Smooth Muscle Myosin Phosphatase Subunit Isoforms

描述（由申请方提供）：肌球蛋白磷酸酶（MP）是平滑肌松弛的主要效应物，也是调节血管张力的信号通路的关键靶标。MP是由催化（PP 1c）、靶向/调节（MYPT 1）和21 kD（M21）亚基组成的异源三聚体。我们的长期目标是了解MP/MYPT 1亚型在发育和疾病中与血管功能相关的调节表达。我们已经表明，MYPT 1的异构体产生的选择性剪接的31 nt外显子（E23）是组织特异性的，发育调控，进化保守和疾病中的调节。E23在具有快速（阶段性）或中间收缩特性的平滑肌组织中剪接，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）平滑肌中跳跃。从E23跳跃到剪接的转换发生在获得快速表型的组织的围产期。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，作为向缓慢表型的一般转变的一部分。E23跳跃编码cGMP依赖性蛋白激酶（cGK 1）二聚化和MP激活所需的MYPT 1 C-末端亮氨酸拉链（LZ）基序，导致力产生的钙脱敏。包含31 nt外显子编码MYPT 1 LZ-同种型.我们已经在许多模型中表明，表达包含E23的/LZ-同种型的组织对NO/cGMP介导的松弛不太敏感，这表明E23的调节剪接是血管平滑肌微调其对NO/cGMP信号传导的敏感性的一种方式。这次更新的重点是MYPT 1 E23剪接的调控。我们建议测试这一假设，即transformer 2b是一种新的调节E23剪接/血管平滑肌表型规格的发展和调制疾病，和Tra 2b可以作为一个新的节点在这条线的调查的推论。Transformer剪接因子最初被认为是果蝇性二型性状的主要调节因子。脊椎动物同系物在表型特化中的作用尚不清楚。在之前的资助期间，我们显示了1）Tra 2b表达与发育和疾病模型中的E23剪接之间的强烈和进化上保守的相关性2）Tra 2b结合E23并反式激活其从迷你基因构建体的剪接。提出了以下目的：目的1：通过突变/缺失/嵌合构建体的产生、结合亲和力的测量和体内结合测定，确定Tra 2b顺式元件在MYPT 1 E23剪接调节中的作用。目标二：测试Tra 2b在体内的作用，其中a）LacZ靶向Tra 2b基因座作为在整个发育过程中不同血管床中Tra 2b表达的报告基因B）VSM中Cre-lox介导的TRa 2b的条件失活（功能丧失）c）疾病模型中肠系膜动脉中Tra 2b的强制表达（功能获得/拯救）。目的3：Tra 2b作为一个新的节点解剖VSM多样化。将在转基因试验中测试Tra 2b第一内含子中的~500 nt超保守序列（UCS）驱动快速平滑肌中组织特异性转录的能力。将通过生物信息学、缺失/突变和候选因子（T3、NFAT）方法对UCS进行解剖。这些研究将提供关于Tra 2b的作用的新见解，以及转录和剪接控制之间的新联系，在血管平滑肌收缩表型多样性的产生和调节。 公共卫生相关性：通过收缩和舒张的小阻力动脉的平滑肌是流向组织的血流的主要调节器。拟议的实验将测试Transformer蛋白质的作用，在苍蝇的性别特征的主要决定因素，在调节基因表达和功能的小动脉的发展和疾病。

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.