GH Receptor Proteolysis and Shedding

描述(由申请人提供)： 生长激素(GH)是一种脑下垂体来源的多肽类激素，具有很强的促体作用和代谢作用。GH与其受体(GHR)结合，GHR是一种细胞表面跨膜蛋白，富含在肝脏、肌肉、脂肪和心脏中，对人类和其他脊椎动物的燃料代谢、肌肉质量和能量动态平衡产生深远影响。在急性和慢性疾病状态下，生长激素敏感性会发生改变，目前还不确定在这种情况下，增强或抑制生长激素作用是否有益；这一点很重要，因为现在既有激动剂，也有拮抗剂。我的实验室一直致力于了解GH敏感性的生理和病理生理决定因素，即GH水平、细胞表面GHR的丰富程度以及GHR激活与细胞内信号通路的耦合。在之前和当前的VA资金周期中，我们发现了一种新的调节GH反应性的机制，涉及由金属蛋白酶TACE(肿瘤坏死因子-裂解酶)和TACE抑制剂TIMP3(金属蛋白酶组织抑制因子-3)组成的蛋白分解系统，通过蛋白水解性脱落受体细胞外域来诱导改变表面GHR的利用率。然而，这种金属蛋白分解系统在生理和病理生理状态下对生长激素信号的调节程度尚不清楚。意识到生长激素水平随营养状况的日变化而变化，我们最近研究了这些因素是否影响生长激素丰度和生长激素敏感性。我们在小鼠身上的初步数据表明，肝脏GHR的可用性和相应的GH敏感性随着TIMP3水平的变化而以一天中的时间依赖的方式变化。此外，饮食诱导的肥胖也影响肝脏GHR的丰度。根据最近的报道，我们认为，了解这些生理和病理生理学变量如何影响生长激素敏感性，对于合理地在危重患者中使用生长激素增强型或拮抗型治疗策略至关重要。在这 建议中，我们将检验两个主要假设：1)肝脏GHR的丰度是以一天中的时间依赖的方式被金属蛋白分解调节的。营养因素也影响肝脏GHR的丰度，但在转录水平上。这两种形式的调节都有助于生长激素的敏感性和稳态下的能量平衡。2)以脓毒症为代表的危重疾病，通过TACE介导的GHR蛋白分解，使肝脏对GH严重减敏。由时间和饮食决定的GHR丰度以及危重疾病的时间和程度的净影响决定了急性肝脏GH的反应性，从而可能影响病程。我们将使用我们开发的新的小鼠模型系统和试剂和技术来追求两个特定的目标：1.在小鼠中检测肝脏GHR水平每日变化的潜在机制，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)注射)，确定一天中的时间和喂养方案如何影响急性疾病的倾向，以调节肝脏GH敏感性。这些研究的成功完成将明确归因于肝脏生长激素对临床相关急性疾病的敏感性，使生长激素轴在脓毒症和其他形式的危重疾病中得到合理的治疗利用。

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.