Transmembrane Proteolytic Induction and Thoracic Aneurysms

 描述(申请人提供)：胸主动脉瘤(TAA)是一种通常无症状的过程，是由于主动脉细胞外基质(ECM)异常重塑的结果，这种重塑削弱了主动脉壁，并导致粗大的扩张，通常进展为破裂。目前，治疗的选择包括外科重建或血管内介入；这两种方法都不能解决导致这种毁灭性疾病的潜在途径。TAAs受到细胞内和细胞外调节基质沉积和降解的机制的影响，部分是通过激活基质金属蛋白酶(MMPs)。此前，我们通过其在细胞周围蛋白分解和细胞内信号转导中的作用，确定了膜型1基质金属蛋白酶(MT1-基质金属蛋白酶)是TAA形成的关键介质。利用小鼠TAA模型，蛋白质水平 MT1-MMPs的表达在TAA发育过程中逐渐增加，并定位于主动脉成纤维细胞。此外，MT1-MMPs活性与主动脉扩张程度呈正相关。重要的是，当在MT1-MMP杂合缺陷小鼠中诱导TAA时，主动脉扩张减弱，这表明它是TAA进展所必需的。然而，关于在TAA发育过程中调节MT1-MMPs的表达、丰度和活性的机制还知之甚少。最近发现，microRNAs(MiRs)是参与蛋白质生产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-133a的表达丢失，miR-133a是一种针对MT1-MMP翻译的有效miR。MiR-133a的这种缺失与主动脉大小呈负相关，并与MT1-MMP蛋白的增加相一致。此外，一些研究表明MT1-MMPs可能被蛋白激酶C磷酸化，并可能通过内吞作用调节MT1-MMPs的活性。然而，这种磷酸化是否可以区分MT1-基质金属蛋白酶在细胞周围蛋白分解和细胞内信号传递中的多种作用仍有待确定。因此，这一建议的中心假设是，调制MT1-MMP蛋白丰度、活性和定位的动态调节介导了TAA的形成和进展，将进行以下特定目的的研究：(1)证明MT1-MMP的丰度和TAA的发展受到主动脉成纤维细胞表型变化的调节，而这是由miR133a表达的变化介导的；(2)证明细胞周围的蛋白降解和细胞内信号转导是由MT1-MMP细胞定位的变化介导的，受C末端残基的磷酸化调节。这些研究将为TAA发展过程中发生的机制变化提供证据，并将重点放在转录后和翻译后机制上，这些机制可能揭示潜在的治疗靶点。

 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.