G-protein-coupled receptor kinase 2 safeguards epithelial phenotype in head and neck squamous cell carcinomas.

Head and neck squamous cell carcinoma (HNSCC) arises from the mucosal lining of the upper aerodigestive tract and display few treatment options in advanced stages. Despite increased knowledge of HNSCC molecular biology, the identification of new players involved in triggering HNSCC recurrence and metastatic disease is needed. We uncover that G-protein-coupled receptor kinase-2 (GRK2) expression is reduced in undifferentiated, high-grade human HNSCC tumors, whereas its silencing in model human HNSCC cells is sufficient to trigger epithelial-to-mesenchymal transition (EMT) phenotypic features, an EMT-like transcriptional program and enhanced lymph node colonization from orthotopic tongue tumors in mice. Conversely, enhancing GRK2 expression counteracts mesenchymal cells traits by mechanisms involving phosphorylation and decreased functionality of the key EMT inducer Snail1. Our results suggest that GRK2 safeguards the epithelial phenotype, whereas its downregulation contributes to the activation of EMT programs in HNSCC.

Adenoviral ßARKct Cardiac Gene Transfer Ameliorates Postresuscitation Myocardial Injury in a Porcine Model of Cardiac Arrest.

OBJECTIVE: The aim of the study was to determine whether the inhibition of the G-protein-coupled receptor kinase 2 by adenoviral ßARKct cardiac gene transfer can ameliorate postresuscitation myocardial injury in pigs with cardiac arrest (CA) and explore the mechanism of myocardial protection. METHODS: Male landrace domestic pigs were randomized into the sham group (anesthetized and instrumented, but ventricular fibrillation was not induced) (n = 4), control group (ventricular fibrillation 8 min, n = 8), and ßARKct group (ventricular fibrillation 8 min, n = 8). Hemodynamic parameters were monitored continuously. Blood samples were collected at baseline, 30 min, 2 h, 4 h, and 6 h after the return of spontaneous circulation (ROSC). Left ventricular ejection fraction was assessed by echocardiography at baseline and 6 h after ROSC. These animals were euthanized, and the cardiac tissue was removed for analysis at 6 h after ROSC. RESULTS: Compared with those in the sham group, left ventricular +dp/dtmax, -dp/dtmax, cardiac output (CO), and ejection fraction (EF) in the control group and the ßARKct group were significantly decreased at 6 h after the restoration of spontaneous circulation. However, the ßARKct treatment produced better left ventricular +dp/dtmax, -dp/dtmax, CO, and EF after ROSC. The ßARKct treatment also produced lower serum cardiac troponin I, CK-MB, and lactate after ROSC. Furthermore, the adenoviral ßARKct gene transfer significantly increased ß1 adrenergic receptors, SERCA2a, RyR2 levels, and decreased GRK2 levels compared to control. CONCLUSIONS: The inhibition of GRK2 by adenoviral ßARKct cardiac gene transfer can ameliorate postresuscitation myocardial injury through beneficial effects on restoring the sarcoplasmic reticulum Ca-handling proteins expression and upregulating the ß1-adrenergic receptor level after cardiac arrest.

Paroxetine alleviates T lymphocyte activation and infiltration to joints of collagen-induced arthritis.

T cell infiltration to synovial tissue is an early pathogenic mechanism of rheumatoid arthritis (RA). In the present work, we reveal that G protein coupled receptor kinase 2 (GRK2) is abundantly expressed in T cells of collagen-induced arthritis (CIA). A GRK2 inhibitor, paroxetine protects the joints from inflammation and destruction, primarily through inhibition of both CD4+ helper T (Th) cell and CD8+ cytotoxic T (Tc) cell migration to synovial tissue. Meanwhile, paroxetine restores the balance of Th/Tc, effector Th (Theff)/ naïve Th (Thnaive) and effector Tc (Tceff)/ naïve Tc (Tcnaive) to equilibrium by elevating the frequency of Thnaive, Tcnaive and regulatory Th cells; reducing the increased Theff, activated Th and Tceff, having a similar effect as methotrexate (MTX). In addition, both serum and synovial IL-1ß, TNF-α and CX3CL1 expression was effectively inhibited in treated rats. In vitro assay confirmed that paroxetine inhibits CX3CL1-induced T cell migration through blocking the activity of GRK2. Among three MAPK families, paroxetine was found to be able to decrease the phosphorylation of ERK. This study elucidates that paroxetine attenuates the symptoms of CIA rats due to its inhibitory effect on T cell activation and infiltration to synovial tissue via suppression of ERK pathway.

The influence of TNF-α and Ang II on the proliferation, migration and invasion of HepG2 cells by regulating the expression of GRK2.

PURPOSE: Hepatocellular carcinoma (HCC) is a common digestive system malignancy that is associated with a poor prognosis. This study researched the interaction of tumor necrosis factor-α (TNF-α) and angiotensin II (Ang II) in HCC cells proliferation, migration and invasion and examined their influence on the expression of G protein-coupled receptor kinase 2 (GRK2) and relevant receptors. METHODS: Cell Counting Kit-8 and Transwell assays were performed to evaluate the effects of TNF-α and Ang II on HepG2 cells proliferation, migration and invasion. Flow cytometry was used to investigate the expression of tumor necrosis factor receptor 1 (TNFR1), angiotensin II type 1 (AT1R) and type 2 receptors (AT2R) on the surface of HepG2 cells. Additionally, Western blot was performed to assess the modulation of GRK2 expression by TNF-α and Ang II in HepG2 cells. Meanwhile, GRK2 siRNA-transfected HepG2 cells were used to confirm the effects of GRK2, TNF-α and Ang II on the proliferation, migration and invasion of GRK2-knockdown HCC cells. Finally, the expression of TNF-α, Ang II, TNFR1, AT1R, AT2R and GRK2 proteins in HCC, tumor-adjacent and normal liver tissues were tested by immunohistochemistry. RESULTS: The data demonstrated that TNF-α and Ang II can enhance the proliferation, migration and invasion of HepG2 cells through suppressing GRK2 expression but that the two reagents combined did not have synergistic effects. Moreover,overexpression of TNFR1 and AT1R perhaps promoted the formation and progression of HCC, while high AT2R expression had the opposite effect. CONCLUSIONS: This study provides new ideas for the prevention and treatment of HCC by researching the interaction and probable mechanism of different bioactive factors associated with HCC.

A Single Intrathecal or Intraperitoneal Injection of CB2 Receptor Agonist Attenuates Bone Cancer Pain and Induces a Time-Dependent Modification of GRK2.

The objective of this study was to explore the potential role of G-protein-coupled receptor kinase 2 (GRK2) in the progression of cannabinoid 2 receptor (CB2) agonist-induced analgesic effects of bone cancer pain. Female Sprague-Dawley rats, weighing 160-180 g, were utilized to establish a model of bone cancer pain induced by intra-tibia inoculation of Walker 256 mammary gland carcinoma cells. JWH-015, a selective CB2 agonist, was injected intrathecally or intraperitoneally on postoperative day 10. Bone cancer-induced pain behaviors-mechanical allodynia and ambulatory pain-were assessed on postoperative days -1 (baseline), 4, 7, and 10 and at post-treatment hours 2, 6, 24, 48, and 72. The expressions of spinal CB2 and GRK2 protein were detected by Western Blotting on postoperative days -1 (baseline), 4, 7, and 10 and at post-treatment hours 6, 24, and 72. The procedure produced prolonged mechanical allodynia, ambulatory pain, and different changes in spinal CB2 and GRK2 expression levels. Intrathecal or intraperitoneal administration of JWH-015 alleviated the induced mechanical allodynia and ambulatory pain, and inhibited the downregulation of spinal GRK2 expression. These effects were in a time-dependent manner and reversed by pretreatment of CB2 selective antagonist AM630. The results affirmed CB2 receptor agonists might serve as new treatment targets for bone cancer pain. Moreover, spinal GRK2 was an important regulator of CB2 receptor agonist-analgesia pathway.

Acute aerobic exercise mediates G protein-coupled receptor kinase 2 expression in human PBMCs.

AIMS: G protein-coupled receptor kinase 2 (GRK2), a cytosolic enzyme desensitizing G protein-couple receptors (e.g., ß-adrenergic receptors [ß-ARs]), is involved in regulation of hypertension, congestive heart failure, and inflammatory response. Since cellular GRK2 levels change quickly in response to exogenous/endogenous stimuli, this study examined whether GRK2 levels in human peripheral blood mononuclear cells (PBMCs) would increase during acute aerobic exercise and be associated with plasma IL-6 and cardiorespiratory fitness levels. MAIN METHODS: Eighteen subjects (8 men and 10 women), ages 18 to 30 years, were recruited to perform a 30-minute bout of acute aerobic exercise at 75% VO2max. KEY FINDINGS: Our results demonstrated that women exhibited significantly greater exercise-induced GRK2 expression in PBMCs compared to men. IL-6 modulation is independent of GRK2 expression. Furthermore, the percent change in GRK2 expression was negatively correlated with cardiorespiratory fitness levels (relative VO2max), but not plasma IL-6. SIGNIFICANCE: Acute aerobic exercise induces a greater GRK2 expression in women than men, while increased cardiorespiratory fitness is associated with exercise-induced GRK2 expression in PBMCs. Gender could be a contributor to regulate this GRK2 responsiveness to acute aerobic exercise.

MFG-E8 inhibits neutrophil migration through αvß3-integrin-dependent MAP kinase activation.

We have previously demonstrated the involvement of milk fat globule-epidermal growth factor-factor 8 (MFG­E8) in reducing neutrophil infiltration in a murine model of acute lung injury (ALI). In the present study, we aimed to delineate the mechanisms through which MFG­E8 attenuates neutrophil migration. Recombinant human MFG­E8 (rhMFG­E8) was expressed and purified in our facility. The human differentiated neutrophil cell line, dHL­60, was treated with rhMFG­E8 and cell migration assay was performed in a Boyden chamber using recombinant interleukin­8 (IL­8) as the chemoattractant. Surface CXCR2 and intracellular G protein­coupled receptor kinase 2 (GRK2) levels were evaluated by flow cytometry or western blot analysis. The levels of mitogen­activated protein (MAP) kinases were determined by western blot analysis. Treatment with rhMFG­E8 resulted in a significant inhibition of dHL­60 cell migration in a dose­dependent manner. There was a 46% decrease in CXCR2 expression in the rhMFG­E8­treated dHL­60 cells, which was associated with a 32% increase in GRK2 expression. In the dHL­60 cells, treatment with rhMFG­E8 promoted the phosphorylation of p38 and extracellular signal-regulated kinase (ERK) within 10­30 min. The use of SB203580, a p38 inhibitor, and PD98059, an ERK inhibitor, resulted in the restoration of dHL­60 cell migration which was significantly inhibited treatment with rhMFG­E8. Furthermore, blocking the MFG­E8 receptors, αvß3/αvß5­integrins, by anti­αv­integrin neutralizing antibody (Ab) inhibited the activation of p38 and ERK, and reversed the rhMFG­E8­induced inhibition of dHL­60 cell migration. Finally, treatment of the dHL­60 cells with SB203580 and PD98059 neutralized the rhMFG­E8­induced downregulation of CXCR2 expression and upregulation of GRK2 expression, as well as the inhibitory effects on cell migration. Our findings reveal a novel mechanism of action of MFG­E8 through which it inhibits neutrophil migration through αvß3-integrin-dependent MAP kinase activation.

A broadly conserved g-protein-coupled receptor kinase phosphorylation mechanism controls Drosophila smoothened activity.

Hedgehog (Hh) signaling is essential for normal growth, patterning, and homeostasis of many tissues in diverse organisms, and is misregulated in a variety of diseases including cancer. Cytoplasmic Hedgehog signaling is activated by multisite phosphorylation of the seven-pass transmembrane protein Smoothened (Smo) in its cytoplasmic C-terminus. Aside from a short membrane-proximal stretch, the sequence of the C-terminus is highly divergent in different phyla, and the evidence suggests that the precise mechanism of Smo activation and transduction of the signal to downstream effectors also differs. To clarify the conserved role of G-protein-coupled receptor kinases (GRKs) in Smo regulation, we mapped four clusters of phosphorylation sites in the membrane-proximal C-terminus of Drosophila Smo that are phosphorylated by Gprk2, one of the two fly GRKs. Phosphorylation at these sites enhances Smo dimerization and increases but is not essential for Smo activity. Three of these clusters overlap with regulatory phosphorylation sites in mouse Smo and are highly conserved throughout the bilaterian lineages, suggesting that they serve a common function. Consistent with this, we find that a C-terminally truncated form of Drosophila Smo consisting of just the highly conserved core, including Gprk2 regulatory sites, can recruit the downstream effector Costal-2 and activate target gene expression, in a Gprk2-dependent manner. These results indicate that GRK phosphorylation in the membrane proximal C-terminus is an evolutionarily ancient mechanism of Smo regulation, and point to a higher degree of similarity in the regulation and signaling mechanisms of bilaterian Smo proteins than has previously been recognized.

Balancing GRK2 and EPAC1 levels prevents and relieves chronic pain.

Chronic pain is a major clinical problem, yet the mechanisms underlying the transition from acute to chronic pain remain poorly understood. In mice, reduced expression of GPCR kinase 2 (GRK2) in nociceptors promotes cAMP signaling to the guanine nucleotide exchange factor EPAC1 and prolongs the PGE2-induced increase in pain sensitivity (hyperalgesia). Here we hypothesized that reduction of GRK2 or increased EPAC1 in dorsal root ganglion (DRG) neurons would promote the transition to chronic pain. We used 2 mouse models of hyperalgesic priming in which the transition from acute to chronic PGE2-induced hyperalgesia occurs. Hyperalgesic priming with carrageenan induced a sustained decrease in nociceptor GRK2, whereas priming with the PKCε agonist ΨεRACK increased DRG EPAC1. When either GRK2 was increased in vivo by viral-based gene transfer or EPAC1 was decreased in vivo, as was the case for mice heterozygous for Epac1 or mice treated with Epac1 antisense oligodeoxynucleotides, chronic PGE2-induced hyperalgesia development was prevented in the 2 priming models. Using the CFA model of chronic inflammatory pain, we found that increasing GRK2 or decreasing EPAC1 inhibited chronic hyperalgesia. Our data suggest that therapies targeted at balancing nociceptor GRK2 and EPAC1 levels have promise for the prevention and treatment of chronic pain.

MCARD-mediated gene transfer of GRK2 inhibitor in ovine model of acute myocardial infarction.

ß-Adrenergic receptor (ßAR) dysfunction in acute myocardial infarction (MI) is associated with elevated levels of the G-protein-coupled receptor kinase-2 (GRK2), which plays a key role in heart failure progression. Inhibition of GRK2 via expression of a peptide ßARKct transferred by molecular cardiac surgery with recirculating delivery (MCARD) may be a promising intervention. Five sheep underwent scAAV6-mediated MCARD delivery of ßARKct, and five received no treatment (control). After a 3-week period, the branch of the circumflex artery (OM1) was ligated. Quantitative PCR data showed intense ßARKct expression in the left ventricle (LV). Circumferential fractional shortening was 23.4 ± 7.1 % (baseline) vs. -2.9 ± 5.2 % (p < 0.05) in the control at 10 weeks. In the MCARD-ßARKct group, this parameter was close to baseline. The same trend was observed with LV wall thickening. Cardiac index fully recovered in the MCARD-ßARKct group. LV end-diastolic volume and LV end-diastolic pressure did not differ in both groups. MCARD-mediated ßARKct gene expression results in preservation of regional and global systolic function after acute MI without arresting progressive ventricular remodeling.

Age-related ß-adrenergic receptor-mediated vasorelaxation is changed by altering G protein receptor kinase 2 expression.

Beta-adrenergic receptor- (ß-AR) mediated vasorelaxation declines with age. This change is likely related to receptor desensitization, rather than down regulation. One kinase responsible for desensitization is G protein receptor kinase 2 (GRK2). We have shown that GRK expression and activity increases with age in Fischer 344 rat aorta. In this study we validated that carotid arteries have similar age-related changes in the ß-AR signaling axis as aorta. This finding allowed use of in vivo infection and delivery of two adenovirus vectors to carotid arteries of 2-month-old (2M) and 12-month-old (12M) male Fischer 344 rats. Adeno-GRK2 was used to overexpress GRK2, and adeno-ß-ARK-ct was used to inhibit GRK2 function. Following a five-day infection, vessels were collected and ex vivo tissue bath was used to evaluate vasoreactivity. We used KCl contracted segments, and determined that overexpression of GRK2 significantly impaired isoproterenol (ISO)-mediated vasorelaxation in both age groups. Maximum relaxation (MAX) to ISO in vessels from 2M decreased from 44% to 21%. MAX to ISO in vessels from 12M decreased from 12% to 6%. Sensitivity (ED50) in vessels from 2M and 12M was also impaired 57%, and 30% respectively. We also determined that expression of adeno-ß-ARK-ct significantly improved ISO-mediated vasorelaxation in both age groups. MAX in vessels from 2M increased from 44% to 58%. MAX in vessels from 12M increased from 15% to 69%. ED50 in vessels from 2M and 12M was also improved 46%, and 50% respectively. These findings further implicate age-related increases in GRK2 expression as an important regulator of the age-related decline in ß-AR-mediated vasorelaxation.

Chronic sympathetic activation promotes downregulation of ß-adrenoceptor-mediated effects in the guinea pig heart independently of structural remodeling and systolic dysfunction.

It is uncertain if downregulation of ß-adrenoceptor signaling pathway is promoted by an enhanced adrenergic tone at an early stage of cardiac disease, or it develops secondary to detrimental local myocardial changes in advanced heart failure. We examined the integrity of ß-adrenoceptor signaling pathway upon chronic infusion of isoproterenol, a ß-adrenoceptor agonist, at a dose producing no structural left ventricular (LV) remodeling and systolic dysfunction. Subcutaneous isoproterenol infusion (400 µg kg(-1) h(-1) over 16 days) to guinea pigs using osmotic minipumps produced no change in cardiac weights, LV internal dimensions, myocyte cross-sectional area, extent of interstitial fibrosis, and basal contractile function. Isolated, perfused heart preparations from isoproterenol-treated guinea pigs exhibited attenuated responsiveness to acute ß-adrenoceptor stimulation, as evidenced by reduced LV developed pressure increase, less shortening of LV epicardial monophasic action potential and effective refractory period, and less myocardial cyclic adenosine monophosphate elevation, in response to isoproterenol exposure, when compared to saline-treated controls. Pharmacological responses to forskolin, an activator of the adenylate cyclase catalytic subunit, were well preserved in isoproterenol-treated hearts. Downregulation of ß-adrenoceptor-mediated effects upon chronic isoproterenol infusion was associated with markedly reduced stimulatory G-protein α-subunit (G(sα)) myocardial expression levels. No change in expression levels of ß-adrenoceptors, G-protein-coupled receptor kinase 2, inhibitory G-protein α-subunit (G(iα2)), and Ca(v)1.2 and K(v)7.1 ion channels was determined in isoproterenol-treated hearts. We therefore conclude that sustained adrenergic overstimulation may promote downregulation of myocardial ß-adrenoceptor-mediated effects independently of structural LV remodeling and systolic failure, an effect attributed to ß-adrenoceptor uncoupling from adenylate cyclase due to reduced G(sα)-protein expression.

ß2-Adrenergics in hypoxia desensitize receptors but blunt inhibition of reabsorption in rat lungs.

Alveolar edema and decreased inspired Po(2) decrease the oxygen supply to alveolar epithelia, impairing ß(2)-adrenergic receptor (ß2AR) signaling and alveolar reabsorption. ß2AR agonists potently stimulate alveolar reabsorption. Thus, hypoxia impairs a major defense mechanism that provides protection from alveolar edema. Because in vivo data on the combined effects of prolonged hypoxia and ß2AR agonist treatment on ß2AR signaling are sparse, we tested whether in vivo hypoxia augments the inactivation of ß2AR during prolonged stimulation. Rats were exposed to normoxia (N) and hypoxia (8% O(2); H), and were also treated with terbutaline (T; 2.5 mg/kg, intraperitoneal, twice daily) or saline (S) for 4 days. ß2AR signaling was studied in alveolar epithelial (ATII) cells and in whole-lung tissue from treated rats. The terbutaline-stimulated formation of cyclic adenosine monophosphate was decreased by approximately 40% in whole lung and in ATII cells of NT, HS, and HT. The effects were not additive. The ß2AR number was increased in HS, but decreased in NT and HT. Treatment increased the G-protein-coupled receptor kinase 2 protein in the plasma membranes of ATII cells, but did not affect G proteins. In vivo hypoxia significantly decreased total and amiloride-sensitive alveolar fluid reabsorption, which was prevented by acute alveolar treatment and 4 days of systemic terbutaline treatment. The αENaC (subunit of epithelial Na channels) protein in plasma membranes was increased in HT, without effects on mRNA. These results indicate that prolonged alveolar hypoxia and treatment with terbutaline impaired ß2AR signaling in alveolar epithelia and in whole lungs, and this signaling was not further impaired by hypoxia. Despite impaired ß2AR signaling, treatment with terbutaline for 4 days prevented the inhibition of alveolar reabsorption caused by in vivo hypoxia.