STRETCH-INDUCED CYCLOOXYGENASE-2 IN UROTHELIAL CELLs

性状（由申请方提供）：前列腺素类对泌尿道产生深远的生理作用，包括伤害感受、诱导平滑肌收缩、增强细胞增殖和血流调节。前列腺素类合成的限速步骤由环氧合酶（考克斯）催化。这种酶以两种同种型存在：考克斯-1（通常为组成型）和考克斯-2（高度诱导型）。考克斯-2的表达主要受泌尿组织扩张和伸展的影响。 一个新的领域的兴趣涉及考克斯活动在尿路已被确定：尿路上皮细胞牵拉诱导考克斯-2的表达。在细胞培养中，尿道上皮牵张在施加牵张的6小时内产生考克斯-2水平的10倍诱导。由于考克斯-2上调将导致可溶性前列腺素类增加，该过程代表了尿路上皮牵张与传入疼痛感知、肌肉收缩性和细胞增殖之间的关键联系。虽然该建议的具体模型涉及从输尿管培养的尿路上皮细胞，但这一新概念在泌尿道的病理生理学中具有广泛的意义。 本项目的目的是确定牵张诱导考克斯-2表达的细胞通路，从而为药物治疗提供几个潜在的靶点。基于其他细胞中的滴定报告和初步数据，该提议的假设是PKC和p38 MAP激酶信号级联的牵张激活诱导尿路上皮细胞中的考克斯-2。具体目标是： I.表征机械拉伸对人尿路上皮细胞中考克斯-2的诱导作用。这将通过确定在人尿路上皮细胞中诱导考克斯-2的最佳持续时间和拉伸程度以及确定拉伸诱导的考克斯-2表达是否在人尿路上皮细胞中转录和/或转录后调节来实现。 二.确定牵张诱导考克斯-2是否通过增加细胞内钙离子浓度[Ca++] i和随后的PKC和p38 MAP激酶激活介导。这将通过确定尿路上皮细胞的牵张是否增加[Ca++] i并激活PKC和p38 MAP激酶，钙、PKC或p38 MAP激酶的抑制是否减弱牵张诱导的考克斯-2，以及p38依赖性转录因子是否参与牵张诱导的考克斯-2表达来实现。 这一探索性建议的发现有很大的潜力扩展到几个动态的研究线，并可能确定新的方法来治疗尿路疾病，如膀胱出口梗阻，肾盂输尿管连接部梗阻，间质性膀胱炎。

DESCRIPTION (provided by applicant): Prostanoids exert profound physiological effects on the urinary tract including nociception, induction of smooth muscle contractility, enhancement of cell proliferation, and blood flow regulation. The rate-limiting step in prostanoid synthesis is catalyzed by cyclooxygenase (COX). This enzyme exists in two isoforms: COX-1 (usually constitutive) and COX-2 (highly inducible). COX-2 is substantially induced with distension and stretch of urinary tissues. A new area of interest involving COX activity in urinary tract has been identified: urothelial cell stretch-induced COX-2 expression. In cell culture, urothelial stretch produces a 10-fold induction in COX-2 levels within 6 hours of applying stretch. Since COX-2 upregulation would lead to an increase in soluble prostanoids, this process represents a critical link between urothelial stretch and afferent pain perception, muscle contractility, and cell proliferation. While the specific model of this proposal involves urothelial cells cultured from the ureter, this novel concept has broad implications in the pathophysiology of the urinary tract. The goal of this project is to identify cellular pathways of stretch-induced COX-2 expression and thereby provide several potential targets for drug therapy. Based on titerature reports in other cells, and preliminary data, the hypothesis of this proposal is that stretch activation of PKC and p38 MAP kinase signaling cascades induces COX-2 in urothelial cells. The Specific Aims are: I. To characterize COX-2 induction in human urothelial cells subjected to mechanical stretch. This will be accomplished by determining the optimal duration and degree of stretch for COX-2 induction in human urothelial cells and determining if stretch-induced COX-2 expression is regulated transcriptionally and/or post-transcriptionally in human urothelial cells. II. To determine if stretch induction of COX-2 is mediated by increased intracellular calcium concentrations [Ca++] i and subsequent PKC and p38 MAP kinase activation. This will be accomplished by determining if stretch of urothelial cells increases [Ca++] i, and activates PKC and p38 MAP kinase, if inhibition of calcium, PKC, or p38 MAP kinase attenuate stretch-induction of COX-2, and if p38-dependent transcriptional factors are involved in stretch-induced COX-2 expression. The findings of this exploratory proposal have great potential to expand into several dynamic lines of research and may identify novel approaches to treating urinary tract disorders such as bladder outlet obstruction, ureteropelvic junction obstruction, and interstitial cystitis.