Regulation of Ras Signaling by Rlf In Hypertrophy

描述（由申请人提供）：来自心脏肥大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大和失代偿性心力衰竭。Ras是一种小的GTP结合蛋白，其通过多种效应物发出信号，包括Raf-MEK-ERK途径、PI 3激酶（PI 3 K）、Ral的鸟嘌呤交换因子（RalGEF）和cJun N-末端激酶（JNK）。在转基因小鼠中，V12 Ras介导的肥大与JNK激活和心功能抑制相关。然而，某些Ras依赖性信号级联，包括ERK和PI 3 K，激活代偿性肥大反应，保持收缩活性。为了鉴定新的Ras效应物，我们使用Ras作为诱饵筛选人心脏cDNA文库并分离Ral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，我们在新生大鼠心室肌细胞（NRVM）中表达V12 Ras与RIf。RIf的表达抑制V12 Ras诱导的JNK活性和ANF表达，但不抑制其他Ras效应物。RIf还抑制了薄荷醇诱导的JNK活化，表明RIf表达负性调节JNK活化以响应多种刺激。这些结果表明，RIf抑制了一个效应子途径，该途径被认为介导了心肌细胞的功能抑制。我们将测试的假设，RIf负调控JNK激活和功能抑制肥厚型心肌病在体内的发展。Ras与JNK激活偶联的信号通路尚不清楚，但可能涉及MEKK 1（MAPWERK激酶激酶1），一种优先激活JNK的Ras结合蛋白。在目标#1中，我们将定义MEKK 1-JKK-JNK在RIf介导的V12 Ras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功能的病理生理学意义。我们将测试RIf对压力超负荷、异丙肾上腺素输注和V12 Ras过表达引起的心肌肥大发病机制的潜在保护作用，并研究相关的信号级联反应。阐明RIf调节肥大信号通路的机制可能会导致新的治疗方法的发展，以防止心功能不全。

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.