Smooth Muscle Myosin Phosphatase Subunit Isoforms

描述（由申请人提供）：肌球蛋白磷酸酶（Myosin phosphatase， MP）是平滑肌松弛的主要效应器，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21 kD （M21）亚基组成的异三聚体。我们的长期目标是了解MP/MYPT1亚型在发育和疾病中与血管功能相关的调控表达。我们已经证明，由31 nt外显子（E23）的选择性剪接产生的MYPT1亚型是组织特异性的，发育调控的，进化保守的，并在疾病中调节。E23在具有快速（相）或中间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，而在大动脉和静脉的慢（强直）平滑肌中跳过。从E23跳跃到剪接的转换发生在围产期获得快速表型的组织中。在血流/血压改变的疾病模型中，PV和肠系膜抵抗动脉转变为E23跳变，这是向缓慢表型普遍转变的一部分。E23跳过编码MYPT1 c端亮氨酸拉链（LZ）基序，这是cgmp依赖性蛋白激酶（cGK1）二聚化和MP激活所必需的，导致钙对力产生的脱敏。MYPT1 LZ-异构体的31 nt外显子编码。我们在许多模型中表明，表达E23-包括/LZ-异构体的组织对NO/cGMP介导的松弛不太敏感，这表明E23的调节剪接可以作为血管平滑肌微调其对NO/cGMP信号敏感性的一种方式。这一更新的重点是MYPT1 E23剪接的调控。我们建议验证Transformer2b是E23剪接/血管平滑肌表型规范在疾病发育和调节中的新调节剂的假设，以及Tra2b可以作为这一研究的新节点的推论。变压器剪接因子最初被确定为苍蝇两性二态性状的主要调节因子。脊椎动物同源物在表型规范中的作用尚不清楚。在之前的研究中，我们发现：1)在发育和疾病模型中，Tra2b表达与E23剪接之间存在强烈且进化保守的相关性；2)Tra2b结合E23并通过一个迷你基因结构反式激活其剪接。目标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试验，确定Tra2b顺式元件在MYPT1 E23剪接调控中的作用。目的2：测试Tra2b在体内的作用：a) LacZ靶向Tra2b位点，作为Tra2b在不同血管床发育过程中表达的报告基因；b) Cre-lox介导的VSM中Tra2b的条件失活（功能丧失）；c)疾病模型中Tra2b在肠系膜动脉中的强制表达（功能获得/挽救）。目的3:Tra2b作为解剖VSM多样化的新节点。Tra2b第一个内含子中的一个~500 nt超保守序列（UCS）将在转基因实验中测试其在快速平滑肌中驱动组织特异性转录的能力。UCS将通过生物信息学、缺失/突变和候选因子（T3，NFAT）方法进行解剖。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控制之间的新联系提供新的见解。

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.