Proactive M2 blockade prevents cognitive decline in GRK5-deficient APP transgenic mice via enhancing cholinergic neuronal resilience.

Alzheimer's disease (AD) poses an immense challenge in healthcare, lacking effective therapies. This study investigates the potential of anthranilamide derivative (AAD23), a selective M2 receptor antagonist, in proactively preventing cognitive impairments and cholinergic neuronal degeneration in G protein-coupled receptor kinase-5-deficient Swedish APP (GAP) mice. GAP mice manifest cognitive deficits by 7 months and develop senile plaques by 9 months. A 6-month AAD23 treatment was initiated at 5 months and stopped at 11 months before behavioral assessments without the treatment. AAD23-treated mice exhibited preserved cognitive abilities and improved cholinergic axonal health in the nucleus basalis of Meynert akin to wildtype mice. Conversely, vehicle-treated GAP mice displayed memory deficits and pronounced cholinergic axonal swellings in the nucleus basalis of Meynert. Notably, AAD23 treatment did not alter senile plaques and microgliosis. These findings highlight AAD23's efficacy in forestalling AD-related cognitive decline in G protein-coupled receptor kinase-5-deficient subjects, attributing its success to restoring cholinergic neuronal integrity and resilience, enhancing resistance against diverse degenerative insults.

Clinical Pharmacogenetics Implementation Consortium Guideline (CPIC) for CYP2D6, ADRB1, ADRB2, ADRA2C, GRK4, and GRK5 Genotypes and Beta-Blocker Therapy.

Beta-blockers are widely used medications for a variety of indications, including heart failure, myocardial infarction, cardiac arrhythmias, and hypertension. Genetic variability in pharmacokinetic (e.g., CYP2D6) and pharmacodynamic (e.g., ADRB1, ADRB2, ADRA2C, GRK4, GRK5) genes have been studied in relation to beta-blocker exposure and response. We searched and summarized the strength of the evidence linking beta-blocker exposure and response with the six genes listed above. The level of evidence was high for associations between CYP2D6 genetic variation and both metoprolol exposure and heart rate response. Evidence indicates that CYP2D6 poor metabolizers experience clinically significant greater exposure and lower heart rate in response to metoprolol compared with those who are not poor metabolizers. Therefore, we provide therapeutic recommendations regarding genetically predicted CYP2D6 metabolizer status and metoprolol therapy. However, there was insufficient evidence to make therapeutic recommendations for CYP2D6 and other beta-blockers or for any beta-blocker and the other five genes evaluated (updates at www.cpicpgx.org).

Porphyromonas gingivalis virulence factors induce toxic effects in SH-SY5Y neuroblastoma cells: GRK5 modulation as a protective strategy.

Periodontitis (PDS) is a chronic inflammatory disease initiated by a dysbiosis of oral pathogenic bacterial species, such as Porphyromonas gingivalis (Pg). These bacteria can penetrate the bloodstream, releasing various endo and exotoxins that fuel the infection, and stimulate toxic inflammation in different compartments, including the brain. However, the specific mechanisms by which PDS/Pg contribute to brain disorders, such as Alzheimer's disease (AD), remain unclear. This study assessed the effects of Pg's virulence factors - lipopolysaccharide (LPS-Pg) and gingipains (gps) K (Kgp) and Rgp - on SH-SY5Y cells. Our results demonstrated that LPS-Pg activated signaling through the Toll-like receptor (TLR)-2/4 induced a significant downregulation of G protein-coupled receptor kinase 5 (GRK5). Additionally, LPS-Pg stimulation resulted in a robust increase in Tau phosphorylation (pTau) and p53 levels, while causing a marked reduction in Bcl2 and increased cell death compared to unstimulated cells (Ns). LPS-Pg also elevated inducible nitric oxide synthase (iNOS) expression, leading to oxidative damage. In cells overexpressing GRK5 via Adenovirus, LPS-Pg failed to increase iNOS and pTau levels compared to GFP control cells. High GRK5 levels also prevented the nuclear accumulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). Furthermore, the overexpression of a GRK5 mutant form lacking the nuclear localization signal (ΔNLS) nearly abolished LPS-Pg induced p53 and iNOS upregulation. Finally, we tested whether Kgp and Rgp mediated similar effects and our data showed that both gps caused a marked downregulation of GRK5 leading to increased p53 and pTau levels. In conclusion, this study provides further insight into the toxic effects elicited by Pg in cells and suggests that preventing GRK5 deficiency may be a valid strategy to mitigate Pg-induced toxic effects (i.e. cell death, oxidative damage, and Tau hyperphosphorylation) in SH-SY5Y cells, which are typical molecular hallmarks of neurodegenerative disorders.

Association of GRK5 variant rs10886471 with the therapeutic effect of repaglinide in patients of type 2 diabetes mellitus in Peshawar, Pakistan.

Type 2 diabetes mellitus (T2DM) is a complex metabolic disorder with a rising global prevalence. The primary objective of this study was to explore the relationship between the GRK5 variant (rs10886471) and the therapeutic effect of repaglinide in patients of T2DM in Peshawar, Pakistan. A quasi-experimental study was designed. The study group consisted of patients with Type 2 Diabetes Mellitus (T2DM) categorized into responders and non-responders based on their HbA1c level reduction in response to repaglinide treatment. After ethical approval, and consent from the participants, sociodemographic and clinical data was collected from 60 T2DM patients. Blood samples were collected followed by DNA extraction and quantification with UV-Vis Spectroscopy. Genotyping for the GRK5 variant rs10886471 was done using the PCR-based method. Among socio-demographic factors family history and BMI showed significant association (P<0.05) with the therapeutic response to repaglinide. The Statistical analyses, including chi-square tests and logistic regression of GRK5 variant rs10886471 exhibited a significant association with the therapeutic response. Variant allele exhibited significant association (OR: 1.2, p=0.049) with the therapeutic response to repaglinide. The study demonstrated a significant relationship between the GRK5 variant (rs10886471) and the therapeutic response to repaglinide in patients of T2DM of Peshawar, Pakistan.

Unveiling G-protein coupled receptor kinase-5 inhibitors for chronic degenerative diseases: Multilayered prioritization employing explainable machine learning-driven multi-class QSAR, ligand-based pharmacophore and free energy-inspired molecular simulation.

GRK5 holds a pivotal role in cellular signaling pathways, with its overexpression in cardiomyocytes, neuronal cells, and tumor cells strongly associated with various chronic degenerative diseases, which highlights the urgent need for potential inhibitors. In this study, multiclass classification-based QSAR models were developed using diverse machine learning algorithms. These models were built from curated compounds with experimentally derived GRK5 inhibitory activity. Additionally, a pharmacophore model was constructed using active compounds from the dataset. Among the models, the SVM-based approach proved most effective and was initially used to screen DrugBank compounds within the applicability domain. Compounds showing significant GRK5 inhibitory potential underwent evaluation for key pharmacophoric features. Prospective compounds were subjected to molecular docking to assess binding affinity towards GRK5's key active site amino acid residues. Stability at the binding site was analyzed through 200 ns molecular dynamics simulations. MM-GBSA analysis quantified individual free energy components contributing to the total binding energy with respect to binding site residues. Metadynamics analysis, including PCA, FEL, and PDF, provided crucial insights into conformational changes of both apo and holo forms of GRK5 at defined energy states. The study identifies DB02844 (S-Adenosyl-1,8-Diamino-3-Thiooctane) and DB13155 (Esculin) as promising GRK5 inhibitors, warranting further in vitro and in vivo validation studies.

GRK5 promoted renal fibrosis via HDAC5/Smad3 signaling pathway.

Renal fibrosis is a common pathological feature of chronic kidney diseases (CKD), poses a significant burden in the aging population, and is a major cause of end-stage renal disease (ESRD). In this study, we investigated the role of G protein-coupled receptor kinases (GRKs) 5 in the pathogenesis of renal fibrosis. GRK5 is a serine/threonine kinase that regulates G protein-coupled receptor (GPCR) signaling. GRK5 has been shown to play a role in various diseases including cardiac disorders and cancer. However, the role of GRK5 in renal fibrosis remains largely unknown. Our finding revealed that GRK5 was significantly overexpressed in renal fibrosis. Specifically, GRK5 was transferred into the nucleus via its nuclear localization sequence to regulate histone deacetylases (HDAC) 5 expression under renal fibrosis. GRK5 acted as an upstream regulator of HDAC5/Smad3 signaling pathway. HDAC5 regulated and prevented the transcriptional activity of myocyte enhancer factor 2A (MEF2A) to repress the transcription of Smad7 which leading to the activation of Smad3. These findings first revealed that GRK5 may be a potential therapeutic target for the treatment of renal fibrosis. Inhibition of GRK5 activity may be a promising strategy to attenuate the progression of renal fibrosis.

Insulin-Like Growth Factor 1 Receptor Deficiency Alleviates Angiotensin II-Induced Cardiac Fibrosis Through the Protein Kinase B/Extracellular Signal-Regulated Kinase/Nuclear Factor-κB Pathway.

Background The renin-angiotensin system plays a crucial role in the development of heart failure, and Ang II (angiotensin II) acts as the critical effector of the renin-angiotensin system in regulating cardiac fibrosis. However, the mechanisms of cardiac fibrosis are complex and still not fully understood. IGF1R (insulin-like growth factor 1 receptor) has multiple functions in maintaining cardiovascular homeostasis, and low-dose IGF1 treatment is effective in relieving Ang II-induced cardiac fibrosis. Here, we aimed to investigate the molecular mechanism of IGF1R in Ang II-induced cardiac fibrosis. Methods and Results Using primary mouse cardiac microvascular endothelial cells and fibroblasts, in vitro experiments were performed. Using C57BL/6J mice and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9)-mediated IGF1R heterozygous knockout (Igf1r+/-) mice, cardiac fibrosis mouse models were induced by Ang II for 2 weeks. The expression of IGF1R was examined by quantitative reverse transcription polymerase chain reaction, immunohistochemistry, and Western blot. Mice heart histologic changes were evaluated using Masson and picro sirius red staining. Fibrotic markers and signal molecules indicating the function of the Akt (protein kinase B)/ERK (extracellular signal-regulated kinase)/nuclear factor-κB pathway were detected using quantitative reverse transcription polymerase chain reaction and Western blot. RNA sequencing was used to explore IGF1R-mediated target genes in the hearts of mice, and the association of IGF1R and G-protein-coupled receptor kinase 5 was identified by coimmunoprecipitation. More important, blocking IGF1R signaling significantly suppressed endothelial-mesenchymal transition in primary mouse cardiac microvascular endothelial cells and mice in response to transforming growth factor-ß1 or Ang II, respectively. Deficiency or inhibition of IGF1R signaling remarkably attenuated Ang II-induced cardiac fibrosis in primary mouse cardiac fibroblasts and mice. We further observed that the patients with heart failure exhibited higher blood levels of IGF1 and IGF1R than healthy individuals. Moreover, Ang II treatment significantly increased cardiac IGF1R in wild type mice but led to a slight downregulation in Igf1r+/- mice. Interestingly, IGF1R deficiency significantly alleviated cardiac fibrosis in Ang II-treated mice. Mechanistically, the phosphorylation level of Akt and ERK was upregulated in Ang II-treated mice, whereas blocking IGF1R signaling in mice inhibited these changes of Akt and ERK phosphorylation. Concurrently, phosphorylated p65 of nuclear factor-κB exhibited similar alterations in the corresponding group of mice. Intriguingly, IGF1R directly interacted with G-protein-coupled receptor kinase 5, and this association decreased ≈50% in Igf1r+/- mice. In addition, Grk5 deletion downregulated expression of the Akt/ERK/nuclear factor-κB signaling pathway in primary mouse cardiac fibroblasts. Conclusions IGF1R signaling deficiency alleviates Ang II-induced cardiac fibrosis, at least partially through inhibiting endothelial-mesenchymal transition via the Akt/ERK/nuclear factor-κB pathway. Interestingly, G-protein-coupled receptor kinase 5 associates with IGF1R signaling directly, and it concurrently acts as an IGF1R downstream effector. This study suggests the promising potential of IGF1R as a therapeutic target for cardiac fibrosis.

Novel roles for G protein-coupled receptor kinases in cardiac injury and repair.

G protein-coupled receptors (GPCRs) are key modulators of cell signaling. Multiple GPCRs are present in the heart where they regulate cardiac homeostasis including processes such as myocyte contraction, heart rate and coronary blood flow. GPCRs are pharmacological targets for several cardiovascular disorders including heart failure (HF) such as beta-adrenergic receptor (ßAR) blockers and angiotensin II receptor (AT1R) antagonists. The activity of GPCRs are finely regulated by GPCR kinases (GRKs), which phosphorylate agonist-occupied receptors and start the process of desensitization. Among the seven members of the GRK family, GRK2 and GRK5 are predominantly expressed in the heart, where they exhibit both canonical and non-canonical functions. Both kinases are known to be increased in cardiac pathologies and contribute to pathogenesis through their roles in different cellular compartments. Lowering or inhibiting their actions mediate cardioprotective effects against pathological cardiac growth and failing heart. Therefore, given their importance in cardiac dysfunction, these kinases are drawing attention as promising targets for the treatment of HF, which needs improved therapies. Over the past three decades, broad knowledge on GRK inhibition in HF has been gained by studies using genetically engineered animal models or through gene therapy with peptide inhibitors or using small molecule inhibitors. In this mini review, we summarize the work focusing on GRK2 and GRK5 but also discuss a couple of the non-abundant cardiac subtypes and their multi-functional roles in the normal and diseased heart and the potential and therapeutic targets.

Targeting GRK2 and GRK5 for treating chronic degenerative diseases: Advances and future perspectives.

The development of small-molecule inhibitors targeting G protein-coupled receptor kinase 2 (GRK2) and G protein-coupled receptor kinase 5 (GRK5) for the treatment of chronic degenerative diseases has attracted wide attention. GRK2 and GRK5 can regulate essential physiological processes by phosphorylating G protein-coupled receptor (GPCR). Alterations in the functional levels of GRK2 and GRK5 have been found in a variety of chronic degenerative diseases, such as cardiovascular diseases, neurodegenerative diseases, cancers, type 2 diabetes, and rheumatoid arthritis (RA). Abnormal GRK2 and GRK5 expression contribute to the development of chronic degenerative diseases through environmental molecular mechanisms, making them promising molecular targets for treating chronic degenerative diseases. To date, many novel GRK2 and GRK5 inhibitors have been reported for the treatment of chronic degenerative diseases. We focus on the recent progress of single and dual-target inhibitors of GRK2/GRK5. This review summarizes the structural optimization rationale, structure-activity relationships (SARs), and the latest application in the treatment of chronic degenerative diseases. We believe it will shed light on the future development of small molecule inhibitors of GRK2 and GRK5, as well as the clinical applications in chronic degenerative diseases.

lncRNA LINC01315 promotes malignancy of triple-negative breast cancer and predicts poor outcomes by modulating microRNA-876-5p/GRK5.

Triple-negative breast cancer (TNBC) is a malignant tumor that threatens women's health. Exploring novel development-associated biomarkers could help improve the survival rate of TNBC. This study evaluated the significance and mechanism of LINC01315 in TNBC progression aiming to identify a potential biomarker. There were 103 TNBC patients that provided clinical tissues in this study. The expression of LINC01315 was assessed by PCR and its association with clinical data was evaluated by statistical analyses. The in vitro cell experiments were conducted to estimate the biological effect of LINC01315 and its molecular mechanism. A significant upregulation of LINC01315 was observed in TNBC, which was associated with disease development and severity of patients. The upregulation of LINC01315 could be a symptom of the poor prognosis of patients. The knockdown of LINC01315 suppressed the main cellular processes of TNBC progression. Additionally, miR-876-5p was demonstrated to be a target of LINC01315 and regulate the expression of GRK5, through which LINC01315 modulated the progression of TNBC. Upregulated LINC01315 in TNBC indicated the malignant development and poor survival rate of patients. Inhibition of LINC01315 might be a potential therapeutic strategy of TNBC.

Heterotrimeric Gq proteins act as a switch for GRK5/6 selectivity underlying ß-arrestin transducer bias.

Signaling-biased ligands acting on G-protein-coupled receptors (GPCRs) differentially activate heterotrimeric G proteins and ß-arrestins. Although a wealth of structural knowledge about signaling bias at the GPCR level exists (preferential engagement of a specific transducer), little is known about the bias at the transducer level (different functions mediated by a single transducer), partly due to a poor understanding of GPCR kinase (GRK)-mediated GPCR phosphorylation. Here, we reveal a unique role of the Gq heterotrimer as a determinant for GRK-subtype selectivity that regulates subsequent ß-arrestin conformation and function. Using the angiotensin II (Ang II) type-1 receptor (AT1R), we show that ß-arrestin recruitment depends on both GRK2/3 and GRK5/6 upon binding of Ang II, but solely on GRK5/6 upon binding of the ß-arrestin-biased ligand TRV027. With pharmacological inhibition or genetic loss of Gq, GRK-subtype selectivity and ß-arrestin functionality by Ang II is shifted to those of TRV027. Single-molecule imaging identifies relocation of AT1R and GRK5, but not GRK2, to an immobile phase under the Gq-inactive, AT1R-stimulated conditions. These findings uncover a previously unappreciated Gq-regulated mechanism that encodes GRK-subtype selectivity and imparts distinct phosphorylation-barcodes directing downstream ß-arrestin functions.

Polymorphic Variants in the GRK5 Gene Promoter Are Associated With Diastolic Dysfunction in Coronary Artery Bypass Graft Surgery Patients.

BACKGROUND: The G-protein-coupled receptor kinase 5 (GRK5) is a mediator of cardiovascular homeostasis and participates in inflammation and cardiac fibrosis, both being involved in the development of diastolic dysfunction (DD). While mechanisms of transcriptional regulation of the GRK5 promoter are unclear, we tested the hypotheses, that (1) GRK5 expression varies depending on functional single nucleotide polymorphisms (SNPs) in the GRK5 promoter and (2) this is associated with DD in patients undergoing coronary artery bypass graft (CABG) surgery. METHODS: We amplified and sequenced the GRK5 promoter followed by cloning, reporter assays, and electrophoretic mobility shift assays (EMSA). GRK5 messenger ribonucleic acid (mRNA) expression was determined in right atrial tissue sampled from 50 patients undergoing CABG surgery. In another prospective study, GRK5 genotypes were associated with determinants of diastolic function using transesophageal echocardiography in 255 patients with CABG with normal systolic left ventricular (LV) function. Specifically, we measured ejection fraction (EF), transmitral Doppler early filling velocity (E), tissue Doppler early diastolic lateral mitral annular velocity (E' lateral), and calculated E/E', E' norm and the difference of E' lateral and E' norm to account for age-related changes in diastolic function. RESULTS: We identified 6 SNPs creating 3 novel haplotypes with the greatest promoter activation in haplotype tagging (ht) SNP T(-678)C T-allele constructs (P < .001). EMSAs showed allele-specific transcription factor binding proving functional activity. GRK5 mRNA expression was greatest in TT genotypes (TT: 131 fg/µg [95% CI, 108-154]; CT: 109 [95% confidence interval {CI}, 93-124]; CC: 83 [95% CI, 54-112]; P = .012). Moreover, GRK5 genotypes were significantly associated with determinants of diastolic function. Grading of DD revealed more grade 3 patients in TT compared to CT and CC genotypes (58% vs 38% vs 4%; P = .023). E´ lateral was lowest in TT genotypes (P = .007) and corresponding E/E' measurements showed 1.27-fold increased values in TT versus CC genotypes (P = .01), respectively. While E' norm values were not different between genotypes (P = .182), the difference between E' lateral and E' norm was significantly higher in TT genotypes compared to CC and CT genotypes (-1.2 [interquartile range {IQR}, 2.7], -0.5 [IQR, 3.4], and -0.4 [IQR, 4.2; P = .035], respectively). CONCLUSIONS: A functional GRK5 SNP results in allele-dependent differences in GRK5 promoter activity and mRNA expression. This is associated with altered echocardiographic determinants of diastolic function. Thus, SNPs in the GRK5 promoter are associated with altered perioperative diastolic cardiac function. In the future, preoperative testing for these and other SNPs might allow to initiate more specific diagnostic and perioperative pathways to benefit patients at risk.

GRK5 Deficiency Causes Mild Cognitive Impairment due to Alzheimer's Disease.

Prevention of Alzheimer's disease (AD) is a high priority mission while searching for a disease modifying therapy for AD, a devastating major public health crisis. Clinical observations have identified a prodromal stage of AD for which the patients have mild cognitive impairment (MCI) though do not yet meet AD diagnostic criteria. As an identifiable transitional stage before the onset of AD, MCI should become the high priority target for AD prevention, assuming successful prevention of MCI and/or its conversion to AD also prevents the subsequent AD. By pulling this string, one demonstrated cause of amnestic MCI appears to be the deficiency of G protein-coupled receptor-5 (GRK5). The most compelling evidence is that GRK5 knockout (GRK5KO) mice naturally develop into aMCI during aging. Moreover, GRK5 deficiency was reported to occur during prodromal stage of AD in CRND8 transgenic mice. When a GRK5KO mouse was crossbred with Tg2576 Swedish amyloid precursor protein transgenic mouse, the resulted double transgenic GAP mice displayed exaggerated behavioral and pathological changes across the spectrum of AD pathogenesis. Therefore, the GRK5 deficiency possesses unique features and advantage to serve as a prophylactic therapeutic target for MCI due to AD.

G protein-coupled receptor kinase 5 (GRK5) contributes to impaired cardiac function and immune cell recruitment in post-ischemic heart failure.

AIMS: Myocardial infarction (MI) is the most common cause of heart failure (HF) worldwide. G protein-coupled receptor kinase 5 (GRK5) is upregulated in failing human myocardium and promotes maladaptive cardiac hypertrophy in animal models. However, the role of GRK5 in ischemic heart disease is still unknown. In this study, we evaluated whether myocardial GRK5 plays a critical role post-MI in mice and included the examination of specific cardiac immune and inflammatory responses. METHODS AND RESULTS: Cardiomyocyte-specific GRK5 overexpressing transgenic mice (TgGRK5) and non-transgenic littermate control (NLC) mice as well as cardiomyocyte-specific GRK5 knockout mice (GRK5cKO) and wild type (WT) were subjected to MI and, functional as well as structural changes together with outcomes were studied. TgGRK5 post-MI mice showed decreased cardiac function, augmented left ventricular dimension and decreased survival rate compared to NLC post-MI mice. Cardiac hypertrophy and fibrosis as well as fetal gene expression were increased post-MI in TgGRK5 compared to NLC mice. In TgGRK5 mice, GRK5 elevation produced immuno-regulators that contributed to the elevated and long-lasting leukocyte recruitment into the injured heart and ultimately to chronic cardiac inflammation. We found an increased presence of pro-inflammatory neutrophils and macrophages as well as neutrophils, macrophages and T-lymphocytes at 4-days and 8-weeks respectively post-MI in TgGRK5 hearts. Conversely, GRK5cKO mice were protected from ischemic injury and showed reduced early immune cell recruitment (predominantly monocytes) to the heart, improved contractility and reduced mortality compared to WT post-MI mice. Interestingly, cardiomyocyte-specific GRK2 transgenic mice did not share the same phenotype of TgGRK5 mice and did not have increased cardiac leukocyte migration and cytokine or chemokine production post-MI. CONCLUSIONS: Our study shows that myocyte GRK5 has a crucial and GRK-selective role on the regulation of leucocyte infiltration into the heart, cardiac function and survival in a murine model of post-ischemic HF, supporting GRK5 inhibition as a therapeutic target for HF.

Circ_0001821 knockdown suppresses growth, metastasis, and TAX resistance of non-small-cell lung cancer cells by regulating the miR-526b-5p/GRK5 axis.

Non-small-cell lung cancer (NSCLC) remains a huge obstacle to human health. Certain circular RNAs endow with crucial regulatory roles in NSCLC progression. Here, we investigated the functional effects of circ_0001821 on cellular behaviors of NSCLC cells and explored the possible mechanism. The expression of circ_0001821, microRNA (miR)-526b-5p, and G protein-coupled receptor kinase 5 (GRK5) was determined by quantitative real-time polymerase chain reaction or Western blot assay. Clonogenicity in NSCLC cells was detected via colony formation assay. Cell migration and invasion were monitored by Transwell assay. Cell sensitivity to paclitaxel (TAX) evaluated by Cell Counting Kit-8 assay. Cell apoptosis was assessed by flow cytometry, caspase-3 activity, and caspase-9 activity. The targeted relationship between miR-526b-5p and circ_0001821 or GRK5 was confirmed by dual-luciferase reporter or RNA pull-down assay. Moreover, the role of circ_0001821 in vivo was examined by xenograft model assay. The results presented that the expression of circ_0001821 and GRK5 was increased, while miR-526b-5p expression was decreased in NSCLC tissues and cells. Circ_0001821 knockdown reduced colony formation ability and metastasis ability but enhanced TAX sensibility and apoptosis of NSCLC cells, which was attenuated by miR-526b-5p inhibition or GRK5 overexpression. Circ_0001821 targeted miR-526b-5p, and miR-526b-5p targeted GRK5. Circ_0001821 could upregulate GRK5 expression by sponging miR-526b-5p. Depletion of circ_0001821 also blocked tumor growth in vivo. In conclusion, the depletion of circ_0001821 inhibited NSCLC progression, at least in part, by modulating the miR-526b-5p/GRK5 axis.

Association between gene polymorphism of GRK5 and breast cancer risk in Chinese population.

PURPOSE: Potential influences of GRK5 polymorphism on cancer risks have been reported. This study aimed to explore the distribution of GRK5 genotypes and alleles in Chinese breast cancer (BCa) patients, and to analyze the association between GRK5 and BCa risk. METHODS: Blood samples were collected from 412 BCa patients and 533 healthy individuals for isolating genomic DNA. GRK5 polymorphisms of Gln41Leu A > T and Arg304His G > A, and their alleles were detected using PCR-RFLP. Their influences on BCa susceptibility and pathological indexes were analyzed using Logistic regression model. RESULTS: No significant differences in age, body mass index (BMI) and smoking status were found between BCa patients and healthy persons, while significant differences were detected in drinking status, family history of cancer, hypertension and diabetes. GRK5Gln41Leu A > T and its allele frequency distribution were correlated to BCa susceptibility, while GRK5 Arg304His G > A was not. Higher risks of GRK5 Gln41Leu A > T and Arg304His G > A indicated a higher susceptibility to BCa. Compared with people carrying 0-1 risk allele, those carrying 2-4 risk alleles of GRK5 Gln41Leu A > T and Arg304His G > A of had a higher susceptibility to BCa, manifesting as worse tumor staging and grading, and higher rates of estrogen receptor (ER) (-), progesterone receptor (PR) (-) and HER2 (-). CONCLUSIONS: Gln41Leu A > T and Arg304His G > A fusion gene polymorphisms of GRK5 are vital genetic susceptibility genes to BCa. Our findings require to be validated in a multicenter study with a high-quality large sample size.

Gut microbiota mediates the effects of curcumin on enhancing Ucp1-dependent thermogenesis and improving high-fat diet-induced obesity.

Due to extremely poor systemic bioavailability, the mechanism by which curcumin increases energy expenditure remains unelucidated. Accumulating evidence suggests a strong association between the gut microbiota (GM) and energy metabolism. We investigated whether the GM mediates the effects of curcumin on improving energy homeostasis. High-fat diet (HFD)-fed wild type, uncoupling protein 1 (Ucp1) knockout and G protein-coupled membrane receptor 5 (TGR5) knockout mice were treated with curcumin (100 mg kg-1 d-1, p.o.). Curcumin-treated HFD-fed mice displayed decreased body weight gain and augmented cold tolerance due to enhanced adaptive thermogenesis as compared with that in control mice. The anti-obesity effects of curcumin were abolished by Ucp1 knockout. 16S ribosomal DNA sequencing analysis revealed that curcumin restructured the GM in HFD-fed mice. Fecal microbiota transplantation (FMT) and endogenous GM depletion indicated that the GM mediated the enhanced effect of curcumin on Ucp1-dependent thermogenesis. Curcumin altered bile acid (BA) metabolism with increased fractions of circulating deoxycholic acid (DCA) and lithocholic acid (LCA), which are the two most potent ligands for TGR5. Consistently, the enhanced effect of curcumin on Ucp1-dependent thermogenesis was eliminated by TGR5 knockout. Curcumin requires the GM and TGR5 to activate the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway in thermogenic adipose tissue. Here, we demonstrated that the GM mediates the effects of curcumin on enhancing Ucp1-dependent thermogenesis and ameliorating HFD-induced obesity by influencing BA metabolism. We disclosed the potential of nutritional and pharmacologic manipulations of the GM to enhance Ucp1-dependent thermogenesis in the prevention and treatment of obesity.

G protein-coupled receptor kinase 5 deletion suppresses synovial inflammation in a murine model of collagen antibody-induced arthritis.

G protein-coupled receptor kinase 5 (GRK5) regulates inflammatory responses via the nuclear factor-kappa B (NF-κB) pathway. This study investigated the functional involvement of GRK5 in the pathogenesis of inflammatory arthritis. Immunohistochemically, rheumatoid arthritis (RA) synovium had a significantly higher proportion of GRK5-positive cells in the synovial lining layer than healthy control synovium. Gene expression and NF-κB activation in lipopolysaccharide-stimulated human SW982 synovial cells were significantly suppressed by silencing of the GRK5 gene. Similarly, GRK5 kinase activity inhibition in human primary RA synovial cells attenuated gene expressions of inflammatory factors. In a murine model of collagen antibody-induced arthritis, arthritis scores and serum IL6 production of GRK5 knockout (GRK5-/-) mice were significantly lower than those of wild-type mice. Histologically, the degree of synovitis and cartilage degeneration in GRK5-/- mice was significantly lower than in wild-type mice. In in vitro analyses using activated murine macrophages and fibroblast-like synoviocytes, gene expression of inflammatory factors and p65 nuclear translocation were significantly lower in GRK5-/- mice compared to wild-type mice. In conclusion, our results suggested that GRK5 is deeply involved in the pathogenesis of inflammatory arthritis, therefore, GRK5 inhibition could be a potential therapeutic target for types of inflammatory arthritis such as RA.

Cannabinoid 2 receptors regulate dopamine 2 receptor expression by a beta-arrestin 2 and GRK5-dependent mechanism in neuronal cells.

We have previously reported that the repeated exposure to cannabinoids upregulates and enhances the activity of serotonin 2A (5-HT2A) and dopamine 2 (D2) receptors and facilitates the formation of D2-5-HT2A receptor heterodimers in the rat prefrontal cortex and two neuronal cell lines. Because the repeated exposure to cannabinoids has been associated with adverse neuropsychiatric disorders, this study investigated the mechanisms that underly the cannabinoid-mediated regulation of D2 receptor expression in a neuronal cell model, CLU213 cells. We initially tested the effects of repeated exposure (72 h) to a non-selective cannabinoid agonist (1 nM CP55940), a selective CB1 receptor agonist (15 nM ACEA), or a selective CB2 receptor drug (1 nM GP1a) on the expression of postsynaptic D2 (D2L) receptors in CLU213 cells. Repeated CP55940, GP1a, or ACEA treatments significantly increased D2L receptor protein levels (99 % ± 7%, 30 % ± 7%, and 39 % ± 5% increases compared with control levels, respectively). Repeated exposure to both GP1a and ACEA increased D2L receptor protein levels by 73 % ± 8%. Interestingly, CP55940 and GP1a, but not ACEA, upregulated D2 mRNA. Using cells that were stably transfected with short-hairpin RNA (shRNA) lentiviral particles targeting CB2 receptors, G protein-coupled receptor kinase 5 (GRK5), and ß-arrestin 2, we found that CB2 receptors regulated D2 expression through a mechanism that is dependent on GRK5, ß-arrestin 2, and extracellular signal-related kinase (ERK)1/2. We also found that repeated exposure to either ACEA or GP1a selectively stimulated the protein and mRNA expression of GRK proteins. ACEA significantly upregulated GRK2 proteins, whereas GP1a upregulated GRK5 protein expression. Our results identified mechanisms associated with the upregulation of D2 receptors in neuronal cells after the repeated exposure to cannabinoids. These data can shed light on the mechanisms that can be targeted to prevent potential adverse effects, while simultaneously determining the therapeutic benefits of cannabinoids.

Targeting GRK5 for Treating Chronic Degenerative Diseases.

G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors and they are responsible for the transduction of extracellular signals, regulating almost all aspects of mammalian physiology. These receptors are specifically regulated by a family of serine/threonine kinases, called GPCR kinases (GRKs). Given the biological role of GPCRs, it is not surprising that GRKs are also involved in several pathophysiological processes. Particular importance is emerging for GRK5, which is a multifunctional protein, expressed in different cell types, and it has been found located in single or multiple subcellular compartments. For instance, when anchored to the plasma membrane, GRK5 exerts its canonical function, regulating GPCRs. However, under certain conditions (e.g., pro-hypertrophic stimuli), GRK5 translocates to the nucleus of cells where it can interact with non-GPCR-related proteins as well as DNA itself to promote "non-canonical" signaling, including gene transcription. Importantly, due to these actions, several studies have demonstrated that GRK5 has a pivotal role in the pathogenesis of chronic-degenerative disorders. This is true in the cardiac cells, tumor cells, and neurons. For this reason, in this review article, we will inform the readers of the most recent evidence that supports the importance of targeting GRK5 to prevent the development or progression of cancer, cardiovascular, and neurological diseases.