Transmembrane Proteolytic Induction and Thoracic Aneurysms

 描述（由申请方提供）：胸主动脉瘤（TAA）通常是一种无症状的过程，是由于主动脉细胞外基质（ECM）的异常重塑而发生的，ECM会削弱主动脉壁并导致大体扩张，通常进展为破裂。目前，治疗方案包括手术重建或血管内介入;这两种方法都没有解决驱动这种毁灭性疾病的潜在途径。TAA受到细胞内和细胞外机制的影响，这些机制部分通过激活基质金属蛋白酶（MMP）来调节基质沉积和降解。在此之前，我们确定了膜1型MMP（MT 1-MMP）作为TAA形成的关键介质，通过其在细胞周围蛋白水解和细胞内信号传导中的作用。利用TAA小鼠模型，蛋白水平 MT 1-MMP在TAA形成过程中表达增加，并定位于主动脉成纤维细胞。此外，MT 1- MMP活性显示与主动脉扩张直接相关。重要的是，当在MT 1-MMP杂合缺陷小鼠中诱导TAA时，主动脉扩张减弱，表明这是TAA进展所必需的。然而，关于在TAA发展过程中调节MT 1-MMP的时间表达、丰度和活性的机制知之甚少。microRNA（miRs）是近年来发现的参与蛋白质转录后调控的上游调节因子。我们已经证明了在TAA发展过程中miR-133 a表达的损失，miR-133 a是一种经验证的靶向MT 1-MMP翻译的miR。miR-133 a的缺失与主动脉大小呈负相关，与MT 1-MMP蛋白的增加一致。此外，一些研究表明，MT 1-MMP可能被蛋白激酶C磷酸化，并可能通过内吞作用在调节MT 1-MMP活性中发挥作用。然而，这种磷酸化是否可以区分MT 1-MMP在细胞周围蛋白水解和细胞内信号传导方面的多种作用仍有待确定。因此，该建议的中心假设是，MT 1-MMP蛋白丰度、活性和定位的动态调节的调节介导TAA的形成和进展，这将用以下具体目的来检验：（1）证明MT 1-MMP丰度和TAA的发展受主动脉成纤维细胞表型变化的调节，所述表型变化由miR 133 a表达的变化介导;（2）证明细胞周围蛋白水解和细胞内信号传导是由MT 1- MMP细胞定位的变化介导的，由C-末端残基的磷酸化调节。这些研究将为TAA发育过程中发生的机制变化提供证据，并将重点关注可能揭示潜在治疗靶点的转录后和翻译后机制。

 DESCRIPTION (provided by applicant): Thoracic aortic aneurysms (TAAs), a generally asymptomatic process, develop as a consequence to abnormal remodeling of the aortic extracellular matrix (ECM) which weakens the aortic wall and leads to gross dilation that typically progresses to rupture. Currently, the treatment options consist of surgical reconstruction or endovascular intervention; neither of which addresses the underlying pathways which drive this devastating disease. TAAs are influenced by both intracellular and extracellular mechanisms that function to regulate matrix deposition and degradation, in part through the activation of the matrix metalloproteinases (MMPs). Previously, we identified membrane type-1 MMP (MT1-MMP) as a key mediator of TAA formation, through its role in both pericellular proteolysis and intracellular signaling. Utilizing murine model of TAA, protein levels of MT1-MMP increased during TAA development and localized to aortic fibroblasts. Moreover, MT1- MMP activity displayed a direct relationship with aortic dilatation. Importantly, when TAAs were induced in MT1-MMP heterozygous deficient mice, aortic dilatation was attenuated, suggesting it is required for TAA progression. Nevertheless, little is known in regards to the mechanisms that regulate the temporal expression, abundance, and activity of MT1-MMP during TAA development. MicroRNAs (miRs) have recently been identified as upstream mediators involved in the post-transcriptional regulation of protein production. We have demonstrated a loss in miR-133a expression, a validated miR that targets MT1-MMP translation, during TAA development. This loss of miR-133a displayed an inverse relationship with aortic size and coincided with an increase in MT1-MMP protein. Furthermore, several studies have suggested that MT1-MMP may be phosphorylated by protein kinase C and may play a role in regulating MT1-MMP activity through endocytosis. However, whether or not this phosphorylation may differentiate the multiple roles of MT1-MMP in regards to pericellular proteolysis and intracellular signaling remains to be defined. Accordingly, the central hypothesis of this proposal is that modulation of the dynamic regulation of MT1-MMP protein abundance, activity, and localization mediates TAA formation and progression, which will be examined with the following specific aims: (1) Demonstrate that MT1-MMP abundance and TAA development are regulated by changes in aortic fibroblast phenotype, mediated by changes in miR133a expression; (2) Demonstrate that pericellular proteolysis and intracellular signaling are mediated by changes in MT1- MMP cellular localization, regulated by phosphorylation of C-terminal residues. These studies will provide evidence for mechanistic changes that occur during TAA development, and will focus on both post-transcriptional and post-translational mechanisms that may reveal potential therapeutic targets.