Regulation of Ras Signaling by Rlf In Hypertrophy

描述（由申请人提供）：来自心脏肥厚实验模型的重要最新进展表明，不同的信号通路调节代偿性肥厚和失代偿性心力衰竭。Ras是一种小的GTP结合蛋白，通过多种效应器发出信号，包括Raf-MEK-ERK途径、PI3K激酶（PI3K）、Ral的鸟嘌呤交换因子（RalGEFs）和cJun n -末端激酶（JNK）。在转基因小鼠中，v12ras介导的肥厚与JNK激活和心功能抑制有关。然而，某些ras依赖的信号级联，包括ERK和PI3K，激活代偿性肥厚反应，保持收缩活性。为了鉴定新的Ras效应物，我们以Ras为诱饵筛选了人心脏cDNA文库，并分离了Ras鸟嘌呤核苷酸交换因子样因子（RIf）。为了研究Ras-RIf相互作用的功能后果，我们在新生儿心室大鼠肌细胞（NRVM）中表达V12Ras和RIf。RIf的表达抑制了v12ras诱导的JNK活性和ANF表达，但对其他Ras效应物没有抑制作用。RIf还抑制山梨醇诱导的JNK激活，表明RIf表达在多种刺激下负调控JNK激活。这些结果表明，RIf抑制了一条可能介导心肌细胞功能抑制的效应通路。我们将在体内验证RIf负性调节JNK激活以及该功能抑制肥厚性心肌病发展的假设。将Ras与JNK激活结合的信号通路尚不清楚，但可能涉及MEKK1 (MAPWERK激酶激酶1)，一种优先激活JNK的Ras结合蛋白。在目的1中，我们将定义MEKK1-JKK-JNK在rif介导的V12Ras-和应激信号传导抑制中的作用。在目标#2和#3中，将在过表达RIf的转基因小鼠中研究RIf功能的病理生理意义。我们将测试RIf在压力过载、异丙肾上腺素输注和V12Ras过表达的心脏肥厚发病机制中的潜在保护作用，并研究相关的信号级联反应。阐明RIf调节肥厚性信号通路的机制可能会导致新的治疗方法的发展，以防止心功能障碍。

DESCRIPTION (provided by applicant): Important recent advances derived from experimental models of cardiac hypertrophy suggest that distinct signaling pathways regulate compensated hypertrophy and decompensated heart failure. Ras is a small GTP binding protein that signals through multiple effectors, including the Raf-MEK-ERK pathway, PI3 kinase (PI3K), guanine exchange factors for Ral (RalGEFs) and cJun N-terminal kinase (JNK). In transgenic mice, V12Ras-mediated hypertrophy is associated with JNK activation and depressed cardiac function. However, certain Ras-dependent signaling cascades, including ERK and PI3K, activate compensatory hypertrophic responses that preserve contractile activity. To identify novel Ras effectors we screened a human heart cDNA library using Ras as bait and isolated Ral guanine nucleotide exchange factor-like factor (RIf). To examine the functional consequence of the Ras-RIf interaction, we expressed V12Ras in concert with RIf in neonatal ventricular rat myocytes (NRVM). Expression of RIf suppressed V12Ras-induced JNK activity and ANF expression but not other Ras effectors. RIf also inhibited sorbitol-induced JNK activation indicating that RIf expression negatively regulates JNK activation in response to multiple stimuli. These results demonstrate that RIf suppresses an effector pathway suggested to mediate depressed function of myocardial cells. We will test the hypothesis that RIf negatively regulates JNK activation and that function inhibits the development of hypertrophic cardiomyopathy in vivo. The signaling pathways that couple Ras to JNK activation are not known but may involve MEKK1 (MAPWERK kinase kinase 1), a Ras binding protein that preferentially activates JNK. In aim #1, we will define the role of MEKK1-JKK-JNK in RIf-mediated inhibition of V12Ras- and stress- signaling. In aims #2 and #3, the pathophysiological implications of RIf function will be investigated in transgenic mice that overexpress RIf. We will test the potential protective role of RIf on the pathogenesis of cardiac hypertrophy in response to pressure overload, isoproterenol infusion and V12Ras overexpression and investigate the signaling cascades involved. Elucidation of the mechanisms by which RIf regulates hypertrophic signaling pathways may lead the development of novel therapeutic approaches to prevent cardiac dysfunction.