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

描述（由申请人提供）：我们在筛选血管损伤相关基因时鉴定了一种新型蛋白质，即含胶原蛋白三重受阻重复序列-1（Cthrc 1）。它是一种从低等脊索动物到哺乳动物高度保守的独特蛋白质，在数据库中没有列出相关分子。Cthrc 1具有意想不到的生物发生，因为它包含用于分泌的信号肽，但仍保留在分化的平滑肌细胞（SMC）的细胞质中。在损伤Cthrc 1后，发现它在细胞外进行蛋白水解加工。 我们预测，细胞贩运涉及蛋白水解加工，因为N末端截短形式的Cthrc 1细胞外发现。Cthrc 1的过度表达导致I型和III型间质胶原的深度抑制。过度表达Cthrc 1的转基因小鼠由于缺乏胶原骨基质而具有脆性骨骼，并且它们的动脉在损伤后对新生内膜损伤形成具有显著抵抗性。Cthrc 1的抗纤维化作用与SMC中TGF-β信号的显著抑制有关。为了进一步评估Cthrc 1在血管形成和动脉重塑中的作用，我们建立了遗传性功能获得和功能丧失小鼠品系。此外，我们已经产生了在PDGFR β启动子控制下表达Cre重组酶的独特转基因小鼠品系。这些线使我们能够控制基因表达，特别是在血管外膜或中膜室以及周细胞。大约15%的杂合子Cthrc 1小鼠在胚胎发育期间或出生后一周内死于严重的血管异常，包括影响小血管和大血管的大规模扩张血管。在受影响小鼠的许多组织中，观察到似乎由畸形血管破裂引起的出血。因此，Cthrc 1是致死性单倍不足的部分突变的必需分子。本提案的目的是：i）使用Cthrc 1的条件性缺失以及平滑肌隔室和外膜中的过表达来表征Cthrc 1在血管形成和动脉重塑中的功能，ii）表征Cthrc 1与TGF-β信号传导途径的相互作用，以及iii）确定由纤溶酶产生的鉴定的Cthrc 1片段的功能。本研究拟采用Cthrc 1功能获得和功能丧失的小鼠模型，结合生物化学和细胞生物学方法研究Cthrc 1的功能机制。公共卫生相关性：我们发现了一种新的蛋白质Cthrc 1，并发现其能够有效抑制胶原蛋白的产生。过度的胶原蛋白产生是与慢性疾病相关的所有纤维化疾病的标志，在美国，45%的死亡最终可归因于这种疾病。由于其抑制胶原蛋白生成的能力，Cthrc 1可能非常适合未来抗纤维化疗法的发展。

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.