Signaling Mechanisms Regulating Cardiac Remodeling

描述（由申请人提供）：我们实验室的长期目标是了解正常和异常心脏发育的信号机制以及可能导致先天性心脏病的机制。事实上，1-2%的新生儿患有某种形式的先天性心脏病，使其成为最常见的先天性出生缺陷。这些心血管缺陷中的许多与心内膜垫组织形成和重塑成心脏的瓣膜和膜间隔有关。我们已经证明，维甲酸X受体α空（RXR α-/-）小鼠是一个很好的模型系统，以帮助我们了解维甲酸信号如何影响心脏发育。胚胎日龄（E）13.5 RXR α-/-小鼠的致死率部分通过流出道中升高的TGF β 2和增强的细胞凋亡来标记。TGF β 2主要通过Smad 2/Smad 3的磷酸化（p）发出信号。pSmad 2和pSmadS在E13.5垫间充质中显示出与凋亡惊人一致的定位，表明它们在TGF β 2介导的程序性细胞死亡和重塑事件中起关键作用。初步的蛋白质印迹数据表明，与单独的每种试剂相比，用视黄酸和TGF β 2共处理分离的胰蛋白酶分散的心脏细胞1小时导致Smad 2/Smad 3的协同磷酸化。这种新的相互作用不是由于RXRa的转录作用，因为这种作用仅在一小时的处理后就观察到了。相反，在RXR α-/-中，免疫组织化学显示流出道中缺乏核定位的pSmad 2/pSmad 3。这些数据表明，在发育中的心脏中，维甲酸和TGF β 2信号级联之间存在一种新的显著串扰，这在以前是没有意识到的。因此，虽然维甲酸信号传导的长期转录效应被充分记录，但该通路的短期激活效应尚未被研究。因此，我们假设视黄酸信号可以在流出道重塑过程中以快速反应的方式调节TGF β 2介导的作用。使用整个小鼠胚胎培养物，原代细胞培养物和遗传杂交，我们提出了三个目的来验证这一假设：1）确定视黄酸对心脏中TGF β 2信号传导的影响，并评估RXR α在这些过程中的作用。2)确定视黄酸信号如何调节TGF β 2诱导的流出道细胞凋亡和重塑。3)确定RXR α是否与流出道中的Smad信号直接相互作用。

DESCRIPTION (provided by applicant): The long-term goal of our laboratory is to understand signaling mechanisms that underlie both normal and abnormal heart development as well as mechanisms that can cause congenital heart disease. Indeed, 1-2% of newborns have some form of congenital heart disease making it the most common congenital birth defect. Many of these cardiovascular defects are related to the formation and remodeling of endocardial cushion tissue into the valves and membranous septa of the heart. We have documented that the retinoid X receptor alpha null (RXRalpha-/-) mouse is an excellent model system to aid our understanding of how retinoic acid signaling influences heart development. Lethality in the embryonic day (E) 13.5 RXRalpha-/- mouse is marked, in part, by elevated TGFbeta2 and enhanced apoptosis in the outflow tract. TGFbeta2 primarily signals through phosphorylation (p) of Smad2/Smad3. pSmad2 and pSmadS show a striking coincident localization with apoptosis in E13.5 cushion mesenchyme suggesting they play a key role in programmed cell death and remodeling events mediated by TGFbeta2. Preliminary Western blot data demonstrates co-treatment of isolated trypsin-dispersed heart cells with retinoic acid and TGFbeta2 for one hour resulted in a synergistic phosphorylation of Smad2/Smad3 compared to each agent alone. This novel interaction is not due to transcriptional effects of RXRa since the effects were seen after only a one-hour treatment. Conversely, in the RXRalpha-/-, immunohistochemistry shows a lack of nuclear localized pSmad2/pSmad3 in the outflow tract. These data demonstrate a novel and significant crosstalk between the retinoic acid and TGFbeta2 signaling cascades in the developing heart that has not previously been realized. Thus, while long-term transcriptional effects of retinoic acid signaling are well documented, effects of short-term activation of the pathway have not been studied. Thus, we hypothesize that retinoic acid signaling can function in a rapid response manner to modulate TGFbeta2-mediated effects during outflow tract remodeling. Using whole mouse embryo cultures, primary cell cultures, and genetic crosses we propose three aims to test this hypothesis: 1) To determine the influence of retinoic acid on TGFbeta2 signaling in the heart and to assess the role for RXRalpha in these processes. 2) To determine how retinoic acid signaling regulates TGFbeta2-induced apoptosis and remodeling in the outflow tract. 3) To determine if RXRalpha directly interacts with Smad signaling in the outflow tract.