Mechanotransduction in bladder smooth muscle

项目摘要尿路纤维增生性重构与多种病理相关，包括继发于出口梗阻、神经源性膀胱和糖尿病。尽管在病理刺激下膀胱壁发生的宏观变化，如膀胱壁增厚和肌肉收缩力丧失，多年来一直被人们所认识，但在分子水平上组织重塑的信号仍然知之甚少。本研究的目的是揭示膀胱平滑肌在病理刺激下调节生长和分化的信号事件。确定这些过程的关键调节因子将揭示治疗干预的新靶点。由于膀胱在中空器官中是独特的药物输送部位，因此这些研究可能直接为泌尿道功能障碍的新疗法提供机会，特别是在肥大的情况下。本研究小组的数据表明，在机械刺激或血小板衍生生长因子（PDGF）治疗的反应中，磷酸肌苷-3-激酶(PI3K)/Akt通路是原发性膀胱平滑肌细胞（BSMC）生长的中介。SMC暴露于体外拉伸或PDGF或体外完整啮齿动物膀胱的膨胀中，会引起丝氨酸-苏氨酸激酶Akt的强烈磷酸化，这是PI3K的主要效应物。原代人BSMC的表达谱显示，stretch是一种高度选择性的基因表达调节剂，在表达的基因组中，有小于0.2%的基因组被确定为机械响应。计算机分析表明AP-1家族成员可能是拉伸诱导的BSMC基因表达的潜在调节因子。虽然Akt和AP-1在机械刺激下被上调，但它们在多大程度上相互作用调节中空器官重塑基本上是完全没有研究的。在本提案中，我们将验证Akt-和ap -1调节的信号介导膀胱平滑肌在机械刺激下的生长，并在细胞内的一个或多个水平上收敛的假设。我们将使用体外BSMC拉伸模型和膀胱膨胀动物模型来解决以下具体目标：(1)确定Akt如何调节体外和体内机械刺激下SMC的生长；(2)确定ap -1介导的基因表达变化如何调控SMC的生长以及Akt的调控程度。我们将使用几种互补的方法来调节体外和体内BSMC中Akt-和ap -1依赖的信号，包括RNA干扰，药物抑制和蛋白质转导技术。我们期望这些实验的发现将为病理膀胱平滑肌生长的机制提供新的见解。由于缺乏对正常和病理情况下调节组织生长的分子信号的理解，膀胱疾病的有效治疗受到阻碍。本实验将探讨Akt和AP-1两个蛋白家族如何相互作用，在机械刺激下调节膀胱平滑肌的生长。我们期望这一分析将揭示膀胱肌肉生理学的基本机制，并可能为新的治疗策略提供新的见解。

DESCRIPTION (provided by applicant): Project Abstract Fibroproliferative remodeling in the urinary tract is associated with a number of pathologies including hypertrophic bladder growth secondary to outlet obstruction, neurogenic bladder and diabetes. Although the macroscopic changes that occur in the bladder wall exposed to pathologic stimulation, such as wall thickening and loss of muscle contractility, have been appreciated for many years, the signals that underlie tissue remodeling at the molecular level are still poorly understood. The goal of the proposed studies is to uncover the signaling events that regulate growth and differentiation of bladder smooth muscle in response to pathologic stimuli. The identification of key regulators of these processes will reveal novel targets for therapeutic intervention. Because the bladder is unique among hollow organs as a privileged site for drug delivery, these studies may lead directly to opportunities for novel therapies for urinary tract dysfunction particularly in the context of hypertrophy. Data from our group have implicated the phosphoinositide-3-kinase (PI3K)/Akt pathway as a mediator of primary bladder smooth muscle cell (BSMC) growth in response to mechanical stimulation or platelet- derived growth factor (PDGF) treatment. Exposure of SMC to stretch or PDGF in vitro or distension of the intact rodent bladder ex vivo elicited robust phosphorylation of the serine-threonine kinase Akt, a principal effector of PI3K. Expression profiling of primary human BSMC revealed stretch to be a highly selective regulator of gene expression, with <0.2% of the expressed genome identified as mechanically responsive. In silico analysis implicated AP-1 family members as potential regulators of stretch-induced BSMC gene expression. Although Akt and AP-1 are upregulated by mechanical stimuli, the extent to which they interact to regulate hollow organ remodeling is essentially completely unstudied. In this proposal we will test the hypothesis that Akt- and AP-1-regulated signals mediate growth of bladder smooth muscle in response to mechanical stimulation and converge at one or more levels within the cell. We will use an in vitro model of BSMC stretch as well as an animal model of bladder distension to address the following specific aims: (1) Determine how Akt regulates growth in SMC exposed to mechanical stimuli in vitro and in vivo; (2) Determine how AP-1-mediated changes in gene expression regulate SMC growth and the extent of regulation by Akt. We will use several complementary approaches to modulate Akt- and AP-1-dependent signaling in BSMC in vitro and in vivo, including RNA interference, pharmacologic inhibition and protein transduction technology. We anticipate that findings from these experiments will provide novel insights into the mechanisms underlying pathologic bladder smooth muscle growth.Project Narrative Effective treatment of bladder diseases is hampered by a lack of understanding about the molecular signals that regulate tissue growth both in normal and pathologic situations. The proposed experiments will investigate how two protein families, Akt and AP-1, interact to regulate the growth of bladder smooth muscle in response to mechanical stimulation. We anticipate this analysis will shed new light on fundamental mechanisms underlying bladder muscle physiology and may also provide insight into new treatment strategies.