Critical role of thrombospondin-1 in promoting intestinal mucosal wound repair
Zachary S. Wilson, Arturo Raya-Sandino, Jael Miranda, Shuling Fan, Jennifer C. Brazil, Miguel Quiros, Vicky Garcia-Hernandez, Qingyang Liu, Chang H. Kim, Kurt D. Hankenson, Asma Nusrat, Charles A. Parkos
Zachary S. Wilson, Arturo Raya-Sandino, Jael Miranda, Shuling Fan, Jennifer C. Brazil, Miguel Quiros, Vicky Garcia-Hernandez, Qingyang Liu, Chang H. Kim, Kurt D. Hankenson, Asma Nusrat, Charles A. Parkos
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Research Article Gastroenterology Inflammation

Critical role of thrombospondin-1 in promoting intestinal mucosal wound repair

Abstract

Thrombospondin-1 (TSP1) is a matricellular protein associated with the regulation of cell migration through direct binding interactions with integrin proteins and by associating with other receptors known to regulate integrin function, including CD47 and CD36. We previously demonstrated that deletion of an epithelial TSP1 receptor, CD47, attenuates epithelial wound repair following intestinal mucosal injury. However, the mechanisms by which TSP1 contributes to intestinal mucosal repair remain poorly understood. Our results show upregulated TSP1 expression in colonic mucosal wounds and impaired intestinal mucosal wound healing in vivo upon intestinal epithelium–specific loss of TSP1 (VillinCre/+ Thbs1fl/fl or Thbs1ΔIEC mice). We report that exposure to exogenous TSP1 enhanced migration of intestinal epithelial cells in a CD47- and TGF-β1–dependent manner and that deficiency of TSP1 in primary murine colonic epithelial cells resulted in impaired wound healing. Mechanistically, TSP1 modulated epithelial actin cytoskeletal dynamics through suppression of RhoA activity, activation of Rho family small GTPase (Rac1), and changes in filamentous-actin bundling. Overall, TSP1 was found to regulate intestinal mucosal wound healing via CD47 and TGF-β1, coordinate integrin-containing cell–matrix adhesion dynamics, and remodel the actin cytoskeleton in migrating epithelial cells to enhance cell motility and promote wound repair.

Authors

Zachary S. Wilson, Arturo Raya-Sandino, Jael Miranda, Shuling Fan, Jennifer C. Brazil, Miguel Quiros, Vicky Garcia-Hernandez, Qingyang Liu, Chang H. Kim, Kurt D. Hankenson, Asma Nusrat, Charles A. Parkos

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Figure 5

TSP1 enhances IEC migration with enhanced focal adhesion formations at the periphery of leading cells.

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TSP1 enhances IEC migration with enhanced focal adhesion formations at t...
(A) Immunofluorescence of p130Cas pY410 (pCas) and actin at the leading cells of migrating WT mouse IEC monolayers following 8 hours of TSP1 treatment (N = 3 independent experiments). Scale bar = 20 μm, 5 μm inset. (B) Quantification of p130Cas pY410 (pCas) focal adhesions. Performed unpaired, 2-tailed Student’s t test. (C) Western blot of phosphorylated p130Cas Y410, total p130Cas, β1 integrin, and GAPDH in response to indicated TSP1 treatment at 24 hours after wounding (representative of N = 3 independent experiments). (D) In situ hybridization (RNAscope) of Thbs1 at the leading edge of migrating monolayers of WT mouse IECs in response to 4 hours TSP1 treatment (representative of N = 3 independent experiments). Scale bar = 50 μm, 20 μm inset. (E) qPCR of thrombospondin-1 mRNA (Thbs1) in CD47-replete and deplete IECs in response to TSP1 treatment 24 hours after wounding (n = 3–4 mice). Performed unpaired, 2-tailed Student’s t test. Box plots show interquartile range, median (line), and minimum and maximum (whiskers). Histograms show mean ± SEM. *P < 0.05, **P < 0.01.