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?

Public Health Relevance

The specific gene that is mutated in the Tsk2/+ mouse is not known, but the result of this mutation creates a disease that closely resembles a type of Scleroderma called the Diffuse-proliferative subset. If we are successful in our gene discovery, gene expression and cell subset investigations, we anticipate that several 'druggable'targets for this devastating disease will be found. At the very least, the Tsk2/+ mouse may become a better alternative as a pre-clinical model of scleroderma than other existing models.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Project (R01)
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Arthritis, Connective Tissue and Skin Study Section (ACTS)
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Tseng, Hung H
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Drexel University
Schools of Medicine
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Long, Kristen B; Li, Zhenghui; Burgwin, Chelsea M et al. (2015) The Tsk2/+ mouse fibrotic phenotype is due to a gain-of-function mutation in the PIIINP segment of the Col3a1 gene. J Invest Dermatol 135:718-27
Long, Kristen B; Artlett, Carol M; Blankenhorn, Elizabeth P (2014) Tight skin 2 mice exhibit a novel time line of events leading to increased extracellular matrix deposition and dermal fibrosis. Matrix Biol 38:91-100
Long, Kristen B; Burgwin, Chelsea M; Huneke, Richard et al. (2014) Tight Skin 2 Mice Exhibit Delayed Wound Healing Caused by Increased Elastic Fibers in Fibrotic Skin. Adv Wound Care (New Rochelle) 3:573-581
Arron, Sarah T; Dimon, Michelle T; Li, Zhenghui et al. (2014) High Rhodotorula sequences in skin transcriptome of patients with diffuse systemic sclerosis. J Invest Dermatol 134:2138-45