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

描述（由申请人提供）：系统性硬化症（SSc）是一种复杂的炎性纤维化疾病，具有几种不同的临床表现。它可能会导致危及生命的并发症，而且无法治愈。SSc患者表现出疾病严重程度的连续性，通常范围从限于四肢的较轻纤维化到更严重且通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突变已被培育到同质近交系（C57Bl/6，或B6）背景上，但Tsk2/+的基因是未知的，对他们疾病的分子基础知之甚少。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和显著的自身免疫。我们发现Tsk2介导的自身免疫和纤维化体征随着年龄的增长而逐渐发展。SSc根据疾病症状的局限性和弥漫性表现被分为不同亚型。与我们的合作者团队一起，我们现在表明，4周的Tsk2/+小鼠在基因表达上与“弥漫性增殖性”人类疾病亚型有显著的重叠。TGF21在SSc中的促病作用是众所周知的，我们发现Tsk2/+小鼠的皮肤样本中TGF21的下游靶点显著增加。在此，我们建议鉴定Tsk2基因并了解其作用机制。这只小鼠提供了一个独特的机会来检查从出生起导致纤维化疾病的多种临床参数的途径。我们的主要假设是，Tsk2/+小鼠准确地代表了皮肤显示TGFb通路激活的弥漫性增生性SSc患者的亚群，并且鉴定Tsk2基因将为这组患者的疾病机制提供独特的见解。我们将从这些小鼠中分离纯合子Tsk2突变体和野生型胚胎成纤维细胞，目的是对两个等位基因间隔进行深度测序以找到该基因。我们将在全球基因谱研究中研究RNA表达，并使用相同的样本，我们将检查由此产生的ECM失调的组织学证据。我们将研究这种特性是细胞自主的还是需要不同细胞亚型的共同努力，特别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化细胞。一旦我们确定了Tsk2基因，我们将在体外测试暴露于Tsk2/+细胞的ECM成分后纤维化细胞行为的显著变化。来自三个不同机构的四位合作者将共同探讨有关SSc和B6的关键问题。Tsk2/+模型：Col3a1是Tsk2/+性状的基因，还是突变发生在一个连锁位点？Tsk2/+模型中的主要刺激事件是什么？ Tsk2等位基因如何引发疾病？TGF21何时以及如何发出ECM失调信号？TGF21信号传导的直接靶点是什么？B6。Tsk2/+小鼠可作为弥漫性增殖性SSc的良好模型

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?