Elucidating the Phosphatase-Independent Roles of PTPN11 in the Heart

描述（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。编码蛋白酪氨酸磷酸酶（PTP） Shp2的基因PTPN11的突变与冠心病有直接关系，导致约50%的努南综合征（NS）和几乎所有的豹综合征（LS）病例。常染色体显性遗传，NS和LS都是等位变异疾病，具有几个共同的表型特征，包括心脏缺陷。尽管有这些相似之处，NS和LS之间Shp2磷酸酶功能的生化特性却有很大的不同；PTPN11 NS突变是功能获得“激活”突变，而LS突变是功能丧失，表现为“显性阴性”。确切地说，相反的PTP催化功能如何导致如此相似的疾病病因尚不清楚。我们认为，NS和LS的差异是由磷酸酶依赖的功能介导的，而相似之处是由PTPN11中磷酸酶独立的功能介导的。我们假设LS突变同时激活了磷酸酶依赖性和非依赖性Shp2功能，在心脏发育过程中诱导异常信号效应，从而导致肥厚性心肌病（HCM）的发作。为了直接解决这个问题，我们产生了表达ls相关Ptpn11 Y279C突变的诱导“敲入”小鼠。当与deleter-Cre杂交时，这些Shp2LS/+小鼠几乎再现了人类LS表型的所有方面，包括进行性HCM。重要的是，Shp2LS/+小鼠显示LS突变在体内没有催化活性。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机制。Shp2LS/+小鼠的心脏裂解物消除了激动剂诱发的Erk/Mapk活性，而Shp2NS/+小鼠的心脏裂解物显示Erk/Mapk信号升高。此外，Shp2LS/+，而不是Shp2NS/+，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。我们认为磷酸酶独立的信号也必须存在，并有助于NS和LS的心脏缺陷。事实上，利用我们已经建立的小鼠模型系统（LS、Shp2杂合缺失、Shp2 floxed和NS小鼠），我们有独特的机会通过研究LS和NS在心脏发育过程中信号的异同，来分析Shp2在LS中依赖磷酸酶和不依赖磷酸酶的作用。我们建议1)确定（并与NS比较）在心脏发育过程中LS在谱系特异性细胞类型中的表达在体内的影响，2)分析Shp2在心脏中磷酸酶依赖性和非依赖性的贡献。我们期望我们的补充体内、离体和体外分析将1)为Shp2（及其突变）在发育中的心脏中的功能提供新的见解；2)阐明先天性HCM发展的功能机制和信号通路；3)提出潜在的新疗法，不仅适用于NS和LS，也适用于其他类型的冠心病患者。

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.