G Protein-coupled Receptor Kinase 2 Expression Research Articles

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.

Potential Regulatory Roles of GRK2 in Endothelial Cell Activity and Pathological Angiogenesis.

G protein-coupled receptor (GPCR) kinase 2 (GRK2) is an integrative node in many signaling network cascades. Emerging evidence indicates that GRK2 can interact with a large number of GPCRs and non-GPCR substrates in both kinase-dependent and -independent modes. Some of these pathways are associated with endothelial cell (EC) activity. The active state of ECs is a pivotal factor in angiogenesis. The occurrence and development of some inflammation-related diseases are accompanied by pathological angiogenesis, but there remains a lack of effective targeted treatments. Alterations in the expression and/or localization of GRK2 have been identified in several types of diseases and have been demonstrated to regulate the angiogenesis process in these diseases. GRK2 as a target may be a promising candidate for anti-angiogenesis therapy.

The role of G‐protein‐coupled receptor kinase 2 in diabetic mechanical hyperalgesia in rats

Previous studies have indicated a negative correlation between GRK2 expression and pain development and transmission. Here, we investigated whether G-protein-coupled receptor kinase 2 (GRK2) was involved in regulating diabetic mechanical hyperalgesia (DMH). The adeno-associated viral vectors containing the GRK2 gene (AAV-GRK2) were used to up-regulate GRK2 protein expression. The expression of GRK2 and exchange protein directly activated by cyclic adenosine monophosphate 1 (Epac1) in the dorsal root ganglion (DRG) of lumbar 4-6 was detected via immunoblotting and immunohistochemistry, and the transfection of the GRK2 gene was detected by immunofluorescence. Low levels of GRK2 were able to sustain STZ-induced pain in DMH rats. Intrathecal injection of AAV-GRK2 vector up-regulated GRK2 expression, providing pain rain to rats with DMH. With an increase in DMH duration, there was a decrease in paw withdrawal threshold (PWT) value, aggravating the pain, resulting in a decreasing pattern in GRK2 protein expression over time, whereas Epac1 protein expression showed an opposite trend. GRK2 expression regulated DMH progression and is expected to play a role in the development of targeted therapy for DMH. GRK2 and Epac1 expressions play a vital role in maintaining pain in DMH rats.

G Protein-Coupled Receptor Kinase 2 (GRK2) Regulates T Cell Response in a Murine Model of House Dust Mite-Induced Asthma.

G protein-coupled receptor kinase 2 (GRK2) is an adapter protein that modulates G protein-coupled receptor (GPCR) signaling. It also regulates the functions and activity of other intracellular proteins in many cell types. Accordingly, GRK2 is thought to contribute to disease progression by a variety of mechanisms related to its multifunctional roles. Indeed, GRK2 levels are enhanced in patient samples as well as in preclinical models of several diseases. We have previously shown that GRK2 regulates mast cell functions, and thereby contributes to exacerbated inflammation during allergic reactions. In the current study, we observed that GRK2 levels are enhanced in the lungs of human asthma patients and in mice sensitized to house dust mite extract (HDME) allergen. Consistent with these findings, interleukin (IL)-4 and IL-13 levels were reduced in the lungs of GRK2+/− mice in a HMDE mouse model of asthma. Because Th2 cells are the major source of these cytokines during asthma, we determined the role of GRK2 in regulating T cell-specific responses in our HMDE mouse model. We observed a significant reduction of airway hyperresponsiveness (AHR), lung eosinophil and lymphocyte counts, serum IgE, Th2 cytokines (IL-4 and IL-13), goblet cell hyperplasia and mucus production in mice that had reduced GRK2 expression specifically in T cells. Collectively, our studies reveal an important role for GRK2 in regulating T cell response during asthma pathogenesis and further elucidation of the mechanisms through which GRK2 modulates airway inflammation will lead to the development of new therapeutic strategies for asthma.

GRK3 deficiency elicits brain immune activation and psychosis

The G protein-coupled receptor kinase (GRK) family member protein GRK3 has been linked to the pathophysiology of schizophrenia and bipolar disorder. Expression, as well as protein levels, of GRK3 are reduced in post-mortem prefrontal cortex of schizophrenia subjects. Here, we investigate functional behavior and neurotransmission related to immune activation and psychosis using mice lacking functional Grk3 and utilizing a variety of methods, including behavioral, biochemical, electrophysiological, molecular, and imaging methods. Compared to wildtype controls, the Grk3−/− mice show a number of aberrations linked to psychosis, including elevated brain levels of IL-1β, increased turnover of kynurenic acid (KYNA), hyper-responsiveness to D-amphetamine, elevated spontaneous firing of midbrain dopamine neurons, and disruption in prepulse inhibition. Analyzing human genetic data, we observe a link between psychotic features in bipolar disorder, decreased GRK expression, and increased concentration of CSF KYNA. Taken together, our data suggest that Grk3−/− mice show face and construct validity relating to the psychosis phenotype with glial activation and would be suitable for translational studies of novel immunomodulatory agents in psychotic disorders.

G-protein coupled receptor kinase 2 (GRK2) regulates T cell response in a murine model of house dust mite-induced asthma

Abstract G protein-coupled receptor kinase 2 (GRK2) is an adapter protein that modulates G protein-coupled receptor (GPCRs) signaling. It also regulates the functions and activity of other intracellular proteins in many cell types. Accordingly, GRK2 is thought to contribute to disease progression by a variety of mechanisms related to its multifunctional roles. Indeed, GRK2 levels are enhanced in patient samples as well as in preclinical models of several diseases. We have previously shown that GRK2 regulates mast cell functions, and thereby contributes to exacerbated inflammation during allergic reactions. In the current study, we observed that GRK2 levels are enhanced in the lungs of human asthma patients and in mice sensitized to house dust mite extract (HDME) allergen. Consistent with these findings, interleukin (IL)-4 and IL-13 levels were reduced in the lungs of GRK2+/− mice in a HMDE mouse model of asthma. Because Th2 cells are the major source of these cytokines during asthma, we determined the role of GRK2 in regulating T cell-specific responses in our HMDE mouse model. We observed a significant reduction of airway hyperresponsiveness (AHR), lung eosinophil and lymphocyte count, serum IgE, Th2 cytokines (IL-4 and IL-13), goblet cell hyperplasia and mucus production in mice that had reduced GRK2 expression specifically in T cells. Collectively, our studies reveal an important role for GRK2 in regulating T cell response during asthma pathogenesis and further elucidation of the mechanisms through which GRK2 modulates airway inflammation will lead to the development of new therapeutic strategies for asthma.

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.

The GRKs Reactome: Role in Cell Biology and Pathology

G protein-coupled receptor kinases (GRKs) are protein kinases that function in concert with arrestins in the regulation of a diverse class of G protein-coupled receptors (GPCRs) signaling. Although GRKs and arrestins are key participants in the regulation of GPCR cascades, the complex regulatory mechanisms of GRK expression, its alternation, and their function are not thoroughly understood. Several studies together with the work from our lab in recent years have revealed the critical role of these kinases in various physiological and pathophysiological processes, including cardiovascular biology, inflammation and immunity, neurodegeneration, thrombosis, and hemostasis. A comprehensive understanding of the mechanisms underlying functional interactions with multiple receptor proteins and how these interactions take part in the development of various pathobiological processes may give rise to novel diagnostic and therapeutic strategies. In this review, we summarize the current research linking the role of GRKs to various aspects of cell biology, pathology, and therapeutics, with a particular focus on thrombosis and hemostasis.

Myeloid GRK2 Regulates Obesity-Induced Endothelial Dysfunction by Modulating Inflammatory Responses in Perivascular Adipose Tissue.

Perivascular adipose tissue (PVAT) is increasingly being regarded as an important endocrine organ that directly impacts vessel function, structure, and contractility in obesity-associated diseases. We uncover here a role for myeloid G protein-coupled receptor kinase 2 (GRK2) in the modulation of PVAT-dependent vasodilation responses. GRK2 expression positively correlates with myeloid- (CD68) and lymphoid-specific (CD3, CD4, and CD8) markers and with leptin in PVAT from patients with abdominal aortic aneurysms. Using mice hemizygous for GRK2 in the myeloid lineage (LysM-GRK2+/−), we found that GRK2 deficiency in myeloid cells allows animals to preserve the endothelium-dependent acetylcholine or insulin-induced relaxation, which is otherwise impaired by PVAT, in arteries of animals fed a high fat diet (HFD). Downregulation of GRK2 in myeloid cells attenuates HFD-dependent infiltration of macrophages and T lymphocytes in PVAT, as well as the induction of tumor necrosis factor-α (TNFα) and NADPH oxidase (Nox)1 expression, whereas blocking TNFα or Nox pathways by pharmacological means can rescue the impaired vasodilator responses to insulin in arteries with PVAT from HFD-fed animals. Our results suggest that myeloid GRK2 could be a potential therapeutic target in the development of endothelial dysfunction induced by PVAT in the context of obesity.

Investigation of G protein‐coupled receptor kinase 2 (GRK2) regulation of D2 dopamine receptor signaling: effects of GRK2 functional domains and pharmacologic and genetic inhibition of GRK2 activity

The D2 dopamine (DA) receptor (D2R) signals through two major pathways, namely activation of G proteins and recruitment of β‐arrestin. Agonist binding stabilizes a receptor state that activates G proteins and facilitates receptor phosphorylation by one or more members of the G protein‐coupled receptor kinase (GRK) family, and this phosphorylation is known to enhance β‐arrestin recruitment and initiate the process of internalization for many GPCRs. Previously, we found that only GRK2 and GRK3 were involved in D2R phosphorylation in response to agonist stimulation (Namkung et al., JBC: 284: 15038, 2009). In this study, we further investigated the influence of GRK2 on receptor signaling, β‐arrestin recruitment, and internalization of the D2R. Overexpression of GRK2 was found to increase DA‐stimulated β‐arrestin recruitment and receptor internalization, whereas it suppressed D2R‐mediated inhibition of cAMP accumulation. In contrast, a catalytically inactive GRK2 mutant (GRK2‐K220R) did not affect any of these functional outputs suggesting that GRK2 kinase activity is required to influence D2R signaling. A disabling mutation of the GRK2 RGS homology (RH) domain (GRK2‐D110A) behaved similarly as WT GRK2, suggesting that this domain is not required for GRK2’s regulatory effects on the D2R. Finally, overexpression of a GRK2 construct containing a mutation in the pleckstrin homology (PH) domain (GRK2‐R587Q), which negates GRK2 interaction with Gβγ subunits, increased DA‐stimulated β‐arrestin recruitment and receptor internalization (more than observed with WT GRK2), but did not affect D2R regulation of cAMP accumulation, indicating that this GRK2 domain is involved in regulating some, but not all, aspects of D2R signaling. To study D2R regulation in the complete absence of GRK2, we utilized a cell line in which GRK2 expression was knocked out using CRISPR‐Cas9 technology. DA‐stimulated β‐arrestin recruitment and receptor internalization were decreased, but not eliminated, in the GRK2 KO cells suggesting that, while GRK2 may be facilitatory, it is not essential for these processes. In parallel studies we characterized the effects of Compound 101, a dual GRK2/3 inhibitor. Compound 101 was found to negate GRK2 interactions with the D2R and attenuate, but not eliminate, β‐arrestin recruitment and receptor internalization. In contrast, in the GRK2 KO cells, Compound 101 had no effect on the residual DA‐stimulated β‐arrestin recruitment. In conclusion, our data suggest that different GRK2 domains affect specific regulatory pathways of the D2R and that both agonist‐induced β‐arrestin recruitment and receptor internalization can occur in the absence of GRK2 interactions with the D2R.Support or Funding InformationNINDS Intramural Research Program

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.

G protein-coupled receptor kinase-2: A potential biomarker for early diabetic cardiomyopathy.

BackgroundG protein‐coupled receptor kinase‐2 (GRK2) has been shown as a key regulator of cardiac function, and the myocardial GRK2 levels are mirrored by the levels in peripheral blood mononuclear cells (PBMCs). In this study, we evaluated the myocardial and PBMCs GRK2 levels in early diabetic cardiomyopathy (DCM).MethodsC57BL/KS‐db/db male diabetic mice at 12 weeks of age, as the type 2 diabetes (T2DM) animal model of early DCM were evaluated. Forty‐four T2DM patients with left ventricular diastolic dysfunction (LVDD), without evidence of hypertension, coronary artery diseases, congestive heart failure, and diabetic complications and without evidence of ischemia in a maximal treadmill exercise test, were recruited as the DM + LVDD group; 30 age‐matched T2DM patients without LVDD were recruited as the DM control group. Left ventricular diastolic function was evaluated by cardiac tissue Doppler. The pseudonormal pattern of ventricular filling and E′/A′ < 1 were regarded as LVDD.ResultsCompared to 8‐week‐old diabetic mice and 12‐week‐old control mice, GRK2‐mRNA level and expression in myocardial tissues of 12‐week‐old diabetic mice were significantly increased, as well as the left ventricular wall thickness and systolic function. And the collagen volume fraction (CVF), collagen‐3 expression, P53 expression, and cell apoptotic rate in the myocardium of 12‐week‐old diabetic mice elevated as well. The GRK2‐mRNA level in PBMCs of DM with LVDD was significantly higher than in DM control without LVDD.ConclusionsGRK2 expression increased in the myocardial tissue and the PBMCs at the early stage of DCM. These data support further research on the role of GRK2 as the clinical biomarker for early DCM.

Degradation of GRK2 and AKT is an early and detrimental event in myocardial ischemia/reperfusion.

BackgroundIdentification of signaling pathways altered at early stages after cardiac ischemia/reperfusion (I/R) is crucial to develop timely therapies aimed at reducing I/R injury. The expression of G protein-coupled receptor kinase 2 (GRK2), a key signaling hub, is up-regulated in the long-term in patients and in experimental models of heart failure. However, whether GRK2 levels change at early time points following myocardial I/R and its functional impact during this period remain to be established.MethodsWe have investigated the temporal changes of GRK2 expression and their potential relationships with the cardioprotective AKT pathway in isolated rat hearts and porcine preclinical models of I/R.FindingsContrary to the maladaptive up-regulation of GRK2 reported at later times after myocardial infarction, successive GRK2 phosphorylation at specific sites during ischemia and early reperfusion elicits GRK2 degradation by the proteasome and calpains, respectively, thus keeping GRK2 levels low during early I/R in rat hearts. Concurrently, I/R promotes decay of the prolyl-isomerase Pin1, a positive regulator of AKT stability, and a marked loss of total AKT protein, resulting in an overall decreased activity of this pro-survival pathway. A similar pattern of concomitant down-modulation of GRK2/AKT/Pin1 protein levels in early I/R was observed in pig hearts. Calpain and proteasome inhibition prevents GRK2/Pin1/AKT degradation, restores bulk AKT pathway activity and attenuates myocardial I/R injury in isolated rat hearts.InterpretationPreventing transient degradation of GRK2 and AKT during early I/R might improve the potential of endogenous cardioprotection mechanisms and of conditioning strategies.

Relationship between p38MAPK and GRK2 in spinal cord in development of persistent postoperative pain in rats

Objective To evaluate the relationship between p38 mitogen-activated protein kinase (p38MAPK) and G protein-coupled receptor kinase 2 (GRK2) in the development of persistent postoperative pain in rats. Methods Pathogen-free healthy male Sprague-Dawley rats, weighing 200-250 g, aged 2 months, were used in this study.Sixty rats in which intrathecal catheters were successfully implanted were divided into 6 groups (n=10 each) using a random number table method: sham operation group (group S), sham operation plus dimethyl sulfoxide (DMSO) group (group D), sham operation plus GRK2 degradation inhibitor MDL28170 group (group M), persistent postoperative pain group (group PPP), persistent postoperative pain plus DMSO group (group PPP+ D) and persistent postoperative pain plus MDL28170 group (group PPP+ M). Persistent postoperative pain was evoked by skin/muscle incision and retraction (SMIR). Immediately after operation and at 1, 2 and 3 days after operation, normal saline 20 μl was intrathecally injected once a day in S and PPP groups, 5% DMSO 10 μl was intrathecally injected once a day in D and PPP+ D groups, and MDL28170 10 μl (50 μg) was intrathecally injected once a day in M and PPP+ M groups.The mechanical paw withdrawal threshold (MWT) was measured at 1 day before operation and 3, 7, 14 and 21 days after operation (T1-4). Four rats in each group were selected after behavioral testing at T3 and sacrificed, and the L4-6 segments of the spinal cord were removed for determination of the expression of phosphorylated p38MAPK (p-p38MAPK) by Western blot. Results There was no significant difference in MWT at each time point or expression of p-p38MAPK among group S, group D and group M (P>0.05). Compared with group S, the MWT was significantly decreased, and the expression of p-p38MAPK was up-regulated in PPP, PPP+ D and PPP+ M groups (P 0.05). Conclusion Down-regulated expression of spinal GRK2 can promote the activation of p38MAPK in the spinal cord and is involved in the development of persistent postoperative pain in rats. Key words: G protein-coupled receptor kinase 2; p38 Mitogen-actived protein kinases; Pain, postoperative; Spinal cord

Inhibition of MicroRNA-15a/16 Expression Alleviates Neuropathic Pain Development through Upregulation of G Protein-Coupled Receptor Kinase 2.

There is accumulating evidence that microRNAs are emerging as pivotal regulators in the development and progression of neuropathic pain. MicroRNA-15a/16 (miR-15a/16) have been reported to play an important role in various diseases and inflammation response processes. However, whether miR-15a/16 participates in the regulation of neuroinflammation and neuropathic pain development remains unknown. In this study, we established a mouse model of neuropathic pain by chronic constriction injury (CCI) of the sciatic nerves. Our results showed that both miR-15a and miR-16 expression was significantly upregulated in the spinal cord of CCI rats. Downregulation of the expression of miR-15a and miR-16 by intrathecal injection of a specific inhibitor significantly attenuated the mechanical allodynia and thermal hyperalgesia of CCI rats. Furthermore, inhibition of miR-15a and miR-16 downregulated the expression of interleukin-1β and tumor-necrosis factor-α in the spinal cord of CCI rats. Bioinformatic analysis predicted that G protein-coupled receptor kinase 2 (GRK2), an important regulator in neuropathic pain and inflammation, was a potential target gene of miR-15a and miR-16. Inhibition of miR-15a and miR-16 markedly increased the expression of GRK2 while downregulating the activation of p38 mitogen-activated protein kinase and NF-κB in CCI rats. Notably, the silencing of GRK2 significantly reversed the inhibitory effects of miR-15a/16 inhibition in neuropathic pain. In conclusion, our results suggest that inhibition of miR-15a/16 expression alleviates neuropathic pain development by targeting GRK2. These findings provide novel insights into the molecular pathogenesis of neuropathic pain and suggest potential therapeutic targets for preventing neuropathic pain development.

Impact of oxidative stress on renal dopamine D(1) receptor dysfunction in offspring of diabetic rat dams

Objective: To explore the effects of oxidative stress on renal dopamine D(1) receptor dysfunction in offspring of diabetic rat dams. Methods: The pregnant Sprague Dawley (SD) rats (n=10) were randomly divided into the diabetic group (a single intraperitoneal injection of 35 mg/kg streptozotocin on day 0 of gestation) and control group (injected with the equal volume of 0.9% saline on day 0 of gestation) according to the random number table (n=5 each group). The offspring rats were divided into 4 groups including offspring of control dams treated with vehicle, offspring of control dams treated with antioxidant, offspring of diabetic dams treated with vehicle and offspring of diabetic dams treated with antioxidant (n=10 each group). After birth, the offspring rats were treated with normal drinking water or antioxidant (tempol, 1.0 mmol/L) from the age of 4 weeks until the end of the study (20 weeks). The blood pressure was monitored continuously by non-invasive tail-cuff method. The renal oxidative markers including superoxide dismutase (SOD) and malondialdehyde (MDA) activity and D(1) receptor agonist (fenoldopam)-mediated urinary and sodium excretion were detected. Furthermore, the protein expression of renal G protein-coupled receptor kinase 2 (GRK2), GRK4, dopamine D(1) receptor and the phosphorylation level of D(1) receptor were detected. Results: The mean arterial pressure of offspring from the diabetic dams treated with vehicle was significantly higher than that of offspring from control dams treated with vehicle (P=0.013), while the mean arterial pressure of offspring from diabetic dams treated with antioxidant was significantly lower than that of offspring from the diabetic dams treated with vehicle (P=0.038). The fenoldopam-mediated urinary flow and urinary sodium excretion rate were significantly lower in offspring of diabetic dams treated with vehicle than those in offspring of control dams treated with vehicle (P<0.01), which were significantly higher in offspring of diabetic dams treated with antioxidant as compared to offspring of diabetic dams treated with vehicle (both P<0.01). There was no significant difference in fenoldopam-mediated urinary flow and urinary sodium excretion rate in offspring of control dams treated with antioxidant or vehicle (urinary flow: P=0.772; urinary sodium excretion rate: P=0.716). Compared with offspring of control dams treated with vehicle, the renal MDA activity was significantly increased, while the SOD activity was significantly decreased in offspring of diabetic dams treated with vehicle (MDA: P<0.01; SOD: P=0.013). The renal MDA activity was significantly decreased, while the SOD activity was significantly increased in offspring of diabetic dams treated with antioxidant in comparison with offspring of diabetic dams treated with vehicle (MDA: P<0.01; SOD: P=0.035).The renal GRK2 and GRK4 protein expression in offspring of diabetic dams treated with vehicle were significantly higher than those in offspring of control dams treated with vehicle (P<0.01), while the expression levels of renal GRK2 and GRK4 in offspring of diabetic dams treated with antioxidant were significantly downregulated compared with offspring of diabetic dams treated with vehicle (P<0.01). There was no significant difference in the protein expression of dopamine D(1) receptor among 4 groups (P=0.735). The level of dopamine D(1) receptor phosphorylation in offspring of diabetic dams treated with vehicle was significantly higher than that in offspring of control dams treated with vehicle (P<0.01), while the dopamine D(1) receptor phosphorylation level was significantly lower in offspring of diabetic dams treated with antioxidant compared to that in offspring of diabetic dams treated with vehicle (P<0.01). Conclusion: Oxidative stress is involved in the dopamine D(1) receptors dysfunction in the offspring of diabetic dams.

Calpains mediate isoproterenol-induced hypertrophy through modulation of GRK2.

Inhibition of the Ca2+-dependent proteases calpains attenuates post-infarction remodeling and heart failure. Recent data suggest that calpain activity is elevated in non-ischemic cardiomyopathies and that upregulation of the key cardiac G-protein-coupled receptor kinase 2 (GRK2) signaling hub promotes cardiac hypertrophy. However, the functional interactions between calpains and GRK2 in this context have not been explored. We hypothesized that calpain modulates GRK2 levels in myocardial hypertrophy of non-ischemic cause, and analyzed the mechanisms involved and the potential therapeutic benefit of inhibiting calpain activity in this situation. The oral calpain inhibitor SNJ-1945 was administered daily to male Sprague-Dawley rats or wild-type and hemizygous GRK2 mice treated with 5mg/Kg/day isoproterenol intraperitoneally for 1week. In isoproterenol-treated animals, calpains 1 and 2 were overexpressed in myocardium and correlated with increased calpain activity and ventricular hypertrophy. Oral co-administration of SNJ-1945 attenuated calpain activation and reduced heart hypertrophy as assessed using morphological and biochemical markers. Calpain activation induced by isoproterenol increased GRK2 protein levels, while genetic downregulation of GRK2 expression prevented isoproterenol-mediated hypertrophy independently of calpain inhibition. GRK2 upregulation was associated to calpain-dependent degradation of the GRK2 ubiquitin ligase MDM2 and to enhanced NF-κB-dependent GRK2 gene expression in correlation with calpain-mediated IĸB proteolysis. These results demonstrate that calpain mediates isoproterenol-induced myocardial hypertrophy by modulating GRK2 protein content through mechanisms involving the control of GRK2 stability and expression. Sustained calpain inhibition attenuates isoproterenol-induced myocardial hypertrophy and could be an effective therapeutic strategy to limit ventricular remodeling of non-ischemic origin.

CP-25 combined with MTX/ LEF ameliorates the progression of adjuvant-induced arthritis by the inhibition on GRK2 translocation

ObjectiveCP-25 attenuates arthritis progression in animal models by inhibiting G protein-coupled receptor kinase 2 (GRK2) membrane expression. This study compared groups treated with high-dose methotrexate (MTX)/leflunomide (LEF) and CP-25 combined with low-dose MTX/LEF in an adjuvant-induced arthritis (AA) rat model and investigated possible mechanisms. MethodsAA was induced in rats via complete Freund’s adjuvant. Experimental groups were divided into a normal group; vehicle group; monotherapy groups, including CP-25 (50 mg/kg), MTX (0.25, 0.5 mg/kg), and LEF (5, 10 mg/kg); and CP-25 (50 mg/kg)-combined MTX (0.25 mg/kg)/LEF (5 mg/kg) groups. We measured cytokine levels, phosphorylation and protein expression, and interactions between proteins. The role of GRK2 on phosphorylated extracellular signal-regulated kinase (p-ERK) was determined via GRK2-siRNA using a high content imaging system. ResultsTherapeutic effects, including pathology and cytokine balance, were equivalent between the CP-25-combination groups and the high-dose MTX/LEF groups. P38, ERK, and c-Jun N-terminal kinase (JNK) activation in AA fibroblast-like synoviocytes (FLS) was reduced in the treatment groups; GRK2 expression was only inhibited in the CP-25 group. Interactions between GRK2 and p-ERK decreased in the vehicle group and were restored in the CP-25 group. GRK2 membrane expression and p-ERK nuclear expression increased in FLS pre-treated with tumour necrosis factor alpha and stimulated with prostaglandin E2. Nuclear expression of p-ERK increased in GRK2-siRNA FLS. ConclusionEquivalent therapeutic effects were observed between CP-25-combination groups and high-dose MTX/LEF groups. CP-25 inhibited p-ERK by reducing the membrane expression of GRK2 in FLS from AA rats.

Exposure to Maternal Diabetes Mellitus Causes Renal Dopamine D1 Receptor Dysfunction and Hypertension in Adult Rat Offspring.

Epidemiological and experimental studies suggest that maternal diabetes mellitus programs hypertension that is associated with impaired sodium excretion in the adult offspring. However, the underlying mechanisms are not clear. Because dopamine receptor function is involved in the pathogenesis of hypertension, we hypothesized that impaired renal dopamine D1 receptor function is also involved in the hypertension in offspring of maternal diabetes mellitus. Maternal diabetes mellitus was induced by a single intraperitoneal injection of streptozotocin (35 mg/kg) to pregnant Sprague-Dawley rats at day 0 of gestation. Compared with the offspring of mothers injected with citrate buffer (control mother offspring), the diabetic mother offspring (DMO) had increased systolic blood pressure and impaired D1 receptor-mediated diuresis and natriuresis, accompanied by increased renal PKC (protein kinase C) expression and activity, GRK-2 (G protein-coupled receptor kinase-2) expression, D1 receptor phosphorylation, D1 receptor/Gαs uncoupling, and loss of D1 receptor-mediated inhibition of Na+-K+-ATPase activity in renal proximal tubule cells from DMO. Inhibition of PKC reduced the increased GRK-2 expression and normalized D1 receptor function in primary cultures of renal proximal tubule cells from DMO. In addition, DMO, relative to control mother offspring, in vivo, had increased oxidative stress, indicated by decreased renal glutathione and increased renal malondialdehyde and urine 8-isoprostane. Normalization of oxidative stress with tempol also normalized the renal D1 receptor phosphorylation, D1 receptor-mediated diuresis and natriuresis, and blood pressure in DMO. Our present study indicates that maternal diabetes mellitus-programed hypertension in the offspring is caused by impaired renal D1 receptor function because of oxidative stress that is mediated by increased PKC-GRK-2 activity.