Richard Z. Lin

Adrenergic Receptors Activate Phosphatidylinositol 3-Kinase in Human Vascular Smooth Muscle Cells ROLE IN MITOGENESIS

Activation of α adrenergic receptors stimulates mitogenesis in human vascular smooth muscle cells (HVSMCs). To examine signaling pathways by which activation of α receptors may induce mitogenesis in HVSMCs, we have found that α receptor stimulated-DNA synthesis and activation of mitogen-activated protein (MAP) kinase are blocked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase). To determine directly if activation of α receptors stimulated PI 3-kinase, in vitro assays of kinase activity were performed in immunocomplexes precipitated by an antibody against the p85α subunit of PI 3-kinase. Noradrenaline stimulated a time- and concentration-dependent activation of PI 3-kinase in the presence of a β adrenergic receptor antagonist. Noradrenaline-stimulated PI 3-kinase activation was blocked by antagonists of α receptors and by pertussis toxin, suggesting that α receptors activate PI 3-kinase via a pertussis toxin-sensitive G protein. Direct activation of protein kinase C by a phorbol ester did not stimulate PI 3-kinase; also, a Ca L-channel blocker did not inhibit noradrenaline-stimulated PI 3-kinase activity. Increased PI 3-kinase activity was detected in both anti-Ras and anti-phosphotyrosine immunoprecipitates from noradrenaline-stimulated HVSMCs. Moreover, noradrenaline stimulated formation of active Ras-GTP complexes. Because blockade of PI 3-kinase by wortmannin inhibited formation of this complex, this result suggests that Ras might be a target of PI 3-kinase. Noradrenaline stimulated tyrosine phosphorylation of the p85 subunit of PI 3-kinase, and a phosphorylated tyrosine protein could be co-immunoprecipitated with anti-p85 of PI 3-kinase. These results demonstrate that stimulation of α receptors activates PI 3-kinase in HVSMCs and that α receptor-activated PI 3-kinase is associated with an increase in active Ras-GTP and activation of tyrosine protein phosphorylation. These pathways may contribute to α receptor-stimulated mitogenic responses including activation of MAP kinase and DNA synthesis in HVSMCs.

Heat shock activates c-Src tyrosine kinases and phosphatidylinositol 3-kinase in NIH3T3 fibroblasts.

There is increasing evidence that cellular responses to stress are in part regulated by protein kinases, although specific mechanisms are not well defined. The purpose of these experiments was to investigate potential upstream signaling events activated during heat shock in NIH3T3 fibroblasts. Experiments were designed to ask whether heat shock activates p60 c-Src tyrosine kinase or phosphatidylinositol 3-kinase (PI 3-kinase). Using in vitro protein kinase activity assays, it was demonstrated that heat shock stimulates c-Src and PI 3-kinase activity in a time-dependent manner. Also, there was increased PI 3-kinase activity in anti-phosphotyrosine and anti-c-Src immunoprecipitated immunocomplexes from heated cells. Heat shock activated mitogen-activated protein kinase (MAPK) and p70 S6 kinase (S6K) in these cells. The role of PI 3-kinase in regulating heat shock activation of MAPK and p70 S6K was investigated using wortmannin, a specific pharmacological inhibitor of PI 3-kinase. The results demonstrated that wortmannin inhibited heat shock activation of p70 S6K but only partially inhibited heat activation of MAPK. A dominant negative Raf mutant inhibited activation of MAPK by heat shock but did not inhibit heat shock stimulation of p70 S6K. Genistein, a tyrosine kinase inhibitor, and suramin, a growth factor receptor inhibitor, both inhibited heat shock stimulation of MAPK activity and tyrosine phosphorylation of MAPK. Furthermore, a selective epidermal growth factor receptor (EGFR) inhibitor, tryphostin AG1478, and a dominant negative EGFR mutant also inhibited heat shock activation of MAPK. Heat shock induced EGFR phosphorylation. These results suggest that early upstream signaling events in response to heat stress may involve activation of PI 3-kinase and tyrosine kinases, such as c-Src, and a growth factor receptor, such as EGFR; activation of important downstream pathways, such as MAPK and p70 S6K, occur by divergent signaling mechanisms similar to growth factor stimulation.

Suppression of Phosphoinositide 3-Kinase Signaling and Alteration of Multiple Ion Currents in Drug-Induced Long QT Syndrome

The dangerous heart arrhythmias that are triggered as a side effect of some drugs are caused by many ion channels, prompting a rethinking of how we screen for these adverse events. Long QT: A Many-Channeled Syndrome To “do no harm” is a key principle of medical ethics, yet the use of some drugs can trigger life-threatening side effects. For example, the anticancer drug nilotinib—a tyrosine kinase inhibitor—can cause sudden death by inducing an irregular heartbeat and, as such, carries a black box warning from the U.S. Food and Drug Administration. Specifically, nilotinib (and other medications) can produce long QT syndrome, in which repolarization of the heart is delayed after a heartbeat. This effect is believed to be caused by direct blockade of the potassium ion channel through which the repolarizing current flows. Because some phosphoinositide 3-kinases (PI3Ks)—intracellular signal-transducing enzymes—are activated by tyrosine kinases, Lu et al. investigated whether the cardiac effects of nilotinib and related drugs might in part be mediated by PI3Ks. In isolated cardiac cells, delayed repolarization is seen as an increase in action potential duration (APD); as expected, treatment with these drugs increased the APD, whereas PI3K activity decreased. (Likewise, a PI3K inhibitor increased the APD.) The addition of the second messenger produced by PI3K normalized the APD, indicating that drug-induced PI3K inhibition is responsible for the increased APD. Lu et al. also showed that both nilotinib and a PI3K inhibitor affected currents through multiple ion channels, including calcium and sodium channels, in addition to the potassium channel originally thought to be responsible for drug-induced long QT syndrome. Furthermore, isolated mouse hearts lacking a PI3K subunit displayed a prolonged QT interval on an electrocardiogram—the sign of long QT syndrome. Although nilotinib increased this interval in wild-type hearts, it had no effect on those lacking the PI3K subunit. New drug candidates are routinely screened for their effects on the QT interval with tests focused on effects on the potassium channel. The findings of Lu et al. may require changes in how new drugs are tested. Many drugs, including some commonly used medications, can cause abnormal heart rhythms and sudden death, as manifest by a prolonged QT interval in the electrocardiogram. Cardiac arrhythmias caused by drug-induced long QT syndrome are thought to result mainly from reductions in the delayed rectifier potassium ion (K+) current IKr. Here, we report a mechanism for drug-induced QT prolongation that involves changes in multiple ion currents caused by a decrease in phosphoinositide 3-kinase (PI3K) signaling. Treatment of canine cardiac myocytes with inhibitors of tyrosine kinases or PI3Ks caused an increase in action potential duration that was reversed by intracellular infusion of phosphatidylinositol 3,4,5-trisphosphate. The inhibitors decreased the delayed rectifier K+ currents IKr and IKs, the L-type calcium ion (Ca2+) current ICa,L, and the peak sodium ion (Na+) current INa and increased the persistent Na+ current INaP. Computer modeling of the canine ventricular action potential showed that the drug-induced change in any one current accounted for less than 50% of the increase in action potential duration. Mouse hearts lacking the PI3K p110α catalytic subunit exhibited a prolonged action potential and QT interval that were at least partly a result of an increase in INaP. These results indicate that down-regulation of PI3K signaling directly or indirectly via tyrosine kinase inhibition prolongs the QT interval by affecting multiple ion channels. This mechanism may explain why some tyrosine kinase inhibitors in clinical use are associated with increased risk of life-threatening arrhythmias.

The class IA phosphatidylinositol 3-kinase p110-β subunit is a positive regulator of autophagy

p110-β associates with the Vps34–Vps15–Beclin 1–Atg14L complex and facilitates generation of PtdIns(3)P to promote autophagy.

Decreased L-type Ca2+ Current in Cardiac Myocytes of Type 1 Diabetic Akita Mice Due to Reduced Phosphatidylinositol 3-kinase Signaling

OBJECTIVE—Contraction of cardiac myocytes is initiated by Ca2+ entry through the voltage-dependent l-type Ca2+ channel (LTCC). Previous studies have shown that phosphatidylinositol (PI) 3-kinase signaling modulates LTCC function. Because PI 3-kinases are key mediators of insulin action, we investigated whether LTCC function is affected in diabetic animals due to reduced PI 3-kinase signaling. RESEARCH DESIGN AND METHODS—We used whole-cell patch clamping and biochemical assays to compare cardiac LTCC function and PI 3-kinase signaling in insulin-deficient diabetic mice heterozygous for the Ins2Akita mutation versus nondiabetic littermates. RESULTS—Diabetic mice had a cardiac contractility defect, reduced PI 3-kinase signaling in the heart, and decreased l-type Ca2+ current (ICa,L) density in myocytes compared with control nondiabetic littermates. The lower ICa,L density in myocytes from diabetic mice is due at least in part to reduced cell surface expression of the LTCC. ICa,L density in myocytes from diabetic mice was increased to control levels by insulin treatment or intracellular infusion of PI 3,4,5-trisphosphate [PI(3,4,5)P3]. This stimulatory effect was blocked by taxol, suggesting that PI(3,4,5)P3 stimulates microtubule-dependent trafficking of the LTCC to the cell surface. The voltage dependence of steady-state activation and inactivation of ICa,L was also shifted to more positive potentials in myocytes from diabetic versus nondiabetic animals. PI(3,4,5)P3 infusion eliminated only the difference in voltage dependence of steady-state inactivation of ICa,L. CONCLUSIONS—Decreased PI 3-kinase signaling in myocytes from type 1 diabetic mice leads to reduced Ca2+ entry through the LTCC, which might contribute to the negative effect of diabetes on cardiac contractility.

A Transgenic Mouse Model of Heart Failure Using Inducible Gαq

Receptors coupled to Gαq play a key role in the development of heart failure. Studies using genetically modified mice suggest that Gαq mediates a hypertrophic response in cardiac myocytes. Gαq signaling in these models is modified during early growth and development, whereas most heart failure in humans occurs after cardiac damage sustained during adulthood. To determine the phenotype of animals that express increased Gαq signaling only as adults, we generated transgenic mice that express a silent Gαq protein (GαqQ209L-hbER) in cardiac myocytes that can be activated by tamoxifen. Following drug treatment to activate Gαq Q209L-hbER, these mice rapidly develop a dilated cardiomyopathy and heart failure. This phenotype does not appear to involve myocyte hypertrophy but is associated with dephosphorylation of phospholamban (PLB), decreased sarcoplasmic reticulum Ca2+-ATPase activity, and a decrease in L-type Ca2+ current density. Changes in Ca2+ handling and decreased cardiac contractility are apparent 1 week after GαqQ209L-hbER activation. In contrast, transgenic mice that express an inducible Gαq mutant that cannot activate phospholipase Cβ (PLCβ) do not develop heart failure or changes in PLB phosphorylation, but do show decreased L-type Ca2+ current density. These results demonstrate that activation of Gαq in cardiac myocytes of adult mice causes a dilated cardiomyopathy that requires the activation of PLCβ. However, increased PLCβ signaling is not required for all of the Gαq-induced cardiac abnormalities.

Phosphorylation of the cAMP Response Element-binding Protein and Activation of Transcription by α1 Adrenergic Receptors

Activation of α1 adrenergic receptors not only stimulates smooth muscle contraction but also modifies gene expression. We wondered if α1 adrenergic receptors could activate transcription of genes regulated by the cAMP response element-binding protein (CREB). Using Rat1 cells stably transfected with each of the three cloned human α1adrenergic receptor subtypes, norepinephrine strongly stimulated CREB phosphorylation in α1A and α1B but more weakly in α1D-transfected cells. Norepinephrine increased the activity of a somatostatin cAMP-regulated enhancer-chloramphenicol acetyltransferase reporter in these cells. α1 adrenergic receptors are known to activate protein kinase C (PKC) and increase [Ca2+ ] i . Nonetheless, neither GF109203X, a PKC inhibitor, nor BAPTA-AM, a calcium chelator, blocked phosphorylation of CREB induced by norepinephrine. In addition, α1adrenergic receptor-induced CREB phosphorylation was not mediated via the mitogen-activated protein kinase pathway because norepinephrine did not stimulate mitogen-activated protein kinase activity in these cells. Activation of α1 adrenergic receptors increased cAMP accumulation in these cells. Norepinephrine-induced cAMP-regulated enhancer-chloramphenicol acetyltransferase activity was inhibited either by expression of the PKA inhibitory peptide or a dominant negative PKA regulatory subunit mutant. These results demonstrate that α1 adrenergic receptors activate the transcription factor CREB by a PKA-dependent pathway.

Inhibition of Mammalian Target of Rapamycin Signaling by 2-(Morpholin-1-yl)pyrimido[2,1-α]isoquinolin-4-one

Signaling through the mammalian target of rapamycin (mTOR) is hyperactivated in many human tumors, including hamartomas associated with tuberous sclerosis complex (TSC). Several small molecules such as LY294002 inhibit mTOR kinase activity, but they also inhibit phosphatidylinositol 3-kinase (PI3K) at similar concentrations. Compound 401 is a synthetic inhibitor of DNA-dependent protein kinase (DNA-PK) that also targets mTOR but not PI3K in vitro (Griffin, R. J., Fontana, G., Golding, B. T., Guiard, S., Hardcastle, I. R., Leahy, J. J., Martin, N., Richardson, C., Rigoreau, L., Stockley, M., and Smith, G. C. (2005) J. Med. Chem. 48, 569-585). We used 401 to test the cellular effect of mTOR inhibition without the complicating side effects on PI3K. Treatment of cells with 401 blocked the phosphorylation of sites modified by mTOR-Raptor and mTOR-Rictor complexes (ribosomal protein S6 kinase 1 Thr389 and Akt Ser473, respectively). By contrast, there was no direct inhibition of Akt Thr308 phosphorylation, which is dependent on PI3K. Similar effects were also observed in cells that lack DNA-PK. The proliferation of TSC1-/- fibroblasts was inhibited in the presence of 401, but TSC1+/+ cells were resistant. In contrast to rapamycin, long-term treatment of TSC1-/- cells with 401 did not up-regulate phospho-Akt Ser473. Because increased Akt activity promotes survival, this may explain why the level of apoptosis was increased in the presence of 401 but not rapamycin. These results suggest that mTOR kinase inhibitors might be more effective than rapamycins in controlling the growth of TSC hamartomas and other tumors that depend on elevated mTOR activity.

Control of cardiac repolarization by phosphoinositide 3-kinase signaling to ion channels.

Upregulation of phosphoinositide 3-kinase (PI3K) signaling is a common alteration in human cancer, and numerous drugs that target this pathway have been developed for cancer treatment. However, recent studies have implicated inhibition of the PI3K signaling pathway as the cause of a drug-induced long-QT syndrome in which alterations in several ion currents contribute to arrhythmogenic drug activity. Surprisingly, some drugs that were thought to induce long-QT syndrome by direct block of the rapid delayed rectifier (IKr) also seem to inhibit PI3K signaling, an effect that may contribute to their arrhythmogenicity. The importance of PI3K in regulating cardiac repolarization is underscored by evidence that QT interval prolongation in diabetes mellitus also may result from changes in multiple currents because of decreased insulin activation of PI3K in the heart. How PI3K signaling regulates ion channels to control the cardiac action potential is poorly understood. Hence, this review summarizes what is known about the effect of PI3K and its downstream effectors, including Akt, on sodium, potassium, and calcium currents in cardiac myocytes. We also refer to some studies in noncardiac cells that provide insight into potential mechanisms of ion channel regulation by this signaling pathway in the heart. Drug development and safety could be improved with a better understanding of the mechanisms by which PI3K regulates cardiac ion channels and the extent to which PI3K inhibition contributes to arrhythmogenic susceptibility.

Gαq Inhibits Cardiac L-type Ca2+ Channels through Phosphatidylinositol 3-Kinase

Cardiac myocyte contractility is initiated by Ca2+ entry through the voltage-dependent L-type Ca2+ channel (LTCC). To study the effect of Gαq on the cardiac LTCC, we utilized two transgenic mouse lines that selectively express inducible Gαq-estrogen receptor hormone-binding domain fusion proteins (GαqQ209L-hbER or GαqQ209L-AA-hbER) in cardiac myocytes. Both of these proteins inhibit phosphatidylinositol (PI) 3-kinase (PI3K) signaling, but GαqQ209L-AA-hbER cannot activate the canonical Gαq effector phospholipase Cβ (PLCβ). L-type Ca2+ current (ICa,L) density measured by whole-cell patch clamping was reduced by more than 50% in myocytes from both Gαq animals as compared with wild-type cells, suggesting that inhibition of the LTCC by Gαq does not require PLCβ. To investigate the role of PI3K in this inhibitory effect, ICa,L was measured in the presence of various phosphoinositides infused through the patch pipette. Infusion of PI 3,4,5-trisphosphate (PI(3,4,5)P3) into wild-type myocytes did not affect ICa,L, but it fully restored ICa,L density in both Gαq transgenic myocytes to wild-type levels. By contrast, PI 4,5-bisphosphate (PI(4,5)P2) or PI 3,5-bisphosphate had no effect. Infusion with p110β/p85α or p110γ PI3K in the presence of PI(4,5)P2 also restored ICa,L density to wild-type levels. Last, infusion of either PTEN, a PI(3,4,5)P3 phosphatase, or the pleckstrin homology domain of Grp1, which