GH Receptor Proteolysis and Shedding

描述（由申请人提供）： 生长激素（GH）是一种垂体源性肽类激素，具有强有力的促生长和代谢作用。GH与其受体（GHR）结合，GHR是一种富含肝脏、肌肉、脂肪和心脏的细胞表面跨膜蛋白，对人类和其他脊椎动物的燃料代谢、肌肉质量和能量稳态产生深远影响。GH敏感性在急性和慢性疾病状态下发生改变，在这种情况下，不确定增强或抑制GH作用是否有益;这一点很重要，因为现在存在激动剂和拮抗剂。我的实验室一直专注于了解GH敏感性的生理和病理生理决定因素，这是GH水平，细胞表面GHR的丰度，以及GHR激活与细胞内信号通路的耦合的综合效应。在之前和当前的VA资助周期中，我们发现了一种调节GH反应性的新机制，涉及由金属蛋白酶TACE（肿瘤坏死因子-a裂解酶）和TACE抑制剂TIMP 3（金属蛋白酶组织抑制剂-3）组成的蛋白水解系统，其通过蛋白水解脱落受体胞外结构域诱导改变表面GHR的可用性。然而，这种金属蛋白水解系统在生理和病理生理状态下调节GH信号传导的程度尚不清楚。认识到生长激素水平的变化，每日和营养状况，我们最近检查这些因素是否影响生长激素受体丰度和生长激素敏感性。我们在小鼠中的初步数据表明，肝脏GHR可用性和相应的GH敏感性以一天中的时间依赖性方式与TIMP 3水平协调变化。此外，饮食诱导的肥胖也影响肝脏GHR丰度。根据最近的报道，我们认为了解这些生理和病理生理变量如何影响GH敏感性将是至关重要的，合理利用GH增强或拮抗治疗策略，在危重病人。在这 根据该提议，我们将检验两个主要假设：1）肝脏GHR丰度以一天中的时间依赖性方式被金属蛋白水解调节。营养因素也影响肝脏GHR丰度，但在转录水平。这两种形式的调节有助于GH的敏感性和能量稳态。2)以败血症为例的危重疾病通过TACE介导的GHR蛋白水解使肝脏对GH急性脱敏。一天中的时间和饮食决定的GHR丰度和危重疾病的时间和程度的净效应决定了急性肝脏GH反应性，因此可能影响疾病的进程。我们将使用我们开发的新型小鼠模型系统和试剂和技术来实现两个具体目标：1.在小鼠中检查肝脏GHR水平昼夜变化的潜在机制以及这种时间效应对肝脏GH敏感性的影响。2.使用脓毒症小鼠模型（脂多糖（LPS）给药），确定一天中的时间和喂养方案如何影响急性疾病调节肝脏GH敏感性的倾向。这些研究的成功完成将明确地将肝脏GH敏感性的反应归因于临床相关的急性疾病，从而能够在败血症和其他形式的危重疾病中合理地利用GH轴进行治疗。

DESCRIPTION (provided by applicant): Growth hormone (GH) is a pituitary-derived peptide hormone with potent somatogenic and metabolic actions. GH binds its receptor (GHR), a cell surface transmembrane protein enriched in liver, muscle, fat, and heart to exert profound effects on fuel metabolism, muscle mass, and energy homeostasis in humans and other vertebrates. GH sensitivity is altered in the setting of acute and chronic disease states and it is uncertain whether enhancing or inhibiting GH action would be salutary in such situations; this is important, as both agonists and antagonists now exist. My laboratory has focused on understanding physiologic and pathophysiologic determinants of GH sensitivity, which is the integrated effect of GH levels, the abundance of cell surface GHR, and the coupling of GHR activation with intracellular signaling pathways. In previous and the current VA funding cycles, we uncovered a novel mechanism modulating GH responsiveness, involving a proteolytic system composed of the metalloprotease TACE (tumor necrosis factor-a cleaving enzyme) and the TACE inhibitor TIMP3 (tissue inhibitor of metalloproteinases-3) that inducibly alters surface GHR availability by proteolytically shedding the receptor extracellular domain. However, the extent to which this metalloproteolytic system modulates GH signaling in physiological and pathophysiological states is unknown. Realizing that GH levels vary diurnally and with nutritional status, we recently examined whether such factors affect GHR abundance and GH sensitivity. Our preliminary data in mice suggest that hepatic GHR availability and corresponding GH sensitivity vary in a time-of-day-dependent fashion coordinate with the level of TIMP3. Furthermore, diet-induced obesity also affects hepatic GHR abundance. In light of recent reports, we believe understanding how these physiologic and pathophysiologic variables affect GH sensitivity will be crucial to rationally exploit GH-enhancing or -antagonistic therapeutic strategies in critically-ill individuals. In this proposal, we will test two main hypotheses: 1) Hepatic GHR abundance is metalloproteolytically modulated in a time-of-day-dependent fashion. Nutritional factors also affect hepatic GHR abundance, but at a transcriptional level. Both forms of regulation contribute to GH sensitivity and energy homeostasis in the steady state. 2) Critical illness, exemplified by sepsis, acutely desensitizes the liver to GH via TACE- mediated GHR proteolysis. Net effects of time-of-day- and diet-determined GHR abundance and timing and degree of critical illness determine acute hepatic GH responsiveness and may thus impact the course of illness. We will use novel mouse model systems and reagents and techniques we have developed to pursue two specific objectives: 1. Examine in mice potential mechanisms of diurnal variation in liver GHR levels and the impact of such time-of-day effects on hepatic GH sensitivity. 2. Determine how the time of day and feeding regimen impact the propensity of acute illness to modulate hepatic GH sensitivity, using a mouse model of sepsis (lipopolysaccharide (LPS) administration). Successful completion of these studies will definitively ascribe the response of hepatic GH sensitivity to clinically relevant acute illness, enabling rational therapeutic exploitation of the GH axis in sepsis and other forms of critical illness.