Β-arrestin Research Articles

Abstract 36: Ablation of T-Lymphocyte β-Arrestins Reduces Inflammation and Increases Salt Retention in the Absence of Blood Pressure Changes Following Angiotensin II Infusion

Introduction: T-cells contribute to hypertension (HT) development. Among several G-protein coupled receptors regulating T-cell function, it has been hypothesized that some subpopulations use the angiotensin II (ANG II) type 1 receptor (AT 1 R) to modulate immune responses in HT. Selective ablation of AT 1 Rs in T-cells paradoxically increases hypertensive end-organ damage (EOD). While AT 1 Rs typically induces classical G-protein signaling, recent studies have uncovered a protective signaling cascade via β-arrestin ( ARRB ) proteins. We hypothesize that T-cell ARRB s mediate an anti-hypertensive and anti-inflammatory role in response to ANG II, decreasing EOD in HT. Methods: Breeding CD4-Cre mice with Arrb1 -Flox or Arrb2 -Flox mice generated lines with T-cell-specific deficiency of Arrb1 or Arrb2 genes. Hypertensive conditions modeling kidney damage consisted of unilateral nephrectomy, 4% salt diet, and continuous infusion of 1 mg/kg/min ANG II (UNX/HSD/ANG). Age/sex-matched Cre - littermates were used as controls (WT). Blood pressure (BP) was measured at baseline and for the following three weeks using radiotelemetry. Results: At baseline, there were no significant differences in systolic BP between groups (WT:124.1±8.0 mmHg; CD4 Arrb1-null :125.4±8.1; CD4 Arrb2-null :128.7±3.1; p =0.37; n=12/10/8). Three weeks of UNX/HSD/ANG equally increased systolic BP in all groups (WT:178.0±14.0 mmHg; CD4 Arrb1-null :174.8±26.3; CD4 Arrb2-null :179.9±11.1; p =0.86; n=7/9/7). Despite similar BPs, CD4 Arrb2-null mice demonstrated less renal inflammation than WT after four weeks of UNX/HSD/ANG, with a reduction in interleukin-1α protein (3.8±3.2 vs. 8.3±2.8 p <0.05; n=4/6) and a trend towards decreased tumor necrosis factor-α transcript (0.63-fold change vs. 1.9, p =0.051; n=10/8). Furthermore, CD4 Arrb1-null mice increased 24-hour voluntary water intake compared to WT (15.7±3.4 mL vs. 12.2±1.6; p <0.05; n=12/7), and 24-hour urine output (10.0±3.7 mL/d vs. 6.8±1.5; p <0.05; n=11/8). However, there was no difference in sodium excretion during this period (1.9±0.5 eq/d vs. 1.7±0.1; p =0.14; n=11/7). Interestingly, 4 hours after intraperitoneal saline injection, CD4 Arrb1-null mice excreted significantly less sodium compared to WT (5.6±3.5% vs . 31.4±10.6%; p <0.05; n=4/5). Conclusion: Contrary to our hypothesis, these data suggest that T-cell Arrb s do not protect against the hypertensive response to ANG II, but instead play a complicated role in the modulation of hydromineral balance and inflammation.

Human and Mouse Nephrin and Their Interactions With 13 Proteins: An In Silico Study.

Background Human nephrin (hNeph) (podocyte protein) has been known to be involvedin both the formation and maintenance of the slit diaphragm (SD) and also acts as a hub protein in the podocyte by modulating cell polarity, cell survival, cell adhesion, cytoskeletal organization, mechano-sensing, and SD turn-over. Methodology In the present investigation, we aimed to analyse the hNeph and mouse nephrin (mNeph) and their interactions with 13 proteins using the molecular docking method. The 13 selected human proteins which include matrix metalloproteinases (MMP 2 and 9), retinol-binding proteins (RBP 3 and 4), kallikrein 1 (KLK 1), uromodulin, insulin-like growth factor binding protein 7 (IGFBP7), cystatin C, podocin, beta arrestin 1, vang-like protein 2 (VANGL2), dynamin 1, and tensin-like C1 domain-containing phosphatase (TENC1) were studied on the docking analysis of hNeph and mNeph by using the HDOCK (protein-protein) docking method. In addition, the physicochemical (PC) properties of 15 proteins were performed using the ProtParam web server. Results In the present investigation, five chosen human proteins, namely, IGFBP7, cystatin C, podocin, VANGL2, and TENC1, have exhibited theoretical isoelectric point (PI) values greater than 7.0. The protein-protein docking analysis has shown that hKLKand hVANGL2 exhibited the maximum docking score of -206.39 kcal/mol and -329.28 (kcal/mol) with the target proteins mNeph and hNeph, respectively. Conclusions Thus, the current finding highlights the interactions of hNeph and mNeph with 13 chosen proteins, which may help in renal disease management.

Adora2A downregulation promotes caffeine neuroprotective effect against LPS-induced neuroinflammation in the hippocampus

Caffeine has been extensively studied in the context of CNS pathologies as many researchers have shown that consuming it reduces pro-inflammatory biomarkers, potentially delaying the progression of neurodegenerative pathologies. Several lines of evidence suggest that adenosine receptors, especially A1 and A2A receptors, are the main targets of its neuroprotective action. We found that caffeine pretreatment 15 min before LPS administration reduced the expression of Il1b in the hippocampus and striatum. The harmful modulation of caffeine-induced inflammatory response involved the downregulation of the expression of A2A receptors, especially in the hippocampus. Caffeine treatment alone promoted the downregulation of the adenosinergic receptor Adora2A; however, this promotion effect was reversed by LPS. Although administering caffeine increased the expression of the enzymes DNA methyltransferases 1 and 3A and decreased the expression of the demethylase enzyme Tet1, this effect was reversed by LPS in the hippocampus of mice that were administered Caffeine + LPS, relative to the basal condition; no significant differences were observed in the methylation status of the promoter regions of adenosine receptors. Finally, the bioinformatics analysis of the expanded network demonstrated the following results: the Adora2B gene connects the extended networks of the adenosine receptors Adora1 and Adora2A; the Mapk3 and Esr1 genes connect the extended Adora1 network; the Mapk4 and Arrb2 genes connect the extended Adora2A network with the extended network of the proinflammatory cytokine Il1β. These results indicated that the anti-inflammatory effects of acute caffeine administration in the hippocampus may be mediated by a complex network of interdependencies between the Adora2B and Adora2A genes.

Dysregulated Cyclic Nucleotide Metabolism in Alcohol-Associated Steatohepatitis: Implications for Novel Targeted Therapies.

Cyclic nucleotides are second messengers, which play significant roles in numerous biological processes. Previous work has shown that cAMP and cGMP signaling regulates various pathways in liver cells, including Kupffer cells, hepatocytes, hepatic stellate cells, and cellular components of hepatic sinusoids. Importantly, it has been shown that cAMP levels and enzymes involved in cAMP homeostasis are affected by alcohol. Although the role of cyclic nucleotide signaling is strongly implicated in several pathological pathways in liver diseases, studies describing the changes in genes regulating cyclic nucleotide metabolism in ALD are lacking. Male C57B/6 mice were used in an intragastric model of alcohol-associated steatohepatitis (ASH). Liver injury, inflammation, and fibrogenesis were evaluated by measuring plasma levels of injury markers, liver tissue cytokines, and gene expression analyses. Liver transcriptome analysis was performed to examine the effects of alcohol on regulators of cyclic AMP and GMP levels and signaling. cAMP and cGMP levels were measured in mouse livers as well as in livers from healthy human donors and patients with alcohol-associated hepatitis (AH). Our results show significant changes in several phosphodiesterases (PDEs) with specificity to degrade cAMP (Pde4a, Pde4d, and Pde8a) and cGMP (Pde5a, Pde6d, and Pde9a), as well as dual-specificity PDEs (Pde1a and Pde10a) in ASH mouse livers. Adenylyl cyclases (ACs) 7 and 9, which are responsible for cAMP generation, were also affected by alcohol. Importantly, adenosine receptor 1, which has been implicated in the pathogenesis of liver diseases, was significantly increased by alcohol. Adrenoceptors 1 and 3 (Adrb), which couple with stimulatory G protein to regulate cAMP and cGMP signaling, were significantly decreased. Additionally, beta arrestin 2, which interacts with cAMP-specific PDE4D to desensitize G-protein-coupled receptor to generate cAMP, was significantly increased by alcohol. Notably, we observed that cAMP levels are much higher than cGMP levels in the livers of humans and mice; however, alcohol affected them differently. Specifically, cGMP levels were higher in patients with AH and ASH mice livers compared with controls. As expected, these changes in liver cyclic nucleotide signaling were associated with increased inflammation, steatosis, apoptosis, and fibrogenesis. These data strongly implicate dysregulated cAMP and cGMP signaling in the pathogenesis of ASH. Future studies to identify changes in these regulators in a cell-specific manner could lead to the development of novel targeted therapies for ASH.

Determinants of Widespread Metastases and of Metastatic Tropism in Patients with Prostate Cancer: A Genomic Analysis of Primary and Metastatic Tumors

A growing body of evidence suggests that metastatic cancer is better described as a spectrum of disease rather than a binarily defined state, ranging from oligometastatic cancer to widespread metastases. Widespread metastases represent the most common cause of cancer-related death among patients with prostate cancer. Therefore, a greater understanding of the genomic features that determine the extent and location of metastatic spread may inform risk stratification, treatment, and monitoring. We identify genomic alterations from primary prostate tumors that are predictive of widespread metastatic potential. Genomic and clinical data for 1,312 patients with primary prostate adenocarcinomas were extracted from the MSK-MET cohort through cBioPortal. Metastatic site counts and overall survival (OS) data were publicly available for all patients. All samples from primary tumors were profiled using the MSK-IMPACT targeted sequencing platform. Our study focused on 58 genes frequently altered in prostate cancer. Cox proportional hazard analyses defined hazard ratios (HRs) and 95% confidence intervals (CIs) for overall mortality in patients with different metastatic outcomes. Patterns of genomic alterations of the primary tumor associated with metastatic extent and location were compared. Out of 1,312 patients, 939 (71%) developed metastases, and 113 (8.6%) had metastases to 5 or more distinct anatomical sites (defining wide-spread metastases, WSM). Bone was the most common site of metastasis (36%), and 80% of patients with liver metastases had 4 or more additional sites of metastasis. Among patients with metastases, increasing number of metastatic sites was associated with increased risk of death (HR:1.8, 95% CI:1.63-1.99, p<0.001). To define genomic determinants of WSM, we characterized genomic alterations in 58 prostate cancer related genes. Alterations in the following genes were enriched in tumors from patients with WSM vs others: TP53 mutation (40% vs 20%, p<0.0001), FOXA1-amplification (8% vs 3%, p = 0.02), AR-amplification (4.4% vs 1%, p = 0.01), RB1-deletion (5.3% vs 0.7%, p = 0.001), and BRCA2-deletion (4.4% vs 0.7%, p = 0.01). In a univariable survival analysis, all these alterations were predictive of OS (p<0.05). However, on multivariable analysis, only TP53 mutations, and FOXA1 and AR amplifications were independent prognostic factors. Amplifications of FOXA1 (n = 37) and AR (n = 13) were mutually exclusive (0 overlap), and we found that patients who have either AR or FOXA1 amplifications experienced very poor OS (HR:3.57, 95% CI:2.26-5.6, p p<0.001]. We identified genomic alterations (TP53 mutations, FOXA1 and AR amplification, RB1 and BRCA2 deletions) from primary prostate tumors that are predictive of wide-spread metastases and poor outcomes.

Determinants of widespread metastases and of metastatic tropism in patients with prostate cancer: A genomic analysis of primary and metastatic tumors.

5067 Background: A growing body of evidence suggests that metastatic cancer is better described as a spectrum of disease rather than a binarily defined state, ranging from oligometastatic cancer to widespread metastases. Widespread metastases represent the most common cause of cancer-related death among patients with prostate cancer. Therefore, a greater understanding of the genomic features that determine the extent and location of metastatic spread may inform risk stratification, treatment, and monitoring. We identify genomic alterations from primary prostate tumors that are predictive of widespread metastatic potential. Methods: Genomic and clinical data for 1,312 patients with primary prostate adenocarcinomas were extracted from the MSK-MET cohort through cBioPortal. Metastatic site counts and overall survival (OS) data were publicly available for all patients. All samples from primary tumors were profiled using the MSK-IMPACT targeted sequencing platform. Our study focused on 58 genes frequently altered in prostate cancer. Cox proportional hazard analyses defined hazard ratios (HRs) and 95% confidence intervals (CIs) for overall mortality in patients with different metastatic outcomes. Patterns of genomic alterations of the primary tumor associated with metastatic extent and location were compared. Results: Out of 1,312 patients, 939 (71%) developed metastases, and 113 (8.6%) had metastases to 5 or more distinct anatomical sites (defining wide-spread metastases, WSM). Bone was the most common site of metastasis (36%), and 80% of patients with liver metastases had 4 or more additional sites of metastasis. Among patients with metastases, increasing number of metastatic sites was associated with increased risk of death (HR:1.8, 95%CI:1.63-1.99, p&lt;0.001). To define genomic determinants of WSM, we characterized genomic alterations in 58 prostate cancer related genes. Alterations in the following genes were enriched in tumors from patients with WSM vs others: TP53 mutation (40% vs 20%, p&lt;0.0001), FOXA1-amplification (8% vs 3%, p=0.02), AR-amplification (4.4% vs 1%, p=0.01), RB1-deletion (5.3% vs 0.7%, p=0.001), and BRCA2-deletion (4.4% vs 0.7%, p=0.01). In a univariable survival analysis, all these alterations were predictive of OS (p&lt;0.05). However, on multivariable analysis, only TP53 mutations, and FOXA1 and AR amplifications were independent prognostic factors. Amplifications of FOXA1 (n=37) and AR (n=13) were mutually exclusive (0 overlap), and we found that patients who have either AR or FOXA1 amplifications experienced very poor OS (HR:3.57, 95%CI:2.26-5.6, p p&lt;0.001]. Conclusions: We identified genomic alterations (TP53 mutations, FOXA1 and AR amplification, RB1 and BRCA2 deletions) from primary prostate tumors that are predictive of wide-spread metastases and poor outcomes.

Molecular docking analysis of the tumor protein beta arrestin-1 with oxadiazole compounds.

Beta arrestins are a family of adaptor proteins that help in the regulation of signaling and trafficking of various G protein coupled receptors (GPCRs). Six oxadiazole derivatives taken from literature are analyzed for anti-cancer properties. The toxicity profiles of all the drugs were similar to Tamoxifen used as control. Data shows that compounds 2, 4, and 6 exhibited comparably significant molecular interactions with the cancerous protein for further consideration.

87-LB: Binding Kinetics, Bias, Receptor Internalization, and Effects on Insulin Secretion for a Novel GLP1R-GIPR Dual Agonist, HISHS-2001

Background and Aims: To characterize a novel incretin analogue which engages glucagon-like peptide-1 (GLP1R) and glucose-dependent insulinotropic peptide (GIPR) receptors.Methods: Potencies for cAMP production were determined at GLP-1R and GIPR in Flp-In T-REx-293 cells. Assessment of SNAP-tagged GLP-1R internalisation by confocal microscopy, incorporation of GLP-1R into lipid rafts, Gs recruitment to GLP-1R and GIPR with NanoBiT™ used INS-1(832/3) beta cells. Ca2+ responses were recorded in mouse islets by time-lapse confocal microscopy using Cal520 AM.Results: HISHS-2001 displayed higher potency versus tirzepatide for cAMP production at both receptors (Table). GLP-1R beta arrestin-2 recruitment was lower with HISHS-2001, indicating increased bias. Efficacy and potency of recruitment of Gs protein to the GLP-1R were lower for HISHS-2001 vs. tirzepatide (Table). HISHS-2001 showed increased potency for Gs recruitment to the GIPR. GLP-1R endocytosis was lower for HISHS-2001 vs. tirzepatide but both agonists showed increased recruitment of GLP-1R to lipid rafts vs. semaglutide. Ca2+ responses, and potentiation of insulin secretion from mouse islets tended to be higher for HISHS-2001 vs. tirzepatide. Both displayed enhanced insulin secretion from human islets vs. semaglutide (Table).Conclusions: HISHS-2001 may provide a useful new GLP1R-GIP dual agonist.View largeDownload slideView largeDownload slide DisclosureY. Manchanda: None. G. A. Rutter: Advisory Panel; Self; Sun Pharmaceutical Industries Ltd. B. Jones: Research Support; Self; Sun Pharmaceutical Industries Ltd. G. Carrat: None. Z. Ramchunder: None. P. Marchetti: Consultant; Self; Menarini Group. I. Leclerc: Consultant; Spouse/Partner; Sun Pharmaceutical Industries Ltd. R. Thennati: None. V. S. Burade: None. A. Tomas: Other Relationship; Self; Sun Pharmaceutical Industries Ltd.FundingSun Pharmaceutical Industries Ltd. (P79787)

Genetic Factors Associated With Pain Severity, Daily Opioid Dose Requirement, and Pain Response Among Advanced Cancer Patients Receiving Supportive Care

BackgroundCurrent understanding of genetic factors associated with pain severity, and improvement of pain with opioids in advanced cancer patients (AC) is inadequate for delivery of personalized pain therapy (PPT). Therefore, the aim of this study was to determine the genetic factors associated with pain severity, daily opioid dose, and pain response in AC patients receiving supportive care. MethodsIn this prospective study, AC patients were eligible if they had cancer pain ≥4/10 on Edmonton Symptom Assessment Scale (ESAS) - Pain Item and needed opioid rotation for pain control by specialist at the outpatient supportive care center. Association of genetic factors with pain phenotype was assessed using logistic regression models and SKATO (Gene-block) analysis. ResultsAbout 174/178 (98%) patient samples were analyzed. After adjustment for demographic and clinical variables, pain severity was negatively associated with intron variant alleles in OPRM1 rs9322446, P = 0.02; rs2270459, P = 0.038; rs62052210, P = 0.038. Opioid daily dose was positively associated NFKBIA rs2233419, P = 0.008; rs2233417, P = 0.007; rs3138054, P = 0.008; rs1050851, P = 0.015; ORPM1 rs9479759, P = 0.046; rs2003185, P = 0.047; rs636433, P = 0.044; COMT (rs9306234, P = 0.014; rs165728, P = 0.014; rs2020917, P = 0.036; rs165728, P = 0.034); ARRB2 (rs1045280, P = 0.045); and pain response to opioids was negatively associated OPRM1 rs1319339, P = 0.024; rs34427887, P = 0.048; and COMT rs4646316, P = 0.03; rs35478083, P = 0.028, respectively. SKATO analysis showed association between pain severity and CXCL8 (P = 0.0056), and STAT6 (P = 0.0297) genes respectively, and pain response with IL-6 (P = 0.00499). ConclusionsThis study identified that SNPs of OPRM1, COMT, NFKBIA, CXCL8, IL-6, STAT6, and ARRB2 genes were associated with pain severity, opioid daily dose, and pain response in AC receiving supportive care. Additional studies are needed to validate our findings for PPT.

Identification of a β-Arrestin 2 Mutation Related to Autism by Whole-Exome Sequencing.

Autism spectrum disorder (ASD) is a complex neurological disease characterized by impaired social communication and interaction skills, rigid behavior, decreased interest, and repetitive activities. The disease has a high degree of genetic heterogeneity, and the genetic cause of ASD in many autistic individuals is currently unclear. In this study, we report a patient with ASD whose clinical features included social interaction disorder, communication disorder, and repetitive behavior. We examined the patient's genetic variation using whole-exome sequencing technology and found new de novo mutations. After analysis and evaluation, ARRB2 was identified as a candidate gene. To study the potential contribution of the ARRB2 gene to the human brain development and function, we first evaluated the expression profile of this gene in different brain regions and developmental stages. Then, we used weighted gene coexpression network analysis to analyze the associations between ARRB2 and ASD risk genes. Additionally, the spatial conformation and stability of the ARRB2 wild type and mutant proteins were examined by simulations. Then, we further established a mouse model of ASD. The results showed abnormal ARRB2 expression in the mouse ASD model. Our study showed that ARRB2 may be a risk gene for ASD, but the contribution of de novo ARRB2 mutations to ASD is unclear. This information will provide references for the etiology of ASD and aid in the mechanism-based drug development and treatment.

AS129 - Crucial role of beta-arrestin-2 in linking fibrogenesis and cancer

AS129 - Crucial role of beta-arrestin-2 in linking fibrogenesis and cancer

MP37-19 CIRCULATING TUMOR DNA TARGETED SEQUENCING REVEALS GERMLINE AND SOMATIC ALTERATIONS THAT CAN GUIDE DECISION MAKING IN METASTATIC CASTRATION-RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) III (MP37)1 Apr 2020MP37-19 CIRCULATING TUMOR DNA TARGETED SEQUENCING REVEALS GERMLINE AND SOMATIC ALTERATIONS THAT CAN GUIDE DECISION MAKING IN METASTATIC CASTRATION-RESISTANT PROSTATE CANCER Baijun Dong*, Liancheng Fan, Jiahua Pan, and Wei Xue Baijun Dong*Baijun Dong* More articles by this author , Liancheng FanLiancheng Fan More articles by this author , Jiahua PanJiahua Pan More articles by this author , and Wei XueWei Xue More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000886.019AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The genomic landscape of metastatic castration-resistant prostate cancer (mCRPC) is dynamic with the application of multiple treatments. The circulating tumor DNA (ctDNA), which reveals germline and somatic alterations, provides a mini-invasive tool for monitoring tumor evolution. Whether ctDNA sequencing could capture potentially clinically actionable genomic alterations and could guide decision making in mCRPC remains unexplored. This study is to investigate the ability of ctDNA profiling to capture potentially clinically actionable genomic alterations in mCRPC patients, and evaluated their association with outcomes. METHODS: We performed an exploratory analysis of 299 ctDNA samples from 8 centers through application of multiple-gene deep targeted sequencing. The Kaplan-Meier method was used to evaluate time to progression of multiple treatments of mCRPC. RESULTS: The most common recurrent genomic alterations were in AR(34.7%), TP53(18.9%), CDK12(15.4%, 100% in somatic), BRCA2(13.3%). The results showed the frequency of AR amplification and TP53 defect significantly increased in post-second and later line treatment group compared with treatment-naive group. AR amplification, TP53 or RB1 defect were associated with resistance to abiraterone or docetaxel. The patients with CDK12 defect showed rapid resistance to abiraterone and limited efficacy of PARPi. However, these patients seemed to benefit from chemotherapy, espeacially platnium-based chemotherapy. Limitations include a relatively small sample size of different treatment cohorts and relatively short follow-up time of this study. CONCLUSIONS: Our study discovers the genomic landscape of mCRPC at different treatment stages and explores the driver genomic alterations on patient response to the most widely used therapies for mCRPC. ctDNA targeted sequencing can help guide clinical decision making in mCRPC throughout the whole treatment process. These findings established genomic drivers associated with resistance to multiple treatments (including PARPi and platinum-based chemotherapy) in mCRPC. CDK12 might be able to be a novel predictive biomarker to guide treatment selection in mCRPC. Source of Funding: No © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e568-e568 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Baijun Dong* More articles by this author Liancheng Fan More articles by this author Jiahua Pan More articles by this author Wei Xue More articles by this author Expand All Advertisement PDF downloadLoading ...

Identification of Candidate Biomarkers for Idiopathic Thrombocytopenic Purpura by Bioinformatics Analysis of Microarray Data.

Idiopathic Thrombocytopenic Purpura (ITP) is a multifactorial disease with decreased count of platelet that can lead to bruising and bleeding manifestations. This study was intended to identify critical genes associated with chronic ITP. The gene expression profile GSE46922 was downloaded from the Gene Expression Omnibus database to recognize Differentially Expressed Genes (DEGs) by R software. Gene ontology and pathway analyses were performed by DAVID. The biological network was constructed using the Cytoscape. Molecular Complex Detection (MCODE) was applied for detecting module analysis. Transcription factors were identified by the PANTHER classification system database and the gene regulatory network was constructed by Cytoscape. One hundred thirty-two DEGs were screened from comparison newly diagnosed ITP than chronic ITP. Biological process analysis revealed that the DEGs were enriched in terms of positive regulation of autophagy and prohibiting apoptosis in the chronic phase. KEGG pathway analysis showed that the DEGs were enriched in the ErbB signaling pathway, mRNA surveillance pathway, Estrogen signaling pathway, and Notch signaling pathway. Additionally, the biological network was established, and five modules were extracted from the network. ARRB1, VIM, SF1, BUB3, GRK5, and RHOG were detected as hub genes that also belonged to the modules. SF1 also was identified as a hub-TF gene. To sum up, microarray data analysis could perform a panel of genes that provides new clues for diagnosing chronic ITP.

Centrality Analysis of Protein-Protein Interaction Networks and Molecular Docking Prioritize Potential Drug-Targets in Type 1 Diabetes.

Type 1 diabetes (T1D) occurs as a consequence of an autoimmune attack against pancreatic β- cells. Due to a lack of a clear understanding of the T1D pathogenesis, the identification of effective therapies for T1D is the active area in the research. The study purpose was to prioritize potential drugs and targets in T1D via systems biology approach. Gene expression data of peripheral blood mononuclear cells (PBMCs) and pancreatic β-cells in T1D were analyzed and differential expressed genes were integrated with protein-protein interactions (PPI) data. Multiple topological centrality parameters of extracted query-query PPI (QQPPI) networks were calculated and the interaction of more central proteins with drugs was investigated. Molecular docking was performed to further predict the interactions between drugs and the binding sites of targets. Central proteins were identified by the analysis of PBMC (MYC, ERBB2, PSMA1, ABL1 and HSP90AA1) and pancreatic β-cells (HSP90AB1, ESR1, RELA, RAC1, NFKB1, NFKB2, IKBKE, ARRB2 and SRC) QQPPI networks. Thirteen drugs which targeted eight central proteins were identified by further analysis of drug-target interactions. Some drugs which investigated for diabetes treatment in the experimental models of T1D were prioritized by literature verification, including melatonin, resveratrol, lapatinib, geldanamycin, eugenol and fostaminib. Finally, according on molecular docking analysis, lapatinib-ERBB2 and eugenol-ESR1 exhibited highest and lowest binding energy, respectively. This study presented promising results for the prioritization of potential drug-targets which might facilitate T1D targeted therapy and its drug discovery process more effectively.

SAT-361 Rapid Glucocorticoid Regulation of Adrenoreceptor Trafficking Desensitizes CRH Neurons to Noradrenergic Activation

Stress activates the hypothalamic-pituitary-adrenal (HPA) axis via excitatory noradrenergic projections from the brainstem to corticotropin releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus (PVN). The resulting systemic release of glucocorticoids feeds back to the hypothalamus to rapidly suppress HPA activation. Patch-clamp electrophysiology experiments revealed a strong increase in excitatory currents via alpha 1 adrenoreceptor (AR1a) activation, which was desensitized after acute restraint stress or glucocorticoid application. This rapid desensitization was reversed by inhibiting endocytosis, suggesting that desensitization was achieved by AR1a internalization. Live cell imaging of AR1a-GFP in a hypothalamic cell line (mHypoE-N42) revealed a glucocorticoid facilitation of norepinephrine (NE)-induced AR1a internalization. FRET interactions between AR1a-GFP and endocytic markers labeled with dsRed revealed that glucocorticoids prevent rapid recycling of AR1a to the plasma membrane, forcing the receptor further down the endocytic pathway. Glucocorticoid treatment also caused an increase in ubiquitination of the trafficking protein beta arrestin, without affecting phosphorylation of the beta arrestin or AR1a. Thus, glucocorticoids likely cause rapid ubiquitin modifications to beta arrestin that shuttle AR1a to the late endosome and prevent it from recycling to the membrane, desensitizing the neurons to NE. The desensitization was found to last at least 4 hours in both ex vivo slice preparations and in vivo stressed animals with elevated blood corticosterone levels. Long term desensitization specifically to noradrenergic inputs could contribute to a systemic stressor-selective negative feedback regulation of the HPA axis.

A pilot study on the effects of hormonal contraceptives on beta‐arrestin1 levels in peripheral blood mononuclear leukocytes

OBJECTIVEWomen are twice as likely as likely as men to develop depression during reproductive years. Use of hormonal contraceptives, during reproductive years, may be causal with development of depression in a subgroup of women because observational studies indicate an association between use of progestin (Depo‐Provera) and development of depressive symptoms. Previously we have reported reduced levels of beta arrestin 1(β‐AR‐1) during premenstrual depressive disorder, may be a potential biomarker of depression. In this study we compared the β‐AR‐1 protein levels and the Hamilton Depression Rating Scores (HDRS) as well, in women on non‐hormonal contraceptives (control), combine‐only contraceptives (COC), and progestin‐only contraceptives (POC).METHODSStudy participants (n=29) were non‐pregnant women between 18–42 years of age, not taking any antidepressants/therapy and at the luteal phase of menstruation. The participants were evaluated with Neuropsychiatric Interview (DSMIV‐TR). The severity of depression were determined by the HDRS. The protein levels of beta‐arrestin1 in the in peripheral blood mononuclear leukocytes (PBMC) were determined by ELISA.RESULTSThe magnitude of the different parameters for Axis 1 mental disorders were significantly higher and beta arrestin1 protein levels in PBMC were significantly lower in women currently using progestin‐only contraceptives as compared to the control.CONCLUSIONThese findings indicate the possibility of reduced levels of β‐AR‐1 and associated symptoms of depression in a subgroup of women may results from the use of POC.Support or Funding InformationClinical Research Education and Career Development in Minority R25RR17577 (NCRR/NIMHD) and U54 MD007593 (NIMHD) and U54 RR026140 (NCRR) at NIH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

P005 A MUCOSAL AND CHEMOKINE LIGAND FOR THE LYMPHOCYTE CHEMOATTRACTANT RECEPTOR GPR15

Lymphocytes are key mediators of inflammation and IBD, including ulcerative colitis (UC). GPR15 is a chemoattractant receptor for lymphocyte homing to the colon and is implicated in mouse models of colitis. It is expressed by subsets of CD4 and CD8 effector and of CD4 regulatory T cells in both human and mouse. In humans the receptor is highly expressed by presumed pathogenic Th2 cells in the colonic lamina propria of UC patients. In mice it is expressed by colon-homing pathogenic Th17 cells, and deficiency of GPR15 suppresses colitis in the naïve T cell transfer model. Thus GPR15 is considered as a promising target to control the recruitment of pathogenic effector T cells to the colon. The pursuit of the receptor as a drug target has been hampered by the lack of understanding of its physiological ligand. Recently, in an exploratory study included in a patent filed by Novartis, the 9 kDa CC-motif containing cationic polypeptide AP57/colon-derived SUSD2 binding factor (CSBF), encoded by C10orf99 in the human and 2610528A11Rik in the mouse, was reported to trigger Ca2+-mediated signaling upon interaction with GPR15. To describe AP57 as the colon-expressed chemokine ligand for GPR15 (GPR15L). In vitro assays of GPR15 activation (beta arrestin) by GPR15L, competitive GPR15L binding of GPR15 and GPR15L-driven chemotactic migration (Transwell). GPR15L binds and activates GPR15, as well as attracts in a GPR15-dependent manner GPR15-expressing T cells including lymphocytes in colon draining lymph nodes and Vγ3+ thymic precursors of dermal epithelial T cells. Identification of the chemotactic activity of GPR15L, and its characterization as the colon-expressed chemotactic ligand for GPR15, adds to its already reported anti-bacterial activities, and suggests the potential of targeting GPR15L-GPR15 interactions for the development of new drugs intended to improve the clinical management of UC.

P005 A MUCOSAL AND CHEMOKINE LIGAND FOR THE LYMPHOCYTE CHEMOATTRACTANT RECEPTOR GPR15

Lymphocytes are key mediators of inflammation and IBD, including ulcerative colitis (UC). GPR15 is a chemoattractant receptor for lymphocyte homing to the colon and is implicated in mouse models of colitis. It is expressed by subsets of CD4 and CD8 effector and of CD4 regulatory T cells in both human and mouse. In humans the receptor is highly expressed by presumed pathogenic Th2 cells in the colonic lamina propria of UC patients. In mice it is expressed by colon-homing pathogenic Th17 cells, and deficiency of GPR15 suppresses colitis in the naïve T cell transfer model. Thus GPR15 is considered as a promising target to control the recruitment of pathogenic effector T cells to the colon. The pursuit of the receptor as a drug target has been hampered by the lack of understanding of its physiological ligand. Recently, in an exploratory study included in a patent filed by Novartis, the 9 kDa CC-motif containing cationic polypeptide AP57/colon-derived SUSD2 binding factor (CSBF), encoded by C10orf99 in the human and 2610528A11Rik in the mouse, was reported to trigger Ca2+-mediated signaling upon interaction with GPR15. To describe AP57 as the colon-expressed chemokine ligand for GPR15 (GPR15L). In vitro assays of GPR15 activation (beta arrestin) by GPR15L, competitive GPR15L binding of GPR15 and GPR15L-driven chemotactic migration (Transwell). GPR15L binds and activates GPR15, as well as attracts in a GPR15-dependent manner GPR15-expressing T cells including lymphocytes in colon draining lymph nodes and Vγ3+ thymic precursors of dermal epithelial T cells. Identification of the chemotactic activity of GPR15L, and its characterization as the colon-expressed chemotactic ligand for GPR15, adds to its already reported anti-bacterial activities, and suggests the potential of targeting GPR15L-GPR15 interactions for the development of new drugs intended to improve the clinical management of UC.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Impact of SNP-SNP interaction among ABCB1, ARRB2, DRD1 and OPRD1 on methadone dosage requirement in Han Chinese patients.

To evaluate the potential association of the genetic polymorphisms in ABCB1, ARRB2, DRD1 and OPRD1 genes with methadone dosage requirement among Han Chinese opioid-dependent patients. Eight SNPs in ABCB1, ARRB2, DRD1 and OPRD1 genes were selected and genotyped using Sequenom MassARRAY platform among 257 methadone maintenance treatment patients. The required information about stable methadone dose, urine analysis for opioid and socio-demographic characteristics was collected. Generalized multifactor dimensionality reduction method was performed to analyze the SNP-SNP interaction. We found that patients carrying the rs529520TG genotype of OPRD1 probably required higher methadone treatment dosage. A 3-locus SNP-SNP interaction pattern (rs1128503 in ABCB1, rs529520 in OPRD1 and rs1045280 in ARRB2) was significantly associated with the methadone dosage requirement (p=0.029). Our results suggested that specific OPRD1 variants and interaction among polymorphisms in ABCB1, OPRD1 and ARRB2 genes contributes to methadone dosage requirement in Han Chinese opioid-dependent patients.

Role of β-arrestin-1 in penehyclidine hydrochloride-induced inhibition of LPS-caused increase in pulmonary microvascular permeability in human pulmonary microvascular endothelial cells

Objective To evaluate the role of β-arrestin-1 in penehyclidine hydrochloride(PHC)-induced inhibition of lipopolysaccharide(LPS)-caused increase in pulmonary microvascular permeability in human pulmonary microvascular endothelial cells(PMVECs). Methods Human PMVECs were seeded in 6-well plates(2 ml/well)or in culture flasks(4 ml/flask)at the density of 1×105 cells/ml and divided into 5 groups(n=15 each)using a random number table: empty plasmid transfection group(group C), LPS plus empty plasmid transfection group(LPS group), PHC plus LPS plus empty plasmid transfection group(P+ LPS group), LPS plus β-arrestin-1 short hairpin RNA(shRNA)transfection group(LPS+ shRNA group)and PHC plus LPS plus β-arrestin-1 shRNA transfection group(P+ LPS+ shRNA group). In LPS and LPS+ shRNA groups, the cells were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1 shRNA, LPS with the final concentration of 0.1 μg/ml was added at 24 h of incubation, and the cells were then incubated for 1 h. In P+ LPS and P+ LPS+ shRNA groups, the cells were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1 shRNA, PHC with the final concentration of 2 μg/ml was added at 24 h of incubation, LPS with the final concentration of 0.1 μg/ml was added at 1 h of incubation, and the cells were then incubated for 1 h. The cell permeability was measured using Transwell chambers.The expression of heat shock protein(HSP27)was detected by immunofluorescence.The expression of β-arrestin-1, p38 mitogen-activated protein kinase(p38MAPK)and phosphorylated p38MAPK(p-p38MAPK)was detected by Western blot.The ratio of p-p38MAPK/p38MAPK was calculated. Results Compared with group C, the cell permeability was significantly increased, the expression of HSP27 was up-regulated, p-p38MAPK/p38MAPK ratio was increased, and the expression of β-arrestin-1 was down-regulated in LPS, LPS+ shRNA and P+ LPS+ shRNA groups(P 0.05). Compared with group LPS, the cell permeability was significantly decreased, the expression of HSP27 was down-regulated, p-p38MAPK/p38MAPK ratio was decreased, and the expression of β-arrestin-1 was up-regulated in group P+ LPS, and p-p38MAPK/p38MAPK ratio was significantly increased(P 0.05). Compared with group P+ LPS, the cell permeability was significantly increased, the expression of HSP27 was up-regulated, p-p38MAPK/p38MAPK ratio was increased, and the expression of β-arrestin-1 was down-regulated in group P+ LPS+ shRNA(P<0.05). Conclusion The mechanism by which PHC inhibits LPS-induced increase in pulmonary microvascular permeability is totally related to β-arrestin-1 in human PMVECs. Key words: Arrestins; Cholinergic antagonists; Endotoxins; Lung; Capillary permeability