Elucidating the Phosphatase-Independent Roles of PTPN11 in the Heart

阐明 PTPN11 在心脏中不依赖磷酸酶的作用

• 批准号: 8828286
• 负责人: Maria I Kontaridis
• 金额: $ 41.91万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8828286
• 关键词: Accounting; Address; Agonist; Apoptotic; Biochemical; Biological; Biological Assay; Biological Models; C-terminal; Cardiac; Cardiac Myocytes; Cardiac development; Cessation of life; Characteristics; Congenital Abnormality; Congenital Heart Defects; Data; Defect; Development; Disease; Dominant-Negative Mutation; Embryo; Endothelial Cells; Endothelium; Etiology; Fibroblasts; Genes; Hand; Health; Heart; Human; Hypertrophic Cardiomyopathy; In Vitro; LEOPARD Syndrome; Lead; Measurable; Mediating; Mitral Valve; Mus; Mutation; Myocardial; Neonatal; Neural Crest; Noonan Syndrome; PTPN11 gene; Pathogenesis; Patients; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation Site; Physiological; Proline; Property; Protein Tyrosine Phosphatase; Role; Signal Pathway; Signal Transduction; Stretching; System; Testing; Tissues; Work; Zebrafish; cell type; congenital heart disorder; gain of function; gain of function mutation; genetic variant; in vivo; insight; loss of function mutation; mouse Cre recombinase; mouse model; mutant; novel; novel therapeutic intervention; recombinase; src Homology Region 2 Domain

DESCRIPTION (provided by applicant): Congenital heart disease (CHD) is the most common type of birth defect worldwide. Mutations in PTPN11, the gene encoding the protein tyrosine phosphatase (PTP) Shp2, are directly implicated in CHD, causing ~50% of Noonan Syndrome (NS) and nearly all LEOPARD Syndrome (LS) cases. Both autosomal dominant, NS and LS are allelic variant disorders that have several phenotypic characteristics in common, including cardiac defects. Despite these similarities, the biochemical properties of Shp2 phosphatase function between NS and LS are very different; whereas PTPN11 NS mutations are gain-of-function "activating" mutations, LS mutations are loss-of-function and behave as "dominant negatives." Precisely how opposing PTP catalytic functions lead to such similar disease etiologies remains unknown. We propose that differences in NS and LS are mediated by phosphatase-dependent functions, whereas similarities are mediated by phosphatase- independent functions in PTPN11. We hypothesize that LS mutations evoke both phosphatase- dependent and -independent functions of Shp2 to induce aberrant signaling effects during cardiac development that lead to the onset of hypertrophic cardiomyopathy (HCM). To address this directly, we generated inducible "knockin" mice expressing the LS-associated Ptpn11 Y279C mutation. When crossed to deleter-Cre, these Shp2LS/+ mice recapitulate nearly all aspects of the human LS phenotype, including progressive HCM. Importantly, Shp2LS/+ mice show LS mutations are catalytically inactive in vivo. Therefore, as expected, there are aberrantly regulated phosphatase-dependent mechanisms associated with LS. Heart lysates from Shp2LS/+ mice have abrogated agonist-evoked Erk/Mapk activity, in contrast to Shp2NS/+ mice, whose heart lysates show elevated Erk/Mapk signaling. Moreover, Shp2LS/+, but not Shp2NS/+, heart lysates have elevated basal and agonist-induced Akt and mTor activity. However, these differences do not account for the similarities in LS and NS phenotypes. We propose that phosphatase-independent signaling must also exist and contribute to the cardiac defects in NS and LS. Indeed, with our established mouse model systems on hand (LS, Shp2 heterozygous null, Shp2 floxed, and NS mice), we have the unique opportunity to parse phosphatase-dependent vs. -independent roles for Shp2 in LS by investigating the signaling similarities/differences in LS and NS during cardiac development. We propose to 1) determine (and compare to NS) the in-vivo effects of LS expression in lineage-specific cell types during cardiac development and 2) parse the phosphatase-dependent vs. -independent contributions of Shp2 in the heart. We expect our complementary in-vivo, ex-vivo and in-vitro analyses will 1) provide novel insights into the function of Shp2 (and its mutations) in the developing heart; 2) elucidate the functional mechanisms and signaling pathways involved in development of congenital HCM, and 3) suggest potential novel therapies, not only for NS and LS, but for patients with other types of CHD as well.

描述（由申请人提供）：先天性心脏病（CHD）是全球最常见的先天缺陷类型。 PTPN11的突变，编码蛋白酪氨酸磷酸酶（PTP）SHP2的基因直接与CHD有关，导致约50％的Noonan综合征（NS）和几乎所有豹综合征（LS）病例。常染色体显性，NS和LS都是等位基因变体疾病，具有多种表型特征，包括心脏缺陷。尽管有这些相似性，但NS和LS之间SHP2磷酸酶功能的生化特性却大不相同。 PTPN11 ns突变是功能的“激活”突变，而LS突变是功能丧失，行为为“主要的负面因素”。恰恰是对相反的PTP催化功能如何导致这种类似的疾病病因仍然未知。我们建议NS和LS的差异是由磷酸酶依赖性功能介导的，而相似性是由PTPN11中的磷酸酶独立函数介导的。我们假设LS突变引起了SHP2的磷酸酶依赖性和非依赖性功能，从而在心脏发育过程中诱导异常信号传导效应，从而导致肥厚性心肌病（HCM）发作。为了直接解决这一问题，我们生成了表达与LS相关的PTPN11 Y279C突变的诱导型“敲击”小鼠。当这些SHP2LS/+小鼠越过DELERE-CRE时，几乎概括了人类LS表型的所有方面，包括进行性HCM。重要的是，SHP2LS/+小鼠显示LS突变在体内催化无活性。因此，正如预期的那样，与LS相关的磷酸酶依赖机制异常受到调节。与SHP2NS/+小鼠相比，SHP2LS/+小鼠的心脏裂解废除了激动剂诱发的ERK/MAPK活动，其心脏裂解物显示出升高的ERK/MAPK信号传导。此外，SHP2LS/+，但没有SHP2NS/+，心脏裂解物具有升高的基底和激动剂诱导的Akt和MTOR活性。但是，这些差异并不能说明LS和NS表型的相似性。我们提出，磷酸酶独立的信号传导也必须存在，并有助于NS和LS中的心脏缺陷。实际上，借助我们既定的小鼠模型系统（LS，SHP2杂合无效，SHP2 Floxed和NS小鼠），我们有独特的机会通过研究信号相似性/与SHP2的磷酸酶相关性与非依赖性磷酸酶的作用。心脏发育过程中LS和NS的差异。我们建议1）确定（并与Ns）确定（并比较）LS表达在心脏发育过程中谱系特异性细胞类型中的体内效应； 2）解析SHP2在心脏中磷酸酶依赖性与非依赖性贡献。我们期望我们的互补体内，前体内和视野分析将1）提供有关发育中心心脏中SHP2（及其突变）功能的新见解； 2）阐明了与先天性HCM发育有关的功能机理和信号通路，3）提出了潜在的新型疗法，不仅针对NS和LS，而且针对具有其他类型CHD的患者。