Myogenic Control Mechanisms of Circular Muscle of the Rectosigmoid Colon

描述(由申请人提供)：膜微域中信号蛋白的转位、磷酸化和联合，强烈表明PKC1和RhoA在结肠运动中的个别重要作用。HSP27的磷酸化是结肠环状平滑肌细胞(CSMC)收缩过程中PKC1和RhoA转位所必需的。CSMC的收缩与HSP27的磷酸化有关，而S16处的HSP20的磷酸化抑制收缩。成人CSMC的初步结果表明：1)脂筏膜组分中存在Cav-1，2)乙酰胆碱(Ach)诱导的PKC_1、磷酸化PKC_1(S657)和HSP27在脂筏中的滞留增加，HSP20移位出脂筏，3)PKC_1和HSP27与CSMC颗粒组分中的小窝蛋白1(Cav-1)结合增加；4)沉默PKC1(PKC1的siRNA)对人结肠CSMC生物工程三维环(3DBR)中Ach诱导的力产生的初始上升和持续阶段均有抑制作用，提示PKC1在力的产生和维持中起作用。来自老年大鼠CSMC的初步数据显示，分离的脂筏部分的Cav-1和随后的磷酸化PKC1(S657)被耗尽，并伴随着PKC1和HSP27与Cav-1的结合减少。此外，转染DNCav-1基因的成年CSMC通过降低PKC1和HSP27与Cav-1的结合来模拟老年CSMC的Ach反应。这与Ach减少老年大鼠CSMC产生3DBR的力有关。这些数据表明，脂筏、Cav-1和HSP27在正常收缩反应和与老化的CSMC相关的小窝形成减少中起着关键作用。小凹的形成减少可能是衰老影响的一个关键因素，也是一个可能的治疗靶点。初步数据表明，wt-Cav-1的异位表达恢复了Ach诱导的3DBR老年结肠的力量产生。因此，我们建议使用多水平功能方法来研究收缩信号通路的复杂性。我们将使用活细胞成像、生化和分子生物学工具，以及使用来自成年、老年和稳定转基因的平滑肌细胞的三维环生物工程技术实时生理监测收缩反应，来研究不同蛋白质的时空重组和重新定位。通过这些方法获得的数据将使我们能够：1)识别与了解结肠运动生理学有关的错综复杂的分子机制；2)了解和确定可能受年龄影响的影响结肠收缩迟缓和收缩的病理生理机制；以及3)识别和测试纠正与年龄相关的病理生理学和结肠运动迟缓的可能靶点。 公共卫生相关性：总而言之，我们将利用我们开发的生化、分子和结构生理学工具来检测由于衰老造成的正常生理运动功能的破坏，并最终设计治疗方法来纠正这些缺陷。

DESCRIPTION (provided by applicant): Translocation, phosphorylation and association of signaling proteins in membrane microdomains, strongly suggest individual imperative roles of PKC1 and RhoA in colonic motility. Phosphorylation of HSP27 is essential for translocation of PKC1 and RhoA during contraction of colonic circular smooth muscle cells (CSMC). Contraction of CSMC is associated with HSP27 phosphorylation, while HSP20 phosphorylation at S16 inhibits contraction. Preliminary results from adult CSMC show: 1) the presence of cav-1 in lipid raft membrane fractions; 2) increased acetylcholine (Ach)-induced sequestration of PKC1, phospho-PKC1 (S657) and HSP27 into the lipid rafts and translocation of HSP20 out of the lipid rafts; 3) increased association of PKC1 and HSP27 with caveolin 1 (cav-1) in the particulate fraction of CSMC; and 4) silencing of PKC1 (siRNA for PKC1) had an inhibitory effect of both the initial rise and the sustained phase of Ach-induced force generation in 3-dimensional rings bioengineered (3DBR) from human colon CSMC treated with siRNA for PKC1 suggesting a role for PKC1 in force generation and its maintenance. Preliminary data from aged rat CSMC show that isolated lipid raft fractions were depleted of cav-1 and consequently of phospho-PKC1 (S657) concomitant with decreased association of PKC1 and HSP27 with cav-1. Further, adult CSMC transfected with DN cav-1 cDNA, mimicked Ach response of aged CSMC by exhibiting reduced association of PKC1 and HSP27 with cav-1. This correlated with reduced Ach-induced force generation of 3DBR from CSMC of aged rats. These data suggest a crucial role for lipid rafts, cav-1, and HSP27 in normal contractile responses and a reduction in caveolae formation associated with aging CSMC. Reduced caveolae formation could be a critical factor affected by aging and a putative therapeutic target. Preliminary data indicates that ectopic expression of wt-cav-1 reinstated Ach-induced force generation in 3DBR from aged colon. Therefore, we propose to use multilevel functional approaches to study the intricacies of contractile signaling pathways. We will study the spatiotemporal reorganization and relocation of different proteins using live cell imaging, biochemical and molecular biology tools and real time physiological monitoring of contractile response using 3-dimensional rings bioengineered from adult, aged, and stably transfected smooth muscle cells. The data obtained through these approaches will allow us to: 1) Discern the intricate molecular mechanisms responsible for the understanding of the physiology of colonic motility; 2) Understand and identify putative disrupted mechanisms affected by aging that contribute to the sluggishness and pathophysiology of contraction of the colon; and 3) Identify and test the possible putative targets to rectify age-related pathophysiology and sluggishness of colonic motility. Public Health Relevance: In summary, we will utilize biochemical, molecular and structural physiological tools we have developed to detect the disruption of normal physiological motor function due to aging and ultimately design therapies to rectify these defects.