Characterization of the cell-autonomous traits of Tsk2/+ mice, a model of systemic sclerosis

Chelsea Marie Burgwin

doi:10.17918/etd-7149

Overview: The tight skin 2 (Tsk2/+) mouse is a genetic model of systemic sclerosis (SSc). This model has several similarities to human disease including tight skin, increased collagen, and alterations in the extracellular matrix. There are multiple goals of this thesis research, including confirming the role of causal mutation of Tsk2/+ in both lethality and fibrosis, determining critical drivers of the cell-autonomous Tsk2/+ phenotype, and quantifying Tsk2/+ dermal health. Approach: We used RNA and DNA sequencing methods to identify Tsk2/+ specific SNPs. The most promising of these SNPs caused a cysteine-to-serine amino acid change in COL3A1. We examined primary dermal Tsk2/+ and WT fibroblasts to discover previously uncharacterized components of the cell-autonomous Tsk2/+ phenotype; then we used this phenotype to evaluate C33S in vitro. Two pathways of interest, TGF-[beta] and TWEAK/FN14, were inhibited in vitro to determine their role in the Tsk2/+ phenotype. Results: To confirm that the C33S mutation is responsible for the lethality of Tsk2/Tsk2 homozygotes, we crossed Tsk2/+ mice to a mouse carrying one copy of the Col3a1-knockout allele (Col3a1HET), and determined that the compound Tsk2/Col3a1- genotype was not viable. We found that in addition to Col1a1, other Tsk2/+ specific cell-autonomous genes were upregulated in primary dermal Tsk2/+ fibroblasts. Transfection of C33S-mutated Col3a1 (Col3a1Tsk2) DNA in plasmid form resulted in increased mRNA expression of pro-fibrotic signatures compared to Col3a1WT transfections. Inhibition of FN14 and TGF-[beta] in vitro significantly decreased the fibrotic signature observed in Col3a1Tsk2 transfections. Surprisingly, breeding Tsk2/+ mice to Fn14KO mice did not improve the Tsk2/+ fibrotic phenotype in vivo. Conclusions: We have proven that the C33S mutation in COL3A1 is responsible for lethality and can induce fibrosis in Tsk2/+ mice. Based on sequence homology between this region in type I, II, and III collagen, we suspect the C33S mutation disrupts the ability of COL3A1 (C33S) to bind and sequester TGF-[beta]. We postulate that failure to bind TGF-[beta] would result in increased TGF-[beta] signaling in Tsk2/+ dermis compared to WT littermates. This suspicion is supported by the ability of FN14 and TGFBR1 inhibitors to downregulate fibrotic signatures induced by Col3a1Tsk2 transfection, implying these pathways are essential for Tsk2/+ cell-autonomous signaling. However, Fn14KO breeding failed to improve the Tsk2/+ mouse phenotype, demonstrating that abrogation of Tsk2/+ fibrosis in vivo may require a multipronged approach.

Characterization of the cell-autonomous traits of Tsk2/+ mice, a model of systemic sclerosis

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Characterization of the cell-autonomous traits of Tsk2/+ mice, a model of systemic sclerosis

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