Inhibition Of G Protein-coupled Receptor Kinase 2 Research Articles

G protein-coupled receptor kinase 2 as a novel therapeutic target for gland fibrosis of Sjögren's syndrome.

Sjogren's syndrome (SS) is a chronic, progressive autoimmune disorder characterized by gland fibrosis. We previously found a close correlation between gland fibrosis and the expression of G protein-coupled receptor kinase 2 (GRK2). In this study we explored the pathological and therapeutic significance of GRK2 in SS. Submandibular gland (SMG) antigen-induced SS mouse model was established in WT and GRK2+/- mice. We showed that the expression levels of GRK2 were significantly up-regulated in glandular tissue and positively correlated with fibrotic morphology in SS patients and mice. Hemizygous knockout of GRK2 significantly inhibited the gland fibrosis. In mouse salivary gland epithelial cells (SGECs), we demonstrated that GRK2 interacted with Smad2/3 to positively regulate the activation of TGF-β-Smad signaling with a TGF-β-GRK2 positive feedback loop contributing to gland fibrosis. Hemizygous knockout of GRK2 attenuated TGF-β-induced collagen I production in SGECs in vitro and hindered gland fibrosis in murine SS though preventing Smad2/3 nuclear translocation. Around 28 days post immunization with SMG antigen, WT SS mice were treated with a specific GRK2 inhibitor paroxetine (Par, 5 mg·kg-1·d-1, i.g. for 19 days). We found that Par administration significantly attenuated gland fibrosis and alleviated the progression of SS in mice. We conclude that genetic knockdown or pharmacological inhibition of GRK2 significantly attenuates gland fibrosis and alleviates the progression of SS. GRK2 binds to Smad2/3 and positively regulates the activation of TGF-β-Smad signaling. A TGF-β-GRK2 positive feedback loop contributes to gland fibrosis. Our research points out that GRK2 could be a promising therapeutic target for treating SS.

GRK2 is critical for the cleavage of the porcine embryo by regulating HSP90 and the AKT pathway.

Among the family of GPCR kinases (GRKs) that regulate receptor phosphorylation and signaling termination, G-protein coupled receptor kinase 2 (GRK2) binds to HSP90 in response to hypoxia or other stresses. In the present study, we investigated the effects of GRK2 knockdown and inhibition on porcine embryonic development from the zygote stage. Immunofluorescence and western blotting were used to determine the localization and expression, respectively, of GRK2 and related proteins. First, GRK2 and p-GRK2 were expressed in both the cytoplasm and membrane and co-localized with HSP90 on the membrane. The mRNA level of GRK2 increased until the 8C- morula stage, suggesting that GRK2 may play an essential role during the early development of the porcine embryos. GRK2 knockdown reduced porcine embryo development capacity and led to significantly decreased blastocyst quality. In addition, inhibition of GRK2 also induced poor ability of embryo development at early stage, indicating that GRK2 is critical for embryonic cleavage in pigs. Knockdown and inhibition of GRK2 reduced HSP90 expression and AKT activation and cAMP levels. Additionally, GRK2 deficiency increased LC3 expression, suggesting enhanced autophagy during embryo development. In summary, we showed that GRK2 binds to HSP90 on the membrane to regulate embryonic cleavage and AKT activation during embryonic development in pigs.

Paroxetine alleviates dendritic cell and T lymphocyte activation via GRK2-mediated PI3K-AKT signaling in rheumatoid arthritis.

G protein-coupled receptor kinase 2 (GRK2) could participate in the regulation of diverse cells via interacting with non-G-protein-coupled receptors. In the present work, we explored how paroxetine, a GRK2 inhibitor, modulates the differentiation and activation of immune cells in rheumatoid arthritis (RA). The blood samples of healthy individuals and RA patients were collected between July 2021 and March 2022 from the First Affiliated Hospital of Anhui Medical University. C57BL/6 mice were used to induce the collagen-induced arthritis (CIA) model. Flow cytometry analysis was used to characterize the differentiation and function of dendritic cells (DCs)/T cells. Co-immunoprecipitation was used to explore the specific molecular mechanism. In patients with RA, high expression of GRK2 in peripheral blood lymphocytes, accompanied by the increases of phosphatidylinositol 3 kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR). In animal model, a decrease in regulatory T cells (Tregs), an increase in the cluster of differentiation 8 positive (CD8+) T cells, and maturation of DCs were observed. Paroxetine, when used in vitro and in CIA mice, restrained the maturation of DCs and the differentiation of CD8+ T cells, and induced the proportion of Tregs. Paroxetine inhibited the secretion of pro-inflammatory cytokines, the expression of C-C motif chemokine receptor 7 in DCs and T cells. Simultaneously, paroxetine upregulated the expression of programmed death ligand 1, and anti-inflammatory cytokines. Additionally, paroxetine inhibited the PI3K-AKT-mTOR metabolic pathway in both DCs and T cells. This was associated with a reduction in mitochondrial membrane potential and changes in the utilization of glucose and lipids, particularly in DCs. Paroxetine reversed PI3K-AKT pathway activation induced by 740 Y-P (a PI3K agonist) through inhibiting the interaction between GRK2 and PI3K in DCs and T cells. Paroxetine exerts an immunosuppressive effect by targeting GRK2, which subsequently inhibits the metabolism-related PI3K-AKT-mTOR pathway of DCs and T cell in RA.

The selective serotonin reuptake inhibitor paroxetine improves right ventricular systolic function in experimental pulmonary hypertension

BackgroundPulmonary hypertension (PH) often leads to right ventricle (RV) failure, a significant cause of morbidity and mortality. Despite advancements in PH management, progression to RV maladaptation and subsequent failure remain a clinical challenge. This study explored the effect of paroxetine, a selective serotonin reuptake inhibitor (SSRI), on RV function in a rat model of PH, hypothesizing that it improves RV function by inhibiting G protein-coupled receptor kinase 2 (GRK2) and altering myofilament protein phosphorylation. MethodsThe Su5416/hypoxia (SuHx) rat model was used to induce PH. Rats were treated with paroxetine and compared to vehicle-treated and control groups. Parameters measured included RV morphology, systolic and diastolic function, myofilament protein phosphorylation, GRK2 activity, and sympathetic nervous system (SNS) markers. ResultsParoxetine treatment significantly improved RV systolic function, evidenced by increased stroke volume, cardiac output, and ejection fraction, without significantly affecting RV hypertrophy, myosin heavy chain/titin isoform switching, or fibrosis. Enhanced phosphorylation of titin and myosin light chain-2 was observed, correlating positively with improved systolic function. Contrary to the hypothesis, improvements occurred independently of GRK2 inhibition or SNS modulation, suggesting an alternate mechanism, potentially involving antioxidant properties of paroxetine. ConclusionParoxetine improves RV systolic function in PH rats, likely through mechanisms beyond GRK2 inhibition, possibly related to its antioxidant effects. This highlights the potential of paroxetine in managing RV dysfunction in PH, warranting further investigation into its detailed mechanisms of action and clinical applicability.

GRK2 mediated degradation of SAV1 initiates hyperplasia of fibroblast-like synoviocytes in rheumatoid arthritis

Hyperplasia and migration of fibroblast-like synoviocytes (FLSs) are the key drivers in the pathogenesis of rheumatoid arthritis (RA) and joint destruction. Abundant Yes-associated protein (YAP), which is a powerful transcription co-activator for proliferative genes, was observed in the nucleus of inflammatory FLSs with unknown upstream mechanisms. Using Gene Expression Omnibus database analysis, it was found that Salvador homolog-1 (SAV1), the pivotal negative regulator of the Hippo-YAP pathway, was slightly downregulated in RA synovium. However, SAV1 protein expression is extremely reduced. Subsequently, it was revealed that SAV1 is phosphorylated, ubiquitinated, and degraded by interacting with an important serine-threonine kinase, G protein-coupled receptor (GPCR) kinase 2 (GRK2), which was predominately upregulated by GPCR activation induced by ligands such as prostaglandin E2 (PGE2) in RA. This process further contributes to the decreased phosphorylation, nuclear translocation, and transcriptional potency of YAP, and leads to aberrant FLSs proliferation. Genetic depletion of GRK2 or inhibition of GRK2 by paroxetine rescued SAV1 expression and restored YAP phosphorylation and finally inhibited RA FLSs proliferation and migration. Similarly, paroxetine treatment effectively reduced the abnormal proliferation of FLSs in a rat model of collagen-induced arthritis which was accompanied by a significant improvement in clinical manifestations. Collectively, these results elucidate the significance of GRK2 regulation of Hippo-YAP signaling in FLSs proliferation and migration and the potential application of GRK2 inhibition in the treatment of FLSs-driven joint destruction in RA.

Cardioprotective Effects of the GRK2 Inhibitor Paroxetine on Isoproterenol-Induced Cardiac Remodeling by Modulating NF-κB Mediated Prohypertrophic and Profibrotic Gene Expression.

Pathological cardiac remodeling is associated with cardiovascular disease and can lead to heart failure. Nuclear factor-kappa B (NF-κB) is upregulated in the hypertrophic heart. Moreover, the expression of the G-protein-coupled receptor kinase 2 (GRK2) is increased and linked to the progression of heart failure. The inhibitory effects of paroxetine on GRK2 have been established. However, its protective effect on IκBα/NFκB signaling has not been elucidated. This study investigated the cardioprotective effect of paroxetine in an animal model of cardiac hypertrophy (CH), focusing on its effect on GRK2-mediated NF-κB-regulated expression of prohypertrophic and profibrotic genes. Wistar albino rats were administered normal saline, paroxetine, or fluoxetine, followed by isoproterenol to induce CH. The cardioprotective effects of the treatments were determined by assessing cardiac injury, inflammatory biomarker levels, histopathological changes, and hypertrophic and fibrotic genes in cardiomyocytes. Paroxetine pre-treatment significantly decreased the HW/BW ratio (p < 0.001), and the expression of prohypertrophic and profibrotic genes Troponin-I (p < 0.001), BNP (p < 0.01), ANP (p < 0.001), hydroxyproline (p < 0.05), TGF-β1 (p < 0.05), and αSMA (p < 0.01) as well as inflammatory markers. It also markedly decreased pIκBα, NFκB(p105) subunit expression (p < 0.05) and phosphorylation. The findings suggest that paroxetine prevents pathological cardiac remodeling by inhibiting the GRK2-mediated IκBα/NF-κB signaling pathway.

Paroxetine protects against bleomycin-induced pulmonary fibrosis by blocking GRK2/Smad3 pathway.

G protein-coupled receptor kinase-2 (GRK2) is involved in TGF-β1-induced activation of lung fibroblasts, which could give rise to the pathogenesis of pulmonary fibrosis. Paroxetine (PRXT) serves as a selective GRK2 inhibitor which is widely used to treat anxiety and depression for several decades. However, whether PRXT could inhibit TGF-β1-induced activation of lung fibroblasts and combat bleomycin-induced pulmonary fibrosis remains unclear. Here, we investigated the effects of PRXT on pulmonary fibrosis in C57/BL6 caused by bleomycin as well as on the activation of murine primary lung fibroblasts stimulated with TGF-β1. The results demonstrated that PRXT markedly improved the pulmonary function and 21-day survival in bleomycin-induced mice. Meanwhile, PRXT significantly decreased collagen deposition, inflammation, and oxidative stress in lung tissues from bleomycin-induced mice. Furthermore, we found that PRXT could inhibit the protein and mRNA expression of GRK2 and Smad3 in lung tissues from bleomycin-induced mice. In vitro experiments also PRXT could inhibit cell activation and collagen synthesis in a concentration-dependent manner in TGF-β1-induced lung fibroblasts. In addition, we found that Smad3 overexpression by adenovirus transfection could offset anti-fibrotic and antioxidative effects from PRXT in TGF-β1-induced lung fibroblasts, which showed no effects on the protein expression of GRK2. In conclusion, PRXT mediates the inhibition of GRK2, which further blocks the transcription of Smad3 in TGF-β1-induced lung fibroblasts, providing an attractive therapeutic target for pulmonary fibrosis.

GRK2 Mediates Macrophage Polarization by Regulating EP4-cAMP-pCREB Signaling in Ulcerative Colitis and the Therapeutic Effect of Paroxetine on Mice with DSS-Induced Colitis.

G protein-coupled receptor kinase 2 (GRK2) is one of the cytosolic enzymes, and GRK2 translocation induces prostaglandin E2 receptor 4 (EP4) over-desensitization and reduces the level of cyclic adenosine monophosphate (cAMP) to regulate macrophage polarization. However, the role of GRK2 in the pathophysiology of ulcerative colitis (UC) remains unclear. In this study, we investigated the role of GRK2 in macrophage polarization in UC, using biopsies from patients, a GRK2 heterozygous mouse model with dextran sulfate sodium (DSS)-induced colitis, and THP-1 cells. The results showed that a high level of prostaglandin E2 (PGE2) stimulated the receptor EP4 and enhanced the transmembrane activity of GRK2 in colonic lamina propria mononuclear cells (LPMCs), resulting in a down-regulation of membrane EP4 expression. Then, the suppression of cAMP-cyclic AMP responsive element-binding (CREB) signal inhibited M2 polarization in UC. Paroxetine is acknowledged as one of the selective serotonin reuptake inhibitors (SSRI), which is also considered as a potent GRK2 inhibitor with a high selectivity for GRK2. We found that paroxetine could alleviate symptoms of DSS-induced colitis in mice by regulating GPCR signaling to affect macrophage polarization. Taken together, the current results show that GRK2 may act as a novel therapeutic target in UC by regulating macrophage polarization, and paroxetine as a GRK2 inhibitor may have therapeutic effect on mice with DSS-induced colitis.

GRK2 participation in cardiac hypertrophy induced by isoproterenol through the regulation of Nrf2 signaling and the promotion of NLRP3 inflammasome and oxidative stress.

In cases of heart failure, cardiac hypertrophy may be caused by the upregulation of G-protein-coupled receptor kinase 2 (GRK2). Both NLRP3 inflammasome and oxidative stress contribute to cardiovascular disease. In this study, we clarified the effect of GRK2 on cardiac hypertrophy in H9c2 cells induced by isoproterenol (ISO) and examined the underlying mechanisms. We randomly categorized H9c2 cells into five groups: an ISO group, a paroxetine plus ISO group, a GRK2 small-interfering RNA (siRNA) plus ISO group, a GRK2 siRNA combined with ML385 plus ISO group, and a control group. To determine the effect of GRK2 on cardiac hypertrophy induced by ISO, we carried out CCK8 assays, RT-PCR, TUNEL staining, ELISA assay, DCFH-DA staining, immunofluorescence staining, and western blotting. By using paroxetine or siRNA to inhibit GRK2, we significantly decreased cell viability; reduced the mRNA levels of ANP, BNP, and β-MHC; and limited the apoptosis rate and protein levels of cleaved caspase-3 and cytochrome c in H9c2 cells treated with ISO. We also found that oxidative stress induced by ISO could be mitigated with paroxetine or GRK2 siRNA. This result was validated by decreased activities of the antioxidant enzymes CAT, GPX, and SOD and increased MDA levels and ROS production. We observed that the protein expression of NLRP3, ASC, and caspase-1 and the intensity of NLRP3 could be inhibited by paroxetine or GRK2 siRNA. Both paroxetine and GRK2 siRNA were able to abolish the increase in GRK2 expression induced by ISO. They also could increase protein levels of HO-1, nuclear Nrf2, and Nrf2 immunofluorescence intensity; however, they could not change the protein level of cytoplasmic Nrf2. By combining treatment with ML385, we were able to reverse GRK2 inhibition on H9c2 cells treated with ISO. According to the results of this study, GRK2 participated in cardiac hypertrophy induced by ISO by mitigating NLRP3 inflammasome and oxidative stress through the signaling of Nrf2 in H9c2 cells.

Vinculin phosphorylation impairs vascular endothelial junctions promoting atherosclerosis.

Atherosclerosis preferentially develops in arterial branches and curvatures where vascular endothelium is exposed to disturbed flow. In this study, the effects of disturbed flow on the regulation of vascular endothelial phosphoproteins and their contribution to therapeutic application in atherogenesis were elucidated. Porcine models, large-scale phosphoproteomics, transgenic mice, and clinical specimens were used to discover novel site-specific phosphorylation alterations induced by disturbed flow in endothelial cells (ECs). A large-scale phosphoproteomics analysis of native endothelium from disturbed (athero-susceptible) vs. pulsatile flow (athero-resistant) regions of porcine aortas led to the identification of a novel atherosclerosis-related phosphoprotein vinculin (VCL) with disturbed flow-induced phosphorylation at serine 721 (VCLS721p). The induction of VCLS721p was mediated by G-protein-coupled receptor kinase 2 (GRK2)S29p and resulted in an inactive form of VCL with a closed conformation, leading to the VE-cadherin/catenin complex disruption to enhance endothelial permeability and atherogenesis. The generation of novel apolipoprotein E-deficient (ApoE-/-) mice overexpressing S721-non-phosphorylatable VCL mutant in ECs confirmed the critical role of VCLS721p in promoting atherosclerosis. The administration of a GRK2 inhibitor to ApoE-/- mice suppressed plaque formation by inhibiting endothelial VCLS721p. Studies on clinical specimens from patients with coronary artery disease (CAD) revealed that endothelial VCLS721p is a critical clinicopathological biomarker for atherosclerosis progression and that serum VCLS721p level is a promising biomarker for CAD diagnosis. The findings of this study indicate that endothelial VCLS721p is a valuable hemodynamic-based target for clinical assessment and treatment of vascular disorders resulting from atherosclerosis.

GRK2 inhibitors, paroxetine and CCG258747, attenuate IgE-mediated anaphylaxis but activate mast cells via MRGPRX2 and MRGPRB2.

G protein-coupled receptor (GPCR) kinase 2 (GRK2), which phosphorylates agonist-occupied GPCRs to promote their desensitization, has been investigated as an attractive therapeutic target for cardiovascular and metabolic diseases. Several GRK2-targeted inhibition strategies have been reported including the use of direct pharmacological inhibitors such as paroxetine (a widely prescribed antidepressant) and its analogs such as compound CCG258747. Cross-linking of high affinity IgE receptor (FcϵRI) on mast cells (MCs) and the resulting degranulation causes anaphylaxis and allergic asthma. Using gene silencing strategy, we recently showed that GRK2 contributes to FcεRI signaling and MC degranulation. The purpose of this study was to determine if the GRK2 inhibitors paroxetine and CCG258747 modulate FcεRI-mediated MC responses in vitro and in vivo. Utilizing rat basophilic leukemia (RBL-2H3) cells and primary mouse lung MCs (LMCs), we found that paroxetine and CCG258747 inhibit FcϵRI-mediated calcium mobilization and degranulation. Furthermore, intravenous administration of paroxetine and CCG258747 in mice resulted in substantial reduction of IgE-mediated passive cutaneous anaphylaxis. Unlike LMCs, human cutaneous MCs abundantly express a novel GPCR known as MRGPRX2 (mouse; MRGPRB2). We found that in contrast to their inhibitory effects on FcεRI-mediated MC responses, both paroxetine and CCG258747 induce calcium mobilization and degranulation in RBL-2H3 cells stably expressing MRGPRX2 but not in untransfected cells. Furthermore, paroxetine and CCG258747 induced degranulation in peritoneal MCs from Wild-type (WT) mice in vitro and caused increased cutaneous vascular permeability in vivo, but these responses were substantially reduced in Mrgprb2−/− mice. Additionally, upon intradermal injection, paroxetine also induced neutrophil recruitment in WT but not Mrgprb2−/− mice. These findings suggest that in addition to their potential therapeutic utility against cardiovascular and metabolic disorders, paroxetine-based GRK2-inhibitors may serve to modulate IgE-mediated anaphylaxis and to enhance cutaneous host defense by harnessing MC’s immunomodulatory property through the activation of MRGPRX2/MRGPRB2.

Early Gβγ-GRK2 Inhibition Ameliorates Osteoarthritis Development by Simultaneous Anti-Inflammatory and Chondroprotective Effects.

The G-protein-coupled receptor kinase 2 (GRK2) is an important regulator of inflammation and pathological macrophage phenotype in a variety of diseases. We hypothesize that Gβγ-GRK2 signaling promotes the early inflammatory response and chondrocyte loss in osteoarthritis (OA). Using the destabilization of the medial meniscus (DMM) model in 12-week-old male C57BL/6 mice, we determined the role of Gβγ-GRK2 signaling in synovitis, macrophage activation, and OA development. We achieved Gβγ-GRK2 inhibition at the time of DMM by administering the Gβγ inhibitor “gallein” and the GRK2 inhibitor “paroxetine” daily, starting from 2 days before DMM surgery, for a duration of 1 or 12 weeks. Synovial and cartilage structural changes were evaluated by histomorphometry, and molecular events and macrophage activation were examined. We studied the direct role of Gβγ-GRK2 in synovitis and macrophage activation in vitro using SW982 and THP1 cells. Continuous Gβγ-GRK2 inhibition initiated at the time of DMM attenuated OA development and decreased chondrocyte loss more effectively than delayed treatment. GRK2 expression and the M1 macrophage phenotype were elevated in the inflamed synovium, while early gallein and paroxetine treatment for 1 and 12 weeks following DMM resulted in their reduction and an upregulated M2 macrophage phenotype. In vitro experiments showed that Gβγ-GRK2 inhibition attenuated synoviocyte inflammation and the M1 phenotype. We show that early Gβγ-GRK2 inhibition is of higher therapeutic efficacy in OA than delayed inhibition, as it prevents OA development by inhibiting the early inflammatory response.

Paroxetine increases delta opioid responsiveness in sensory neurons.

There are currently no Food and Drug Administration (FDA)-approved δ-opioid receptor (DOR)-selective agonists, despite having fewer side effects in rodents and nonhuman primates compared with traditional μ-opioid receptor (MOR) therapeutics (Vanderah, 2010). Targeting peripheral receptors is an attractive strategy to reduce abuse potential. However, peripheral opioid receptors do not readily respond to agonists unless primed by inflammation, which would limit their efficacy in noninflammatory pain patients (Stein et al., 1989). It was recently identified that G-protein-coupled receptor kinase 2 (GRK2) maintains DOR incompetence in noninflamed nociceptors (Brackley et al., 2016, 2017). Here, we report that paroxetine, a selective serotonin reuptake inhibitor (SSRI) and potent GRK2 inhibitor (Thal et al., 2012), reduces chronic GRK2 association with membrane DOR, thereby enhancing peripheral DOR-mediated analgesic competence in the absence of inflammation. Interestingly, paroxetine’s effects on GRK2 in vivo are limited to peripheral tissues in the male rat. The effects of paroxetine on DOR competence are notably antagonized by GRK2 overexpression. This is the first study to suggest that paroxetine induces peripheral DOR analgesic competence through a GRK2-dependent mechanism, improving analgesic efficacy in noninflamed tissue. Because paroxetine targets the protein that governs peripheral opioid receptor responsiveness, and does so in the absence of inflammation, we propose that paroxetine may be suitable as a co-therapy with peripherally-restrictive doses of opioids to improve analgesic efficacy in noninflammatory pain conditions.

Mitochondrial GRK2 is a Novel Regulator of Cardiac Energetics

G protein‐coupled receptor (GPCR) kinase 2 (GRK2) is highly expressed in the heart, where during injury or heart failure (HF), both its levels and activity increase. GRK2 is canonically studied in the context of GPCR phosphorylation; however, noncanonical activities of GRK2 have emerged and it is now appreciated that GRK2 has a large non‐GPCR interactome. For example, in cardiac myocytes, GRK2 translocates from the cytosol to mitochondria (mtGRK2) following oxidative stress or ischemia injury, and this pool of mtGRK2 is associated with negative effects on metabolism and also induces myocyte cell death. However, the mechanisms by which mtGRK2 contributes to cardiac dysfunction and HF are not fully understood. We hypothesized that mtGRK2 could have novel substrates and phosphorylate proteins involved in mitochondrial bioenergetics, thus contributing to our previously established post‐injury phenotype. Stress‐induced mitochondrial translocation of cytosolic GRK2 was validated in cell and animal models and the mtGRK2 interactome was identified using liquid chromatography‐mass spectroscopy (LCMS). Proteomics analysis identified mtGRK2 interacting proteins which were involved in mitochondrial dysfunction, bioenergetics, and OXPHOS, particularly complexes I, II, IV and V of the electron transport chain (ETC). Specifically, mtGRK2 interactions with Complex V (ATP synthase) subunits were particularly increased following stress. We established that mtGRK2 phosphorylates ATP synthase on the F1 catalytic barrel, which is critical for oxidative phosphorylation and ATP production. We have also determined that alterations in either the levels or activity of GRK2 appear to alter ATP synthase enzymatic activity in vitro. Excitingly, in vivo data suggest that reducing levels of GRK2 in a mouse model of myocardial infarction prevents the post‐injury reduction in ATP synthesis. We are currently assessing the ability of the SSRI drug paroxetine, a GRK2 inhibitor, to preserve mitochondrial bioenergetics in a transgenic GRK2 mouse model. Thus, phosphorylation of the ATP synthesis machinery by mtGRK2 may contribute to the impaired mitochondrial phenotype observed in injured or failing heartssuch as reduced fatty acid metabolism and substrate utilization. These data uncover a druggable, novel target for rescuing cardiac function in patients with injured and/or failing hearts.

Activation of APJ Receptors by CMF‐019, but not Apelin, Causes Endothelium‐Dependent Relaxation of Spontaneously Hypertensive Rat Coronary Arteries

Hypertension is a major risk factor for coronary artery disease, but the pathophysiological mechanisms linking these diseases are not completely understood. Apelin is a novel vasoactive adipokine. Receptors for apelin (APJ receptors) are G‐protein‐coupled receptors and are widely expressed throughout the cardiovascular system. APJ receptors can also signal via G‐protein‐independent pathways, including G‐protein‐coupled‐receptor kinase 2 (GRK2), which inhibits nitric oxide synthase (eNOS) activity and nitric oxide (NO) production in endothelial cells. Apelin causes endothelium‐dependent, NO‐mediated relaxation of coronary arteries from normotensive animals, but the effects of activating APJ receptor signaling pathways in hypertensive coronary arteries are unknown. Since endothelium‐dependent relaxations are often blunted in arteries from hypertensive animals, we tested the hypothesis that apelin‐induced relaxation is impaired in coronary arteries from spontaneously hypertensive rats (SHR). Moreover, we hypothesized that endothelium‐dependent relaxations in response to CMF‐019, an APJ receptor G‐protein biased agonist with little effect on GRK2, would be unaffected in SHR coronary arteries. Western blot and RT‐qPCR analysis demonstrated that APJ receptor protein and mRNA expression did not differ between normotensive WKY (Wistar‐Kyoto) and SHR coronary arteries. eNOS protein expression was also similar between SHR and WKY coronary arteries, but GRK2 expression was significantly increased in SHR coronary endothelial cells, as compared to WKY. In myograph studies, apelin (10‐9‐10‐6 M) caused endothelium‐dependent relaxation of isolated WKY coronary arterial rings (pD2= 7.00 ± 0.11; Emax= 54 ± 4% relaxation; n=6), but had no effect in arteries from SHR. By contrast, CMF‐019 (10‐9‐10‐6 M)‐induced relaxation did not differ between normotensive and hypertensive coronary arteries. The relaxation response to CMF‐019 was abolished by the APJ receptor antagonist, F13A (10‐7 M), by removal of the endothelium, and by inhibition of eNOS with nitro‐ L‐arginine (3 x 10‐5 M). Moreover, the GRK2 inhibitor, CMPD 101 (3 x 10‐5 M), partially restored the relaxation response to apelin in SHR coronary arteries (pD2= 6.56 ± 0.22; Emax= 46 ± 6% relaxation; n=6). Immunoblot analysis of SHR coronary endothelial cells demonstrated that CMF‐019 (10‐7 M) significantly increased eNOS phosphorylation whereas apelin (10‐7 M) had no effect. We conclude that apelin failed to cause relaxation in hypertensive coronary arteries, likely due to impaired eNOS activity resulting from APJ receptor signaling via the GRK2 pathway and decreased eNOS phosphorylation. APJ receptor activation by the biased agonist, CMF‐019, caused similar endothelium‐dependent relaxations in WKY and SHR coronary arteries. Thus, APJ receptor‐biased agonists, such as CMF‐019, may be more effective than apelin in causing vasodilation of hypertensive coronary arteries.

GRK2 promotes activation of lung fibroblast cells and contributes to pathogenesis of pulmonary fibrosis through increasing Smad3 expression.

Pulmonary fibrosis is a chronic, progressive, and irreversible interstitial lung disease. Transforming growth factor-β1 (TGF-β1) plays a major role in lung fibroblast cell differentiation to myofibroblast cells and production of extracellular matrix, which are hallmarks of pulmonary fibrosis. G protein-coupled receptor kinase-2 (GRK2) has been shown to play controversial roles in TGF-β1-induced signal transduction in different cell types; however, the role of GRK2 in TGF-β1-induced activation of lung fibroblast cells and development of pulmonary fibrosis has not been revealed. In this study, we found that GRK2 levels were increased in lungs and isolated fibroblast cells in a murine model of pulmonary fibrosis, as well as TGF-β1-treated lung fibroblasts. GRK2 levels were not changed in lungs in the injury phase of pulmonary fibrosis. Posttreatment with GRK2 inhibitor reduced extracellular matrix (ECM) accumulation in lungs in bleomycin-challenged mice, suggesting that GRK2 activation contributes to the progressive phase of pulmonary fibrosis. Inhibition or downregulation of GRK2 attenuates fibronectin, collagen, and α-smooth muscle actin expression in TGF-β1-induced lung fibroblast cells or myofibroblast cells isolated from patients with pulmonary fibrosis. Furthermore, we showed that GRK2 regulates Smad3 expression, indicating that inhibition of GRK2 attenuates ECM accumulation through downregulation of Smad3 expression. This study reveals that GRK2 is a therapeutic target in treating pulmonary fibrosis and inhibition of GRK2 dampens pulmonary fibrosis by suppression of Smad3 expression, eventually attenuating TGF-β1 signal pathway and ECM accumulation.

G protein-coupled receptor kinase-2 confers isoform-specific calcium sensitivity to dopamine D2 receptor desensitization.

The dopamine D2 receptor (D2 R) functions as an autoreceptor on dopaminergic cell bodies and terminals and as a postsynaptic receptor on a variety of neurons in the central nervous system. As a result of alternative splicing, the D2 R is expressed as two isoforms: long (D2L R) and short (D2S R) differing by a stretch of 29 residues in the third intracellular loop, with D2S R being the predominant presynaptic isoform. Recent reports described a Ca2+ sensitivity of the desensitization time course of potassium currents elicited via D2S R, but not via D2L R, when either isoform was selectively expressed in dopaminergic neurons. Here, we aimed to study the mechanism behind this subtype-specific Ca2+ sensitivity. Thus, we measured the desensitization of potassium channel responses evoked by D2L R and D2S R using two-electrode voltage clamp in Xenopus oocytes in the absence and presence of different amounts of β-arrestin2 and G protein-coupled receptor kinase-2 (GRK2), both of which are known to play important roles in D2 R desensitization in native cells. We found that co-expression of both GRK2 and β-arrestin2 was necessary for reconstitution of the Ca2+ sensitivity of D2S R desensitization, while D2L R did not display Ca2+ sensitivity under these conditions. The effect of Ca2+ chelation by BAPTA-AM to slow the rate of D2S R desensitization was mimicked by the GRK2 inhibitor, Cmpd101, and by the kinase-inactivating GRK2 mutation, K220R, but not by the PKC inhibitor, Gö6976, nor by the calmodulin antagonist, KN-93. Thus, Ca2+ -sensitive desensitization of D2S R appears to be mediated via a GRK2 phosphorylation-dependent mechanism.

IGF1-driven induction of GPCR kinase 2 in the primary afferent neuron promotes resolution of acute hyperalgesia

Dynamic regulation of G-protein-coupled receptor (GPCR) kinase 2 (GRK2) expression restores cellular function by protecting from overstimulation via GPCR and non-GPCR signaling. In the primary afferent neurons, GRK2 negatively regulates nociceptive tone. The present study tested the hypothesis that induction of GRK2 in the primary afferent neurons contributes to the resolution of acute pain after tissue injury.GRK2 expression in the dorsal root ganglion (DRG) was analyzed at 1 and 7 days after the incision. Intraperitoneal administration of a GRK2 inhibitor was performed 7 days post-incision in male Sprague-Dawley rats who underwent plantar incisions to analyze the pain-related behavioral effect of the GRK2 inhibitor. Separately, GRK2 expression was analyzed after injecting insulin-like growth factor 1 (IGF1) into the rat hind paw. In addition, an IGF1 receptor (IGF1R) inhibitor was administered in the plantar incision rats to determine its effect on the incision-induced hyperalgesia and GRK2 expression.Plantar incision induced an increase in GRK2 in the DRG at 7 days, but not at 1 day post-incision. Acute hyperalgesia after the plantar incision disappeared by 7 days post-incision. Intraperitoneal injection of the GRK2 inhibitor at this time reinstated mechanical hyperalgesia, although the GRK2 inhibitor did not produce hyperalgesia in naive rats. After the incision, IGF1 expression increased in the paw, but not in the DRG. Intraplantar injection of IGF1 increased GRK2 expression in the ipsilateral DRG. IGF1R inhibitor administration prevented both the induction of GRK2 and resolution of hyperalgesia after the plantar incision.These findings demonstrate that induction of GRK2 expression driven by tissue IGF1 has potent analgesic effects and produces resolution of hyperalgesia after tissue injury. Dysregulation of IGF1-GRK2 signaling could potentially lead to failure of the spontaneous resolution of acute pain and, hence, development of chronic pain after surgery.

G protein-coupled receptor kinase 2 is essential to enable vasoconstrictor-mediated arterial smooth muscle proliferation

Hypertension is associated with increased production and circulation of vasoconstrictors, resulting in enhanced signalling through their cognate G protein-coupled receptors (GPCR). Prolonged vasoconstrictor GPCR signalling increases arterial contraction and stimulates signalling pathways that promote vascular smooth muscle cell (VSMC) proliferation, contributing to the development of atherosclerotic plaques, re-stenosis lesions and vascular remodelling. GPCR signalling through phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) promotes VSMC proliferation. In VSMC, G protein-coupled receptor kinase 2 (GRK2) is known to regulate numerous vasoconstrictor GPCRs and their downstream signalling pathways. As GRK2 is implicated in controlling various aspects of cellular growth, we examined whether GRK2 could affect VSMC proliferation. Using two indices of cell growth, we show that PI3K inhibition and depletion of GRK2 expression produced a similar ablation of pro-proliferative vasoconstrictor-stimulated VSMC growth. Furthermore, GRK2-knockdown ablated the sustained phase of endothelin-1 and angiotensin-II-stimulated Akt phosphorylation, whilst the peak (5 min) phase was unaffected. Conversely, the GRK2 inhibitor compound 101 did not affect vasoconstrictor-driven Akt phosphorylation. Vasoconstrictor-stimulated phosphorylation of the Akt substrates GSK3α and GSK3β was ablated following RNAi-mediated GRK2 depletion, or after PI3K inhibition. Moreover, GRK2 knockdown prevented endothelin-1 and angiotensin-II from increasing cyclin D1 expression.These data suggest GRK2 expression is essential to facilitate vasoconstrictor-driven VSMC proliferation through its ability to promote efficient prolonged PI3K-Akt signalling, and thus relieve the GSK3-mediated block on cell cycling. Considering VSMC GRK2 expression increases early in the development of hypertension, this highlights the potential for GRK2 to promote VSMC growth and exacerbate hypertensive pathophysiological vascular remodelling.

Apelin‐Induced Relaxation is Impaired in Coronary Arteries from Spontaneously Hypertensive Rats

Hypertension is a major modifiable risk factor for coronary artery disease. The pathophysiological mechanisms underlying hypertension as a risk factor for coronary artery disease are not fully understood. Apelin is a vasoactive peptide that binds to APJ receptors, which are highly expressed throughout the cardiovascular system, including coronary arteries. APJ receptors signal via G-protein-dependent and -independent pathways, including activation of G-protein-coupled-receptor kinase 2 (GRK2), which can potentially lead to inhibition of eNOS and nitric oxide (NO) production. We have previously shown that apelin causes endothelium-dependent, NO-mediated relaxation of coronary arteries from normotensive animals (JPET 366:265, 2018), but its effects in hypertensive coronary arteries are unknown. Here, we tested the hypothesis that apelin-induced relaxation is impaired in coronary arteries from spontaneously hypertensive rats (SHR). Western blot analysis demonstrated increased expression of GRK2 in cultured coronary endothelial cells from SHR in comparison with normotensive WKY (control) rats. Isolated coronary arterial rings were mounted in wire myographs for isometric tension recording and contracted with 5-HT (10-7 M). Apelin (10-9-10-6 M) caused endothelium-dependent relaxation of WKY coronary arteries (pD2 = 7.00 ± 0.11; Emax = 54 ± 4% relaxation; n=6), but had no effect in rings from SHR. Conversely, endothelium-dependent relaxations to an APJ receptor biased agonist, CMF-019 (10-9-10-5 M), which preferentially activates the G-protein-dependent pathway with minimal effect on GRK2, did not differ between SHR and WKY. In the presence of apelin (10-7 M), endothelium-dependent relaxation to acetylcholine (ACh) (10-9-10-5 M) was impaired in SHR coronary arteries (pD2 = 6.82 ± 0.10 vs 6.52 ± 0.18, and Emax = 81 ± 8 vs 60 ± 10% relaxation, in the absence and presence of apelin, respectively; n=5; p<0.05); however, apelin had no effect on ACh-induced relaxation of WKY coronary arteries. By contrast, ACh-induced relaxation of SHR and WKY coronary arterial rings was unaffected by CMF-019 (10-7 M). Moreover, the GRK2 inhibitor, CMPD 101 (3 × 10-5 M), partially restored the relaxation response to apelin in SHR coronary arteries (pD2 = 6.24 ± 0.30; Emax = 42 ± 13% relaxation; n=3). Relaxations in response to the NO-donor, DEA NONOate (10-9-10-4 M), were not altered by apelin (10-7 M) treatment in either WKY or SHR coronary arteries. Taken together, the data indicate that apelin failed to cause relaxation in hypertensive coronary arteries, likely due to impaired production or release of NO from endothelial cells rather than interfering with the action of NO on coronary smooth muscle cells. In SHR coronary arteries, apelin instead actively inhibited relaxation to another endothelium-dependent vasodilator; i.e. ACh. That the effects of the G-protein biased agonist, CMF-019, did not differ between WKY and SHR coronary arteries is consistent with a role for GRK2 activation in the altered response to apelin in hypertensive arteries. APJ receptor signaling via the GRK2 pathway may contribute to both the loss of relaxation to apelin itself, as well as the ability of apelin to inhibit endothelium-dependent relaxation to ACh in coronary arteries from SHR.