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

• 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
• 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

 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(申请人提供)：肿瘤坏死因子α是一种主要的促炎细胞因子，在2型糖尿病和肥胖症中显著升高，这两种因素是已知的共病心血管危险因素。众所周知，接触肿瘤坏死因子α会导致心脏降压的负性变力作用。尽管有这样的观察，但人们对其潜在机制知之甚少。我们最近发现，肿瘤坏死因子α通过G蛋白偶联受体激酶2导致β肾上腺素能受体(βAR)功能障碍，这可能是肿瘤坏死因子α的心脏负性变力作用的基础。β受体是心功能最强大的调节器之一，βAR脱敏(即儿茶酚胺减少)反应是心力衰竭的标志。β对儿茶酚胺的AR反应减弱是由于β受体的磷酸化，这种磷酸化主要是由GRK2介导的，而GRK2在心脏应激时明显升高。有趣的是，我们的研究表明，肿瘤坏死因子α上调GRK2介导的βAR脱敏，因为GRK2的siRNA击倒使βAR功能正常化，而不是肿瘤坏死因子α。小鼠心肌细胞经肿瘤坏死因子α预处理后，其对β受体激动剂异丙肾上腺素的收缩作用受到抑制，这种收缩作用在α缺失的心肌细胞中有明显的保留作用，提示GRK2在肿瘤坏死因子受体α介导的β受体功能中起一定作用。由于GRK2的募集是由G蛋白的Gβγ亚基介导的，我们确定了α介导的βAR功能能否通过表达Gβγ隔离肽GRK2-ct(βark-ct)来挽救。GRK2-ct虽然保留了β对ISO的AR功能，但不能保留β对α的AR功能。此外，在心脏表达GRK2-ct的转基因小鼠中应用肿瘤坏死因子α可导致与βAR脱敏相关的心功能障碍，尽管儿茶酚胺没有变化。令人惊讶的是，即使在α受体拮抗剂普萘洛尔存在的情况下，肿瘤坏死因子-β-β治疗也能显著地引起β-2AR的磷酸化。这些数据表明，肿瘤坏死因子α以激动剂和Gβγ非依赖的方式介导了GRK2对βAR的脱敏作用，与目前的βAR脱敏模式相反。除了肿瘤坏死因子α的升高，肥胖的小鼠模型(高脂饮食或脂肪分解缺陷脂肪甘油三酯脂肪酶缺失的小鼠(ATGL-/-))的特征是心脏β2AR磷酸化增加和GRK2上调。基于这些令人兴奋的观察结果，我们假设，肿瘤坏死因子α通过非传统的β受体复合体募集βα参与心脏βAR脱敏，提示肿瘤坏死因子GRK2和GRK2信号之间存在尚未确定的串扰。因此，为了从机制上理解肿瘤坏死因子α诱导的βAR脱敏，我们设计了以下研究：具体目的1：确定肿瘤坏死因子α-TNFR 1/2-TRAF 2轴是否促进Gβγ非依赖性的βAR脱敏。特异目的2：明确TRAF2依赖的GRK2向β受体复合体募集的机制。具体目的3：证明条件性心肌细胞GRK2消融(GKR2 Del)是否能改善肥胖小鼠模型的心功能障碍/肥厚。了解这一途径可能提供新的治疗靶点/策略，重要的是，也可能是对肿瘤坏死因子α反应使其他GPCR脱敏的普遍现象。1