Gq-coupled Receptors Inhibit PI 3-kinase/Akt Signaling Pathway

描述(由申请人提供)：本提案的目的是了解IA类磷脂酰肌醇3-激酶(PI3K)p110a和p110？在调节胰腺和骨骼肌中的病理和生理细胞生长中的作用。这两个IA类PI3K被认为控制蛋白质的合成和细胞的生长和存活。然而，由于胚胎的致死性，利用全身基因缺失来研究这些酶的生物学作用是不可能的。为了克服这一实验问题，产生了在特定组织中可以选择性地缺失两个PI3K基因的小鼠品系。利用这些动物，这项提案解决了四个研究问题。目的1确定胰腺特异性消融p110a或p110是否能阻断成分活性KrasG12D诱导的胰腺肿瘤的形成。胰腺特异表达KrasG12D的小鼠会发展成人类胰腺癌常见的恶性上皮内病变的全谱。此外，荧光光谱技术被用来测量激活的Kras与含有p110a或p110的PI3K复合体之间的结合亲和力，这可能提供对两个基因敲除菌株的表型的机械性洞察。目的2研究胰岛素样生长因子-1(IGF-1)在肌肉特异性p110a基因敲除小鼠的肌管中激活哺乳动物雷帕霉素靶点(MTOR)信号的作用。IGF-1激活PI3K，继而激活mTOR被认为是刺激肌肉生长的中枢调节信号。这些研究追求的分子机制解释了在p110a缺失的肌肉中意外发现响应IGF-1的mTOR信号增强的分子机制，尽管Akt的激活大大减少。目的3研究消融p110a或p110‘是否影响后肢脱负荷或重建后肌肉再生所致的骨骼肌萎缩。这个动物模型模拟了住院期间可能发生的肌肉卸载和重新加载的过程。肌肉萎缩的程度和随后的再生是通过对同一动物肌肉块的显微CT扫描进行监测的。AIM 4还使用MicroCT扫描来确定瘦肉精--一种已知可促进人类和啮齿动物肌肉生长的肾上腺素能受体激动剂--是否仍能刺激肌肉特异性p110a或p110基因敲除小鼠的肌肉肥大。克伦特罗对mTOR的信号也在从基因敲除小鼠制备的肌管中进行了研究。从这些实验中获得的知识很重要，因为抑制PI3K的药物已经在癌症治疗的临床试验中进行了测试。识别对这种靶向信号转导治疗有反应的癌症患者仍然是一个重大挑战。此外，如果PI3K酶在调节器官功能(包括维持肌肉质量)中发挥重要作用，全身抑制PI3K有产生不良副作用的风险。 公共卫生相关性：胰腺癌是一种致命的疾病，几乎没有有效的治疗方法。这个项目的一个目标是了解磷脂酰肌醇-3激酶(PI3K)是否在这种癌症的发生发展中起作用。肌肉萎缩是一个主要的健康问题，其影响范围从体力活动减少到行动能力严重受损，可能会给患者及其家人带来严重的医疗和经济后果。这个项目的另一个目标是了解PI3K是否在控制肌肉损耗和生长方面发挥作用。这些研究将增加我们对PI3K信号变化如何促进这些健康问题的发展并导致对这些疾病的更好治疗的了解。

DESCRIPTION (provided by applicant): The goal of this proposal is to understand the role of class IA phosphatidylinositol 3-kinase (PI3K) p110a and p110¿ in regulating pathological and physiological cell growth in the pancreas and skeletal muscle. These two class IA PI3Ks are postulated to control protein synthesis and cell growth and survival. However, it has not been possible to investigate the biological roles of these enzymes using whole-body gene deletion due to embryonic lethality. To overcome this experimental problem, mouse strains in which the two PI3K genes can be selectively deleted in specific tissues were generated. Using these animals, this proposal addresses four research questions. Aim 1 determines if pancreas-specific ablation of p110a or p110¿ blocks the formation of pancreatic tumors induced by constitutively active KrasG12D. Mice with pancreas-specific expression of KrasG12D develop the full spectrum of malignant intraepithelial lesions commonly found in human pancreatic cancer. In addition, a fluorescence spectroscopy technique is used to measure the binding affinity between activated Kras and PI3K complexes containing p110a or p110¿, which might provide mechanistic insight into the phenotypes seen in the two knockout strains. Aim 2 investigates how insulin-like growth factor-1 (IGF-1) activates mammalian target of rapamycin (mTOR) signaling in myotubes prepared from muscle-specific p110a knockout mice. IGF-1 activation of PI3K and then mTOR is thought to be a central regulatory signal for stimulating muscle growth. These studies pursue the molecular mechanisms that explain the unexpected finding of enhanced mTOR signaling in response to IGF-1 in p110a-null muscle, even though Akt activation is greatly reduced. Aim 3 investigates if ablation of p110a or p110¿ affects skeletal muscle atrophy caused by hindlimb unloading or muscle regrowth following reambulation. This animal model mimics the process of muscle unloading and reloading that can occur during hospitalization. The degree of muscle atrophy and subsequent regrowth is monitored by microCT scans of muscle mass in the same animal. Aim 4 also uses microCT scans to determine if clenbuterol, a ¿2 adrenergic receptor agonist known to promote muscle growth in humans and rodents, can still stimulate muscle hypertrophy in muscle-specific p110a or p110¿ knockout mice. Clenbuterol signaling to mTOR is also investigated in myotubes prepared from knockout mice. Knowledge gained from these experiments is important because drugs that inhibit PI3K are already being tested in clinical trials for the treatment of cancer. The identification of cancer patients who will respond to this targeted signal transduction therapy remains a major challenge. Moreover, systemic inhibition of PI3K runs the risk of adverse side effects if these enzymes play important roles in regulating organ function, including maintenance of muscle mass. PUBLIC HEALTH RELEVANCE: Pancreatic cancer is a deadly disease with little efficacious treatment. One goal of this project is to understand if phosphatidylinositol-3 kinase (PI3K) plays a role in the development of this cancer. Muscle atrophy is a major health problem, with effects ranging from reduced physical activity to severely impaired mobility that can have severe medical and financial consequences on patients and their families. Another goal of this project is to understand if PI3K plays a role in controlling muscle wasting and growth. These studies will increase our knowledge about how alterations in PI3K signaling contribute to the development of these health problems and lead to better treatment of these conditions.