor substrate-2 (IRS2), Src homology 2 domain-containing inositol 5'-phosphatase (SHIP), Grb2-associated binder-1 (Gab1), and the Epo receptor (EpoR). Using different in vitro systems, we demonstrate that 3 alternative pathways independently lead to Epo-induced activation of PI 3-kinase and phosphorylation of its downstream effectors, Akt, FKHRL1, and P70S6 kinase: through direct association of PI 3-kinase with the last tyrosine residue (Tyr479) of the Epo receptor (EpoR), through recruitment and phosphorylation of Gab proteins via either Tyr343 or Tyr401 of the EpoR, or through phosphorylation of IRS2 adaptor protein. The mitogen-activated protein (MAP) kinase pathway was also activated by Epo in erythroid progenitors, but we found that this process is independent of PI 3-kinase activation. In erythroid progenitors, the functional role of PI 3-kinase was both to prevent apoptosis and to stimulate cell proliferation in response to Epo stimulation. Finally, our results show that PI 3-kinase-mediated proliferation of erythroid progenitors in response to Epo occurs mainly through modulation of the E3 ligase SCF(SKP2), which, in turn, down-regulates p27(Kip1) cyclin-dependent kinase (CDK) inhibitor via proteasome degradation.     

CoenzymeA glutathione disulfide is a potent modulator of angiotensin II-induced vasoconstriction

CoenzymeA glutathione disulfide (CoASSG) has recently been isolated from bovine adrenal glands and is assumed to play an important role in blood pressure (BP) control. We used the isolated perfused rat kidney to investigate the modulating effects of CoASSG on angiotensin II (AngII)-induced vasoconstriction. Permanent perfusion with CoASSG (1 micromol/L) for 60 min induced a significant (P < .05) shift to the left in the dose-response curve for AngII (about 3.1-fold), whereas the dose-response curve for norepinephrine (NE) was unaffected. During continuous perfusion with 1 micromol/L CoASSG, the repetitive application of 10 pmol AngII significantly increased its vasoconstriction by 170% +/- 14% (P < .05) and 235% +/- 50% (P < .05) for 60 and 120 min, respectively. The potentiation of AngII by permanent perfusion with CoASSG is dose- and time-dependent and shows a plateau at 120 min. Glutathione, oxidized coenzymeA, and coenzymeA (each 1 micromol/L) are not able to enhance the vasoconstriction induced by AngII. We conclude that CoASSG is able to potentiate the vasoactive properties of AngII, and that CoASSG might play an important role in BP regulation via modulating effects of AngII.     

Evidence for down-regulation of phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR)-dependent translation regulatory signaling pathways in Ames dwarf mice

How growth hormone (GH) stimulates protein synthesis is unknown. Phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathways balance anabolic and catabolic activities in response to nutrients and growth factor signaling. As a test of GH signaling, immunoassays of two downstream translation regulatory proteins were compared in ad libitum-fed 2-month-old normal and Ames (Prop1df) dwarf mice. Phosphorylation of the p70 and p85 isoforms of S6 kinase 1 in liver and the p70 isoform in gastrocnemius muscle were significantly decreased in dwarfs. Messenger RNA (mRNA) Cap-binding demonstrated significantly higher levels of translation repressor 4E-BP1/eukaryotic initiation factor 4E (eIF4E) (coprecipitates) from dwarf livers, but not muscle. Consistent with these binding data, significantly less phosphorylation of 4E-BP1 was documented in dwarf liver. These data suggest a link between GH signaling and translation control in a model of extended longevity.     

Soluble epoxide hydrolase is a main effector of angiotensin II-induced hypertension

The soluble epoxide hydrolase (sEH) metabolizes vasodilatory epoxyeicosatrienoic acids (EETs) to their di-hydroxy derivatives. We hypothesized that the metabolism of EETs by the sEH contributes to angiotensin II-induced hypertension and tested the effects of a water-soluble sEH inhibitor, 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA) on blood pressure. AUDA (130 microg/mL in drinking water) did not affect blood pressure in normotensive animals but markedly lowered it in mice with angiotensin II-induced hypertension (1 mg/kg per day). The effect of AUDA was accompanied by an increase in urinary salt and water excretion. Intravenous application of AUDA (8 mg/kg) acutely lowered blood pressure and heart rate in animals with angiotensin II-induced hypertension but failed to affect blood pressure in animals with phenylephrine-induced hypertension (29 mg/kg per day). AUDA (0.1 micromol/L) selectively lowered vascular resistance in an isolated perfused kidney preparation from angiotensin II-pretreated mice but not from control mice. In the perfused hind limb and in isolated carotid arteries from angiotensin II-treated mice, AUDA was without effect. The omega-hydroxylase inhibitor N-methylsulfonyl-12,12-dibromododec-11-enamide, which attenuates formation of the potent vasoconstrictor 20-hydroxyeicosatetraenoic acid, decreased tone in carotid arteries from angiotensin II-treated but not from control mice. These data demonstrate that the decrease in blood pressure observed after sEH inhibition in angiotensin II-induced hypertension can be attributed to an initial reduction in heart rate followed by pressure diuresis resulting from increased perfusion of the kidney. Direct vasodilatation of resistance arteries in skeletal muscles does not appear to contribute to the antihypertensive effects of sEH inhibition in mice.     

Glutathione peroxidase-1 plays a major role in protecting against angiotensin II-induced vascular dysfunction

Levels of reactive oxygen species, including hydrogen peroxide(,) increase in blood vessels during hypertension and in response to angiotensin II (Ang II). Although glutathione peroxidases are known to metabolize hydrogen peroxide, the role of glutathione peroxidase during hypertension is poorly defined. We tested the hypothesis that glutathione peroxidase-1 protects against Ang II-induced endothelial dysfunction. Responses of carotid arteries from Gpx1-deficient (Gpx1(+/-) and Gpx1(-/-)) and Gpx1 transgenic mice, and their respective littermate controls, were examined in vitro after overnight incubation with either vehicle or Ang II. Under control conditions, relaxation to acetylcholine (ACh; an endothelium-dependent agonist) was similar in control, Gpx1(+/-), and Gpx1 transgenic mice, whereas in Gpx1(-/-) mice, responses to ACh were impaired. In control mice, ACh-induced vasorelaxation was not affected by 1 nmol/L of Ang II. In contrast, relaxation to ACh in arteries from Gpx1(+/-) mice was inhibited by approximately 60% after treatment with 1 nmol/L of Ang II, indicating that Gpx1 haploinsufficiency markedly enhances Ang II-induced endothelial dysfunction. A higher concentration of Ang II (10 nmol/L) selectively impaired relaxation to ACh in arteries from control mice, and this effect was prevented in arteries from Gpx1 transgenic mice or in arteries from control mice treated with polyethylene glycol-catalase (which degrades hydrogen peroxide). Thus, genetic and pharmacological evidence suggests a major role for glutathione peroxidase-1 and hydrogen peroxide in Ang II-induced effects on vascular function.     

Review of aldosterone- and angiotensin II-induced target organ damage and prevention

Aldosterone is well recognized as a cause of sodium reabsorption, water retention, and potassium and magnesium loss; however, it also produces a variety of other actions that lead to progressive target organ damage in the heart, vasculature, and kidneys. Aldosterone interacts with mineralocorticoid receptors to promote endothelial dysfunction, facilitate thrombosis, reduce vascular compliance, impair baroreceptor function, and cause myocardial and vascular fibrosis. Although angiotensin II has been considered the major mediator of cardiovascular damage, increasing evidence suggests that aldosterone may mediate and exacerbate the damaging effects of angiotensin II. While angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor blockers reduce plasma aldosterone levels initially, aldosterone rebound, or 'escape' may occur during long-term therapy. Therefore, aldosterone blockade is required to reduce the risk of progressive target organ damage in patients with hypertension and heart failure. This may be achieved nonselectively with spironolactone or with use of the selective aldosterone blocker eplerenone. While both agents have been demonstrated to be effective antihypertensive agents, eplerenone may produce improved target organ protection as witnessed in a variety of clinical settings, without the antiandrogenic and progestational effects commonly observed with spironolactone.     

Embryonic liver fordin is involved in glucose glycolysis of hepatic stellate cell by regulating PI3K/Akt signaling

AIMTo investigate the role of embryonic liver fordin (ELF) in liver fibrosis by regulating hepatic stellate cells (HSCs) glucose glycolysis.METHODSThe expression of ELF and the glucose glycolysis-related proteins were evaluated in activated HSCs. siRNA was used to silence ELF expression in activated HSCs in vitro and the subsequent changes in PI3K/Akt signaling and glucose glycolysis-related proteins were observed.RESULTSThe expression of ELF increased remarkably in HSCs of the fibrosis mouse model and HSCs that were cultured for 3 wk in vitro. Glucose glycolysis-related proteins showed an obvious increase in the activated HSCs, such as phosphofructokinase, platelet and glucose transporter 1. ELF-siRNA, which perfectly silenced the expression of ELF in activated HSCs, led to the induction of glucose glycolysis-related proteins and extracellular matrix (ECM) components. Moreover, pAkt, which is an important downstream factor in PI3K/Akt signaling, showed a significant change in response to the ELF silencing. The expression of glucose glycolysis-related proteins and ECM components decreased remarkably when the PI3K/Akt signaling was blocked by Ly294002 in the activated HSCs.CONCLUSIONELF is involved in HSC glucose glycolysis by regulating PI3K/Akt signaling.     

Proliferation of cardiomyocytes derived from human embryonic stem cells is mediated via the IGF/PI 3-kinase/Akt signaling pathway

Cardiomyocytes from common experimental animals rapidly exit the cell cycle upon isolation, impeding studies of basic cell biology and applications such as myocardial repair. Here we examined proliferation of cardiomyocytes derived from human and mouse embryonic stem (ES) cells. While mouse ES cell-derived cardiomyocytes showed little proliferation, human cardiomyocytes were highly proliferative under serum-free conditions (15-25% BrdU+/sarcomeric actin+). The cells exhibited only a small serum dose-response, and proliferation gradually slowed with increasing differentiation of the cells. Neither cell density nor different matrix attachment factors affected cardiomyocyte proliferation. Blockade of phosphatidylinositol 3-kinase (PI 3-kinase) and Akt significantly reduced cardiomyocyte proliferation, whereas MEK inhibition had no effect. Antibody blocking of the insulin-like growth factor-1 (IGF-1) receptor significantly inhibited cardiomyocyte proliferation, while addition of IGF-1 or IGF-2 stimulated cardiomyocyte proliferation in a dose-dependent manner. Thus, cardiomyocytes derived from human ES cells proliferate extensively in vitro, and their proliferation appears to be mediated primarily via the PI 3-kinase/Akt signaling pathway, using the IGF-1 receptor as one upstream activator. This system should permit identification of regulatory pathways for human cardiomyocyte proliferation and may facilitate expansion of cardiomyocytes from human ES cells for therapeutic purposes.     

Essential role of Smad3 in angiotensin II-induced vascular fibrosis

Angiotensin II (Ang II) plays a pivotal role in vascular fibrosis, which leads to serious complications in hypertension and diabetes. However, the underlying signaling mechanisms are largely unclear. In hypertensive patients, we found that arteriosclerosis was associated with the activation of Smad2/3. This observation was further investigated in vitro by stimulating mouse primary aorta vascular smooth muscle cells (VSMCs) with Ang II. There were several novel findings. First, Ang II was able to activate an early Smad signaling pathway directly at 15 to 30 minutes. This was extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) dependent but transforming growth factor-beta (TGF-beta) independent because Ang II-induced Smad signaling was blocked by addition of ERK1/2 inhibitor and by dominant-negative (DN) ERK1/2 but not by DN-TGF-beta receptor II (TbetaRII) or conditional deletion of TbetaRII. Second, Ang II was also able to activate the late Smad2/3 signaling pathway at 24 hours, which was TGF-beta dependent because it was blocked by the anti-TGF-beta antibody and DN-TbetaRII. Finally, activation of Smad3 but not Smad2 was a key and necessary mechanism of Ang II-induced vascular fibrosis because Ang II induced Smad3/4 promoter activities and collagen matrix expression was abolished in VSMCs null for Smad3 but not Smad2. Thus, we concluded that Ang II induces vascular fibrosis via both TGF-beta-dependent and ERK1/2 MAPK-dependent Smad signaling pathways. Activation of Smad3 but not Smad2 is a key mechanism by which Ang II mediates arteriosclerosis.