MiR-483-3p promotes dental pulp stem cells osteogenic differentiation via the MAPK signaling pathway by targeting ARRB2.

Human dental pulp stem cells (DPSCs) have become an important component for bone tissue engineering and regenerative medicine due to their ability to differentiate into osteoblast precursors. Two miRNA chip datasets (GSE138180 and E-MTAB-3077) of DPSCs osteogenic differentiation were analyzed respectively to find the expression of miR-483-3p significantly increased in the differentiated groups. We further confirmed that miR-483-3p continued to overexpress during osteogenic differentiation of DPSCs, especially reaching its peak on the 7th day. Moreover, miR-483-3p could significantly promote the expression of osteogenic markers including RUNX2 and OSX, and activate MAPK signaling pathway by inducing phosphorylation of ERK, p38, and JNK. In addition, as a significant gene within the MAPK signaling pathway, ARRB2 was identified as the target gene of miR-483-3p by bioinformatic prediction and experimental verification. In conclusion, we identified miR-483-3p could promote osteogenic differentiation of DPSCs via the MAPK signaling pathway by targeting ARRB2.

GLP-1 and GIP receptors signal through distinct ß-arrestin 2-dependent pathways to regulate pancreatic ß cell function.

Glucagon-like peptide 1 (GLP-1R) and glucose-dependent insulinotropic polypeptide (GIPR) receptors are G-protein-coupled receptors involved in glucose homeostasis. Diabetogenic conditions decrease ß-arrestin 2 (ARRB2) levels in human islets. In mouse ß cells, ARRB2 dampens insulin secretion by partially uncoupling cyclic AMP (cAMP)/protein kinase A (PKA) signaling at physiological doses of GLP-1, whereas at pharmacological doses, the activation of extracellular signal-related kinase (ERK)/cAMP-responsive element-binding protein (CREB) requires ARRB2. In contrast, GIP-potentiated insulin secretion needs ARRB2 in mouse and human islets. The GIPR-ARRB2 axis is not involved in cAMP/PKA or ERK signaling but does mediate GIP-induced F-actin depolymerization. Finally, the dual GLP-1/GIP agonist tirzepatide does not require ARRB2 for the potentiation of insulin secretion. Thus, ARRB2 plays distinct roles in regulating GLP-1R and GIPR signaling, and we highlight (1) its role in the physiological context and the possible functional consequences of its decreased expression in pathological situations such as diabetes and (2) the importance of assessing the signaling pathways engaged by the agonists (biased/dual) for therapeutic purposes.

A Common Variant of ARRB2 Promoter Region Associated with the Prognosis of Heart Failure.

INTRODUCTION: The role of ARRB2 in cardiovascular disease has recently gained increasing attention. However, the association between ARRB2 polymorphisms and heart failure (HF) has not yet been investigated. METHODS: A total of 2,386 hospitalized patients with chronic HF were enrolled as the first cohort and followed up for a mean period of 20.2 months. Meanwhile, ethnically and geographically matched 3,000 individuals without evidence of HF were included as healthy controls. We genotyped the common variant in ARRB2 gene to identify the association between variant and HF. A replicated independent cohort enrolling 837 patients with chronic HF was applied to validate the observed association. A series of function analyses were conducted to illuminate the underlying mechanism. RESULTS: We identified a common variant rs75428611 associated with the prognosis of HF in two-stage population: adjusted p = 0.001, hazard ratio (HR) = 1.31 (1.11-1.54) in additive model and adjusted p = 0.001, HR = 1.39 (1.14-1.69) in dominant model in first-stage population; adjusted p = 0.04, HR = 1.41 (1.02-1.95) in additive model and adjusted p = 0.03, HR = 1.51 (1.03-2.20) in dominant model in replicated stage. However, rs75428611 did not significantly associate with the risk of HF. Functional analysis indicated that rs75428611-G allele increased the promoter activity and the mRNA expression level of ARRB2 by facilitating transcription factor SRF binding but not the A allele. CONCLUSIONS: Our findings demonstrated that rs75428611 in promoter of ARRB2 was associated with the risk of HF mortality. It is a promising potential treatment target for HF.

MiR-365-3p is a negative regulator in IL-17-mediated asthmatic inflammation.

Background: Interleukin-17, the major proinflammatory cytokine secreted by Th17 cells, makes essential contribution to pathogenesis of severe asthma, while the detailed mechanisms, especially the involvement of microRNAs which are also important participants in asthma progression, remains largely unclear. Methods: In this study, we established a house dust mite (HDM) extract-induced murine asthmatic models and the miRNA expression in the lung tissues of mice were profiled by miRNA microarray assay. The effect of miR-365-3p on IL-17-mediated inflammation was examined by qRT-PCR and immunoblotting analysis. The involvement of ARRB2 as target gene of miR-365-3p was verified by overexpression or RNA interference. Results: HDM extract-induced asthmatic inflammation was proved to be IL17-mediated and miR-365-3p was screened out to be the only miRNA exclusively responsive to IL-17. miR-365-3p, whose expression was significantly downregulated upon IL-17 stimulation, was demonstrated to exert remarkable anti-inflammatory effect to decrease IL-17-provoked inflammatory cytokines (KC/IL-8 and IL-6) in both airway epithelial cells and macrophages of murine and human origins, verifying its universal antagonizing activity against IL-17-initiated inflammation across the two species. ARRB2 was characterized as the key target of miR-365-3p to negate IL-17-induced inflammatory cytokines. Conclusion: Taken together, our data supported the notion that miR-365-3p, which was diminished by IL-17 in murine and human asthmatic pathogenesis, functioned as an essential negative mediator in IL-17-stimuated inflammatory response by targeting ARRB2, which would shed new light to the understanding and therapeutics thereof of asthmatic inflammation.

LncRNA GATA3-AS1 promoted invasion and migration in human endometrial carcinoma by regulating the miR-361/ARRB2 axis.

Endometrial carcinoma (EC) is a kind of fatal female malignancy. lncRNA GATA3-AS1 has been identified as an oncogene in various cancers. However, the functions and mechanisms of GATA3-AS1 in EC remain to be explored. Human EC tissues and four EC cell lines were used. Western blotting and quantitative real-time PCR (qRT-PCR) were used to evaluate the expression of GATA3-AS1, miR-361, and ARRB2. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to validate the interaction among GATA3-AS1, miR-361, and ARRB2. Flow cytometry, colony formation assay, scratch assay, and transwell assay were used to examine the cell apoptosis, proliferation, migration, and invasion of EC cells, respectively. In vivo tumor growth was monitored in nude mice. GATA3-AS1 and ARRB2 were upregulated while miR-361 was downregulated in human EC tissues and EC cells. GATA3-AS1 knockdown constrained cell proliferation, invasion, migration, and EMT while promoting the apoptosis of EC cells by upregulating miR-361. GATA3-AS1 negatively regulated miR-361 expression. ARRB2 was the direct target of miR-361 and could activate the Src/Akt pathway. In vivo, GATA3-AS1 knockdown suppressed tumor progression by upregulating the miR-361 expression. lncRNA GATA3-AS1 promoted EC invasion and migration by the miR-361/ARRB2 axis, which indicated that GATA3-AS1 might be a promising therapeutic option for advanced EC progression. KEY MESSAGES: GATA3-AS1 knockdown suppressed EC proliferation, invasion, and migration. GATA3-AS1 directly inhibited miR-361 as a ceRNA. MiR-361 knockdown reversed the tumor suppressive effect caused by GATA3-AS1 knockdown. MiR-361 bound to ARRB2 directly and suppressed its expression. The GATA3-AS1/miR-361/ARRB2 axis regulated EC cell proliferation, invasion, and migration.

ß-arrestin2 mediates the hippocampal dopaminergic system in autistic mouse through the ERK signaling pathway.

Autism is a complex neurodevelopmental disease that may be caused by genetic and environmental factors, that are incompletely understood. Overactivation of dopaminergic receptors can lead to autistic-like behavior. ß-arrestin2 (Arrb2) is a scaffolding protein of the arrestin family, which function as cytosolic multifunctional adapter proteins that activate cell signal transduction and mediate the signal termination and endocytosis of G-protein-coupled receptors (GPCRs) complexes. In this study, we established an Arrb2 knockout (Arrb2-/-) mouse to explore the biological function of Arrb2 in autistic-like behavior caused by abnormality in the dopaminergic system. We found that Arrb2-/- mice did not exhibit the autistic-like behavior normally induced by SKF38393, an agonist of the dopamine receptor 1 (D1R). Compared with wild-type (WT) untreated mice, the SKF38393-treated WT mice and Arrb2-/- mice, with or without SKF38393 treatment, showed abnormalities on electroencephalography (EEG) and increased stimulation of the phosphorylated form of extracellular signal-regulated kinase (p-ERK) via the PKA/Rap1/B-Raf/MEK pathway. These results demonstrated that Arrb2 regulated the dopaminergic system through the ERK signaling pathway in the occurrence and development of autism, and that targeted deletion of Arrb2 impeded the development of autistic-like behavior.

Association of polymorphisms in ARRB2 and clinical response to methadone for pain in advanced cancer.

Background: The prescription of methadone in advanced cancer poses multiple challenges due to the considerable interpatient variation seen in effective dose and toxicity. Previous reports have suggested that ARRB2 influences the response to methadone in opioid substitution therapy. Associations with opioid response for pain management in advanced cancer are conflicting, with no studies including methadone as the primary intervention. Methods: In a prospective, multicenter, open-label dose-individualization study, we investigated whether polymorphisms in ARRB2 were associated with methadone dose requirements and pain severity. Results: Significant associations were found for rs3786047, rs1045280, rs2036657 and pain score. Conclusion: While studies are few and the sample size small, ARRB2 genotyping may assist in individualized management of the most feared symptom in advanced cancer.

Arrb2 causes hepatic lipid metabolism disorder via AMPK pathway based on metabolomics in alcoholic fatty liver.

BACKGROUND AND AIMS: Alcoholic fatty liver (AFL) is an early form of alcoholic liver disease (ALD) that usually manifests as lipid synthesis abnormalities in hepatocytes. ß-arrestin2 (Arrb2) is involved in multiple biological processes. The present study aimed to explore the role of Arrb2 in the regulation of lipid metabolism in AFL and the underlying mechanism and identify potential targets for the treatment of AFL. METHODS: The expression of Arrb2 was detected in liver tissues obtained from AFL patients and Gao-binge AFL model mice. In addition, we specifically knocked down Arrb2 in AFL mouse liver in vivo and used Arrb2-siRNA or pEX3-Arrb2 to silence or overexpress Arrb2 in AML-12 cells in vitro to explore the functional role and underlying regulatory mechanism of Arrb2 in AFL. Finally, we investigated whether Arrb2 could cause changes in hepatic lipid metabolites, thereby leading to dysregulation of lipid metabolism based on liquid chromatography-mass spectrometry (LC-MS) analysis. RESULTS: Arrb2 was up-regulated in the livers of AFL patients and AFL mice. The in vivo and in vitro results confirmed that Arrb2 could induce lipid accumulation and metabolism disorders. Mechanistically, Arrb2 induced hepatic metabolism disorder via AMP-activated protein kinase (AMPK) pathway. The results of LC-MS analysis revealed that hepatic lipid metabolites with the most significant differences were primary bile acids. CONCLUSIONS: Arrb2 induces hepatic lipid metabolism disorders via AMPK pathway in AFL. On one hand, Arrb2 increases fatty acid synthesis. On the other hand, Arrb2 could increase the cholesterol synthesis, thereby leading to the up-regulation of primary bile acid levels.

ARRB2 promotes colorectal cancer growth through triggering WTAP.

Colorectal cancer (CRC) is one of the most lethal cancers worldwide. The expression of ß-arrestin2 (ß-Arr2, ARRB2) in CRC has been well investigated; however, its exact mechanism causing the cancer progression remains unclear. In this study, we discovered that the expression level of ARRB2 was significantly upregulated in CRC as compared to the normal tissues by employing the Cancer Genome Atlas (TCGA) data, western blot analysis, and immunohistochemistry. Furthermore, the level of ARRB2 was correlated with the patients' overall survival by Kaplan-Meier analysis. The higher expression of ARRB2 promoted CRC cell growth, enhanced the cell motility, and blocked cell apoptosis, which is crucial for tumor growth. Lastly, the suppression of ARRB2 expression was enough to attenuate the progression of CRC induced by azoxymethane/dextran sodium sulfate. Interestingly, we also found that the knockdown of ARRB2 decreased several cancer pathways mediated by the expression of Wilms tumor 1 associated protein (WTAP), which led to the inhibition of cell proliferation and migration. Altogether, our results demonstrated that ARRB2 promoted the growth and migration of CRC cells by regulating the WTAP expression.

The Role of ß-Arrestins in Regulating Stem Cell Phenotypes in Normal and Tumorigenic Cells.

ß-Arrestins (ARRBs) are ubiquitously expressed scaffold proteins that mediate inactivation of G-protein-coupled receptor signaling, and in certain circumstances, G-protein independent pathways. Intriguingly, the two known ARRBs, ß-arrestin1 (ARRB1) and ß-Arrestin2 (ARRB2), seem to have opposing functions in regulating signaling cascades in several models in health and disease. Recent evidence suggests that ARRBs are implicated in regulating stem cell maintenance; however, their role, although crucial, is complex, and there is no universal model for ARRB-mediated regulation of stem cell characteristics. For the first time, this review compiles information on the function of ARRBs in stem cell biology and will discuss the role of ARRBs in regulating cell signaling pathways implicated in stem cell maintenance in normal and malignant stem cell populations. Although promising targets for cancer therapy, the ubiquitous nature of ARRBs and the plethora of functions in normal cell biology brings challenges for treatment selectivity. However, recent studies show promising evidence for specifically targeting ARRBs in myeloproliferative neoplasms.

The Tumor Suppressor TGFBR3 Blocks Lymph Node Metastasis in Head and Neck Cancer.

The TGF-ß type III receptor (TGFBR3) is an essential constituent of the TGF-ß signaling. In this study, we observed a down-regulation of TGFBR3 in oral cancer, a subtype of head and neck cancer (HNC), and patients with low TGFBR3 had poor clinical outcomes. Ectopic expression of TGFBR3 decreased migration and invasion of oral cancer cells and lymph node metastasis of tumors, whereas depletion of TGFBR3 had the opposite effect. In SMAD4-positive OC-2 oral cancer cells, TGFBR3-mediated suppression requires both of its cytoplasmic interacting partners ARRB2 and GIPC1. We demonstrated that TGFBR3 induces the abundance of secreted angiogenin (ANG), a known pro-angiogenic factor, and ANG is essential and sufficient to mediate TGFBR3-dependent inhibition of migration and invasion of oral cancer cells. Notably, in SMAD4-deficient CAL-27 oral cancer cells, only GIPC1 is essential for TGFBR3-induced suppressive activity. Accordingly, HNC patients with low expressions of both TGFBR3 and GIPC1 had the poorest overall survival. In summary, we conclude that TGFBR3 is as a tumor suppressor via SMAD4-dependent and -independent manner in both tumor and stromal cells during oral carcinogenesis. Our study should facilitate the possibility of using TGFBR3-mediated tumor suppression for HNC treatment.

Repeated social defeat promotes persistent inflammatory changes in splenic myeloid cells; decreased expression of ß-arrestin-2 (ARRB2) and increased expression of interleukin-6 (IL-6).

BACKGROUND: Previous studies suggest that persistent exposure to social stress in mammals may be associated with multiple physiological effects. Here, we examine the effects of social stress in rats, i.e. repeated social defeat, on behavior, hypothalamic-pituitary-adrenal (HPA)-axis and immune system. METHODS: A resident-intruder paradigm, where an intruder rat was exposed to social stress by a dominant resident rat for 1 hour each day for 7 consecutive days was used. The day after the last stress exposure in the paradigm the data were analyzed. Variation in social interaction was observed manually, whereas locomotion was analyzed off-line by a purpose-made software. Gene expression in the pituitary gland, adrenal gland and myeloid cells isolated from the spleen was measured by qPCR. RESULTS: The exposure to social stress induced decreased weight gain and increased locomotion. An increased nuclear receptor subfamily group C number 1 (NR3C1) expression in the pituitary gland was also shown. In myeloid cells harvested from the spleen, we observed decreased expression of the ß2-adrenergic receptor (ADRB2) and ß-arrestin-2 (ARRB2), but increased expression of interleukin-6 (IL-6). Subsequent analyses in the same cells showed that ARRB2 was negatively correlated with IL-6 following the stress exposure. CONCLUSION: Our results show that that the experience of social stress in the form of repeated social defeat in rats is a potent stressor that in myeloid cells in the spleen promotes persistent inflammatory changes. Future research is needed to examine whether similar inflammatory changes also can explain the impact of social stress, such as bullying and harassment, among humans.

Age-dependent effects of dopamine receptor inactivation on cocaine-induced behaviors in male rats: Evidence of dorsal striatal D2 receptor supersensitivity.

N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), which irreversibly inactivates dopamine (DA) receptors, causes pronounced age-dependent behavioral effects in rats. For example, EEDQ either augments or does not affect the DA agonist-induced locomotor activity of preweanling rats while attenuating the locomotion of adolescent and adult rats. The twofold purpose of this study was to determine whether EEDQ would: (a) potentiate or attenuate the cocaine-induced locomotor activity of preweanling, adolescent, and adult rats; and (b) alter the sensitivity of surviving D2 receptors. Rats were treated with vehicle or EEDQ (2.5 or 7.5 mg/kg) on postnatal day (PD) 17, PD 39, and PD 84. In the behavioral experiments, saline- or cocaine-induced locomotion was assessed 24 hr later. In the biochemical experiments, dorsal striatal samples were taken 24 hr after vehicle or EEDQ treatment and later assayed for NPA-stimulated GTPγS receptor binding, G protein-coupled receptor kinase 6 (GRK6), and ß-arrestin-2 (ARRB2). GTPγS binding is a direct measure of ligand-induced G protein activation, while GRK6 and ARRB2 modulate the internalization and desensitization of D2 receptors. Results showed that EEDQ potentiated the locomotor activity of preweanling rats, while attenuating the locomotion of older rats. NPA-stimulated GTPγS binding was elevated in EEDQ-treated preweanling rats, relative to adults, indicating enhanced functional coupling between the G protein and receptor. EEDQ also reduced ARRB2 levels in all age groups, which is indicative of increased D2 receptor sensitivity. In sum, the present results support the hypothesis that D2 receptor supersensitivity is a critical factor mediating the locomotor potentiating effects of EEDQ in cocaine-treated preweanling rats.

ß-Arrestin 2 Promotes Hepatocyte Apoptosis by Inhibiting Akt Pathway in Alcoholic Liver Disease.

Alcoholic liver disease (ALD) is a complex process that includes a wide range of hepatic lesions, from steatosis to cirrhosis, and even hepatocellular carcinoma (HCC). Accumulating evidence shows that the cytotoxic effects of ethanol metabolism lead to cell apoptosis and necrosis in ALD. Recently, several studies revealed that multifunctional protein ß-arrestin 2 (Arrb2) modulated cell apoptosis in liver fibrosis and HCC, but its role in ALD has not been fully understood. The aim of this study is to explore the function and underlying mechanism of Arrb2 in hepatocyte survival and apoptosis in ALD. In our study, the primary hepatocytes were isolated from the livers of C57BL/6 mice fed EtOH-containing diet, it showed an increased level of Arrb2. EtOH also significantly up-regulated Arrb2 production in AML-12 cells in vitro. Furthermore, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) and FCM results demonstrated that knockdown of Arrb2 could inhibit hepatocyte apoptosis induced by EtOH in vivo and vitro while over-expression of Arrb2 induced apoptosis in ALD. In addition, western blot results revealed that Arrb2 remarkably suppressed the Akt signaling. Taken together, our data suggested that Arrb2 may serve as a potential therapeutic target for ALD by promoting hepatocyte apoptosis via Akt suppression.

The association of ß-arrestin2 polymorphisms with response to antidepressant treatment in depressed patients.

The study of genetic polymorphisms involved in antidepressants (AD) response is essential to provide a personalized medicine approach in the field of depression. ß-arrestin 2 (ARRB2) is a candidate gene in the pharmacogenetics of AD as it is involved in the signaling cascade downstream of numerous neurotransmitter receptors. We investigated the association between five ARRB2 single nucleotide polymorphisms (SNPs): rs1045280, rs2036657, rs4790694, rs3786047 and rs452246, and response to AD treatment in a sample of 569 patients with a major depressive episode treated for 6months. We show that GG/GT patients for rs4522461 (n=534) and AA/AC patients for rs4790694 (n=244) have a lower response to AD than other genotype groups (HDRS score of 10.9 vs 8.0 after 6months, multivariate analysis: p=0.03; 12.2 vs 9.6, p=0.02, respectively). These data provide additional evidence that ß-arrestin 2 is a regulator of intracellular signal transduction processes involved in AD treatment.

Breeding and genotype identification of Arrb2 gene knockout mice / 中国药理学通报

Aim To explore the optimal way of breeding and genotype identification of Arrb2 knockout mice, and to find a simple and quick polymerase chain reaction ( PCR) method for the genotyping of Arrb2 knockout mice. Methods Breeding homozygote genotype of Arrb2 gene knockout mice were copula-ted with wild-type C57BL/6J mice, and then the heterozygous mating were used for mating. The growth and development of off-spring were observed. The genomic DNA was extracted from the tail of two-week-old mice. PCR was employed to amplify the Arrb2 gene fragment, and electrophoresis was used to present the gene type. Results The breeding and reproducing were successful and three genotype offspring, including wild-type,heterozygous and homozygous knockout mice were obtained. Agarose gel electrophoresis results showed the size of PCR prod-ucts was about 186 bp and 224 bp, which was consistent with the expected target gene fragment, and identified Arrb2 gene knockout mice of different genotypes successfully. Western blot analysis demonstrated the lack ofβ-arrestin2 protein in the major organs from Arrb2 -/ - mice compared with Arrb2 +/ + and Arrb2 +/ - mice. Conclusions It is feasible to obtain the homo-zygous Arrb2 knockout mice by inbreeding heterozygotes. It is simple, rapid and reliable to identify mouse genetype by PCR.

Association between β-arrestin2 genetic polymorphism and response to methadone maintenance treatment in heroin-dependent patients in Han population in Hunan province / 中华流行病学杂志

Objective To study the distributions of three single nucleotide polymorphisms (SNPs) in β-arrestin2 (ARRB2) which including rs3786047,rs1045280 and rs2036657 and to elucidate the relationship between these SNPs and response to methadone maintenance treatment (MMT) among heroin-dependent patients of Han ethnicity population in Hunan.Methods Han MMT patients were recruited in four random-chosen MMT clinics from Hunan province.Demographics,history of drug-use and MMT were recorded.ARRB2 SNPs were genotyped to determine the association between SNPs and response to MMT.Results Distributions of the three SNPs were in Hardy-Weinberg equilibrium in both groups (responders vs.non-responders).There was no statistical significance in the distribution frequency of genotype on rs3786047 (x2=0.486 2,P=0.784),rs1045280 (x2=1.591 9,P=0.451) and rs2036657 (x2=1.061 5,P=0.588) in ARRB2 among the responders or the non-responders.Conclusion Associations between the ARRB2 genotypes,rs3786047,rs1045280 and rs2036657,and MMT response in Han MMT patients in Hunan province did not appear.

Association between β-arrestin2 genetic polymorphism and response to methadone maintenance treatment in heroin-dependent patients in Han population in Hunan province / 中华流行病学杂志

Objective To study the distributions of three single nucleotide polymorphisms (SNPs) in β-arrestin2 (ARRB2) which including rs3786047,rs1045280 and rs2036657 and to elucidate the relationship between these SNPs and response to methadone maintenance treatment (MMT) among heroin-dependent patients of Han ethnicity population in Hunan.Methods Han MMT patients were recruited in four random-chosen MMT clinics from Hunan province.Demographics,history of drug-use and MMT were recorded.ARRB2 SNPs were genotyped to determine the association between SNPs and response to MMT.Results Distributions of the three SNPs were in Hardy-Weinberg equilibrium in both groups (responders vs.non-responders).There was no statistical significance in the distribution frequency of genotype on rs3786047 (x2=0.486 2,P=0.784),rs1045280 (x2=1.591 9,P=0.451) and rs2036657 (x2=1.061 5,P=0.588) in ARRB2 among the responders or the non-responders.Conclusion Associations between the ARRB2 genotypes,rs3786047,rs1045280 and rs2036657,and MMT response in Han MMT patients in Hunan province did not appear.