Reconsideration of the laminin receptor 67LR in colorectal cancer cells.

The 67 kDa laminin receptor (67LR) was identified as the first laminin receptor shown to be involved in the carcinogenesis of various cancers, including colorectal cancer. While the exact composition of this 67 kDa receptor remains unknown, it has been reported to be formed by the 37 kDa ribosomal protein SA (RPSA) covalently attached to another unidentified protein. The goal of this study was to clarify the molecular structure of 67LR to enhance our understanding of its role in malignancies. Using cell fractionation of colorectal cancer cells, the 67 kDa immunoreactive protein corresponding to 67LR was found in the soluble protein fraction, while some of the 37 kDa RPSA exhibited plasma membrane-like properties. Proteomic analysis of the 67 kDa fraction revealed the absence of RPSA but identified the ß-galactosidase-related 67 kDa elastin-binding protein (67EBP), another laminin binding receptor which presents amino acid sequence similarities that can explain the immune cross reactivity with RPSA. The downregulation of ß-galactosidase through short hairpin RNA (shRNA) led to a reduction in both 67LR and 67EBP immunoreactive proteins, confirming the misidentification of 67LR and 67EBP in colorectal cancer cells. Based on these findings, we propose to redefine the 67LR as the RPSA-containing laminin receptor (RCLR) to avoid confusion with the 67EBP.

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.

Primary Cilium Identifies a Quiescent Cell Population in the Human Intestinal Crypt.

Primary cilia are sensory antennae located at the cell surface which mediate a variety of extracellular signals involved in development, tissue homeostasis, stem cells and cancer. Primary cilia are found in an extensive array of vertebrae cells but can only be generated when cells become quiescent. The small intestinal epithelium is a rapidly self-renewing tissue organized into a functional unit called the crypt-villus axis, containing progenitor and differentiated cells, respectively. Terminally differentiated villus cells are notoriously devoid of primary cilia. We sought to determine if intestinal crypts contain a quiescent cell population that could be identified by the presence of primary cilia. Here we show that primary cilia are detected in a subset of cells located deep in the crypts slightly above a Paneth cell population. Using a normal epithelial proliferative crypt cell model, we show that primary cilia assembly and activity correlate with a quiescent state. These results provide further evidence for the existence of a quiescent cell population in the human small intestine and suggest the potential for new modes of regulation in stem cell dynamics.

Fibrin(ogen) Is Constitutively Expressed by Differentiated Intestinal Epithelial Cells and Mediates Wound Healing.

Fibrinogen is a large molecule synthesized in the liver and released in the blood. Circulating levels of fibrinogen are upregulated after bleeding or clotting events and support wound healing. In the context of an injury, thrombin activation drives conversion of fibrinogen to fibrin. Fibrin deposition contains tissue damage, stops blood loss, and prevents microbial infection. In most circumstances, fibrin needs to be removed to allow the resolution of inflammation and tissue repair, whereas failure of this may lead to the development of various disorders. However, the contribution of fibrinogen to tissue inflammation and repair is likely to be context-dependent. In this study, the concept that fibrin needs to be removed to allow tissue repair and to reduce inflammation is challenged by our observations that, in the intestine, fibrinogen is constitutively produced by a subset of intestinal epithelial cells and deposited at the basement membrane as fibrin where it serves as a substrate for wound healing under physiological conditions such as epithelial shedding at the tip of the small intestinal villus and surface epithelium of the colon as well as under pathological conditions that require rapid epithelial repair. The functional integrity of the intestine is ensured by the constant renewal of its simple epithelium. Superficial denuding of the epithelial cell layer occurs regularly and is rapidly corrected by a process called restitution that can be influenced by various soluble and insoluble factors. Epithelial cell interaction with the extracellular matrix greatly influences the healing process by acting on cell morphology, adhesion, and migration. The functional contribution of a fibrin(ogen) matrix in the intestine was studied under physiological and pathological contexts. Our results (immunofluorescence, immunoelectron microscopy, and quantitative PCR) show that fibrin(ogen) is a novel component of the basement membrane associated with the differentiated epithelial cell population in both the small intestine and colon. Fibrin(ogen) alone is a weak ligand for epithelial cells and behaves as an anti-adhesive molecule in the presence of type I collagen. Furthermore, the presence of fibrin(ogen) significantly shortens the time required to achieve closure of wounded epithelial cell monolayers and co-cultures in a PI3K-dependent manner. In human specimens with Crohn's disease, we observed a major accumulation of fibrin(ogen) throughout the tissue and at denuded sites. In mice in which fibrin formation was inhibited with dabigatran treatment, dextran sulfate sodium administration provoked a significant increase in the disease activity index and pathological features such as mucosal ulceration and crypt abscess formation. Taken together, these results suggest that fibrin(ogen) contributes to epithelial healing under both normal and pathological conditions.

Non-integrin laminin receptors in epithelia.

The interactions between cells and the extracellular matrix (ECM) play a major role in normal and pathological conditions. The ECM can modulate several biological functions including cell proliferation, adhesion, differentiation and survival through its interactions with cell receptors. Laminins are one of the most important glycoproteins present in basement membranes, a type of ECM. The pattern of expression of its different isoforms depends on the spatiotemporal organization of each tissue. While integrins are the most studied laminin receptors, other non-integrin laminin receptors are also involved. This review focuses on two particular non-integrin laminin receptors in the epithelial context: dystroglycan and 37/67 laminin receptor (37/67LR). Dystroglycan is a two-subunit protein discovered in the muscle as part of the dystrophin-associated glycoprotein complex. This protein can also be found in many epithelia where its roles are variable. The 37/67LR is a still incompletely understood laminin receptor that is important to regulate intestinal epithelial cell function and could be involved in various pathological conditions.