Cthrc1 function as a novel TGF-beta antagonist in the vasculature

描述（由申请人提供）：我们在与血管损伤相关的基因筛选中发现了新的蛋白，胶原三螺旋重复包含-1 （Cthrc1）。它是一种独特的蛋白质，从低等脊索动物到哺乳动物都高度保守，数据库中没有列出相关分子。Cthrc1具有意想不到的生物发生机制，因为它含有一种用于分泌的信号肽，但仍然存在于分化的平滑肌细胞（SMC）的细胞质中。损伤后，它在细胞外被发现，在那里它进行蛋白水解加工。我们预测细胞运输与蛋白水解加工有关，因为在细胞外发现了N末端截断形式的Cthrc1。过表达Cthrc1导致I型和III型间质性胶原蛋白的严重抑制。过表达Cthrc1的转基因小鼠由于缺乏胶原骨基质而骨质脆性，其动脉在损伤后对内膜病变的形成具有显著的抵抗力。Cthrc1的抗纤维化作用与SMC中TGF-ss信号的显著抑制有关。为了进一步评估Cthrc1在血管形成和动脉重塑中的作用，我们建立了基因功能获得和功能丧失的小鼠品系。此外，我们在PDGFRss启动子的控制下，产生了独特的表达Cre重组酶的转基因小鼠系。这些细胞系使我们能够控制血管外膜或内侧隔室以及周细胞中的基因表达。大约15%的杂合型cthrc1小鼠在胚胎发生期间或出生后一周内死亡，原因是严重的血管异常，包括影响小血管和大血管的血管大量扩张。在受感染小鼠的许多组织中，可以观察到出血，这似乎是由畸形血管破裂引起的。因此，Cthrc1是致死性单倍不足的重要分子。本研究的目的是：i)利用Cthrc1的条件缺失以及在平滑肌室和外膜中的过表达来表征Cthrc1在血管形成和动脉重构中的功能；ii)表征Cthrc1与TGF-ss信号通路的相互作用；iii)确定已鉴定的由纤溶酶产生的Cthrc1片段的功能。本课题拟采用Cthrc1功能获得和功能丧失小鼠模型，结合生物化学和细胞生物学方法研究Cthrc1功能的作用机制。公共卫生相关性：我们鉴定了新的蛋白Cthrc1，并发现其有效抑制胶原蛋白产生的能力。过多的胶原蛋白生成是所有与慢性疾病相关的纤维化疾病的标志，美国45%的死亡最终可归因于此类疾病。由于其抑制胶原蛋白生成的能力，Cthrc1可能非常适合未来抗纤维化治疗的发展。

DESCRIPTION (provided by applicant): We identified the novel protein, Collagen Triple Helix Repeat-Containing-1 (Cthrc1), in a screen for genes associated with vascular injury. It is a unique protein that is highly conserved from lower chordates to mammals, and there are no related molecules listed in the databases. Cthrc1 has an unexpected biogenesis because as it contains a signal peptide for secretion yet remains in the cytoplasm of differentiated smooth muscle cells (SMC). Upon injury Cthrc1, it is found extracellularly where it undergoes proteolytic processing. We predict that cellular trafficking relates to proteolytic processing, because N terminally truncated forms of Cthrc1 are found extracellularly. Overexpression of Cthrc1 leads to profound suppression of interstitial collagen types I and III. Transgenic mice overexpressing Cthrc1 have brittle bones due a lack of collagenous bone matrix, and their arteries are remarkably resistant to neointimal lesion formation upon injury. The antifibrotic effects of Cthrc1 are associated with dramatic inhibition of TGF-ss signaling in SMC. To further assess the role of Cthrc1 in blood vessel formation and arterial remodeling, we have established genetic gain-of-function and loss-of-function mouse strains. In addition, we have generated unique transgenic mouse lines expressing Cre recombinase under the control of the PDGFRss promoter. These lines allow us to control gene expression specifically in the adventitial or medial compartment of blood vessels as well as in pericytes. Approximately 15% of heterozygous Cthrc1mice die during embryogenesis or within one week after birth as the result of severe vascular abnormalities, which include massively dilated vessels affecting small as well as larger vessels. In many tissues of affected mice, bleeding is observed that appears to be caused by rupture of malformed vessels. Thus Cthrc1 is an essential molecule with partial penetrance of lethal haploinsufficiency. The objectives of this proposal are i) to characterize the function of Cthrc1 in blood vessel formation and arterial remodeling, using conditional deletion of Cthrc1 as well as overexpression in the smooth muscle compartment and the adventitia, ii) to characterize the interaction of Cthrc1 with the TGF-ss signaling pathway, and iii) to determine the function of the identified Cthrc1 fragments generated by plasmin. This proposal will employ mouse models of Cthrc1 gain-of-function and loss-of function, in combination with biochemical and cell biological methods to investigate the mechanism of Cthrc1 function. PUBLIC HEALTH RELEVANCE: We identified the novel protein Cthrc1 and discovered its ability to potently suppress collagen production. Excessive collagen production is a hallmark of all fibrosing disorders associated with chronic diseases, and 45% of all deaths in the USA are ultimately attributable to such disorders. With its ability to inhibit collagen production, Cthrc1 could be very suitable for the development of future antifibrotic therapies.