Integrin α7ß1 represses intestinal absorptive cell differentiation.

Intestinal epithelial cell differentiation is a highly controlled and orderly process occurring in the crypt so that cells migrating out to cover the villi are already fully functional. Absorptive cell precursors, which originate from the stem cell population located in the lower third of the crypt, are subject to several cycles of amplification in the transit amplifying (TA) zone, before reaching the terminal differentiation compartment located in the upper third. There is a large body of evidence that absorptive cell differentiation is halted in the TA zone through various epigenetic, transcriptional and intracellular signalling events or mechanisms allowing the transient expansion of this cell population but how these mechanisms are themself regulated remains obscure. One clue can be found in the epithelial cell-matrix microenvironment located all along the crypt-villus axis. Indeed, a previous study from our group revealed that α5-subunit containing laminins such as lamimin-511 and 512 inhibit early stages of differentiation in Caco-2/15 cells. Among potential receptors for laminin 511/512 is the integrin α7ß1, which has previously been reported to be expressed in the human intestinal crypts and in early stages of Caco-2/15 cell differentiation. In this study, the effects of knocking down ITGA7 in Caco-2/15 cells were studied using shRNA and CRISPR/Cas9 strategies. Abolition of the α7 integrin subunit resulted in a significant increase in the level of differentiation and polarization markers as well as the morphological features of intestinal cells. Activities of focal adhesion kinase and Src kinase were both reduced in α7-knockdown cells while the three major intestinal pro-differentiation factors CDX2, HNFα1 and HNF4α were overexpressed. Two epigenetic events associated with intestinal differentiation, the reduction of tri-methylated lysine 27 on histone H3 and the increase of acetylation of histone H4 were also observed in α7-knockdown cells. On the other hand, the ablation of α7 had no effect on cell proliferation. In conclusion, these data indicate that integrin α7ß1 acts as a major repressor of absorptive cell terminal differentiation in the Caco-2/15 cell model and suggest that the laminin-α7ß1 integrin interaction occurring in the transit amplifying zone of the adult intestine is involved in the transient halting of absorptive cell terminal differentiation.

In vivo multivesicular bodies and their exosomes in the absorptive cells of the zebrafish (Danio Rerio) gut.

Intercellular communication of gut epithelial cells is critical to gut mucosal homeostasis. Exosomes are important intercellular mediators in communication between cell to cell. Although many literature focus on the immunologic roles in the gut by the exosomes, the biological process of exosomes in the absorptive cells remains unknown. Uncovering the distribution, classification and formation process of multivesicular bodies (MVBs) and their exosomes in the absorptive cells of the zebrafish gut, is urgently needed to establish a platform for immunological research of fish gut exosomes. The expression levels of CD63 and TSG101 were different among the three segments of the gut, and they were enriched at the apex of the mid gut villi. The characteristics of MVBs and their exosomes in the absorptive cells were further revealed by transmission electron microscopy (TEM). Early endosomes (ee) were mainly present in the apical and basal cytoplasm of absorptive cells. Late endosomes (le) were mostly distributed with the supranuclear part of these cells. "Heterogeneous" MVBs were detected underlying the apical membranes of absorptive cells. Many exosomes with some MVB-like structures occurred in the lumen, indicating that the release process was mainly through apical secretion. Various MVBs with exosomes and the endosome-heterogeneous MVB-exosome complex existed widely in the mid gut absorptive cells, concluding that zebrafish as a potential model for in vivo MVBs and their exosomes research. All the results were summarized in a schematic diagram illustrating the morphological characteristics of gut MVBs and their exosomes in zebrafish.

Lgr5 positive stem cells sorted from small intestines of diabetic mice differentiate into higher proportion of absorptive cells and Paneth cells in vitro.

Intestinal epithelial stem cells (IESCs) can differentiate into all types of intestinal epithelial cells (IECs) and Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) is a marker for IESC. Previous studies reported enhanced proliferation of IECs in diabetic mice. In this study, the in vitro differentiation of Lgr5 positive IESCs sorted from diabetic mice was further investigated. The diabetic mouse model was induced by streptozotocin (STZ), and crypt IECs were isolated from small intestines. Subsequently, Lgr5 positive IESCs were detected by flow cytometry (FCM) and sorted by magnetic activated cell sorting (MACS). Differentiation of the sorted IESCs was investigated by detecting the IEC markers in the diabetic mice using immunostaining, quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR), and Western blot analysis, which was compared with normal mice. We found that the proportion of Lgr5 positive cells in the crypt IECs of diabetic mice was higher than that of control mice (P < 0.05). Lgr5 positive IESCs could be significantly enriched in Lgr5 positive cell fraction sorted by MACS. Furthermore, the absorptive cell marker sucrase-isomaltase (SI) and the Paneth cell marker lysozyme 1 (Lyz1) were more highly expressed in the differentiated cells derived from Lgr5 positive IESCs of diabetic mice in vitro (P < 0.05). We demonstrate that the number of Lgr5 positive IESCs is significantly increased in the small intestines of STZ-induced diabetic mice. Lgr5 positive IESCs sorted from the diabetic mice can differentiate into a higher proportion of absorptive cells and Paneth cells in vitro. We characterized the expression of Lgr5 in the small intestine of diabetic mice, and sorted Lgr5 positive intestinal epithelial stem cells (IESCs) for investigating their differentiation in vitro. We proved that the quantity of Lgr5 positive IESCs was significantly increased in the small intestines of diabetic mice. IESCs sorted from the diabetic mice can differentiate into a higher proportion of absorptive cells and Paneth cells in vitro.