Mechanotransduction in bladder smooth muscle

项目简介(申请人提供)：尿路纤维增生性重塑与多种病理有关，包括出口梗阻继发的肥厚性膀胱生长、神经原性膀胱和糖尿病。尽管多年来人们已经认识到暴露在病理刺激下的膀胱壁的宏观变化，如壁厚和肌肉收缩能力的丧失，但在分子水平上支持组织重塑的信号仍然知之甚少。本研究的目的是揭示在病理刺激下调节膀胱平滑肌生长和分化的信号事件。确定这些过程的关键调控因素将揭示治疗干预的新靶点。由于膀胱在中空器官中是独一无二的，作为药物输送的特权部位，这些研究可能直接导致治疗尿路功能障碍的新疗法的机会，特别是在肥大的情况下。本课题组的研究表明，磷脂酰肌醇-3-激酶(PI3K)/Akt通路是原代膀胱平滑肌细胞(BSMC)在机械刺激或血小板衍生生长因子(PDGF)治疗下的生长调节因子。SMC在体外暴露于拉伸或PDGF，或在体外扩张完整的啮齿动物膀胱，可引起PI3K的主要效应者-丝氨酸苏氨酸激酶Akt的强烈磷酸化。原代人BSMC的表达谱显示STRAND是基因表达的高度选择性调节因子，0.2%的表达基因组被鉴定为机械响应。在计算机分析中，AP-1家族成员可能是牵张诱导的BSMC基因表达的调节因子。虽然Akt和AP-1被机械刺激上调，但它们相互作用调节中空器官重塑的程度基本上是完全未知的。在这个方案中，我们将检验这样的假设，即Akt和AP-1调节的信号介导了机械刺激下膀胱平滑肌的生长，并在细胞内的一个或多个水平上收敛。我们将使用体外BSMC拉伸模型和膀胱扩张动物模型来解决以下特定目标：(1)确定Akt如何在体外和体内调节机械刺激下SMC的生长；(2)确定AP-1介导的基因表达变化如何调控SMC生长以及Akt调节的程度。我们将使用几种互补的方法在体外和体内调节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。我们期待这些实验的发现将为病理性膀胱平滑肌生长的机制提供新的见解。项目叙事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了解而阻碍了对膀胱疾病的有效治疗。这项拟议的实验将研究两个蛋白质家族Akt和AP-1如何相互作用来调节机械刺激下膀胱平滑肌的生长。我们预计这一分析将为揭示膀胱肌肉生理学的基本机制提供新的线索，并可能为新的治疗策略提供洞察力。

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.