The Tsk2/+ mouse fibrotic phenotype is due to a gain-of-function mutation in the PIIINP segment of the Col3a1 gene

Abstract

Systemic sclerosis (SSc) is a polygenic, autoimmune disorder of unknown etiology, characterized by the excessive accumulation of extracellular matrix (ECM) proteins, vascular alterations, and autoantibodies. The tight skin (Tsk)2/+ mouse model of SSc demonstrates signs similar to SSc including tight skin and excessive deposition of dermal ECM proteins. By linkage analysis, we mapped the Tsk2 gene mutation to <3 megabases on chromosome 1. We performed both RNA sequencing of skin transcripts and genome capture DNA sequencing of the region spanning this interval in Tsk2/+ and wild-type littermates. A missense point mutation in the procollagen III amino terminal propeptide segment (PIIINP) of collagen, type III, alpha 1 (Col3a1) was found to be the best candidate for Tsk2; hence, both in vivo and in vitro genetic complementation tests were used to prove that this Col3a1 mutation is the Tsk2 gene. All previously documented mutations in the human Col3a1 gene are associated with the Ehlers-Danlos syndrome, a connective tissue disorder that leads to a defect in type III collagen synthesis. To our knowledge, the Tsk2 point mutation is the first documented gain-of-function mutation associated with Col3a1, which leads instead to fibrosis. This discovery provides insight into the mechanism of skin fibrosis manifested by Tsk2/+ mice.

Figure 1

Tsk2 lies between and not including 44.67 – 46.27Mb Mb on chromosome 1 (A) The Tsk2 interval was narrowed by genotyping back-crossed mice on the B6 and B6.chr 1-A/J backgrounds. Grey bars (101/H) depict the original parental strain, bearing Tsk2. White bars depict the B6 genome. Indefinite areas between typed markers are grey. Recombinants A – G bear additional recombination sites. The phenotypes are tight (T – Tsk2/+) or loose (L – WT). (B) With the use of additional markers (arrows, see text), the current interval comprises Col3a1, Col5a2, Wdr75, Slc40a1, part of Gulp1, and part of Dnahc7b; the five latter genes do not have coding region mutations. The elements of the Gulp1 gene above 44.67 Mb are excluded by the recombination in mouse F, and Dnahc7b below 46.27 is excluded by Mouse G.

Figure 2

Col3a1 is the only interval gene expressed at high levels in the skin of Tsk2/+ mice. (A) This graph shows gene expression for the seven Tsk2 interval genes, as determined from the RNA-Seq abundance results. (B) Heat map for seven Tsk2 interval genes detected as transcripts in RNA-Seq. (C + D) Distribution of nucleotide calls in heterozygous Tsk2/+ and homozygous WT mice for Col3a1 and Gulp1.

Figure 3

Mouse Col3a1-KO fibroblasts transfected with mutant Col3a1^Tsk2 express a more fibrotic protein profile than Col3a1^WT transfectants. (A) Culture supernatants assayed by Western blot for COL1A1. Col3a1^Tsk2 transfectants produced 34% more COL1A1 than Col3a1^WT (p<0.001) or mock transfectants (p<0.0001). (B) Col1a1 mRNA is more highly expressed in Col3a1-KO fibroblasts transfected with Col3a1^Tsk2 than with Col3a1^WT (p<0.0001). (C) There was no significant difference in efficiency of plasmid transfection between Col3a1^Tsk2 and Col3a1^WT. (D) Col3a1^−/− fibroblasts transfected with Col3a1^Tsk2 show a significant increase in Gene Ontology (GO) terms associated with fibrosis. (E) Expression of the genes that contributed most to the ECM enrichment results in in Col3a1^Tsk2 vs. Col3a1^WT transfected mice fibroblasts or in 4-week old female Tsk2/+ vs. WT mice. (F) Expression of genes that contributed to integrin binding term. (G) Expression of genes that contributed to transmembrane receptor protein kinase activity term.

Figure 4

Tsk2/+ mice have increased reticular fiber accumulation and COL3A1 in skin compared WT littermates. (A) Reticular fiber staining was performed on mice of the indicated ages (2–23 weeks). Stars mark the location of the epidermis. COL3A1 fibers (black staining) are much thicker and more abundant at each life stage in Tsk2/+ than in WT. Fibers were found to be especially pronounced in the panniculus carnosus region of the tissue; increased staining of COL3A1 in the dermis was also noted. The dermal reticular fibers are composed entirely of COL3A1 protein as this protein is receptive to silver impregnation, and they are increased in Tsk2/+ mice. All images were taken at 200X magnification. Bar size = 100 μM. (B + C) Skin lysates were analyzed for COL3A1 content (both bands) relative to beta-actin (not shown) by western blot analysis. Tsk2/+ mouse skin has significantly more COL3A1 protein than WT mouse skin (p=0.0025, ANOVA).