Gq-Coupled Receptors Inhibit PI 3-Kinase/Akt Signaling Pathway

本提案的目的是了解IA类磷脂酰肌醇3激酶（PI3K） p110 α和p110 β在调节胰腺和骨骼肌病理和生理细胞生长中的作用。这两种IA类pi3k被认为控制蛋白质合成和细胞生长和存活。然而，由于胚胎致死性而使用整个身体基因缺失的方法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，产生了两个PI3K基因可以在特定组织中选择性删除的小鼠品系。利用这些动物，该提案解决了四个研究问题。目的1确定胰腺特异性消融p110alpha或p110beta是否阻断组成型活性KrasG12D诱导的胰腺肿瘤的形成。KrasG12D在胰腺中特异性表达的小鼠出现了人类胰腺癌中常见的全谱恶性上皮内病变。此外，荧光光谱技术用于测量活化Kras与含有p110 α或p110 β的PI3K复合物之间的结合亲和力，这可能为两种敲除菌株的表型提供机制见解。目的2研究胰岛素样生长因子1 （IGF‐1）如何激活由肌肉特异性p110 α敲除小鼠制备的肌管中雷帕霉素（mTOR）信号的哺乳动物靶标。IGF&#8208；1激活PI3K，然后mTOR被认为是刺激肌肉生长的中心调节信号。这些研究追求的分子机制解释了在p110alpha和null肌中IGF&#8208；1的反应中mTOR信号的意外发现，尽管Akt的激活大大降低。目的3研究p110 α或p110 β的消融是否影响后肢卸载或肌肉再生引起的骨骼肌萎缩。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质量的微ct扫描来监测的。Aim 4还使用微ct扫描来确定盐酸克仑特罗（一种已知能促进人类和啮齿动物肌肉生长的β 2肾上腺素受体激动剂）是否仍能刺激肌肉特异性p110 α或p110 β敲除小鼠的肌肉肥大。在敲除小鼠制备的肌管中也研究了盐酸克仑特罗对mTOR的信号传导。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。确定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，如果这些酶在调节器官功能（包括维持肌肉质量）中发挥重要作用，那么全身抑制PI3K可能会产生不良副作用。

The goal of this proposal is to understand the role of class IA phosphatidylinositol 3‐kinase (PI3K) p110alpha and p110beta 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 p110alpha or p110beta 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 p110alpha or p110beta, 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 p110alpha 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 p110alpha‐null muscle, even though Akt activation is greatly reduced. Aim 3 investigates if ablation of p110alpha or p110beta 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 beta2 adrenergic receptor agonist known to promote muscle growth in humans and rodents, can still stimulate muscle hypertrophy in muscle‐specific p110alpha or p110beta 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.