Non-canonical regulation of GRK2 by TNFalpha impairs BetaAR function

TNFα 对 GRK2 的非规范调节会损害 BetaAR 功能

基本信息

批准号：
9243307
负责人：
Sathyamangla V Prasad
金额：
$ 39.63万
依托单位：
CLEVELAND CLINIC LERNER COM-CWRU
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-01 至 2020-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9243307
关键词：
ADRBK1 gene Ablation Adenylate Cyclase Adipose tissue Adrenergic beta-Agonists Adrenergic beta-Antagonists Adult Agonist Cardiac Cardiac Myocytes Catecholamines Cells Co-Immunoprecipitations Complement Complex Coupled Coupling Cyclic AMP Data Diabetes Mellitus Fatty acid glycerol esters Functional disorder G protein coupled receptor kinase G-Protein-Coupled Receptors G-substrate GTP-Binding Proteins Heart Heart Hypertrophy Heart failure High Fat Diet Hypertrophy Impairment In Vitro Inflammation Inflammatory Isoproterenol Knock-out Knockout Mice Knowledge Link Lipase Lipolysis Maps Measures Mediating Metabolic syndrome Mus Muscle Cells Myocardial dysfunction Myocarditis Neonatal Non-Insulin-Dependent Diabetes Mellitus Obese Mice Obesity Pathology Pathway interactions Patients Peptides Phosphorylation Phosphotransferases Process Propranolol Proteins Receptor Signaling Recruitment Activity Regulation Role Scaffolding Protein Sepsis Small Interfering RNA Stress TNF gene TNFRSF1A gene TNFRSF1B gene Transgenic Mice Triglycerides Tumor Necrosis Factor Receptor Up-Regulation base beta-adrenergic receptor beta-arrestin cardiovascular risk factor cytokine deletion analysis desensitization design knock-down mouse model new therapeutic target protein expression public health relevance receptor receptor function response

项目摘要

DESCRIPTION (provided by applicant): Tumor necrosis factor alpha (TNFα) is a major pro-inflammatory cytokine that is significantly elevated in Type 2 diabetes and obesity which are known co-morbid cardiovascular risk factors. It is known that TNFα exposure leads to cardio-depressant negative inotropic effects. Despite this observation, little is understood about the underlying mechanisms. We have recently shown that TNFα causes β-adrenergic receptor (βAR) dysfunction through G-protein coupled receptor kinase 2 (GRK2) which may underlie cardio-depressant negative inotropic effects of TNFα. βARs are one of the most powerful regulators of cardiac function and βAR desensitization (i.e., diminished catecholamine) response is a hallmark of heart failure. Reduced βAR response to catecholamines is due to phosphorylation of βARs that is predominantly mediated by GRK2 which is markedly elevated in cardiac stress. Interestingly, our studies show that TNFα up-regulates GRK2 mediating βAR desensitization as siRNA knock down of GRK2 normalizes βAR function despite TNFα. TNFα pre-treatment of murine cardiomyocytes inhibits contractility to βAR agonist isoproterenol (ISO) that is remarkably preserved in GRK2 null myocytes indicating a role for GRK2 in TNFα mediated βAR function. Since GRK2 recruitment is mediated by Gβγ subunits of G-proteins, we determined whether TNFα mediated βAR function could be rescued by expression of Gβγ sequestering peptide GRK2-ct (βARK-ct). Although GRK2-ct preserved βAR function to ISO, it could not preserve βAR function to TNFα. Furthermore, TNFα administration in GRK2-ct transgenic mice with cardiac expression of GRK2-ct resulted in cardiac dysfunction associated with βAR desensitization despite no changes in catecholamines. Surprisingly, TNFα treatment resulted in marked β2AR phosphorylation even in the presence of βAR antagonist propranolol. These data suggest that TNFα mediates βAR desensitization by GRK2 in an agonist- and Gβγ-independent manner contrary to the current paradigm of βAR desensitization. In addition to elevation in TNFα, mouse models of obesity (mice on high fat-diet or lipolysis deficient adipose triglyceride lipase null mice (ATGL-/-)) are characterized by increased cardiac β2AR phosphorylation and upregulation of GRK2. Based on these exciting observations, we hypothesize that TNFα contributes to cardiac βAR desensitization by non-traditional GRK2 recruitment to the βAR complex suggesting a yet, unidentified cross-talk between TNFα and βAR signaling. Thus, to mechanistically understand TNFα- induced βAR desensitization, we have designed the following studies: Specific Aim 1: To determine whether TNFα-TNFR1/2-TRAF2 axis facilitates Gβγ-independent GRK2-mediated βAR desensitization. Specific Aim 2: To identify the mechanism of TRAF2-dependent GRK2 recruitment to βAR complex. Specific Aim 3: To demonstrate whether conditional cardiomyocyte GRK2 ablation (GKR2 del) in mice will ameliorate cardiac dysfunction/hypertrophy in mouse models of obesity. Understanding this pathway may provide novel therapeutic targets/strategies and importantly could also be a universal phenomenon of desensitizing other GPCRs in response to TNFα. 1

描述（由申请人提供）：肿瘤坏死因子α（TNFα）是一种主要的促炎细胞因子，在2型糖尿病和肥胖症中显着升高，已知TNFα暴露会导致心脏病。 - 抑制负性肌力作用。尽管如此，但我们最近发现 TNFα 通过 G 蛋白受体偶联激酶 2 导致 β-肾上腺素能受体 (βAR) 功能障碍。 (GRK2) 可能是 TNFα 的心脏抑制负性肌力作用的基础，它是心脏功能最强大的调节剂之一，βAR 脱敏（即儿茶酚胺）反应减少是心力衰竭的标志。我们的研究表明，βAR 的磷酸化主要由 GRK2 介导，GRK2 在心脏应激时显着升高。尽管 TNFα 预处理小鼠心肌细胞会抑制对 βAR 激动剂异丙肾上腺素 (ISO) 的收缩性，但 siRNA 敲低 GRK2 可使 βAR 功能正常化，从而上调 GRK2 介导的 βAR 脱敏，这表明 GRK2 在 TNFα 中发挥作用。由于 GRK2 募集是由 G 蛋白的 Gβγ 亚基介导的，因此我们确定 TNFα 介导的 βAR 功能是否可以通过 Gβγ 隔离肽 GRK2-ct (βARK-ct) 的表达来挽救，尽管 GRK2-ct 保留了 ISO 的 βAR 功能，但它不能保留 TNFα 的 βAR 功能。心脏表达 GRK2-ct 的 ct 转基因小鼠导致与 βAR 脱敏相关的心脏功能障碍，尽管儿茶酚胺没有变化，但令人惊讶的是，TNFα 治疗导致了心脏功能障碍。即使在存在 βAR 拮抗剂普萘洛尔的情况下，β2AR 也显着磷酸化。这些数据表明，TNFα 以不依赖于激动剂和 Gβγ 的方式介导 βAR 脱敏，这与目前的 βAR 脱敏模式相反。肥胖症（高脂肪饮食的小鼠或脂肪分解缺陷的脂肪甘油三酯脂肪酶缺失小鼠（ATGL-/-））的特征基于这些令人兴奋的观察结果，我们发现 TNFα 通过非传统的 GRK2 募集到 βAR 复合物来促进心脏 βAR 脱敏，这表明 TNFα 和 βAR 信号传导之间存在尚未确定的相互作用。，为了从机制上理解 TNFα 诱导的 βAR 脱敏，我们设计了以下研究：具体目标 1：确定是否TNFα-TNFR1/2-TRAF2 轴促进 Gβγ 独立的 GRK2 介导的 βAR 脱敏。具体目标 2：确定 TRAF2 依赖性 GRK2 募集到 βAR 复合物的机制。具体目标 3：证明条件性心肌细胞 GRK2 消融（GKR2 del）是否存在。）在小鼠中将改善肥胖小鼠模型的心脏功能障碍/肥大。了解这一途径可能会提供新的治疗靶点/策略。重要的是，其他 GPCR 对 TNFα 1 的反应也可能是一种普遍现象。