Krenz Maike, Yutzey Katherine E, Robbins Jeffrey
Cincinnati Children's Hospital Medical Center, Division of Molecular Cardiovascular Biology, Cincinnati, Ohio 45229-3039, USA.
Circ Res. 2005 Oct 14;97(8):813-20. doi: 10.1161/01.RES.0000186194.06514.b0. Epub 2005 Sep 15.
The molecular pathways regulating valve development are only partially understood. Recent studies indicate that dysregulation of mitogen-activated protein kinase (MAPK) signaling might play a major role in the pathogenesis of congenital valvular malformations, and, in this study, we explored the role of extracellular signal-regulated kinase (ERK) 1/2 activation in valve primordia expressing the Noonan syndrome mutation Q79R-Shp2. Noonan syndrome is an autosomal dominant disease characterized by dysmorphic features and cardiac abnormalities, with frequent pulmonic stenosis. The Q79R mutation of PTPN11 previously identified in Noonan syndrome families results in a gain-of-function of the encoded protein tyrosine phosphatase Shp2. We compared the effects of wild-type Shp2 and Q79R-Shp2 on endocardial cushion development. Atrioventricular and outflow tract endocardial cushions were excised from chick embryos, infected with wild-type Shp2 or Q79R-Shp2 adenovirus and embedded in a gel matrix. Q79R-Shp2, but not wild-type-Shp2, expression resulted in increased outgrowth of cells into the gel. The dependence of the Q79R-Shp2 effect on ERK1/2 and p38 MAPK signaling was then determined. The MAPK/ERK kinase (MEK)-1 inhibitor U0126, but not the p38-MAPK pathway inhibitor SB203580, abolished the effect of Q79R-Shp2 on cushion outgrowth. Coinfection with Q79R-Shp2 and dominant negative MEK-1 prevented enhanced endocardial cushion outgrowth, whereas expression of constitutively active MEK-1 mimicked the effect of Q79R-Shp2. Furthermore, dissociated cushion cells displayed increased 5-bromodeoxyuridine incorporation when infected with Q79R-Shp2 but not with wild-type Shp2. This promitotic effect was eliminated by U0126. Our results demonstrate that ERK1/2 activation is both necessary and sufficient to mediate the hyperproliferative effect of a gain-of-function mutation of Shp2 on mesenchymal cells in valve primordia.
调节瓣膜发育的分子途径仅得到部分了解。最近的研究表明，丝裂原活化蛋白激酶（MAPK）信号失调可能在先天性瓣膜畸形的发病机制中起主要作用，在本研究中，我们探讨了细胞外信号调节激酶（ERK）1/2激活在表达努南综合征突变Q79R-Shp2的瓣膜原基中的作用。努南综合征是一种常染色体显性疾病，其特征为畸形体征和心脏异常，常伴有肺动脉狭窄。先前在努南综合征家族中鉴定出的PTPN11的Q79R突变导致编码的蛋白酪氨酸磷酸酶Shp2功能增强。我们比较了野生型Shp2和Q79R-Shp2对心内膜垫发育的影响。从鸡胚中切除房室和流出道心内膜垫，用野生型Shp2或Q79R-Shp2腺病毒感染并包埋在凝胶基质中。Q79R-Shp2而非野生型Shp2的表达导致细胞向凝胶中的生长增加。然后确定Q79R-Shp2效应对ERK1/2和p38 MAPK信号传导的依赖性。MAPK/ERK激酶（MEK）-1抑制剂U0126而非p38-MAPK途径抑制剂SB203580消除了Q79R-Shp2对心内膜垫生长的影响。Q79R-Shp2与显性负性MEK-1共感染可阻止心内膜垫生长增强，而组成型活性MEK-1的表达模拟了Q79R-Shp2的效应。此外，解离的心内膜垫细胞在感染Q79R-Shp2而非野生型Shp2时显示5-溴脱氧尿苷掺入增加。这种促有丝分裂效应被U0126消除。我们的结果表明，ERK1/2激活对于介导Shp2功能获得性突变对瓣膜原基中间充质细胞的过度增殖作用既是必要的也是充分的。
