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。许多这些心血管缺陷与心内膜缓冲组织形成和重塑到心脏瓣膜和膜间隔有关。我们已经证明，类维甲酸X受体α null （rxrα -/-）小鼠是一个很好的模型系统，可以帮助我们理解维甲酸信号传导如何影响心脏发育。胚胎日(E) 13.5 RXRalpha-/-小鼠的致死性在一定程度上是通过TGFbeta2升高和流出道细胞凋亡增强来标志的。TGFbeta2主要通过Smad2/Smad3的磷酸化(p)发出信号。pSmad2和pSmadS在E13.5缓冲间质中显示出与凋亡惊人的一致定位，这表明它们在TGFbeta2介导的程序性细胞死亡和重塑事件中发挥关键作用。初步的Western blot数据表明，与单独使用这两种药物相比，将分离的胰蛋白酶分散的心脏细胞与维甲酸和TGFbeta2共同处理1小时可导致Smad2/Smad3的协同磷酸化。这种新的相互作用不是由于RXRa的转录作用，因为仅在治疗一小时后就看到了这种作用。相反，在rxrα -/-中，免疫组织化学显示流出道缺乏核定位的pSmad2/pSmad3。这些数据表明，维甲酸和TGFbeta2信号级联之间存在一种新的、重要的串扰，这在发育中的心脏中是前所未有的。因此，虽然维甲酸信号的长期转录作用已被充分记录，但该途径的短期激活的影响尚未被研究。因此，我们假设维甲酸信号可以在流出道重塑过程中以快速反应的方式调节tgfbeta2介导的作用。通过全鼠胚胎培养、原代细胞培养和遗传杂交，我们提出了三个目标来验证这一假设：1)确定维甲酸对心脏中tgfbet2信号传导的影响，并评估RXRalpha在这些过程中的作用。2)研究维甲酸信号如何调控tgfbeta2诱导的流出道细胞凋亡和重塑。3)确定RXRalpha是否直接与流出道的Smad信号相互作用。

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.