Human Shp2 (Ptpn11) mutations and cardiac valve development

描述（申请人提供）：先天性心脏病（CHD）是最常见的出生缺陷类型。心内膜垫发育异常导致的瓣膜间隔缺损占冠心病的三分之一，也是成人发病率/死亡率的重要原因。尽管在描述干扰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，以及正常发育如何被疾病相关突变改变，在很大程度上仍然未知。蛋白酪氨酸磷酸化是由蛋白酪氨酸激酶（PTKs）和蛋白酪氨酸磷酸酶（PTP）控制的关键细胞调控机制。许多生长因子受体是跨膜PTKs，其中一些与瓣膜发生有关。由心肌细胞产生的血管内皮生长因子（VEGF）抑制心内膜-间质转化（EMT），这是心内膜特化的关键初始事件。细胞转化为间充质细胞，侵入缓冲基质并增殖。ErbB3，最有可能与ErbB2 （HER2）合作并响应heregulin (HRG)，促进EMT和/或间质增殖。相反，肝素结合EGF （HB-EGF）通过EGFR （ErbB1）起作用，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）在瓣膜发育中也起着重要作用。Shp2缺乏增强小鼠EGFR功能丧失的影响，导致瓣膜异常增厚。此外，常染色体显性遗传病Nponan综合征（NS）是冠心病最常见的非染色体原因，约50%的病例是由PTPN11突变引起的。结构、酶学和生化研究以及我们实验室建立的小鼠NS模型表明，NS突变是增强心脏缓冲中Erk MAP激酶激活的功能获得等位基因。PTPN11突变也会引起LEOPARD综合征（LS），它表现出重叠的心脏缺陷谱。但我们最近发现，与NS等位基因不同，LS突变体是ptp失活的，并且作为显性负突变体损害Erk的激活。基于这些数据，我们假设NS和LS突变在瓣膜形成的不同时间以相反的方式作用于不同的RTK通路，从而导致类似的心脏瓣膜缺陷。我们提出结合生化、细胞生物学和遗传学的方法来解决由人类PTPN11突变引起的心脏缺陷的发病机制的关键问题。目的1将使用诱导敲入方法来确定细胞类型和发育间隔，其中NS突变体起作用导致瓣膜间隔缺陷。在Aim 2中，我们将研究一系列Shp2敲入等位基因，研究特定的PTPN11突变是否会影响NS心脏表型，并建立LS敲入小鼠模型，以验证LS突变在瓣膜发生后期抑制EGFR信号传导/重塑的假设。最后，Aim 3将结合小鼠模型、外植体实验和药物来验证NS等位基因增强ErbB2/3向Ras/Erk通路的信号，从而增强EMT和间充质细胞增殖的假设。我们的研究结果将为冠心病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.