FAK signaling in cardiac growth and hypertrophy

描述(申请人提供)：最近的遗传证据表明，整合素类的纤维连接蛋白结合黏附受体(5E1和其他)可以调节心脏的形态和功能。整合素连接推动许多结构分子和信号分子重新聚集到腹侧质膜，统称为“焦点黏附”，用于将细胞内产生力量的肌动蛋白细胞骨架连接到细胞外基质(ECM)，并协调下游信号通路的激活。非受体酪氨酸激酶，粘着斑激酶(FAK)被整合素和生长因子强烈激活，在生长发育过程中可能整合这些不同途径的下游信号。事实上，FAK的胚系缺失导致中胚层缺陷和e7.5-10之间的胚胎死亡，类似于在纤维连接蛋白基因缺失和D5基因缺失的小鼠中观察到的表型。尽管FAK在心脏发育中的直接作用尚未得到研究，但来自FAK缺失胚胎的心脏显示缺乏单独的心内膜和中层，这表明心肌细胞成熟存在缺陷。有趣的是，我们和其他人最近的工作清楚地表明，FAK在培养的心肌细胞中被各种肥大刺激激活，包括苯肾上腺素(PE)、内皮素I(ET-1)、血管紧张素II(AII)和低渗应激，并且在肥厚的心脏中可以观察到体内心脏FAK活性的增加。FAK激活在心肌细胞肥大的发展中起直接作用的观点是显而易见的，我们的开创性发现是PE刺激的培养细胞肥大需要FAK的激活，其他人的类似发现也表明FAK是最大的ET-1和牵张诱导的体外肥大所必需的。该方案的实验目标是验证FAK在体内调节心脏发育和病理性肥大的假说，并确定参与这些过程的FAK依赖的信号通路。我们将使用Cre/loxP技术产生转基因小鼠，在其中FAK将以时间和心脏受限的方式被删除，以研究FAK在心脏生长中的功能作用。我们还将建立心肌细胞培养模型，以确定FAK依赖的信号和受肥大刺激差异调控的靶基因。

DESCRIPTION (provided by applicant): Recent genetic evidence indicates that the integrin class of fibronectin-binding adhesion receptors (5E1 and others) can regulate both the form and function of the heart. Integrin ligation drives recruitment of a number of structural and signaling molecules to the ventral plasma membrane collectively termed a "focal adhesion" which serves to link the force-generating actin cytoskeleton inside the cell to the extracellular matrix (ECM), and to coordinate activation of downstream signaling pathways. The non-receptor tyrosine kinase, Focal Adhesion Kinase (FAK) is strongly activated by both integrins and growth factors, and is a likely candidate to integrate downstream signals from these diverse pathways during growth and development. Indeed, germline deletion of FAK results in mesodermal defects and embryonic lethality between E7.5-10 similar to the phenotype observed in both fibronectin-, and D5-null mice. Although a direct role for FAK in cardiac development has yet to be examined, hearts from FAK-null embryos revealed a lack of separate mesocardial and endocardial layers, indicative of a defect in cardiomyocyte maturation. Interestingly, recent work by our group and others clearly indicate that FAK is activated in cultured cardiomyocytes by a variety of hypertrophic stimuli including, phenylephrine (PE), endothelin I (ET-1), angiotensin II (AII), and hypo-osmotic stress, and that increased cardiac FAK activity is observed in vivo in hypertrophic hearts. The idea that FAK activation plays a direct role in the development of cardiomyocyte hypertrophy is evident from our seminal findings that the activation of FAK is required for PE-stimulated hypertrophy of cultured cells and similar findings from others that FAK is required for maximal ET-1 and stretch-induced hypertrophy in vitro. The experimental goals of this proposal are to test the hypothesis that FAK regulates cardiac development and pathological hypertrophy in vivo and to identify the FAK-dependent signaling pathways involved in these processes. We will generate genetically modified mice in which FAK will be deleted in a temporal and cardiac-restricted fashion using Cre/LoxP technology to examine a functional role for FAK in cardiac growth. We will also establish a cardiac cell culture model to identify FAK-dependent signals and target genes that are differentially regulated by hypertrophic stimuli.