Cysteine-rich domain of type III collagen N-propeptide inhibits fibroblast activation by attenuating TGFß signaling.

TGFß is a key regulator of the dynamic reciprocity between cells and the extracellular matrix that drives physiologic and pathologic responses in both tissue repair and tumor microenvironments. Our studies define type III Collagen (Col3) as a suppressor of scar formation and desmoplasia through its effects, in part, on myofibroblasts. TGFß stimulates activation of myofibroblasts, and here, we demonstrate that cultured Col3-deficient fibroblasts have increased TGFß signaling compared to wild-type fibroblasts. Moreover, kinetic binding studies show that a synthetic peptide containing a Col3 cysteine-rich (CR) domain found within its N-propeptide binds in a dose-dependent manner to TGFß1, while a CR control peptide with mutated cysteines does not, suggesting that Col3 attenuates TGFß signaling in part through the N-propeptide CR domain. Consistent with this hypothesis, the CR peptide attenuates TGFß signaling in fibroblasts and 4T1 breast cancer cells and suppresses fibroblast activation and contraction, as assessed by α-smooth-muscle actin staining, cell wrinkling of deformable silicone, and stressed-fibroblast populated collagen lattice contraction assays. Finally, CR peptide treatment of orthotopically injected breast cancer cells (4T1) suppresses intratumoral fibroblast activation and inhibits primary tumor growth compared to CR control. Treatment with the CR peptide decreases both intratumoral canonical and non-canonical downstream TGFß signaling targets, consistent with its extracellular binding to TGFß. Taken together, our results suggest that the Col3 N-propeptide CR domain binds TGFß1 and attenuates (but importantly does not eliminate) TGFß signaling in fibroblasts and cancer cells. Expanding on our previous work, this study demonstrates an additional mechanism by which Col3 regulates cell behaviors in post-injury and tumor microenvironments and suggests that novel Col3-targeted strategies could effectively control biologic responses in vivo and improve anti-scarring/fibrosis and oncologic therapies.

Identification of Optimal Mouse Models of Systemic Sclerosis by Interspecies Comparative Genomics.

OBJECTIVE: Understanding the pathogenesis of systemic sclerosis (SSc) is confounded by considerable disease heterogeneity. Animal models of SSc that recapitulate distinct subsets of disease at the molecular level have not been delineated. We applied interspecies comparative analysis of genomic data from multiple mouse models of SSc and patients with SSc to determine which animal models best reflect the SSc intrinsic molecular subsets. METHODS: Gene expression measured in skin from mice with sclerodermatous graft-versus-host disease (GVHD), bleomycin-induced fibrosis, Tsk1/+ or Tsk2/+ mice was mapped to human orthologs and compared to SSc skin biopsy-derived gene expression. Transforming growth factor ß (TGFß) activation was assessed using a responsive signature in mice, and tumor necrosis factor receptor superfamily member 12A (TNFRSF12A) expression was measured in SSc patient and mouse skin. RESULTS: Gene expression in skin from mice with sclerodermatous GVHD and bleomycin-induced fibrosis corresponded to that in SSc patients in the inflammatory molecular subset. In contrast, Tsk2/+ mice showed gene expression corresponding to the fibroproliferative SSc subset. Enrichment of a TGFß-responsive signature was observed in both Tsk2/+ mice and mice with bleomycin-induced skin fibrosis. Expression of TNFRSF12A (the TWEAK receptor/fibroblast growth factor-inducible 14) was elevated in skin from patients with fibroproliferative SSc and the skin of Tsk2/+ mice. CONCLUSION: This study reveals similarities in cutaneous gene expression between distinct mouse models of SSc and specific molecular subsets of the disease. Different pathways underlie the intrinsic subsets including TGFß, interleukin-13 (IL-13), and IL-4. We identify a novel target, Tnfrsf12a, with elevated expression in skin from patients with fibroproliferative SSc and Tsk2/+ mice. These findings will inform mechanistic and translational preclinical studies in SSc.

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

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.

Tight Skin 2 Mice Exhibit Delayed Wound Healing Caused by Increased Elastic Fibers in Fibrotic Skin.

Rationale: The Tight Skin 2 (Tsk2) mouse model of systemic sclerosis (SSc) has many features of human disease, including tight skin, excessive collagen deposition, alterations in the extracellular matrix (ECM), increased elastic fibers, and occurrence of antinuclear antibodies with age. A tight skin phenotype is observed by 2 weeks of age, but measurable skin fibrosis is only apparent at 10 weeks. We completed a series of wound healing experiments to determine how fibrosis affects wound healing in Tsk2/+ mice compared with their wild-type (WT) littermates. Method: We performed these experiments by introducing four 4 mm biopsy punched wounds on the back of each mouse, ventral of the midline, and observed wound healing over 10 days. Tsk2/+ mice showed significantly delayed wound healing and increased wound size compared with the WT littermates at both 5 and 10 weeks of age. We explored the potential sources of this response by wounding Tsk2/+ mice that were genetically deficient either for the NLRP3 inflammasome (a known fibrosis mediator), or for elastic fibers in the skin, using a fibulin-5 knockout. Conclusion: We found that the loss of elastic fibers restores normal wound healing in the Tsk2/+ mouse and that the loss of the NLRP3 inflammasome had no effect. We conclude that elastic fiber dysregulation is the primary cause of delayed wound healing in the Tsk2/+ mouse and therapies that promote collagen deposition in the tissue matrix in the absence of elastin deposition might be beneficial in promoting wound healing in SSc and other diseases.

Identification of specific inhibitors of human RAD51 recombinase using high-throughput screening.

RAD51 is a key protein of homologous recombination that plays a critical role in the repair of DNA double-strand breaks (DSB) and interstrand cross-links (ICL). To better understand the cellular function(s) of human RAD51, we propose to develop specific RAD51 inhibitors. RAD51 inhibitors may also help to increase the potency of anticancer drugs that act by inducing DSBs or ICLs, e.g., cisplatin or ionizing radiation. In vitro, RAD51 promotes DNA strand exchange between homologous ss- and dsDNA. Here, we developed a DNA strand exchange assay based on fluorescence resonance energy transfer and used this assay to identify RAD51 inhibitors by high-throughput screening of the NIH Small Molecule Repository (>200,000 compounds). Seventeen RAD51 inhibitors were identified and analyzed for selectivity using additional nonfluorescent DNA-based assays. As a result, we identified a compound (B02) that specifically inhibited human RAD51 (IC(50) = 27.4 µM) but not its E. coli homologue RecA (IC(50) > 250 µM). Two other compounds (A03 and A10) were identified that inhibited both RAD51 and RecA but not the structurally unrelated RAD54 protein. The structure-activity relationship (SAR) analysis allowed us to identify the structural components of B02 that are critical for RAD51 inhibition. The described approach can be used for identification of specific inhibitors of other human proteins that play an important role in DNA repair, e.g., RAD54 or Bloom's syndrome helicase.