Identifying the Gene for Scleroderma in Tsk/2 mice that model Human SSc Subsets

鉴定模拟人类 SSc 亚群的 Tsk/2 小鼠中的硬皮病基因

• 批准号: 8331376
• 负责人: Elizabeth P Blankenhorn
• 金额: $ 27.83万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-10 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8331376
• 关键词: 101/H Mouse; Address; Affect; Age; Alleles; Animal Model; Appearance; Area; Autoimmune Diseases; Autoimmune Process; Autoimmunity; Backcrossings; Birth; Breeding; Cells; Classification; Clinical; Collagen; Collection; Complex; DNA; Dendritic Cells; Deposition; Development; Diffuse; Disease; Embryo; Etiology; Event; Exposure to; Extracellular Matrix; Fibroblasts; Fibrosis; Gene Expression; Gene Expression Profile; Gene Mutation; Genes; Genetic; Genetic Transcription; Genome; Genomics; Human; Immunohistochemistry; In Vitro; Inbred Strains Mice; Inbreeding; Inflammatory; Institution; Investigation; Laboratories; Lead; Life; Limb structure; Link; Lung; Mediating; Modeling; Molecular; Molecular Profiling; Mus; Mutate; Mutation; Pathogenesis; Pathway interactions; Patients; Pattern; Phenotype; Pre-Clinical Model; Precipitating Factors; Predisposition; Process; Relative (related person); Role; Sampling; Scientist; Scleroderma; Severities; Severity of illness; Signal Transduction; Skin; Symptoms; Systemic Scleroderma; Testing; Time; base; cDNA Arrays; cell behavior; congenic; consomic; gene discovery; genome-wide; human disease; insight; keratinocyte; mRNA Expression; mast cell; molecular marker; mouse model; mutant; skin disorder; therapeutic target; trait

DESCRIPTION (provided by applicant): Systemic sclerosis (SSc) is a complex inflammatory fibrotic disease, with several distinct clinical manifestations. It can have life-threatening complications and there is no cure. SSc patients show a continuum of disease severity, generally ranging from less severe fibrosis limited to the extremities, to a more severe and often fatal diffuse disease. Tsk2/+ mice are a model for SSc. The Tsk2 mutation has been bred onto a homogeneous inbred (C57Bl/6, or B6) background in our laboratory, but the gene for Tsk2/+ is unknown, and little is understood about the molecular basis for their disease. B6.Tsk2/+ mice have many features of the human disease, including tight skin, dysregulated extracellular matrix deposition, and significant autoimmunity. We have found that Tsk2- mediated autoimmune and fibrotic signs develop progressively with age. SSc is classified into subtypes based on the limited vs. disseminated appearance of disease symptoms. With our team of collaborators, we now show that the Tsk2/+ mice at four weeks have a significant overlap in gene expression with the "diffuse- proliferative" human disease sub-type. The disease-promoting role of TGF21 in SSc is well-known, and we find that skin samples from Tsk2/+ mice have substantial increases in downstream targets of TGF21. Herein we propose to identify the Tsk2 gene and understand its mechanism of action. This mouse affords a unique opportunity to examine the pathways leading to the multiple clinical parameters of fibrotic disease from birth onward. Our lead hypothesis is that the Tsk2/+ mouse accurately represents the subset of diffuse- proliferative SSc patients whose skin shows activation of the TGFb pathway, and that identifying the Tsk2 gene will provide unique insight into the mechanism of disease in this group of patients. We will isolate homozygous Tsk2 mutant and wild type embryonic fibroblasts from these mice for purposes of deep sequencing the two allelic intervals to find the gene. We will study both RNA expression in global gene profiling studies and with the same samples, we will examine the resulting histological evidence of ECM dysregulation. We will study whether the trait is cell-autonomous or whether it requires the concerted efforts of different cell subtypes, especially dendritic cells or mast cells, and the fibroblasts and keratinocytes that are in the affected areas. Once we have the identity of the Tsk2 gene, we will test for significant changes in fibrotic cell behavior after exposure to ECM components from the Tsk2/+ cells in vitro. Together, the four collaborators 26 from three different institutions will address key questions about SSc and our B6.Tsk2/+ model: Is Col3a1 the gene underlying the Tsk2/+ traits or has the mutation occurred in a linked locus? What is the major inciting event in the Tsk2/+ model and how does the Tsk2 allele initiate disease? When and how does TGF21 signal ECM dysregulation? What is the direct target of TGF21 signaling? Can B6.Tsk2/+ mice be used as a good model for diffuse-proliferative SSc?

描述（由申请人提供）：系统性硬化症（SSC）是一种复杂的炎症性纤维化疾病，具有几种不同的临床表现。它可能会有威胁生命的并发症，并且无法治愈。 SSC患者表现出疾病严重程度的连续性，通常从不太严重的纤维化限制到四肢，到更严重且经常致命的弥漫性疾病。 TSK2/+小鼠是SSC的模型。在我们的实验室中，TSK2突变已繁殖到同质的近交（C57BL/6或B6）背景上，但是TSK2/+的基因尚不清楚，对其疾病的分子基础知识很少。 B6.TSK2/+小鼠具有许多人类疾病的特征，包括皮肤紧绷，细胞外基质沉积失调以及明显的自身免疫性。我们发现TSK2-介导的自身免疫和纤维化迹象随着年龄的增长而逐渐发展。基于疾病症状的有限与传播外观，SSC分为亚型。与我们的合作者团队一起，我们现在表明，四周时TSK2/+小鼠的基因表达与“弥漫性增殖”人类疾病亚型具有显着重叠。 TGF21在SSC中促进疾病的作用是众所周知的，我们发现来自TSK2/+小鼠的皮肤样本在TGF21的下游靶标有很大的增加。 在此，我们建议识别TSK2基因并了解其作用机理。该小鼠提供了一个独特的机会来检查导致从出生开始的纤维化疾病多个临床参数的途径。我们的主要假设是，TSK2/+小鼠准确地表示皮肤显示TGFB途径激活的弥漫性增生性SSC患者的子集，并且鉴定TSK2基因将提供对这组患者疾病机制的独特见解。我们将从这些小鼠中分离纯合TSK2突变体和野生型胚胎成纤维细胞，以深入测序两个等位基因间隔以找到该基因。 我们将研究全球基因分析研究中的RNA表达，并且通过相同的样品，我们将研究ECM失调的组织学证据。我们将研究该性状是细胞自主的，还是需要不同细胞亚型（尤其是树突状细胞或肥大细胞）以及受影响区域的成纤维细胞和角质形成细胞的共同努力。一旦我们具有TSK2基因的身份，我们将在体外暴露于TSK2/+细胞的ECM成分后测试纤维化细胞行为的显着变化。总之，来自三个不同机构的四个合作者26将解决有关SSC和我们的B6.TSK2/+模型的关键问题：COL3A1是TSK2/+特征的基因还是该突变发生在链接的轨迹中？ TSK2/+模型中的主要煽动性事件是什么？TSK2等位基因如何引发疾病？ TGF21何时以及如何信号ECM失调？ TGF21信号的直接目标是什么？ B6.TSK2/+小鼠可以用作弥漫性增强SSC的良好模型吗？