Understanding the biological activity of Cthrc1 cleavage fragments

了解 Cthrc1 切割片段的生物活性

• 批准号: 7394648
• 负责人: Renee LeClair
• 金额: $ 4.96万
• 依托单位: MAINEHEALTH
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7394648
• 关键词: Address; Animals; Biochemical; Biological; Biological Assay; Blood Vessels; Bos taurus; Cattle; Cell Line; Cell Proliferation; Cells; Characteristics; Cleaved cell; Clinical; Collagen; Defect; Deposition; Development; Disease; Embryo; Equilibrium; Event; Excision; Family member; Fibroblasts; Fibrosis; Gene Targeting; Generations; Genes; Goals; Harvest; Histology; In Vitro; Injury; Investigation; Knock-out; Length; Lesion; Location; Luciferases; Molecular; Mus; Organ; Pattern; Plasmin; Play; Process; Protein Overexpression; Proteins; Proteolytic Processing; Regulation; Reporter; Reporting; Role; S100A8 gene; Series; Signal Pathway; Signal Transduction; Smooth Muscle Myocytes; Societies; TGF-beta type I receptor; Time; Tissues; Transforming Growth Factors; Transgenic Mice; Transgenic Model; Translating; Vascular remodeling; Western Blotting; Work; base; cell motility; clinical application; clinically relevant; human TGFBR2 protein; in vivo; inhibitor/antagonist; member; mortality; novel; receptor; repaired; research study; therapy development; tool; triple helix

DESCRIPTION (provided by applicant): Collagen Triple Helix Repeat Containing protein 1 (Cthrd) is a gene product with novel biochemical activities and its ability to reduce collagen deposition by inhibition of Smad 2/3 activation could have major clinical applications. Both overexpression and knockout of Cthrd result in various levels of lethality, while the surviving animals are characteristic of vascular defects. These observations suggest that the characterization of Cthrd will be instructive in understanding vascular development, repair and fibrosis. We hypothesize that Cthrd expression in vascular cells regulate transforming growth factor-p (TGF-B) responsiveness, thereby impacting TGF-P target genes, including collagens, and regulation or activation of Cthrd is strictly controlled in these cells. The proposed experiments specifically address the following hypotheses: 1) Removal of the Cthrd propeptide is essential for the inhibition of TGF-B signaling via Smad 2/3 signaling and 2) Cthrd directly or indirectly inhibits TGF-B signaling by blocking signal transduction via TGF- 3 receptors. Plasmin is capable of cleaving Cthrd in two locations and the activity of these fragments will be assessed in vitro using a collagen deposition assay in smooth muscle cells and luciferase reporter assay using PAC1 cells stably transfected with the TGF-B sensitive (CAGA) 12-Luc reporter. The generation of transgenic mouse lines overexpressing each of these fragments will be instrumental in addressing the effect of these processed Cthrd fragments during development. Harvesting of various embryonic time points and histology will allow us to examine the phenotypic consequences of overexpression of cleaved forms of Cthrd. The potential mechanism of collagen regulation is hypothesized to be a novel interaction of Cthrd with the TGF-B signaling pathway. Specific aim two will begin to examine the location of Cthrd inhibition of TGF-B signaling. The effect of Cthrd (both full length, and the plasmin generated fragments), on levels of Smad 2/3 activation in cell lines stably transected with a series of constitutively active TGF-B receptors will allow us to address this question. Using various luciferase reporter constructs we also seek to determine whether Cthrd is specific to inhibition of signaling via Smad 2 or 3. Determining Cthrd activation (or inactivation) and addressing the mechanism of TGF-B inhibition are key aspects to translating this work into clinically relevant applications. Diseases involving fibrosis involve nearly every major organ and without effective treatment, often result in mortality. The development of therapies involving regulation of Cthrd has the potential to impact society and the biochemical characterization of this gene is the first step towards achieving these goals.

描述（由申请人提供）：含有蛋白质1（CTHRD）的胶原蛋白三重螺旋重复是具有新型生化活性的基因产物及其通过抑制SMAD 2/3激活减少胶原沉积的能力，可能具有主要的临床应用。 CTHRD的过表达和敲除都导致各种致死性，而幸存的动物是血管缺陷的特征。这些观察结果表明，CTHRD的表征将具有理解血管发育，修复和纤维化的启发性。我们假设血管细胞中的CTHRD表达调节转化生长因子-P（TGF-B）响应性，从而影响TGF-P靶基因，包括胶原，以及CTHRD的调节或激活在这些细胞中受到严格控制。提出的实验专门针对以下假设：1）去除CTHRD丙肽对于通过SMAD 2/3信号抑制TGF-B信号传导至关重要。纤溶酶能够在两个位置裂解CTHRD，并将使用胶原沉积测定法在平滑肌细胞中的胶原沉积测定法和荧光素酶报告基因测定法中使用PAC1细胞在体外进行评估，并使用TGF-B敏感（CAGA）12-luc Reporter稳定转染的PAC1细胞进行评估。过表达这些片段中每一个的转基因小鼠系的产生将有助于解决这些过程中这些处理后的CTHRD片段的效果。收集各种胚胎时间点和组织学将使我们能够检查CTHRD裂解形式过表达的表型后果。假设胶原蛋白调节的潜在机制是CTHRD与TGF-B信号通路的新型相互作用。具体目标两个将开始检查CTHRD抑制TGF-B信号传导的位置。 CTHRD（全长和纤溶酶产生的片段）的影响，对与一系列组成型活性TGF-B受体稳定转移的细胞系中SMAD 2/3激活的效果将使我们能够解决这个问题。使用各种荧光素酶报告基因构建体，我们还试图确定CTHRD是否特定于通过SMAD 2或3的信号抑制。确定CTHRD激活（或灭活）并解决TGF-B抑制的机制是将这项工作转化为临床相关应用的关键方面。涉及纤维化的疾病几乎涉及每个主要器官，没有有效的治疗，通常会导致死亡率。涉及调节CTHRD的疗法的发展具有影响社会的潜力，并且该基因的生化特征是实现这些目标的第一步。