Smooth Muscle Myosin Phosphatase Subunit Isoforms

描述(申请人提供)：肌球蛋白磷酸酶(MP)是平滑肌松弛的主要效应者，也是调节血管张力的信号通路的关键靶点。MP是由催化(PP1c)、靶向/调节(MYPT1)和21kD(M21)亚基组成的杂三聚体。我们的长期目标是了解MP/MYPT1亚型的调控表达与血管功能在发育和疾病中的关系。我们已经证明，通过选择性剪接31个核苷酸外显子(E23)而产生的MYPT1亚型是组织特异性的、发育调节的、进化上保守的和在疾病中调节的。E23在具有快速(时相)或中等收缩特性的平滑肌组织中拼接，例如门静脉和肠系膜阻力动脉，并在大动脉和静脉的慢速(紧张性)平滑肌中跳过。在获得快速表型的组织中，从E23跳过到剪接的转换发生在围产期。在血流/压力改变的疾病模型中，PV和肠系膜阻力动脉转变为E23跳跃，这是向缓慢表型转变的普遍转变的一部分。E23跳过了MYPT1 C末端亮氨酸拉链(LZ)基序的编码，这是cGMP依赖的蛋白激酶(CGK1)二聚体和MP激活所必需的，导致力量产生的钙脱敏。包括MYPT1 LZ-亚型的31个核苷酸外显子编码。我们已经在许多模型中表明，表达E23-包含/LZ-异构体的组织对NO/cGMP介导的松弛不那么敏感，这表明E23的调节剪接是血管平滑肌微调其对NO/cGMP信号敏感性的一种方式。此次更新的重点是MYPT1 E23剪接的调控。我们建议检验这一假设，即Transformer2b是E23剪接/血管平滑肌在疾病发生和调控中的新的表型规范的调节因子，以及Tra2b可以作为这一研究领域的一个新的结点的推论。变形金刚剪接因子最初被认为是果蝇性别二型性特征的主要调节因子。脊椎动物同源物在表型规范中的作用尚不清楚。在之前的资助期间，我们显示了：1)在发育和疾病模型中，Tra2b的表达和E23剪接之间存在强烈的进化保守的相关性；2)Tra2b结合E23并反式激活其从微基因结构的剪接。目的1：通过突变/缺失/嵌合构建体的产生、结合亲和力的测定和体内结合的测定，确定Tra2b顺式元件在调节MYPT1 E23剪接中的作用。目的：研究Tra2b在体内的作用：a)靶向Tra2b基因的LacZ报告Tra2b在发育过程中在不同血管床中的表达；b)Cre-lox介导的TRa2b在VSM中的条件性失活；c)疾病模型(功能获得/挽救)中肠系膜动脉中Tra2b的被迫表达。目的3：Tra2b作为解剖VSM多样化的新结点。将在转基因实验中测试Tra2b第一内含子中一个~500nT的超保守序列(USC)，以确定其在快速平滑肌中驱动组织特异性转录的能力。我们将通过生物信息学、缺失/突变和候选因子(T3，NFAT)等方法对该基因进行解剖。这些研究将为Tra2b在血管平滑肌收缩表型多样性的产生和调节中的作用以及转录和剪接调控之间的新联系提供新的见解。 与公共健康相关：通过收缩和松弛的小阻力动脉的平滑肌肉是流向组织的血液的主要调节器。拟议中的实验将测试转化蛋白在调节小动脉发育和疾病中的基因表达和功能方面的作用。转化蛋白是果蝇性别特征的主要决定因素。

DESCRIPTION (provided by applicant): Myosin phosphatase (MP) is the primary effector of smooth muscle relaxation and a key target of signaling pathways that regulate vessel tone. MP is a hetero-trimer composed of catalytic (PP1c), targeting/regulatory (MYPT1) and 21 kD (M21) subunits. Our long term goals are to understand the regulated expression of MP/MYPT1 isoforms in relation to vascular function in development and disease. We have shown that isoforms of MYPT1 generated by alternative splicing of a 31 nt exon (E23) are tissue-specific, developmentally regulated, evolutionarily conserved and modulate in disease. E23 is spliced in smooth muscle tissues with fast (phasic) or intermediate contractile properties, e.g. portal vein and mesenteric resistance arteries, and skipped in the slow (tonic) smooth muscle of the large arteries and veins. A switch from E23 skipping to splicing occurs in the perinatal period in tissues that acquire a fast phenotype. In disease models of altered blood flow/pressure, the PV and mesenteric resistance arteries shift to E23 skipping as part of a generalized shift towards the slow phenotype. E23 skipping codes for a MYPT1 C-terminus leucine zipper (LZ) motif required for cGMP-dependent protein kinase (cGK1) dimerization and activation of MP, resulting in calcium de-sensitization of force production. Inclusion of the 31 nt exon codes for the MYPT1 LZ- isoform. We have shown in a number of models that tissues that express the E23-included/LZ- isoform are less sensitive to NO/cGMP-mediated relaxation, suggesting that the regulated splicing of E23 serves as a way for vascular smooth muscle to fine tune its sensitivity to NO/cGMP signaling. This renewal focuses on the regulation of MYPT1 E23 splicing. We propose to test the hypothesis that Transformer2b is a novel regulator of E23 splicing/vascular smooth muscle phenotypic specification in development and modulation in disease, and the corollary that Tra2b can be used as a novel nodal point in this line of investigation. The Transformer splicing factors were originally identified as master regulators of sexually dimorphic traits in the fly. The role of the vertebrate homologues in phenotypic specification is unknown. In the previous funding period we showed 1) a strong and evolutionarily conserved correlation between Tra2b expression and E23 splicing in developmental and disease models 2) Tra2b binds E23 and trans-activates its splicing from a mini-gene construct. The following aims are proposed: Aim 1: Define the role of Tra2b cis-element in the regulation of MYPT1 E23 splicing through the generation of mutation/deletion/chimeric constructs, measurements of binding affinity, and assay for binding in vivo. Aim 2: Test the role of Tra2b in vivo with a) LacZ targeting the Tra2b locus as a reporter for the expression of Tra2b in different vascular beds throughout development b) Cre-lox mediated conditional inactivation of TRa2b in VSM (loss-of-function) c) forced expression of Tra2b in the mesenteric arteries in the disease models (gain-of-function/rescue). Aim 3: Tra2b as a novel nodal point for the dissection of VSM diversification. An ~500 nt ultra-conserved sequence (UCS) in the first intron of Tra2b will be tested for its ability to drive tissue-specific transcription in fast smooth muscle in a transgenic assay. The UCS will be dissected through bio-informatics, deletion/mutation, and candidate factor (T3,NFAT) approaches. These studies will provide novel insights regarding the role of Tra2b, and novel links between transcriptional and splicing controls, in the generation and modulation of vascular smooth muscle contractile phenotypic diversity. PUBLIC HEALTH RELEVANCE: The smooth muscle of the small resistance arteries by contracting and relaxing is the primary regulator of blood flow to the tissues. The proposed experiments will test the role of Transformer proteins, master determinants of sexual features in flies, in regulating gene expression and function of the small arteries in development and disease.