Human Shp2 (Ptpn11) mutations and cardiac valve development

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。由异常内膜垫发育引起的瓣膜间隔缺损占CHD的三分之一，也是成人发病/死亡的重要原因。虽然在描述扰乱小鼠和人类瓣膜发生的单基因缺陷方面取得了进展，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，在很大程度上仍然未知。蛋白质酪氨酸磷酸化是一种重要的细胞调节机制，由蛋白质酪氨酸激酶（PTKs）和蛋白质酪氨酸磷酸酶（PTP）控制。许多生长因子受体是跨膜PTK，并且几种与瓣膜发生有关。血管内皮生长因子（VEGF），由心肌细胞产生，抑制心内膜-间质转变（EMT），这是一个关键的初始事件，其中专门的心内膜！细胞转化为间充质细胞，其侵入垫基质并增殖。ErbB 3，最有可能与ErbB 2（HER 2）合作，并响应heregulin（HRG），促进EMT和/或间充质增殖。相反，肝素结合EGF（HB-EGF）通过EGFR（ErbB 1）起作用，终止间充质细胞增殖，并在瓣膜重塑中起关键作用。非受体PTP，Shp 2（PTPN 11），也在瓣膜发育中发挥重要作用。Shp 2缺陷增强小鼠EGFR功能丧失的作用，导致异常瓣膜增厚。此外，约50%的常染色体显性遗传病Nponan综合征（NS）病例是由PTPN 11突变引起的，NS是CHD最常见的非染色体原因。结构，酶学和生化研究，以及在我们的实验室中产生的小鼠NS模型表明，NS突变是功能获得性等位基因，增强ERK MAP激酶激活在发展中的心脏垫。PTPN 11突变也会导致LEOPARD综合征（LS），其表现出心脏缺陷的重叠谱。但我们最近发现，与NS等位基因不同，LS突变体是PTP失活的，并作为显性负突变体损害Erk激活。基于这些数据，我们假设NS和LS突变通过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途径而导致相似的心脏瓣膜缺陷。我们提出了一种生物化学、细胞生物学和遗传学相结合的方法来解决由人类PTPN 11突变引起的心脏缺陷发病机制的关键问题。目的1将使用诱导敲入的方法，以确定细胞类型和发育间隔，其中NS突变体的行为，造成瓣膜间隔缺损。在目标2中，我们将研究Shp 2敲入等位基因的等位基因系列，询问特定PTPN 11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突变体在瓣膜发生期间稍后作用以抑制EGFR信号传导/重塑的假设。最后，目标3将使用小鼠模型，外植体测定和药理学试剂的组合来测试NS等位基因增强Ras/Erk通路的ErbB 2/3信号传导，从而增强EMT和可能的间充质增殖的假设。我们的研究结果将为CHD的发病机制提供新的见解，并可能对NS和LS的治疗具有重要意义。

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.