No abstract

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.

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.

Background The 67 kDa laminin receptor (67LR) was the first non-integrin cell surface receptor for laminin isolated on laminin affinity columns from cancer cells in the 1980’s. Initially, 67LR is found as a cytoplasmic precursor of a 37 kDa protein, named 37LR, associated with the small ribosomal subunit. In human cells, 37LR allows the formation of the polyribosome complex and plays a key role in the initiation of translation. The mechanism by which the ribosomal protein becomes the 67LR membrane receptor is still unclear. It is presumed that the process involves post-translational modifications combined with homo or hetero-dimerization with non-associated ribosomal proteins. It has been shown that 37/67LR regulates adhesion and proliferation of normal human intestinal epithelial cells. Interestingly, overexpression of 37/67LR is correlated with aggressiveness and a poor prognosis in a wide variety of cancers. Aims The aim of this study was to confirm the overexpression of 37/67LR at the membrane of colorectal cancer cells and to identify its homo or heterodimerization partners. Methods To detect the expression of 37/67LR in colorectal cancer we performed an indirect immunofluorescence on tissues from normal and diseased colons. To confirm the presence of 67LR at the membrane of Caco-2 cells we used a cellular fractionation extraction protocol combined with ultracentrifugation and detergent treatment to separate ribosome-containing fractions from the membranes and isolate the membrane associated 67LR. Mass spectrometry analysis to study the molecular identity of 67LR was performed on immunoreactive bands corresponding to 37LR and 67LR. Results Immunolocalization of 37/67LR revealed an overexpression in colorectal cancer tissues. Following analysis by western blotting, immunoreactive 67LR protein was found in the soluble fraction after ultracentrifugation at 210,000 x g while 37LR was detected in the insoluble counterpart which was solubilized after treatment with detergent, suggesting that 37LR is associated with the membrane. Mass spectrometry analysis of these fractions indicated that 37LR was not identified in the immunoreactive bands of 67LR in the soluble fraction but identified the 67 kDa elastin binding protein, another 67 kDa cell surface laminin receptor. Conclusions These results indicate that 37LR is overexpressed in colorectal adenocarcinoma cells. Further characterization of the receptor by cell fractionation and mass spectrometry indicated that the 67 kDa immunoreactive form is not related to 37LR. Supported by CIHR. Funding Agencies CIHR

Background The interaction between cells and extracellular matrix components like laminin or elastin is a crucial step in cancer development and the metastasis process. The 67 kDa Laminin Receptor (67LR) was one of the first cell surface receptors for laminin to be discovered in the 1980s. The 67LR has been reported to be the cytoplasmic 37 kDa small ribosomal subunit protein RPSA (40S ribosomal protein SA) combined with another yet to be identified component to become the 67 kDa membrane receptor. The mechanism by which the ribosomal protein becomes the 67LR membrane receptor is still unclear, but it is presumed that the process involves post-translational modifications combined with homo or hetero-dimerization with non-associated ribosomal proteins. Interestingly, the 67LR confers aggressiveness and a poor prognosis to a wide variety of cancers. Aims The aim of this study was to confirm the overexpression of 67LR at the membrane of colorectal cancer cells (CRC) and to identify its homo or hetero-dimerization partners. Methods To detect the expression of 67LR in colorectal cancer we performed indirect immunofluorescence on tissues from normal and diseased colons. To confirm the presence of 67LR at the membrane of CRC cells we used a cellular fractionation protocol combined with ultracentrifugation and detergent treatment to separate ribosome-containing fractions from the membranes to isolate membrane associated 67LR-related components. Mass spectrometry analysis to study the molecular identity of 67LR was performed on immunoreactive bands corresponding to RPSA and 67LR from different subcellular fractions. Results Immunolocalization of 67LR revealed an overexpression in colorectal cancer tissues. Following analysis by western blotting a 67 kDa immunoreactive protein was found in the supernatant of the 210,000 x g centrifugation while a 37 kDa protein was detected in the pellet solubilized with detergent, suggesting that the 37 kDa form is associated with the membrane. Furthermore, mass spectrometry analysis of the 67 kDa immunoreactive band present in the soluble fraction did not identify any RPSA-related peptides but found a 67 kDa elastin binding receptor (67EBP) related peptide, an elastin receptor that can also bind laminin. The identity of the 67EBP was then confirmed using a shRNA knockdown approach. Conclusions These results suggest a possible confusion between the 67 kDa laminin receptor (67LR) and 67 kDa elastin receptor (67EBP) and the possibility that the 37 kDa protein acts as a cell membrane laminin receptor. Funding Agencies CIHR