Myogenic Control Mechanisms of Circular Muscle of the Rectosigmoid Colon

描述（由申请人提供）：膜微域信号蛋白的易位，磷酸化和关联，强烈表明PKC1和RhoA在结肠运动中的个体重要作用。在结肠循环平滑肌细胞（CSMC）收缩过程中，HSP27的磷酸化对于PKC1和RhoA的易位至关重要。CSMC的收缩与HSP27的磷酸化有关，而HSP20在S16的磷酸化则抑制收缩。成人CSMC的初步结果表明：1)cav-1存在于脂筏膜组分中；2)乙酰胆碱（Ach）诱导PKC1、磷酸化PKC1 （S657）和HSP27被隔离到脂筏中，HSP20被移出脂筏；3) CSMC颗粒组分中PKC1和HSP27与cav-1的关联增加；4) PKC1（用于PKC1的siRNA）的沉默对人结肠CSMC三维环生物工程（3DBR）中乙酰胆碱诱导的力产生的初始上升和持续阶段都有抑制作用，这表明PKC1在力的产生和维持中起作用。老龄大鼠CSMC的初步数据显示，分离的脂质筏组分中cav-1和磷酸化PKC1 （S657）的缺失，同时PKC1和HSP27与cav-1的关联降低。此外，转染DN cav-1 cDNA的成人CSMC通过显示PKC1和HSP27与cav-1的关联降低，模仿老年CSMC的Ach反应。这与老年大鼠CSMC的3DBR疼痛诱导的力产生减少相关。这些数据表明脂筏、cav-1和HSP27在正常收缩反应和与衰老CSMC相关的小泡形成减少中起关键作用。小泡形成的减少可能是受衰老影响的关键因素，也是一个假定的治疗靶点。初步数据表明，wt-cav-1的异位表达恢复了老年结肠3DBR中疼痛诱导的力产生。因此，我们建议使用多层次的功能方法来研究收缩信号通路的复杂性。我们将利用活细胞成像、生化和分子生物学工具，以及利用成人、老年和稳定转染的平滑肌细胞的三维环生物工程对收缩反应进行实时生理监测，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)辨别负责理解结肠运动生理学的复杂分子机制；2)理解和识别可能受衰老影响的破坏机制，这些机制导致结肠收缩迟缓和病理生理；3)确定并测试可能的靶点，以纠正与年龄相关的病理生理和结肠运动迟缓。

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.