Recombinant Porcine R-Spondin 1 Facilitates Intestinal Stem Cell Expansion along the Crypt–Villus Axis through Potentiating Wnt/β-Catenin Signaling in Homeostasis and Deoxynivalenol Injury

Abstract

R-spondin 1 (RSPO1) is a ligand for the intestinal stem cell (ISC) marker Lgr5 in the crypt, which functions to amplify canonical Wnt signaling to stimulate the division of ISCs. Despite the crucial role of recombinant human RSPO1 (rhRSPO1) in homeostasis and regeneration, little is known about RSPO1 among different species. Here, we cloned the porcine RSPO1 (pRSPO1) gene and obtained rpRSPO1 protein through the expression system of the recombinant Escherichia coli Rosetta (DE3) chemical competent cells. Using the in vitro IPEC-J2 model that combines cell proliferation evaluation approaches, we identified the rpRSPO1 activity in stimulating jejunal epithelial cells. And upon deoxynivalenol challenge in mice, we found that rpRSPO1 ameliorated their growth retardation and jejunal epithelial integrity. Importantly, the ISCs in the jejunum had greater proliferation and differentiation potential that was accompanied by Wnt/β-catenin pathway activation after rpRSPO1 modulation. Subsequently, the jejunal organoids expanded from these ISCs ex vivo presented robust growth advantages. And the rpRSPO1 was able to guide Wnt/β-catenin activity to increase ISC activity. Our work systematically demonstrates that rpRSPO1 facilitates ISC expansion by potentiating Wnt/β-catenin signaling during homeostasis and responding to deoxynivalenol perturbations.

Mulberry Leaf-Derived Morin Activates β-Catenin by Binding to Frizzled7 to Promote Intestinal Stem Cell Expansion upon Heat-Stable Enterotoxin b Injury

“…secreted by surrounding Paneth cells, mesenchymal cells, and subepithelial fibroblasts, which enable rapid intestinal development at homeostasis and during injury . Multiple studies have demonstrated that the Wnt/β-catenin pathway promotes ISC proliferation in vivo and IO expansion ex vivo. , And, β-catenin inhibition by reducing or blocking its upstream signaling leads to crypt atrophy and intestinal epithelial failure, which highlights the essential role of Wnt/β-catenin in ISCs. , Our previous study has revealed that STp (a type of STa) halts ISC expansion and suppresses canonical Wnt signals . In line with this finding, the current data demonstrated a robust reduction of β-catenin and its downstream target genes (TCF7L2 and c-Myc) in the STb group, indicating that the ISC niche is sensitive to STb.…”

“…Intestinal epithelium regenerates rapidly through the proliferation and produces mature cell lineages (e.g., enterocytes, goblet cells, enteroendocrine cells, and Paneth cells) of intestinal stem cells (ISCs), which are located at the base of the crypt, orchestrated by an ample repertoire of mitogens within the crypt niche and modulated by complex and volatile lumen environment. , Of note, Wnt-driven ISCs exert acute sensibilities under severely stressful conditions to toxins from bacteria or fungi. − For example, heat-stable enterotoxin a (STa) secreted by enterotoxigenic Escherichia coli (ETEC) causes diarrhea in humans (mainly infants) and neonatal animals worldwide by opening ion channels, as well as disrupting ISC expansion by inhibiting Wnt/β-catenin signaling. , Likewise, heat-stable enterotoxin b (STb), another enterotoxin produced by ETEC, is capable of stabilizing in the digestive tract and causing abnormal intestinal function. , Surprisingly, only a few investigations have involved STb stimulating the cystic fibrosis transmembrane regulator (CFTR) by recognizing the sulfatide on the bilayer membranes to induce water and electrolytes into the intestinal lumen. , Current observations of STb damage to the intestine have been limited to describing the crypt-villus axis architecture. It thus remains largely unclear whether ISC fates are perturbed under STb-induced pathological contexts.…”

Novel recombinant R-spondin1 promotes hair regeneration by targeting the Wnt/β-catenin signaling pathway

“…As described previously by our laboratory [17] , rRSPO1 was expressed in the Escherichia coli Rosetta strain, separated rapidly by Ni-chelating affinity chromatography (Sangon, Shanghai, China) and further purified. The protein was lyophilized and then reconstituted in sterile phosphate-buffered saline (PBS) prior to use.…”

Deoxynivalenol: Emerging Toxic Mechanisms and Control Strategies, Current and Future Perspectives

“…69−71 For example, R-reactive protein 1 and methionine can enhance Wnt/β-catenin activity to retard growth and improve the jejunal epithelial integrity of mice, and alleviate DON-induced intestinal injury. 69,70 Moreover, hydrolyzed wheat gluten and zinc L-aspartate can alleviate DON-induced intestinal injury by improving intestinal stem cell expansion and reactivating Wnt/ β-catenin signaling. 71,72 Although most studies have shown that DON inhibits Wnt/ β-catenin signaling pathway activity to exert toxic effects, another study has reported that exposure to DON (0.4 μg/ mL) can promote the activation of the Wnt/β-catenin pathway.…”

“…DON can suppress the Wnt/β-catenin signaling pathway, inhibiting the proliferation of human renal cells (DON: 50 ng/mL) and intestinal adenocarcinoma cells (DON: 1 μg/mL) . Studies have found that a variety of substances can alleviate DON toxicity by targeting the Wnt/β-catenin signaling pathway. − For example, R-reactive protein 1 and methionine can enhance Wnt/β-catenin activity to retard growth and improve the jejunal epithelial integrity of mice, and alleviate DON-induced intestinal injury. , Moreover, hydrolyzed wheat gluten and zinc l -aspartate can alleviate DON-induced intestinal injury by improving intestinal stem cell expansion and reactivating Wnt/β-catenin signaling. , …”