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 6

IEC migration in response to TSP1 is TGF-β1 dependent.

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IEC migration in response to TSP1 is TGF-β1 dependent. (A) Western blot ...
(A) Western blot of wounded murine IECs treated with TSP1 for 24 hours at the indicated concentration probed for phosphorylated SMAD3 S423/425, SMAD2/3, and GAPDH (representative of N = 3 independent experiments). (B) Western blot of WT and Cd47–/– wounded murine IECs treated with TGF-β1 (2 ng/mL) for 24 hours probed for phosphorylated SMAD3 S423/425, SMAD2/3, and cytokeratin-8 (representative of N = 4 independent experiments). (C) Wound closure rates at 24 hours after wounding in a scratch wound assay of murine IECs treated with and without TSP1, SMAD3 inhibitor SIS3, and TGF-β1 inhibitor ITD1. Performed 2-way ANOVA with Holm-Šídák multiple-comparison test. (D) Wound closure rates at 24 hours after wounding in a scratch wound assay of T84 IECs treated with and without TSP1, SIS3, and ITD1. Performed 2-way ANOVA with Holm-Šídák multiple-comparison test. (E) Western blot of wounded murine IECs treated for 24 hours with and without TSP1, SIS3, and ITD1 (representative of N = 2 independent experiments). (F) qPCR of mRNA from TGF-β1–related genes Tgfbr1, Tgfbr2, Tgfb1, and Thbs1 in WT and Cd47–/– murine IECs treated with TGF-β1 (2 ng/mL) 4 hours after wounding (N = 3–4 independent experiments). Performed 1-way ANOVA with Tukey’s multiple comparison test. (G) qPCR of Cd47 mRNA in WT murine IECs treated with TGF-β1 (2 ng/mL) 4 hours after wounding (N = 3 independent experiments). Performed unpaired, 2-tailed Student’s t test. (H) qPCR of Tgfbr1 mRNA after biopsy-based colonic mucosal wound repair at indicated time points (n = 3–4 mice). Performed 1-way ANOVA with Tukey’s multiple-comparison test. Box plots show interquartile range, median (line), and minimum and maximum (whiskers). Histograms show mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001.