Human Shp2 (Ptpn11) mutations and cardiac valve development

人类 Shp2 (Ptpn11) 突变与心脏瓣膜发育

• 批准号: 7319031
• 负责人: BENJAMIN G. NEEL
• 金额: $ 42.5万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-16 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7319031
• 关键词: Address; Adult; Affect; Alleles; Apoptosis; Binding; Biochemical; Biological; Biological Assay; Cardiac; Cardiac Jelly; Cell Proliferation; Cell Shape; Cells; Complex; Congenital Abnormality; Congenital Heart Defects; Data; Defect; Development; Disease; Dominant-Negative Mutation; EGF gene; ERBB2 gene; Endocardium; Epidermal Growth Factor Receptor; Epithelial; ErbB Receptor Family Protein; ErbB4 gene; Event; Exhibits; Extracellular Matrix; Genes; Genetic; Growth Factor Receptors; Heart Atrium; Heart Valves; Heparin Binding; Heregulin; Human; Integral Membrane Protein; Invaded; Knock-in Mouse; LEOPARD Syndrome; Laboratories; MAP Kinase Modules; Medical; Mesenchymal; Mesenchyme; Mitogen-Activated Protein Kinases; Modeling; Morbidity - disease rate; Mus; Mutation; Myocardial; Myocardium; Newborn Infant; Noonan Syndrome; PTPN11 gene; Pathogenesis; Pathway interactions; Peptides; Pharmaceutical Preparations; Phenotype; Play; Process; Production; Proliferating; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Proteins; Publishing; Receptor Protein-Tyrosine Kinases; Research Personnel; Role; Series; Signal Pathway; Signal Transduction; Single-Gene Defect; Specific qualifier value; Structure; Syndrome; Testing; Time; Tyrosine Phosphorylation; Variant; Vascular Endothelial Growth Factors; Ventricular septum; base; cell transformation; cell type; congenital heart disorder; gain of function mutation; human PTPRT protein; insight; loss of function; man; mortality; mouse model; mutant; notch protein; programs; receptor; recombinase; src Homology Region 2 Domain

DESCRIPTION (provided by applicant): Congenital heart disease (CHD) is the most common type of birth defect. Valvuloseptal defects, which result from aberrant endocardial cushion development, comprise up to one third of CHD, and also are a significant cause of adult morbidity/mortality. Although progress has been made in delineating single gene defects that perturb valvulogenesis in mouse and man, how these genes regulate development, and how normal development is altered by disease-associated mutations, remain largely unknown. Protein tyrosine phosphorylation, controlled by protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTP), is a key cellular regulatory mechanism., Many growth factor receptors are transmembrane PTKs, and several are implicated in valvulogenesis. Vascular endothelial growth factor (VEGF), produced by myocardjal cells, inhibits endocardial-mesechymal transition (EMT), a key initial event in which specialized endocardia! cells transform into mesenchymal cells that invade the cushion matrix and proliferate. ErbB3, most likely partnering with ErbB2 (HER2) and responding to heregulin (HRG), promotes EMT and/or mesenchymal proliferation. Conversely, heparin-binding EGF (HB-EGF), acting via the EGFR (ErbB1), terminates mesenchymal cell proliferation and plays a key role in valve remodeling. The non-receptor PTP, Shp2 (PTPN11), also plays an essential role in valve development. Shp2 deficiency enhances the effect of EGFR loss of function in mice, resulting in abnormal valve thickening. Furthermore, ~50% of cases of the autosomal dominant disorder Nponan syndrome (NS), the most common non-chromosomal cause of CHD, is caused by PTPN11 mutations. Structural, enzymologic, and biochemical studies, and a mouse NS model generated in our laboratory, indicate that NS mutations are gain-of-function alleles that enhance Erk MAP kinase activation in developing cardiac cushions. PTPN11 mutations also cause LEOPARD syndrome (LS), which exhibits an overlapping spectrum of cardiac defects. But we showed recently that unlike NS alleles, LS mutants are PTP-inactive, and act as dominant negative mutants that impair Erk activation. Based on these data, we hypothesize that NS and LS mutations cause similar cardiac valve defects by acting in opposing ways on distinct RTK pathways at different times during valvulogenesis. We propose a combined biochemical, cell biological and genetic approach to address key questions about the pathogenesis of cardiac defects caused by human PTPN11 mutations. Aim 1 will use an inducible knock-in approach to determine the cell type and developmental interval in which NS mutants act to cause valvuloseptal defects. In Aim 2, we will study an allelic series of Shp2 knock-in alleles, asking if the specific PTPN11 mutation affects the NS cardiac phenotype, and generate a knock-in mouse model of LS to test the hypothesis that LS mutants act later during valvulogenesis to inhibit EGFR signaling/remodeling. Finally, Aim 3 will use a combination of mouse models, explant assays, and pharmacologic agents to test the hypothesis that NS alleles enhance ErbB2/3 signaling to the Ras/Erk pathway, thereby enhancing EMT and possibly mesenchymal proliferation. Our results should yield new insights into the pathogenesis of CHD, and may have important implications for the therapy of NS and LS.

描述（由申请人提供）：先天性心脏病（CHD）是最常见的出生缺陷类型。瓣膜间隔缺陷是由心内膜垫发育异常引起的，占冠心病的三分之一，也是成人发病/死亡的重要原因。尽管在描述干扰小鼠和人类瓣膜形成的单基因缺陷方面已经取得了进展，但这些基因如何调节发育，以及疾病相关突变如何改变正常发育，仍然很大程度上未知。由蛋白酪氨酸激酶 (PTK) 和蛋白酪氨酸磷酸酶 (PTP) 控制的蛋白酪氨酸磷酸化是关键的细胞调节机制。许多生长因子受体是跨膜 PTK，其中一些与瓣膜形成有关。心肌细胞产生的血管内皮生长因子 (VEGF) 抑制心内膜间充质转化 (EMT)，这是心内膜特化的关键初始事件！细胞转化为侵入垫基质并增殖的间充质细胞。 ErbB3 最有可能与 ErbB2 (HER2) 合作并响应调蛋白 (HRG)，促进 EMT 和/或间质增殖。相反，肝素结合 EGF (HB-EGF) 通过 EGFR (ErbB1) 发挥作用，终止间充质细胞增殖，并在瓣膜重塑中发挥关键作用。非受体 PTP Shp2 (PTPN11) 在瓣膜发育中也发挥着重要作用。 Shp2 缺陷会增强小鼠 EGFR 功能丧失的影响，导致瓣膜异常增厚。此外，约 50% 的常染色体显性遗传疾病 Nponan 综合征 (NS) 是 CHD 最常见的非染色体原因，是由 PTPN11 突变引起的。结构、酶学和生化研究以及我们实验室生成的小鼠 NS 模型表明，NS 突变是功能获得性等位基因，可增强心脏垫发育过程中的 Erk MAP 激酶激活。 PTPN11 突变还会导致 LEOPARD 综合征 (LS)，该综合征表现出一系列重叠的心脏缺陷。但我们最近表明，与 NS 等位基因不同，LS 突变体是 PTP 无活性的，并且作为显性失活突变体损害 Erk 激活。基于这些数据，我们假设 NS 和 LS 突变在瓣膜形成过程中的不同时间以相反的方式作用于不同的 RTK 途径，从而导致类似的心脏瓣膜缺陷。我们提出了一种结合生化、细胞生物学和遗传学的方法来解决有关人类 PTPN11 突变引起的心脏缺陷发病机制的关键问题。目标 1 将使用诱导敲入方法来确定 NS 突变体引起瓣膜间隔缺陷的细胞类型和发育间隔。在目标 2 中，我们将研究一系列 Shp2 敲入等位基因，询问特定的 PTPN11 突变是否影响 NS 心脏表型，并生成 LS 敲入小鼠模型，以检验 LS 突变体在瓣膜形成过程中稍后起作用的假设抑制 EGFR 信号/重塑。最后，Aim 3 将结合使用小鼠模型、外植体测定和药物制剂来测试 NS 等位基因增强 Ras/Erk 通路的 ErbB2/3 信号传导，从而增强 EMT 和可能的间充质增殖的假设。我们的结果应该会对 CHD 的发病机制产生新的见解，并且可能对 NS 和 LS 的治疗具有重要意义。