Elucidating the Phosphatase-Independent Roles of PTPN11 in the Heart

描述（由申请人提供）：先天性心脏病（CHD）是世界上最常见的出生缺陷类型。PTPN 11（编码蛋白酪氨酸磷酸酶（PTP）Shp 2的基因）的突变与CHD直接相关，导致约50%的努南综合征（NS）和几乎所有的狮子综合征（LS）病例。常染色体显性遗传、NS和LS都是等位基因变异型疾病，具有几个共同的表型特征，包括心脏缺陷。尽管有这些相似性，NS和LS之间Shp 2磷酸酶功能的生物化学性质非常不同;而PTPN 11 NS突变是功能获得“激活”突变，LS突变是功能丧失，表现为“显性阴性”。“确切地说，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。我们提出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PTPN 11中的磷酸酶非依赖性功能介导的。我们假设LS突变引起Shp 2的磷酸酶依赖性和非依赖性功能，在心脏发育过程中诱导异常信号传导效应，导致肥厚型心肌病（HCM）的发作。为了直接解决这一问题，我们产生了表达LS相关Ptpn 11 Y279 C突变的诱导型“敲入”小鼠。当与deleter-Cre杂交时，这些Shp 2LS/+小鼠几乎重现了人类LS表型的所有方面，包括进行性HCM。重要的是，Shp 2LS/+小鼠显示LS突变在体内无催化活性。因此，正如预期的那样，存在与LS相关的异常调节的磷酸酶依赖性机制。来自Shp 2LS/+小鼠的心脏裂解物具有消除激动剂诱发的Erk/Mapk活性，与Shp 2NS/+小鼠相反，其心脏裂解物显示升高的Erk/Mapk信号传导。此外，Shp 2LS/+而不是Shp 2NS/+心脏裂解物具有升高的基础和激动剂诱导的Akt和mTor活性。然而，这些差异并不能解释LS和NS表型的相似性。我们提出，磷酸酶非依赖性信号也必须存在，并有助于NS和LS的心脏缺陷。事实上，我们建立的小鼠模型系统（LS，Shp 2杂合无效，Shp 2 floxed和NS小鼠），我们有独特的机会，通过研究心脏发育过程中LS和NS的信号相似性/差异来解析LS中Shp 2的磷酸酶依赖性与非依赖性作用。我们建议1）确定（并与NS比较）在心脏发育期间LS表达在谱系特异性细胞类型中的体内作用，以及2）解析心脏中Shp 2的磷酸酶依赖性贡献与磷酸酶非依赖性贡献。我们预计我们的互补体内、离体和体外分析将1）为Shp 2（及其突变）在发育中的心脏中的功能提供新的见解; 2）阐明参与先天性HCM发展的功能机制和信号通路，和3）提出潜在的新疗法，不仅针对NS和LS，而且针对其他类型的CHD患者。

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.