Hypoxia Sensing of ß-Adrenergic Receptor Is Regulated by Endosomal PI3Kγ.

BACKGROUND: Impaired beta-adrenergic receptor (ß1 and ß2AR) function following hypoxia underlies ischemic heart failure/stroke. Activation of PI3Kγ (phosphoinositide 3-kinase γ) by beta-adrenergic receptor leads to feedback regulation of the receptor by hindering beta-adrenergic receptor dephosphorylation through inhibition of PP2A (protein phosphatase 2A). However, little is known about PI3Kγ feedback mechanism in regulating hypoxia-mediated ß1 and ß2AR dysfunction and cardiac remodeling. METHODS: Human embryonic kidney 293 cells or mouse adult cardiomyocytes and C57BL/6 (WT) or PI3Kγ knockout (KO) mice were subjected to hypoxia. Cardiac plasma membranes and endosomes were isolated and evaluated for ß1 and ß2AR density and function, PI3Kγ activity and ß1 and ß2AR-associated PP2A activity. Metabolic labeling was performed to assess ß1 and ß2AR phosphorylation and epinephrine/norepinephrine levels measured post-hypoxia. RESULTS: Hypoxia increased ß1 and ß2AR phosphorylation, reduced cAMP, and led to endosomal accumulation of phosphorylated ß2ARs in human embryonic kidney 293 cells and WT cardiomyocytes. Acute hypoxia in WT mice resulted in cardiac remodeling and loss of adenylyl cyclase activity associated with increased ß1 and ß2AR phosphorylation. This was agonist-independent as plasma and cardiac epinephrine and norepinephrine levels were unaltered. Unexpectedly, PI3Kγ activity was selectively increased in the endosomes of human embryonic kidney 293 cells and WT hearts post-hypoxia. Endosomal ß1- and ß2AR-associated PP2A activity was inhibited upon hypoxia in human embryonic kidney 293 cells and WT hearts showing regulation of beta-adrenergic receptors by PI3Kγ. This was accompanied with phosphorylation of endogenous inhibitor of protein phosphatase 2A whose phosphorylation by PI3Kγ inhibits PP2A. Increased ß1 and ß2AR-associated PP2A activity, decreased beta-adrenergic receptor phosphorylation, and normalized cardiac function was observed in PI3Kγ KO mice despite hypoxia. Compared to WT, PI3Kγ KO mice had preserved cardiac response to challenge with ß1AR-selective agonist dobutamine post-hypoxia. CONCLUSIONS: Agonist-independent activation of PI3Kγ underlies hypoxia sensing as its ablation leads to reduction in ß1- and ß2AR phosphorylation and amelioration of cardiac dysfunction.

Inhibition of protein phosphatase 2A activity by PI3Kγ regulates ß-adrenergic receptor function.

Phosphoinositide 3-kinase γ (PI3Kγ) is activated by G protein-coupled receptors (GPCRs). We show here that PI3Kγ inhibits protein phosphatase 2A (PP2A) at the ß-adrenergic receptor (ßAR, a GPCR) complex altering G protein coupling. PI3Kγ inhibition results in significant increase of ßAR-associated phosphatase activity leading to receptor dephosphorylation and resensitization preserving cardiac function. Mechanistically, PI3Kγ inhibits PP2A activity at the ßAR complex by phosphorylating an intracellular inhibitor of PP2A (I2PP2A) on serine residues 9 and 93, resulting in enhanced binding to PP2A. Indeed, enhanced phosphorylation of ß2ARs is observed with a phosphomimetic I2PP2A mutant that was completely reversed with a mutant mimicking dephosphorylated state. siRNA depletion of endogenous I2PP2A augments PP2A activity despite active PI3K resulting in ß2AR dephosphorylation and sustained signaling. Our study provides the underpinnings of a PI3Kγ-mediated regulation of PP2A activity that has significant consequences on receptor function with broad implications in cellular signaling.

Phosphorylation inactivation of endothelial nitric oxide synthesis in pulmonary arterial hypertension.

The impairment of vasodilator nitric oxide (NO) production is well accepted as a typical marker of endothelial dysfunction in vascular diseases, including in the pathophysiology of pulmonary arterial hypertension (PAH), but the molecular mechanisms accounting for loss of NO production are unknown. We hypothesized that low NO production by pulmonary arterial endothelial cells in PAH is due to inactivation of NO synthase (eNOS) by aberrant phosphorylation of the protein. To test the hypothesis, we evaluated eNOS levels, dimerization, and phosphorylation in the vascular endothelial cells and lungs of patients with PAH compared with controls. In mechanistic studies, eNOS activity in endothelial cells in PAH lungs was found to be inhibited due to phosphorylation at T495. Evidence pointed to greater phosphorylation/activation of protein kinase C (PKC) α and its greater association with eNOS as the source of greater phosphorylation at T495. The presence of greater amounts of pT495-eNOS in plexiform lesions in lungs of patients with PAH confirmed the pathobiological mechanism in vivo. Transfection of the activating mutation of eNOS (T495A/S1177D) restored NO production in PAH cells. Pharmacological blockade of PKC activity by ß-blocker also restored NO formation by PAH cells, identifying one mechanism by which ß-blockers may benefit PAH and cardiovascular diseases through recovery of endothelial functions.

Gßγ-independent recruitment of G-protein coupled receptor kinase 2 drives tumor necrosis factor α-induced cardiac ß-adrenergic receptor dysfunction.

BACKGROUND: Proinflammatory cytokine tumor necrosis factor-α (TNFα) induces ß-adrenergic receptor (ßAR) desensitization, but mechanisms proximal to the receptor in contributing to cardiac dysfunction are not known. METHODS AND RESULTS: Two different proinflammatory transgenic mouse models with cardiac overexpression of myotrophin (a prohypertrophic molecule) or TNFα showed that TNFα alone is sufficient to mediate ßAR desensitization as measured by cardiac adenylyl cyclase activity. M-mode echocardiography in these mouse models showed cardiac dysfunction paralleling ßAR desensitization independent of sympathetic overdrive. TNFα-mediated ßAR desensitization that precedes cardiac dysfunction is associated with selective upregulation of G-protein coupled receptor kinase 2 (GRK2) in both mouse models. In vitro studies in ß2AR-overexpressing human embryonic kidney 293 cells showed significant ßAR desensitization, GRK2 upregulation, and recruitment to the ßAR complex following TNFα. Interestingly, inhibition of phosphoinositide 3-kinase abolished GRK2-mediated ßAR phosphorylation and GRK2 recruitment on TNFα. Furthermore, TNFα-mediated ßAR phosphorylation was not blocked with ßAR antagonist propranolol. Additionally, TNFα administration in transgenic mice with cardiac overexpression of Gßγ-sequestering peptide ßARK-ct could not prevent ßAR desensitization or cardiac dysfunction showing that GRK2 recruitment to the ßAR is Gßγ independent. Small interfering RNA knockdown of GRK2 resulted in the loss of TNFα-mediated ßAR phosphorylation. Consistently, cardiomyocytes from mice with cardiac-specific GRK2 ablation normalized the TNFα-mediated loss in contractility, showing that TNFα-induced ßAR desensitization is GRK2 dependent. CONCLUSIONS: TNFα-induced ßAR desensitization is mediated by GRK2 and is independent of Gßγ, uncovering a hitherto unknown cross-talk between TNFα and ßAR function, providing the underpinnings of inflammation-mediated cardiac dysfunction.

miRNA-548c: a specific signature in circulating PBMCs from dilated cardiomyopathy patients.

High fidelity genome-wide expression analysis has strengthened the idea that microRNA (miRNA) signatures in peripheral blood mononuclear cells (PBMCs) can be potentially used to predict the pathology when anatomical samples are inaccessible like the heart. PBMCs from 48 non-failing controls and 44 patients with relatively stable chronic heart failure (ejection fraction of ≤ 40%) associated with dilated cardiomyopathy (DCM) were used for miRNA analysis. Genome-wide miRNA-microarray on PBMCs from chronic heart failure patients identified miRNA signature uniquely characterized by the downregulation of miRNA-548 family members. We have also independently validated downregulation of miRNA-548 family members (miRNA-548c & 548i) using real time-PCR in a large cohort of independent patient samples. Independent in silico Ingenuity Pathway Analysis (IPA) of miRNA-548 targets shows unique enrichment of signaling molecules and pathways associated with cardiovascular disease and hypertrophy. Consistent with specificity of miRNA changes with pathology, PBMCs from breast cancer patients showed no alterations in miRNA-548c expression compared to healthy controls. These studies suggest that miRNA-548 family signature in PBMCs can therefore be used to detect early heart failure. Our studies show that cognate networking of predicted miRNA-548 targets in heart failure can be used as a powerful ancillary tool to predict the ongoing pathology.

Long-term α1A-adrenergic receptor stimulation improves synaptic plasticity, cognitive function, mood, and longevity.

The role of α(1)-adrenergic receptors (α(1)ARs) in cognition and mood is controversial, probably as a result of past use of nonselective agents. α(1A)AR activation was recently shown to increase neurogenesis, which is linked to cognition and mood. We studied the effects of long-term α(1A)AR stimulation using transgenic mice engineered to express a constitutively active mutant (CAM) form of the α(1A)AR. CAM-α(1A)AR mice showed enhancements in several behavioral models of learning and memory. In contrast, mice that have the α(1A)AR gene knocked out displayed poor cognitive function. Hippocampal brain slices from CAM-α(1A)AR mice demonstrated increased basal synaptic transmission, paired-pulse facilitation, and long-term potentiation compared with wild-type (WT) mice. WT mice treated with the α(1A)AR-selective agonist cirazoline also showed enhanced cognitive functions. In addition, CAM-α(1A)AR mice exhibited antidepressant and less anxious phenotypes in several behavioral tests compared with WT mice. Furthermore, the lifespan of CAM-α(1A)AR mice was 10% longer than that of WT mice. Our results suggest that long-term α(1A)AR stimulation improves synaptic plasticity, cognitive function, mood, and longevity. This may afford a potential therapeutic target for counteracting the decline in cognitive function and mood associated with aging and neurological disorders.

Respiratory syncytial virus induces ß2-adrenergic receptor dysfunction in human airway smooth muscle cells.

Pharmacologic agonism of the ß2-adrenergic receptor (ß2AR) induces bronchodilation by activating the enzyme adenylyl cyclase to generate cyclic adenosine monophosphate (cAMP). ß2AR agonists are generally the most effective strategy to relieve acute airway obstruction in asthmatic patients, but they are much less effective when airway obstruction in young patients is triggered by infection with respiratory syncytial virus (RSV). Here, we investigated the effects of RSV infection on the abundance and function of ß2AR in primary human airway smooth muscle cells (HASMCs) derived from pediatric lung tissue. We showed that RSV infection of HASMCs resulted in proteolytic cleavage of ß2AR mediated by the proteasome. RSV infection also resulted in ß2AR ligand-independent activation of adenylyl cyclase, leading to reduced cAMP synthesis compared to that in uninfected control cells. Last, RSV infection caused stronger airway smooth muscle cell contraction in vitro due to increased cytosolic Ca2+ concentrations. Thus, our results suggest that RSV infection simultaneously induces loss of functional ß2ARs and activation of multiple pathways favoring airway obstruction in young patients, with the net effect of counteracting ß2AR agonist-induced bronchodilation. These findings not only provide a potential mechanism for the reported lack of clinical efficacy of ß2AR agonists for treating virus-induced wheezing but also open the path to developing more precise therapeutic strategies.

Cardiac expression of microRNA-7 is associated with adverse cardiac remodeling.

Although microRNA-7 (miRNA-7) is known to regulate proliferation of cancer cells by targeting Epidermal growth factor receptor (EGFR/ERBB) family, less is known about its role in cardiac physiology. Transgenic (Tg) mouse with cardiomyocyte-specific overexpression of miRNA-7 was generated to determine its role in cardiac physiology and pathology. Echocardiography on the miRNA-7 Tg mice showed cardiac dilation instead of age-associated physiological cardiac hypertrophy observed in non-Tg control mice. Subjecting miRNA-7 Tg mice to transverse aortic constriction (TAC) resulted in cardiac dilation associated with increased fibrosis bypassing the adaptive cardiac hypertrophic response to TAC. miRNA-7 expression in cardiomyocytes resulted in significant loss of ERBB2 expression with no changes in ERBB1 (EGFR). Cardiac proteomics in the miRNA-7 Tg mice showed significant reduction in mitochondrial membrane structural proteins compared to NTg reflecting role of miRNA-7 beyond the regulation of EGFR/ERRB in mediating cardiac dilation. Consistently, electron microscopy showed that miRNA-7 Tg hearts had disorganized rounded mitochondria that was associated with mitochondrial dysfunction. These findings show that expression of miRNA-7 in the cardiomyocytes results in cardiac dilation instead of adaptive hypertrophic response during aging or to TAC providing insights on yet to be understood role of miRNA-7 in cardiac function.

Unique microRNA profile in end-stage heart failure indicates alterations in specific cardiovascular signaling networks.

It is well established that gene expression patterns are substantially altered in cardiac hypertrophy and heart failure, but the reasons for such differences are not clear. MicroRNAs (miRNAs) are short noncoding RNAs that provide a novel mechanism for gene regulation. The goal of this study was to comprehensively test for alterations in miRNA expression using human heart failure samples with an aim to build signaling pathway networks using predicted targets for the miRNAs and to identify nodal molecules that control these networks. Genome-wide profiling of miRNAs was performed using custom-designed miRNA microarray followed by validation on an independent set of samples. Eight miRNAs are significantly altered in heart failure of which we have identified two novel miRNAs that are yet to be implicated in cardiac pathophysiology. To gain an unbiased global perspective on regulation by altered miRNAs, predicted targets of eight miRNAs were analyzed using the Ingenuity Pathways Analysis network algorithm to build signaling networks and identify nodal molecules. The majority of nodal molecules identified in our analysis are targets of altered miRNAs and are known regulators of cardiovascular signaling. A heart failure gene expression data base was used to analyze changes in expression patterns for these target nodal molecules. Indeed, expression of nodal molecules was altered in heart failure and inversely correlated to miRNA changes validating our analysis. Importantly, using network analysis we have identified a limited number of key functional targets that may regulate expression of the myriad proteins in heart failure and could be potential therapeutic targets.

alpha1-Adrenergic receptors regulate neurogenesis and gliogenesis.

The understanding of the function of alpha(1)-adrenergic receptors in the brain has been limited due to a lack of specific ligands and antibodies. We circumvented this problem by using transgenic mice engineered to overexpress either wild-type receptor tagged with enhanced green fluorescent protein or constitutively active mutant alpha(1)-adrenergic receptor subtypes in tissues in which they are normally expressed. We identified intriguing alpha(1A)-adrenergic receptor subtype-expressing cells with a migratory morphology in the adult subventricular zone that coexpressed markers of neural stem cell and/or progenitors. Incorporation of 5-bromo-2-deoxyuridine in vivo increased in neurogenic areas in adult alpha(1A)-adrenergic receptor transgenic mice or normal mice given the alpha(1A)-adrenergic receptor-selective agonist, cirazoline. Neonatal neurospheres isolated from normal mice expressed a mixture of alpha(1)-adrenergic receptor subtypes, and stimulation of these receptors resulted in increased expression of the alpha(1B)-adrenergic receptor subtype, proneural basic helix-loop-helix transcription factors, and the differentiation and migration of neuronal progenitors for catecholaminergic neurons and interneurons. alpha(1)-Adrenergic receptor stimulation increased the apoptosis of astrocytes and regulated survival of neonatal neurons through phosphatidylinositol 3-kinase signaling. However, in adult normal neurospheres, alpha(1)-adrenergic receptor stimulation increased the expression of glial markers at the expense of neuronal differentiation. In vivo, S100-positive glial and betaIII tubulin neuronal progenitors colocalized with either alpha(1)-adrenergic receptor subtype in the olfactory bulb. Our results indicate that alpha(1)-adrenergic receptors can regulate both neurogenesis and gliogenesis that may be developmentally dependent. Our findings may lead to new therapies to treat neurodegenerative diseases.

GPCRs and Insulin Receptor Signaling in Conversation: Novel Avenues for Drug Discovery.

Type 2 diabetes is a major health issue worldwide with complex metabolic and endocrine abnormalities. Hyperglycemia, defects in insulin secretion and insulin resistance are classic features of type 2 diabetes. Insulin signaling regulates metabolic homeostasis by regulating glucose and lipid turnover in the liver, skeletal muscle and adipose tissue. Major treatment modalities for diabetes include the drugs from the class of sulfonyl urea, Insulin, GLP-1 agonists, SGLT2 inhibitors, DPP-IV inhibitors and Thiazolidinediones. Emerging antidiabetic therapeutics also include classes of drugs targeting GPCRs in the liver, adipose tissue and skeletal muscle. Interestingly, recent research highlights several shared intermediates between insulin and GPCR signaling cascades opening potential novel avenues for diabetic drug discovery.

G Protein-Coupled Receptor Resensitization Paradigms.

Cellular responses to extracellular milieu/environment are driven by cell surface receptors that transmit the signal into the cells resulting in a synchronized and measured response. The ability to provide such exquisite responses to changes in external environment is mediated by the tight and yet, deliberate regulation of cell surface receptor function. In this regard, the seven transmembrane G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors that regulate responses like cardiac contractility, vision, and olfaction including platelet activation. GPCRs regulate these plethora of events through GPCR-activation, -desensitization, and -resensitization. External stimuli (ligands or agonists) activate GPCR initiating downstream signals. The activated GPCR undergoes inactivation or desensitization by phosphorylation and binding of ß-arrestin resulting in diminution of downstream signals. The desensitized GPCRs are internalized into endosomes, wherein they undergo dephosphorylation or resensitization by protein phosphatase to be recycled back to the cell membrane as naïve GPCR ready for the next wave of stimuli. Despite the knowledge that activation, desensitization, and resensitization shoulder an equal role in maintaining GPCR function, major advances have been made in understanding activation and desensitization compared to resensitization. However, increasing evidence shows that resensitization is exquisitely regulated process, thereby contributing to the dynamic regulation of GPCR function. In recognition of these observations, in this chapter we discuss the key advances on the mechanistic underpinning that drive and regulate GPCR function with a focus on resensitization.

A unique microRNA profile in end-stage heart failure indicates alterations in specific cardiovascular signaling networks.

It is well established that the gene expression patterns are substantially altered in cardiac hypertrophy and heart failure, however, less is known about the reasons behind such global differences. MicroRNAs (miRNAs) are short non-coding RNAs that can target multiple molecules to regulate wide array of proteins in diverse pathways. The goal of the study was to profile alterations in miRNA expression using end-stage human heart failure samples with an aim to build signaling network pathways using predicted targets for the altered miRNA and to determine nodal molecules regulating individual networks. Profiling of miRNAs using custom designed microarray and validation with an independent set of samples identified eight miRNAs that are altered in human heart failure including one novel miRNA yet to be implicated in cardiac pathology. To gain an unbiased perspective on global regulation by top eight altered miRNAs, functional relationship of predicted targets for these eight miRNAs were examined by network analysis. Ingenuity Pathways Analysis network algorithm was used to build global signaling networks based on the targets of altered miRNAs which allowed us to identify participating networks and nodal molecules that could contribute to cardiac pathophysiology. Majority of the nodal molecules identified in our analysis are targets of altered miRNAs and known regulators of cardiovascular signaling. Cardio-genomics heart failure gene expression public data base was used to analyze trends in expression pattern for target nodal molecules and indeed changes in expression of nodal molecules inversely correlated to miRNA alterations. We have used NF kappa B network as an example to show that targeting other molecules in the network could alter the nodal NF kappa B despite not being a miRNA target suggesting an integrated network response. Thus, using network analysis we show that altering key functional target proteins may regulate expression of the myriad signaling pathways underlying the cardiac pathology.

An assessment of the role of reactive oxygen species and redox signaling in norepinephrine-induced apoptosis and hypertrophy of H9c2 cardiac myoblasts.

Cardiac myocytes, upon exposure to increasing doses of norepinephrine (NE), transit from hypertrophic to apoptotic phenotype. Since reactive oxygen species (ROS) generation is attributed to both phenomena, the authors tested whether an elevation in intracellular ROS level causes such transition. H9c2 cardiac myoblasts upon treatment with hypertrophic and apoptotic doses of NE (2 and 100 microM, respectively) transiently induced intracellular ROS at a comparable level, while 200 microM H(2)O(2), another proapoptotic agonist, showed robust and sustained ROS generation. Upon analysis of a number of redox-responsive transcription factors as the downstream targets of ROS signaling, the authors observed that NE (2 and 100 microM) and H(2)O(2) (200 microM) were ineffective in inducing NF-kappaB while both the agonists upregulated AP-1 and Nrf-2. However, the extents of induction of AP-1 and Nrf-2 were not in direct correlation with the respective ROS levels. Also, AP-1 activities induced by two doses of NE were intrinsically different, since at 2 microM, it primarily induced FosB, and at 100 microM it activated Fra-1. Differential induction of FosB and Fra-1 was also reiterated in adult rat myocardium injected with increasing doses of NE. Therefore, NE induces hypertrophy and apoptosis in cardiac myocytes by distinct redox-signaling rather than a general surge of ROS.

Potential Role of microRNAs in Cardiovascular Disease: Are They up to Their Hype?

PURPOSE OF REVIEW: Cardiovascular diseases remain the foremost cause of mortality globally. As molecular medicine unravels the alterations in genomic expression and regulation of the underlying atherosclerotic process, it opens new vistas for discovering novel diagnostic biomarkers and therapeutics for limiting the disease process. miRNAs have emerged as powerful regulators of protein translation by regulating gene expression at the post-transcriptional level. RECENT FINDINGS: Overexpression and under-expression of specific miRNAs are being evaluated as a novel approach to diagnosis and treatment of cardiovascular disease. This review sheds light on the current knowledge of the miRNA evaluated in cardiovascular disease. CONCLUSION: In this review we summarize the data, including the more recent data, regarding miRNAs in cardiovascular disease and their potential role in future in diagnostic and therapeutic strategies.

Bidirectional cross-regulation between ErbB2 and ß-adrenergic signalling pathways.

AIMS: Despite the observation that ErbB2 regulates sensitivity of the heart to doxorubicin or ErbB2-targeted cancer therapies, mechanisms that regulate ErbB2 expression and activity have not been studied. Since isoproterenol up-regulates ErbB2 in kidney and salivary glands and ß2AR and ErbB2 complex in brain and heart, we hypothesized that ß-adrenergic receptors (AR) modulate ErbB2 signalling status. METHODS AND RESULTS: ErbB2 transfection of HEK293 cells up-regulates ß2AR, and ß2AR transfection of HEK293 up-regulates ErbB2. Interestingly, cardiomyocytes isolated from myocyte-specific ErbB2-overexpressing (ErbB2(tg)) mice have amplified response to selective ß2-agonist zinterol, and right ventricular trabeculae baseline force generation is markedly reduced with ß2-antagonist ICI-118 551. Consistently, receptor binding assays and western blotting demonstrate that ß2ARs levels are markedly increased in ErbB2(tg) myocardium and reduced by EGFR/ErbB2 inhibitor, lapatinib. Intriguingly, acute treatment of mice with ß1- and ß2-AR agonist isoproterenol resulted in myocardial ErbB2 increase, while inhibition with either ß1- or ß2-AR antagonist did not completely prevent isoproterenol-induced ErbB2 expression. Furthermore, inhibition of ErbB2 kinase predisposed mice hearts to injury from chronic isoproterenol treatment while significantly reducing isoproterenol-induced pAKT and pERK levels, suggesting ErbB2's role in transactivation in the heart. CONCLUSION: Our studies show that myocardial ErbB2 and ßAR signalling are linked in a feedback loop with ßAR activation leading to increased ErbB2 expression and activity, and increased ErbB2 activity regulating ß2AR expression. Most importantly, ErbB2 kinase activity is crucial for cardioprotection in the setting of ß-adrenergic stress, suggesting that this mechanism is important in the pathophysiology and treatment of cardiomyopathy induced by ErbB2-targeting antineoplastic drugs.

Defective Resensitization in Human Airway Smooth Muscle Cells Evokes ß-Adrenergic Receptor Dysfunction in Severe Asthma.

ß2-adrenergic receptor (ß2AR) agonists (ß2-agonist) are the most commonly used therapy for acute relief in asthma, but chronic use of these bronchodilators paradoxically exacerbates airway hyper-responsiveness. Activation of ßARs by ß-agonist leads to desensitization (inactivation) by phosphorylation through G-protein coupled receptor kinases (GRKs) which mediate ß-arrestin binding and ßAR internalization. Resensitization occurs by dephosphorylation of the endosomal ßARs which recycle back to the plasma membrane as agonist-ready receptors. To determine whether the loss in ß-agonist response in asthma is due to altered ßAR desensitization and/or resensitization, we used primary human airway smooth muscle cells (HASMCs) isolated from the lungs of non-asthmatic and fatal-asthmatic subjects. Asthmatic HASMCs have diminished adenylyl cyclase activity and cAMP response to ß-agonist as compared to non-asthmatic HASMCs. Confocal microscopy showed significant accumulation of phosphorylated ß2ARs in asthmatic HASMCs. Systematic analysis of desensitization components including GRKs and ß-arrestin showed no appreciable differences between asthmatic and non-asthmatic HASMCs. However, asthmatic HASMC showed significant increase in PI3Kγ activity and was associated with reduction in PP2A activity. Since reduction in PP2A activity could alter receptor resensitization, endosomal fractions were isolated to assess the agonist ready ß2ARs as a measure of resensitization. Despite significant accumulation of ß2ARs in the endosomes of asthmatic HASMCs, endosomal ß2ARs cannot robustly activate adenylyl cyclase. Furthermore, endosomes from asthmatic HASMCs are associated with significant increase in PI3Kγ and reduced PP2A activity that inhibits ß2AR resensitization. Our study shows that resensitization, a process considered to be a homeostasis maintaining passive process is inhibited in asthmatic HASMCs contributing to ß2AR dysfunction which may underlie asthma pathophysiology and loss in asthma control.

Phosphoinositide 3-kinase γ inhibits cardiac GSK-3 independently of Akt.

Activation of cardiac phosphoinositide 3-kinase α (PI3Kα) by growth factors, such as insulin, or activation of PI3Kγ downstream of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors stimulates the activity of the kinase Akt, which phosphorylates and inhibits glycogen synthase kinase-3 (GSK-3). We found that PI3Kγ inhibited GSK-3 independently of the insulin-PI3Kα-Akt axis. Although insulin treatment activated Akt in PI3Kγ knockout mice, phosphorylation of GSK-3 was decreased compared to control mice. GSK-3 is activated when dephosphorylated by the protein phosphatase 2A (PP2A), which is activated when methylated by the PP2A methyltransferase PPMT-1. PI3Kγ knockout mice showed increased activity of PPMT-1 and PP2A and enhanced nuclear export of the GSK-3 substrate NFATc3. GSK-3 inhibits cardiac hypertrophy, and the hearts of PI3Kγ knockout mice were smaller compared to those of wild-type mice. Cardiac overexpression of a catalytically inactive PI3Kγ (PI3Kγ(inact)) transgene in PI3Kγ knockout mice reduced the activities of PPMT-1 and PP2A and increased phosphorylation of GSK-3. Furthermore, PI3Kγ knockout mice expressing the PI3Kγ(inact) transgene had larger hearts than wild-type or PI3Kγ knockout mice. Our studies show that a kinase-independent function of PI3Kγ could directly inhibit GSK-3 function by preventing the PP2A-PPMT-1 interaction and that this inhibition of GSK-3 was independent of Akt.