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

描述(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。它可能会有危及生命的并发症，而且没有治愈的方法。SSc患者表现出一系列的疾病严重程度，一般从仅限于四肢的较轻的纤维化到更严重的、往往是致命的弥漫性疾病。Tsk2/+小鼠是SSC的模型。Tsk2突变在我们实验室的同源近交系(C57BL/6或B6)背景下培育出来，但Tsk2/+的基因尚不清楚，对其发病的分子基础也知之甚少。B6.Tsk2/+小鼠具有人类疾病的许多特征，包括皮肤紧凑，细胞外基质沉积失调，以及显著的自身免疫。我们发现Tsk2介导的自身免疫和纤维化征象随着年龄的增长而逐渐发展。根据疾病症状的局限性和弥漫性表现，SSc被分为不同的亚型。在我们的合作者团队的帮助下，我们现在发现，四周大的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重叠。TGF21在SSC中的促病作用是众所周知的，我们发现来自Tsk2/+小鼠的皮肤样本中TGF21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导致纤维化疾病从出生起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途径激活的弥漫性增生性SSC患者的子集，识别Tsk2基因将为这一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的Tsk2突变和野生型胚胎成纤维细胞，以便对这两个等位基因区间进行深度测序，以找到该基因。我们将在全球基因图谱研究中研究RNA的表达，并使用相同的样本，检验由此产生的ECM失调的组织学证据。我们将研究这一特征是否是细胞自主的，或者是否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质形成细胞。一旦我们确定了Tsk2基因，我们将测试在体外暴露于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26将共同解决关于SSC和我们的B6的关键问题。Tsk2/+模型：Col3a1是Tsk2/+特征的潜在基因还是突变发生在一个连锁的位置？Tsk2/+模型中的主要激发事件是什么？Tsk2等位基因是如何引发疾病的？TGF21何时以及如何发出ECM失调信号？TGF21信号的直接靶点是什么？B6.Tsk2/+小鼠能否作为弥漫性增殖性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?