Spatiotemporal Modulation of PIP[3]Signaling in Prostatic Tubulogenesis

描述(由申请人提供)：本建议书描述了一位在泌尿系统病理学方面具有临床专业知识的内科科学家的五年职业发展计划，他致力于建立一个专注于前列腺上皮形态发生和迁移的独立实验室。她将在她的科学培训中增加发育生物学、细胞迁移和尖端活细胞成像技术的课程。这项研究将在细胞动力学中心进行，这是约翰·霍普金斯医学院的一个最先进的多学科中心，汇集了研究活细胞中空间和时间调节的分子事件的专家。这项拟议的研究项目将集中于阐明驱动前列腺上皮形态发生的信号通路，并可能在成人前列腺生长异常疾病中重新激活。这个项目将汇集她的两位科学导师的长处：丹尼斯·蒙特尔博士和大卫·伯曼博士，前者是上皮细胞迁移基础科学的国际专家，后者是泌尿科病理学家，拥有专注于前列腺发育和疾病的实验室。尽管PIP3(磷脂酰肌醇[3，4，5]-三磷酸)信号在调节细胞极性方面具有高度保守的作用，但PIP3信号在哺乳动物上皮形态发生中的作用尚不清楚。根据初步数据，我们假设PIP3信号的时空调制对驱动前列腺小管形成的两个关键过程至关重要：前列腺管延长和顶基底部极性指定。利用我们实验室建立的三维前列腺上皮培养系统，以及体外前列腺器官培养和最先进的延时成像技术，我们将从三个相互关联的目的来研究PIP3对前列腺上皮形态发生的调控作用：1)我们将通过转基因PIP3生物传感器和异构体特异性PI3K抑制剂的小鼠来确定PIP3信号在前列腺导管延长过程中的细胞内定位和需求；2)我们将通过检测PI3K抑制对前列腺上皮迁移、增殖和凋亡的特异性影响来确定PIP3信号调节前列腺导管延长的细胞机制；3)利用本实验室建立的转基因小鼠新品系，通过调节PI3K和PTEN(磷酸酶和张力蛋白同源物)的活性和水平，在体外和体内研究PIP3在腺泡形态发生过程中的调节作用。通过阐明在发育过程中驱动前列腺生长的信号网络，我们将更好地理解这些通路的失调如何导致成人的前列腺疾病。 与公共卫生相关：越来越多的证据表明，在胚胎发育期间调节前列腺生长的许多信号通路在成人疾病中重新激活，如良性前列腺增生和癌症。我们有初步的数据表明PIP3信号通路在前列腺发育过程中很重要，我们建议研究它的激活如何使前列腺上皮管生长。这一途径特别令人感兴趣，因为有许多药物可用于调节其活性，一旦我们完全了解这一途径的工作原理，这些药物可能对治疗人类前列腺疾病有用。

DESCRIPTION (provided by applicant): This proposal describes the five-year career development plan of a physician-scientist with clinical expertise in urologic pathology who is committed to establishing an independent laboratory focusing on prostatic epithelial morphogenesis and migration. She will add to her scientific training with coursework in developmental biology, cellular migration, and cutting-edge live-cell imaging techniques. The research will take place in the Center for Cell Dynamics, a state-of-the-art multidisciplinary center at the Johns Hopkins School of Medicine that brings together experts studying spatially and temporally regulated molecular events in living cells. The proposed research project will focus on elucidating the signaling pathways that drive prostatic epithelial morphogenesis and are potentially re-activated in adult diseases of abnormal prostate growth. This project will bring together the strengths of her two scientific mentors, Dr. Denise Montell, an international expert in the basic science of epithelial cell migration, and Dr. David Berman, a urologic pathologist with a laboratory focused on prostate development and disease. Despite the highly conserved role of PIP3 (phosphatidylinositol [3,4,5]- trisphosphate) signaling in regulating cellular polarity, the role of PIP3 signaling in mammalian epithelial morphogenesis remains unknown. Based on preliminary data, we hypothesize that spatiotemporal modulation of PIP3 signaling is critical for two key processes driving prostatic tubulogenesis: prostatic ductal elongation and apicobasal polarity specification. Taking advantage of a 3D prostate epithelial culture system developed in our laboratory, as well as in vitro prostate organ culture and state-of-the-art time-lapse imaging techniques, we will examine the effects of PIP3 modulation on prostatic epithelial morphogenesis in three inter-related Aims: 1)We will establish the intracellular localization of and requirement for PIP3 signaling during prostatic ductal elongation using mice transgenic for a PIP3 biosensor and isoform-specific PI3K (phosphoinositide-3-kinase) inhibitors; 2)We will define the cellular mechanism by which PIP3 signaling regulates prostatic ductal elongation by examining the specific effects of PI3K inhibition on prostatic epithelial migration, proliferation and apoptosis; 3)We will examine the role of PIP3 in regulating apicobasal polarity by modulating the activity and levels of PI3K and PTEN (phosphatase and tensin homologue) in vitro during acinar morphogenesis and in vivo using a novel transgenic mouse line established in our laboratory. By elucidating the signaling networks that drive prostatic growth during development, we will better understand how dysregulation of these pathways may lead to prostatic disease in the adult. PUBLIC HEALTH RELEVANCE: There is increasing evidence that many of the signaling pathways that regulate prostatic growth during embryonic development are reactivated in adult diseases, such as benign prostatic hyperplasia and cancer. We have preliminary data that suggests the PIP3 signaling pathway is important during prostatic development, and we propose to study how its activation may enable the growth of prostatic epithelial tubules. This pathway is of particular interest because there are numerous drugs available for modulating its activity that may be useful for the treatment of human prostate diseases once we fully understand how the pathway works.