Abstract
Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) and B-cell-specific Moloney murine leukemia virus insertion site 1 (BMI1) are markers of fast-cycling and quiescent intestinal stem cells, respectively. To determine the functions of these proteins in large animals, we investigated their effects on the proliferation of intestinal epithelial cells from pigs. Our results indicated that LGR5 and BMI1 are highly conserved proteins and that the pig proteins have greater homology with the human proteins than do mouse proteins. Overexpression of either LGR5 or BMI1 promoted cell proliferation and WNT/β-catenin signaling in pig intestinal epithelial cells (IPEC-J2). Moreover, the activation of WNT/β-catenin signaling by recombinant human WNT3A protein increased cell proliferation and LGR5 and BMI1 protein levels. Conversely, inhibition of WNT/β-catenin signaling using XAV939 reduced cell proliferation and LGR5 and BMI1 protein levels. This is the first report that LGR5 and BMI1 can increase proliferation of pig intestinal epithelial cells by activating WNT/β-catenin signaling.
Highlights
The intestinal epithelium, which is covered by a monolayer of epithelial cells, completely renews itself every four or five days, driven by intestinal stem cells (ISCs) [1]
Bioinformatics performed with DNASTAR revealed that the signal peptide of the pig Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) protein is MDTSSVGVLLSLPVLFQLAAG
The LGR5 overexpression vector was verified by reverse transcription-PCR (Figure 1E) and identified through enzyme digestion (Figure 1F)
Summary
The intestinal epithelium, which is covered by a monolayer of epithelial cells, completely renews itself every four or five days, driven by intestinal stem cells (ISCs) [1]. Two populations of stem cells—fast-cycling stem cells known as crypt base columnar (CBC) cells, and quiescent stem cells known as +4 stem cells—have been found in the intestinal crypts [2,3]. Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a surface molecular marker used to accurately locate and isolate pure CBC cells [2,4], whereas B-cell-specific Moloney murine leukemia virus insertion site 1 (BMI1) is a marker of +4 stem cells [5]. The coding sequence of pig LGR5 has not been obtained, and the effects of LGR5 and BMI1 on the proliferation of intestinal epithelial cells from large animals remain unknown
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