Small heat shock proteins in smooth muscle plasticity

描述(由申请人提供)：肺实质细胞环境的慢性变化导致结构和功能的适应，导致呼吸道阻塞和高反应性。气道平滑肌通过从收缩细胞转变为分泌细胞或增殖和迁移细胞来适应炎症。这被称为“表型可塑性”，需要在数小时、数天或数周的时间内改变基因和蛋白质的表达。呼吸道平滑肌也经历了更快的“机械可塑性”，这种可塑性在几分钟内就会发生。可以在非常宽的组织长度范围内产生几乎恒定的等长力。在这两种情况下，可塑性都被定义为细胞结构或功能的持续变化，以应对环境的变化。引发肌肉可塑性的环境刺激包括神经递质、前列腺素、细胞因子和机械张力。这些信号通过多个蛋白激酶信号级联传递，其中一些针对肌动蛋白细胞骨架。假说：主要的假说是，人呼吸道平滑肌表型和机械可塑性所需的肌动蛋白细胞骨架的动态变化部分是由小的热休克蛋白HSP27、HSP22和HSP20介导的。这些蛋白与横纹肌和平滑肌肉中的肌动蛋白细丝和肌动蛋白附着结构有关，但它们的确切功能尚不清楚。热休克蛋白的磷酸化和同源或异质聚合物的形成被认为调节了它们与肌动蛋白细丝的结合，从而改变了肌动蛋白在细胞膜和细胞质致密小体上的附着位置和数量。方法：为了验证这一假说，我们将使用机械刺激和化学刺激来刺激体外培养的气道平滑肌。细胞和组织力学、细胞附着和扩散、增殖和存活将作为机械和表型适应的读数进行分析。腺病毒、逆转录病毒和质粒介导的基因转移技术将用于获得功能和显性负过表达的方法。具体目的1是：(1)确定调节HSP27在人气道平滑肌细胞中的聚合物大小和肌动蛋白结合的信号和蛋白激酶；(2)通过免疫细胞化学、免疫沉淀和酵母2-杂交筛选确定HSP27的结合伙伴；(3)确定HSP27磷酸化在组织和细胞力学、细胞附着和扩散、细胞增殖和细胞存活中的功能。具体目的2是：(1)确定HSP20是否可被细胞因子和机械应激源诱导；(2)确定HSP20是否与HSP27共聚；(3)确定HSP20磷酸化对收缩、组织僵硬、细胞附着和铺展的必要性。这一结果将直接检验这样一种观点，即小的热休克蛋白是呼吸道平滑肌机械和表型适应的重要参与者。

DESCRIPTION (provided by applicant): Chronic changes in the environment of lung parenchymal cells induce structural and functional adaptations that contribute to airway obstruction and hyperreactivity. Airway smooth muscle adapts to inflammation by changing from contractile cells to secretory or proliferating and migrating cells. This is termed "phenotypic plasticity" which requires changes in gene and protein expression over a period of many hours, days or weeks. Airway smooth muscle also undergoes a more rapid "mechanical plasticity" that occurs in a matter of minutes. Nearly constant isometric force can be generated over a very wide range of tissue lengths. In both cases, plasticity is defined as a persistent change in cell structure or function in response to a change in the environment. Environmental stimuli that trigger muscle plasticity include neurotransmitters, prostanoids, cytokines and mechanical strain. These signals are transduced by multiple protein kinase signaling cascades, some of which target the actin cytoskeleton. Hypothesis: The major hypothesis is that dynamic changes in the actin cytoskeleton required for phenotypic and mechanical plasticity of human airway smooth muscle are mediated in part by small heat shock proteins HSP27, HSP22 and HSP20. These proteins associate with actin filaments and actin attachment structures in striated and smooth muscles, but their precise functions are poorly defined. Phosphorylation and formation of homo- and heteropolymers among the heat shock proteins are thought to regulate their binding to actin filaments, which could alter the number and position of actin attachment sites at the cell membrane and at cytoplasmic dense bodies. Approach: To test this hypothesis, mechanical and chemical stimuli will be used to stimulate airway smooth muscle in vitro. Cell and tissue mechanics, cell attachment and spreading, proliferation and survival will be assayed as readouts of mechanical and phenotypic adaptations. Adenoviral, retroviral and plasmid-mediated gene transfer techniques will be used in gain of function and dominant negative overexpression approaches. Specific Aim 1 is to: (1) Define the signals and protein kinases that regulate polymer size and actin binding of HSP27 in human airway smooth muscle cells; (2) Define the binding partners for HSP27 using immunocytochemistry, immunoprecipitation and yeast 2-hybrid screening (3) Define the functions of HSP27 phosphorylation in tissue and cell mechanics, cell attachment and spreading, cell proliferation and cell survival. Specific Aim 2 is to: (1) Determine whether HSP20 is inducible by cytokines and mechanical stressors, (2) Determine whether HSP20 copolymerizes with HSP27 and (3) Define the necessity of HSP20 phosphorylation for contraction, tissue stiffness, cell attachment and spreading. The results will test directly the notion that small heat shock proteins are important participants in mechanical and phenotypic adaptations of airway smooth muscle.