Obesity-induced cardiac lipid accumulation in adult mice is modulated by G protein-coupled receptor kinase 2 levels.

BACKGROUND: The leading cause of death among the obese population is heart failure and stroke prompted by structural and functional changes in the heart. The molecular mechanisms that underlie obesity-related cardiac remodeling are complex, and include hemodynamic and metabolic alterations that ultimately affect the functionality of the myocardium. G protein-coupled receptor kinase 2 (GRK2) is an ubiquitous kinase able to desensitize the active form of several G protein-coupled receptors (GPCR) and is known to play an important role in cardiac GPCR modulation. GRK2 has also been recently identified as a negative modulator of insulin signaling and systemic insulin resistance. METHODS: We investigated the effects elicited by GRK2 downregulation in obesity-related cardiac remodeling. For this aim, we used  9 month-old wild type (WT) and GRK2+/- mice, which display circa 50% lower levels of this kinase, fed with either a standard or a high fat diet (HFD) for 30 weeks. In these mice we studied different parameters related to cardiac growth and lipid accumulation. RESULTS: We find that GRK2+/- mice are protected from obesity-promoted cardiac and cardiomyocyte hypertrophy and fibrosis. Moreover, the marked intracellular lipid accumulation caused by a HFD in the heart is not observed in these mice. Interestingly, HFD significantly increases cardiac GRK2 levels in WT but not in GRK2+/- mice, suggesting that the beneficial phenotype observed in hemizygous animals correlates with the maintenance of GRK2 levels below a pathological threshold. Low GRK2 protein levels are able to keep the PKA/CREB pathway active and to prevent HFD-induced downregulation of key fatty acid metabolism modulators such as Peroxisome proliferator-activated receptor gamma co-activators (PGC1), thus preserving the expression of cardioprotective proteins such as mitochondrial fusion markers mitofusin MFN1 and OPA1. CONCLUSIONS: Our data further define the cellular processes and molecular mechanisms by which GRK2 down-regulation is cardioprotective during diet-induced obesity, reinforcing the protective effect of maintaining low levels of GRK2 under nutritional stress, and showing a role for this kinase in obesity-induced cardiac remodeling and steatosis.

Cardiac Fibroblast GRK2 Deletion Enhances Contractility and Remodeling Following Ischemia/Reperfusion Injury.

RATIONALE: G protein-coupled receptor kinase 2 (GRK2) is an important molecule upregulated after myocardial injury and during heart failure. Myocyte-specific GRK2 loss before and after myocardial ischemic injury improves cardiac function and remodeling. The cardiac fibroblast plays an important role in the repair and remodeling events after cardiac ischemia; the importance of GRK2 in these events has not been investigated. OBJECTIVE: The aim of this study is to elucidate the in vivo implications of deleting GRK2 in the cardiac fibroblast after ischemia/reperfusion injury. METHODS AND RESULTS: We demonstrate, using Tamoxifen inducible, fibroblast-specific GRK2 knockout mice, that GRK2 loss confers a protective advantage over control mice after myocardial ischemia/reperfusion injury. Fibroblast GRK2 knockout mice presented with decreased infarct size and preserved cardiac function 24 hours post ischemia/reperfusion as demonstrated by increased ejection fraction (59.1±1.8% versus 48.7±1.2% in controls; P<0.01). GRK2 fibroblast knockout mice also had decreased fibrosis and fibrotic gene expression. Importantly, these protective effects correlated with decreased infiltration of neutrophils to the ischemia site and decreased levels of tumor necrosis factor-α expression and secretion in GRK2 fibroblast knockout mice. CONCLUSIONS: These novel data showing the benefits of inhibiting GRK2 in the cardiac fibroblast adds to previously published data showing the advantage of GRK2 ablation and reinforces the therapeutic potential of GRK2 inhibition in the heart after myocardial ischemia.

G-protein-coupled receptor kinase 2-mediated desensitization of adiponectin receptor 1 in failing heart.

BACKGROUND: Phosphorylative desensitization of G-protein-coupled receptors contributes significantly to post-myocardial infarction (MI) remodeling and heart failure (HF). Here, we determined whether adiponectin receptors (AdipoRs) 1 and 2 (the 7-transmembrane domain-containing receptors mediating adiponectin functions) are phosphorylatively modified and functionally impaired after MI. METHODS AND RESULTS: Post-MI HF was induced by coronary artery occlusion. Receptor phosphorylation, kinase expression, and adiponectin function were determined via in vivo, ex vivo, and in vitro models. AdipoR1 and AdipoR2 are not phosphorylated in the normal heart. However, AdipoR1 was significantly phosphorylated after MI, peaking at 7 days and remaining significantly phosphorylated thereafter. The extent of post-MI AdipoR1 phosphorylation positively correlated with the expression level of GPCR kinase (GRK) 2, the predominant GRK isoform upregulated in the failing heart. Cardiac-specific GRK2 knockout virtually abolished post-MI AdipoR1 phosphorylation, whereas virus-mediated GRK2 overexpression significantly phosphorylated AdipoR1 and blocked adiponectin metabolic-regulatory/anti-inflammatory signaling. Mass spectrometry identified serine-7, threonine-24, and threonine-53 (residues located in the n-terminal intracellular AdipoR1 region) as the GRK2 phosphorylation sites. Ex vivo experiments demonstrated that adenosine monophosphate-activated protein kinase activation and the anti-tumor necrosis factor-α effect of adiponectin were significantly inhibited in cardiomyocytes isolated from nonischemic area 7 days after MI. In vivo experiments demonstrated that acute adiponectin administration-induced cardiac GLUT4 translocation and endothelial nitric oxide synthase phosphorylation were blunted 7 days after MI. Continuous adiponectin administration beginning 7 days after MI failed to protect the heart from adverse remodeling and HF progression. Finally, cardiac-specific GRK2 knockdown restored the cardioprotective effect of adiponectin. CONCLUSION: AdipoR1 is phosphorylatively modified and desensitized by GRK2 in failing cardiomyocytes, contributing to post-MI remodeling and HF progression.

GRK2 targeted knock-down results in spontaneous hypertension, and altered vascular GPCR signaling.

Hypertension, elevated arterial pressure, occurs as the consequence of increased peripheral resistance. G protein-coupled receptors (GPCRs) contribute to the regulation of vasodilator and vasoconstrictor responses, and their activity is regulated by a family of GPCR kinases (GRKs). GRK2 expression is increased in hypertension and this facilitates the development of the hypertensive state by increasing the desensitization of GPCRs important for vasodilation. We demonstrate here, that genetic knockdown of GRK2 using a small hairpin (sh) RNA results in altered vascular reactivity and the development of hypertension between 8-12 weeks of age in shGRK2 mice due to enhanced Gαq/11 signaling. Vascular smooth muscle cells (VSMCs) cultured from shGRK2 knockdown mice show increases in GPCR-mediated Gαs and Gαq/11 signaling, as the consequence of reduced GRK2-mediated desensitization. In addition, agonists and biased agonists exhibited age-dependent alterations in ERK1/2 and Akt signaling, as well as cell proliferation and migration responses in shGRK2 knockdown VSMCs when cultured from mice that are either 3 months or 6 months of age. Changes in angiotensin II-stimulated ERK1/2 phosphorylation are observed in VSMCs derived from 6-week-old shGRK2 mice prior to the development of the hypertensive phenotype. Thus, our findings indicate that the balance between mechanisms regulating vascular tone are shifted to favor vasoconstriction in the absence of GRK2 expression and that this leads to the age-dependent development of hypertension, as a consequence of global alterations in GPCR signaling. Consequently, therapeutic strategies that target GRK2 activity, not expression, may be more effective for the treatment of hypertension.

si-RNA-Mediated Silencing of ADRBK1 Gene Attenuates Breast Cancer Cell Proliferation.

Abstract Breast cancer is the most prominent cause of cancer-related deaths among women worldwide. It has been found that genetic mutations play distinct roles in the onset and progression of breast cancer. Androgenic, beta, receptor kinase 1 (ADRBK1) has been reported to possess oncogenic characteristics vital for cancer cell viability. This study was designed to investigate the effects of small interference RNA (si-RNA)-mediated ADRBK1 knockdown on breast cancer cell growth in vitro. High-expression levels of ADRBK1 were observed in all tested breast cancer cell lines (MDA-MB-231, MCF-7, T-47D, and BT-474). ADRBK1 si-RNA was delivered to breast cancer cells using lentivirus delivery system. Depletion of ADRBK1 significantly attenuated the cell viability and colony-formation ability. Flow cytometry analysis further demonstrated that ADRBK1 silencing led to MDA-MB-231 cell arrest in the G0/G1 phase. Collectively, these results indicate that knockdown of ADRBK1 gene has detrimental effects on breast cancer cell growth, which may be a potential therapeutic approach for the treatment of breast cancer.

Developmental and tumoral vascularization is regulated by G protein-coupled receptor kinase 2.

Tumor vessel dysfunction is a pivotal event in cancer progression. Using an in vivo neovascularization model, we identified G protein-coupled receptor kinase 2 (GRK2) as a key angiogenesis regulator. An impaired angiogenic response involving immature vessels was observed in mice hemizygous for Grk2 or in animals with endothelium-specific Grk2 silencing. ECs isolated from these animals displayed intrinsic alterations in migration, TGF-ß signaling, and formation of tubular networks. Remarkably, an altered pattern of vessel growth and maturation was detected in postnatal retinas from endothelium-specific Grk2 knockout animals. Mouse embryos with systemic or endothelium-selective Grk2 ablation had marked vascular malformations involving impaired recruitment of mural cells. Moreover, decreased endothelial Grk2 dosage accelerated tumor growth in mice, along with reduced pericyte vessel coverage and enhanced macrophage infiltration, and this transformed environment promoted decreased GRK2 in ECs and human breast cancer vessels. Our study suggests that GRK2 downregulation is a relevant event in the tumoral angiogenic switch.

Endothelial G protein-coupled receptor kinase 2 regulates vascular homeostasis through the control of free radical oxygen species.

OBJECTIVE: The role of endothelial G protein-coupled receptor kinase 2 (GRK2) was investigated in mice with selective deletion of the kinase in the endothelium (Tie2-CRE/GRK2(fl/fl)). APPROACH AND RESULTS: Aortas from Tie2-CRE/GRK2(fl/fl) presented functional and structural alterations as compared with control GRK2(fl/fl) mice. In particular, vasoconstriction was blunted to different agonists, and collagen and elastic rearrangement and macrophage infiltration were observed. In primary cultured endothelial cells deficient for GRK2, mitochondrial reactive oxygen species was increased, leading to expression of cytokines. Chronic treatment with a reactive oxygen species scavenger in mice corrected the vascular phenotype by recovering vasoconstriction, structural abnormalities, and reducing macrophage infiltration. CONCLUSIONS: These results demonstrate that GRK2 removal compromises vascular phenotype and integrity by increasing endothelial reactive oxygen species production.

The chemokine receptors CXCR1 and CXCR2 couple to distinct G protein-coupled receptor kinases to mediate and regulate leukocyte functions.

The chemokine receptors, CXCR1 and CXCR2, couple to Gαi to induce leukocyte recruitment and activation at sites of inflammation. Upon activation by CXCL8, these receptors become phosphorylated, desensitized, and internalized. In this study, we investigated the role of different G protein-coupled receptor kinases (GRKs) in CXCR1- and CXCR2-mediated cellular functions. To that end, short hairpin RNA was used to inhibit GRK2, 3, 5, and 6 in RBL-2H3 cells stably expressing CXCR1 or CXCR2, and CXCL8-mediated receptor activation and regulation were assessed. Inhibition of GRK2 and GRK6 increased CXCR1 and CXCR2 resistance to phosphorylation, desensitization, and internalization, respectively, and enhanced CXCL8-induced phosphoinositide hydrolysis and exocytosis in vitro. GRK2 depletion diminished CXCR1-induced ERK1/2 phosphorylation but had no effect on CXCR2-induced ERK1/2 phosphorylation. GRK6 depletion had no significant effect on CXCR1 function. However, peritoneal neutrophils from mice deficient in GRK6 (GRK6(-/-)) displayed an increase in CXCR2-mediated G protein activation but in vitro exhibited a decrease in chemotaxis, receptor desensitization, and internalization relative to wild-type (GRK6(+/+)) cells. In contrast, neutrophil recruitment in vivo in GRK6(-/-) mice was increased in response to delivery of CXCL1 through the air pouch model. In a wound-closure assay, GRK6(-/-) mice showed enhanced myeloperoxidase activity, suggesting enhanced neutrophil recruitment, and faster wound closure compared with GRK6(+/+) animals. Taken together, the results indicate that CXCR1 and CXCR2 couple to distinct GRK isoforms to mediate and regulate inflammatory responses. CXCR1 predominantly couples to GRK2, whereas CXCR2 interacts with GRK6 to negatively regulate receptor sensitization and trafficking, thus affecting cell signaling and angiogenesis.

MicroRNA-124 as a novel treatment for persistent hyperalgesia.

BACKGROUND: Chronic pain is often associated with microglia activation in the spinal cord. We recently showed that microglial levels of the kinase G protein-coupled receptor kinase (GRK)2 are reduced in models of chronic pain. We also found that mice with a cell-specific reduction of around 50% in GRK2 level in microglia/macrophages (LysM-GRK2+/- mice) develop prolonged inflammatory hyperalgesia concomitantly with ongoing spinal microglia/macrophage activation. The microRNA miR-124 is thought to keep microglia/macrophages in brain and spinal cord in a quiescent state. In the present study, we investigated the contribution of miR-124 to regulation of hyperalgesia and microglia/macrophage activation in GRK2-deficient mice. In addition, we investigated the effect of miR-124 on chronic inflammatory and neuropathic pain in wild-type (WT) mice. METHODS: Hyperalgesia was induced by intraplantar IL-1ß in WT and LysM-GRK2+/- mice. We determined spinal cord microglia/macrophage miR-124 expression and levels of pro-inflammatory M1 and anti-inflammatory M2 activation markers. The effect of intrathecal miR-124 treatment on IL-1ß-induced hyperalgesia and spinal M1/M2 phenotype, and on carrageenan-induced and spared nerve injury-induced chronic hyperalgesia in WT mice was analyzed. RESULTS: Transition from acute to persistent hyperalgesia in LysM-GRK2+/- mice is associated with reduced spinal cord microglia miR-124 levels. In our LysM-GRK2+/- mice, there was a switch towards a pro-inflammatory M1 phenotype together with increased pro-inflammatory cytokine production. Intrathecal administration of miR-124 completely prevented the transition to persistent pain in response to IL-1ß in LysM-GRK2+/- mice. The miR-124 treatment also normalized expression of spinal M1/M2 markers of LysM-GRK2+/- mice. Moreover, intrathecal miR-124 treatment reversed the persistent hyperalgesia induced by carrageenan in WT mice and prevented development of mechanical allodynia in the spared nerve injury model of chronic neuropathic pain in WT mice. CONCLUSIONS: We present the first evidence that intrathecal miR-124 treatment can be used to prevent and treat persistent inflammatory and neuropathic pain. In addition, we show for the first time that persistent hyperalgesia in GRK2-deficient mice is associated with an increased ratio of M1/M2 type markers in spinal cord microglia/macrophages, which is restored by miR-124 treatment. We propose that intrathecal miR-124 treatment might be a powerful novel treatment for pathological chronic pain with persistent microglia activation.

Cardiac G-protein-coupled receptor kinase 2 ablation induces a novel Ca2+ handling phenotype resistant to adverse alterations and remodeling after myocardial infarction.

BACKGROUND: G-protein-coupled receptor kinase 2 (GRK2) is a primary regulator of ß-adrenergic signaling in the heart. G-protein-coupled receptor kinase 2 ablation impedes heart failure development, but elucidation of the cellular mechanisms has not been achieved, and such elucidation is the aim of this study. METHODS AND RESULTS: Myocyte contractility, Ca(2+) handling and excitation-contraction coupling were studied in isolated cardiomyocytes from wild-type and GRK2 knockout (GRK2KO) mice without (sham) or with myocardial infarction (MI). In cardiac myocytes isolated from unstressed wild-type and GRK2KO hearts, myocyte contractions and Ca(2+) transients were similar, but GRK2KO myocytes had lower sarcoplasmic reticulum (SR) Ca(2+) content because of increased sodium-Ca(2+) exchanger activity and inhibited SR Ca(2+) ATPase by local protein kinase A-mediated activation of phosphodiesterase 4 resulting in hypophosphorylated phospholamban. This Ca(2+) handling phenotype is explained by a higher fractional SR Ca(2+) release induced by increased L-type Ca(2+) channel currents. After ß-adrenergic stimulation, GRK2KO myocytes revealed significant increases in contractility and Ca(2+) transients, which were not mediated through cardiac L-type Ca(2+) channels but through an increased SR Ca(2+). Interestingly, post-MI GRK2KO mice showed better cardiac function than post-MI control mice, which is explained by an improved Ca(2+) handling phenotype. The SR Ca(2+) content was better maintained in post-MI GRK2KO myocytes than in post-MI control myocytes because of better-maintained L-type Ca(2+) channel current density and no increase in sodium-Ca(2+) exchanger in GRK2KO myocytes. An L-type Ca(2+) channel blocker, verapamil, reversed some beneficial effects of GRK2KO. CONCLUSIONS: These data argue for novel differential regulation of L-type Ca(2+) channel currents and SR load by GRK2. G-protein-coupled receptor kinase 2 ablation represents a novel beneficial Ca(2+) handling phenotype resisting adverse remodeling after MI.

Microglial GRK2: a novel regulator of transition from acute to chronic pain.

Pain is a hallmark of tissue damage and inflammation promoting tissue protection and thereby contributing to repair. Therefore, transient acute pain is an important feature of the adaptive response to damage. However, in a significant number of cases, pain persists for months to years after the problem that originally caused the pain has resolved. Such chronic pain is maladaptive as it no longer serves a protective aim. Chronic pain is debilitating, both physiologically and psychologically, and treatments to provide relief from chronic pain are often ineffective. The neurobiological mechanisms underlying the transition from adaptive acute pain to maladaptive chronic pain are only partially understood. In this review, we will summarize recent evidence that a kinase known as G protein-coupled receptor kinase (GRK2) is a key regulator of the transition from acute to chronic inflammatory pain. Our recent studies have shown that mice with a reduction in the cellular level of GRK2 develop chronic hyperalgesia in response to inflammatory mediators that induce only transient hyperalgesia in WT mice. This finding is clinically relevant because rodent models of chronic pain are associated with reduced cellular levels of GRK2. We propose that GRK2 is a newly discovered major player in the regulation of chronic pain. The pathways regulated by this kinase may open up new avenues for development of treatment strategies that target the cause, and not the symptoms of chronic pain.

Microglial/macrophage GRK2 determines duration of peripheral IL-1beta-induced hyperalgesia: contribution of spinal cord CX3CR1, p38 and IL-1 signaling.

Chronic pain associated with inflammation is a major clinical problem, but the underlying mechanisms are incompletely understood. Recently, we reported that GRK2(+/-) mice with a approximately 50% reduction of GRK2 develop prolonged hyperalgesia following a single intraplantar injection of the pro-inflammatory cytokine interleukin-1beta (IL-1beta). Here we show that spinal microglia/macrophage GRK2 is reduced during chronic inflammation-induced hyperalgesia. Next, we applied CRE-Lox technology to create mice with low GRK2 in microglia/macrophages/granulocytes (LysM-GRK2(f/+)), or sensory neurons or astrocytes. Only mice deficient in microglial/macrophage/granulocyte GRK2 display prolonged IL-1beta-induced hyperalgesia that lasts up to 8days. Two days after intraplantar IL-1beta, increased microglial/macrophage activity occurs in the lumbar but not thoracic spinal cord of GRK2-deficient mice. Intrathecal pre-treatment with minocycline, an inhibitor of microglia/macrophage activation, accelerates resolution of hyperalgesia independent of genotype and prevents transition to chronic hyperalgesia in GRK2(+/-) mice. Ongoing hyperalgesia in GRK2(+/-) mice is reversed by minocycline administration at days 1 and 2 after IL-1beta injection. Similarly, IL-1beta-induced hyperalgesia in LysM-GRK2(f/+) mice is attenuated by intrathecal administration of anti-CX3CR1 to abrogate fractalkine signaling, the p38 inhibitor SB239063 and the IL-1 antagonist IL-1ra. These data establish that chronic inflammatory hyperalgesia is associated with reduced GRK2 in microglia/macrophages and that low GRK2 in these cells is sufficient to markedly prolong hyperalgesia after a single intraplantar injection of IL-1beta. Ongoing hyperalgesia is maintained by spinal microglial/macrophage activity, fractalkine signaling, p38 activation and IL-1 signaling. We propose that chronic inflammation decreases spinal microglial/macrophage GRK2, which prevents silencing of microglia/macrophage activity and thereby contributes to prolonged hyperalgesia.