Myogenic Control Mechanisms of Circular Muscle of the Rectosigmoid Colon

描述（由申请人提供）：膜微结构域中信号蛋白的易位、磷酸化和缔合强烈表明PKC 1和RhoA在结肠运动中的个体必要作用。结肠环形平滑肌细胞（CSMC）收缩过程中HSP 27的磷酸化是PKC 1和RhoA转位的关键。CSMC的收缩与HSP 27磷酸化相关，而S16处的HSP 20磷酸化抑制收缩。结果表明：（1）cav-1存在于CSMC的脂筏膜组分中，（2）增加乙酰胆碱（Ach）诱导的PKC_1、磷酸化PKC_1（S657）和HSP_（27）向脂筏内的螯合和HSP_（20）向脂筏外的移位，（3）增加PKC_1和HSP_（27）与Caveolin_1（cav-1）的结合，（4）增加CSMC颗粒组分中PKC_1和HSP_（27）的结合，（5）增加CSMC颗粒组分中PKC_（1）和HSP_（27）的结合，（6）增加CSMC颗粒组分中PKC_（1）和HSP_（27）的结合。和4）PKC 1的沉默（针对PKC 1的siRNA）对来自用针对PKC 1的siRNA处理的人结肠CSMC的三维环生物工程（3DBR）中乙酰胆碱诱导的力产生的初始上升和持续阶段都具有抑制作用，这表明PKC 1在力产生和维持中的作用。来自老年大鼠CSMC的初步数据显示，分离的脂筏组分耗尽cav-1，因此耗尽磷酸化PKC 1（S657），伴随着PKC 1和HSP 27与cav-1的结合减少。此外，成年CSMC转染DN cav-1 cDNA，模仿Ach反应的老年CSMC表现出减少协会的PKC 1和HSP 27与cav-1。这与老年大鼠CSMC的3DBR的乙酰胆碱诱导力产生减少相关。这些数据表明，脂筏，cav-1，和HSP 27在正常的收缩反应和减少与老化CSMC相关的小窝形成的关键作用。减少小窝形成可能是受衰老影响的关键因素，也是公认的治疗靶点。初步数据表明，异位表达的wt-cav-1恢复乙酰胆碱诱导的力产生在3DBR从老年结肠。因此，我们建议使用多层次功能的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、生物化学和分子生物学工具和收缩反应的真实的时间生理监测，研究不同蛋白质的时空重组和重新定位，所述收缩反应使用从成人、老年和稳定转染的平滑肌细胞生物工程化的三维环。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）理解和识别受衰老影响的假定破坏机制，其导致结肠收缩的迟缓和病理生理学;和3）鉴定和测试可能的假定靶点以纠正年龄相关的病理生理学和结肠运动的迟缓。 公共卫生相关性：总之，我们将利用我们开发的生物化学，分子和结构生理学工具来检测由于衰老而导致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.