Disruption of Caldesmon Gene Expression in Bladder Myocytes

膀胱肌细胞中 Caldesmon 基因表达的破坏

• 批准号: 7023791
• 负责人: SAMUEL K. CHACKO
• 金额: $ 35.02万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7023791
• 关键词: RNA interference; actins; adenosinetriphosphatase; biological models; caldesmon; cell differentiation; cell line; gene expression; gene targeting; genetically modified animals; laboratory mouse; model design /development; muscle cells; muscle contraction; myosins; phosphorylation; protein purification; smooth muscle; transfection; urinary bladder; western blottings

DESCRIPTION (provided by applicant): The goal of this proposal is to develop and characterize two tools to study the physiological function for caldesmon (CaD), an actin/calmodulin-binding protein associated with smooth muscle thin filaments and thought to play a role in the maintenance of smooth muscle contractile apparatus and the regulation of contraction. These tools are 1) a knockout (KO) mouse model with disruption of the gene that encodes CaD and 2) a rabbit bladder smooth muscle cell line (BSM) which maintains the smooth muscle phenotype, including the ability to contract in response to agonists. We have been successful in making a CaD mouse KO model by homologous recombination. This CaD KO model lacks the C-terminal functional domains, important for inhibition of actin-activated ATP hydrolysis by myosin, actin-and tropomyosin-binding and tethering of myosin to actin. This model requires further characterization with respect to the effect of deletion of functional domains on the structure and function of CaD. Using myocytes dissociated from urinary bladders, muscle strips and whole bladders, we have generated preliminary data, which reveal changes in the structural and functional phenotypes in the KO mouse when compared with that of the wild-type (WT). Similarly, our preliminary studies on the BSM cell line show that the overexpression of smooth muscle specific CaD (h-CaD) in h-CaD-deficient myocytes favors the assembly of cytoplasmic filaments, including myosin filaments. The effect of deletion of CaD C-terminal functional domains on force, ATPase, and organization of cytoplasmic filaments will be studied using myocytes from CaD KO and the BSM cells after silencing the expression of CaD with siRNA or after over- or under-expression of intact or truncated CaD by transfection with CaD cDNA. Characterization of these two models (the transgenic mouse and bladder myocyte cell models) and the data from proposed studies would help to establish a role for CaD in the maintenance of the contractile apparatus and contraction. This is particularly important, since CaD is present in all urologic smooth muscles and its expression is altered in diseases associated with contractile dysfunction. The expected data would also show what effects the deletion of the CaD functional domains has on the differentiated state of the myocyte and the function of organs which depend on smooth muscle contraction. Loss of the differentiated state of the myocyte and alterations in function are correlated with decreased smooth muscle contraction in diseases such as bladder outlet obstruction, urinary incontinence, and erectile dysfunction. Furthermore, this reagent mouse and cell line would be beneficial to other investigators to study not only the role of CaD in bladder smooth muscle but also to study the signal transduction, structure and contractile function in various regions of the urogenital system.

描述（由申请人提供）：本提案的目标是开发和表征两种工具来研究钙结合蛋白 (CaD) 的生理功能，钙结合蛋白是一种与平滑肌细丝相关的肌动蛋白/钙调蛋白结合蛋白，被认为在平滑肌收缩装置的维持和收缩的调节。这些工具是 1) 编码 CaD 的基因被破坏的敲除 (KO) 小鼠模型，以及 2) 维持平滑肌表型（包括响应激动剂而收缩的能力）的兔膀胱平滑肌细胞系 (BSM)。我们已经通过同源重组成功制作了CaD小鼠KO模型。该 CaD KO 模型缺乏 C 端功能域，这对于抑制肌球蛋白激活的肌动蛋白 ATP 水解、肌动蛋白和原肌球蛋白结合以及肌球蛋白与肌动蛋白的束缚非常重要。该模型需要进一步表征功能域删除对 CaD 结构和功能的影响。使用从膀胱、肌肉条和整个膀胱分离的肌细胞，我们生成了初步数据，这些数据揭示了与野生型（WT）小鼠相比，KO 小鼠的结构和功能表型的变化。同样，我们对 BSM 细胞系的初步研究表明，h-CaD 缺陷的肌细胞中平滑肌特异性 CaD (h-CaD) 的过度表达有利于细胞质丝的组装，包括肌球蛋白丝。将使用 CaD KO 的肌细胞和用 siRNA 沉默 CaD 的表达后或在通过用 CaD cDNA 转染完整或截短的 CaD。这两个模型（转基因小鼠和膀胱肌细胞模型）的表征以及拟议研究的数据将有助于确定 CaD 在维持收缩装置和收缩中的作用。这一点尤其重要，因为 CaD 存在于所有泌尿系统平滑肌中，并且其表达在与收缩功能障碍相关的疾病中发生改变。预期数据还将显示 CaD 功能域的缺失对肌细胞的分化状态和依赖于平滑肌收缩的器官功能有何影响。肌细胞分化状态的丧失和功能的改变与膀胱出口梗阻、尿失禁和勃起功能障碍等疾病中平滑肌收缩减少相关。此外，该试剂小鼠和细胞系将有利于其他研究者不仅研究CaD在膀胱平滑肌中的作用，而且有利于研究泌尿生殖系统各个区域的信号转导、结构和收缩功能。