Mice overexpressing CD97 in intestinal epithelial cells provide a unique model for mammalian postnatal intestinal cylindrical growth

Postnatal enlargement of the mammalian intestine comprises cylindrical and luminal growth, associated with crypt fission and crypt/villus hyperplasia, respectively, which subsequently predominate before and after weaning. The bipartite adhesion G protein–coupled receptor CD97 shows an expression gradient along the crypt–villus axis in the normal human intestine. We here report that transgenic mice overexpressing CD97 in intestinal epithelial cells develop an upper megaintestine. Intestinal enlargement involves an increase in length and diameter but does not affect microscopic morphology, as typical for cylindrical growth. The megaintestine is acquired after birth and before weaning, independent of the genotype of the mother, excluding altered availability of milk constituents as driving factor. CD97 overexpression does not regulate intestinal growth factors, stem cell markers, and Wnt signaling, which contribute to epithelial differentiation and renewal, nor does it affect suckling-to-weaning transition. Consistent with augmented cylindrical growth, suckling but not adult transgenic mice show enlarged crypts and thus more crypt fissions caused by a transient increase of the crypt transit-amplifying zone. Intestinal enlargement by CD97 requires its seven-span transmembrane/cytoplasmic C-terminal fragment but not the N-terminal fragment binding partner CD55. In summary, ectopic expression of CD97 in intestinal epithelial cells provides a unique model for intestinal cylindrical growth occurring in breast-fed infants.     

Comprehensive gene expression profiling of Peyer's patch M cells, villous M-like cells, and intestinal epithelial cells

Separate populations of M cells have been detected in the follicle-associated epithelium of Peyer's patches (PPs) and the villous epithelium of the small intestine, but the traits shared by or distinguishing the two populations have not been characterized. Our separate study has demonstrated that a potent mucosal modulator cholera toxin (CT) can induce lectin Ulex europaeus agglutinin-1 and our newly developed M cell-specific mAb NKM 16-2-4-positive M-like cells in the duodenal villous epithelium. In this study, we determined the gene expression of PP M cells, CT-induced villous M-like cells, and intestinal epithelial cells isolated by a novel approach using FACS. Additional mRNA and protein analyses confirmed the specific expression of glycoprotein 2 and myristoylated alanine-rich C kinase substrate (MARCKS)-like protein by PP M cells but not CT-induced villous M-like cells. Comprehensive gene profiling also suggested that CT-induced villous M-like cells share traits of both PP M cells and intestinal epithelial cells, a finding that is supported by their unique expression of specific chemokines. The genome-wide assessment of gene expression facilitates discovery of M cell-specific molecules and enhances the molecular understanding of M cell immunobiology.     

Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

Background and aims

The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour.

Methods

Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence.

Results

We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids.

Conclusions

The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.     

Distinct fucosylation of M cells and epithelial cells by Fut1 and Fut2, respectively, in response to intestinal environmental stress

The intestinal epithelium contains columnar epithelial cells (ECs) and M cells, and fucosylation of the apical surface of ECs and M cells is involved in distinguishing the two populations and in their response to commensal flora and environmental stress. Here, we show that fucosylated ECs (F-ECs) were induced in the mouse small intestine by the pro-inflammatory agents dextran sodium sulfate and indomethacin, in addition to an enteropathogen derived cholera toxin. Although F-ECs showed specificity for the M cell-markers, lectin Ulex europaeus agglutinin-1 and our monoclonal antibody NKM 16-2-4, these cells also retained EC-phenotypes including an affinity for the EC-marker lectin wheat germ agglutinin. Interestingly, fucosylation of Peyer’s patch M cells and F-ECs was distinctly regulated by α(1,2)fucosyltransferase Fut1 and Fut2, respectively. These results indicate that Fut2-mediated F-ECs share M cell-related fucosylated molecules but maintain distinctive EC characteristics, Fut1 is, therefore, a reliable marker for M cells.     

Intectin, a novel small intestine-specific glycosylphosphatidylinositol-anchored protein, accelerates apoptosis of intestinal epithelial cells

Intestinal epithelial cells undergo rapid turnover and exfoliation especially at the villus tips. This process is modulated by various nutrients especially fat. Apoptosis is one of the important regulatory mechanisms of this turnover. Therefore, identification of the factors that control epithelial cell apoptosis should help us understand the mechanism of intestinal mucosal turnover. Here, we report the identification of a novel small intestine-specific member of the Ly-6 family, intectin, by signal sequence trap method. Intectin mRNA expression was exclusively identified in the intestine and localized at the villus tips of intestinal mucosa, which is known to undergo apoptosis. Intectin mRNA expression was modulated by nutrition. Intestinal epithelial cells expressing intectin were more sensitive to palmitate-induced apoptosis, compared with control intestinal epithelial cells, and such effect was accompanied by increased activity of caspase-3. Intectin expression also reduced cell-cell adhesion of intestinal epithelial cells.     

Sodium-dependent, concentrative nucleoside transport in cultured intestinal epithelial cells

In a simple salts medium, monolayers of IEC-6 intestinal cells achieved concentrations of unmetabolized formycin B (an analog of inosine) about 6-fold higher than in the medium. Rates of formycin B influx were a saturable function of Na+ concentrations in the medium. Although IEC-6 cells possess sites with high affinity for nitrobenzylthioinosine, a potent inhibitor of equilibrative (facilitated diffusion) nucleoside transport systems in certain cell types, the inhibitor had only minor effects on formycin B uptake in IEC-6 cells, but reduced efflux of the analog from these cells. These findings indicate the joint presence in IEC-6 cells of nucleoside transporters of two types, one that is concentrative and Na+-dependent, and another that is sensitive to nitrobenzylthioinosine and apparently equilibrative.     

Mammalian intestinal epithelial cells in primary culture: a mini-review

Epithelial cells lining the digestive tract represent a highly organized system built up by multipotent stem cells. A process of asymmetric mitosis produces a population of proliferative cells that are rapidly renewed and migrate along the crypt-villus axis, differentiating into functional mature cells before dying and exfoliating into the intestinal lumen. Isolated crypts or epithelial cells retaining high viability can be prepared within a few h after tissue sampling. After cells are cultured in serum-free media, short-term studies (16-48 h) can be conducted for endocrinology, energy metabolism, or programmed cell death. However, long-term primary culture of intestinal cells (up to 10 d) is still difficult despite progress in isolation methodologies and manipulation of the cell microenvironment. The main problem in developing primary culture is the lack of structural markers specific to the stem cell compartment. The design of a microscopic multidimensional analytic system to record the expression profiles of biomarkers all along the living intestinal crypt should improve basic knowledge of the survival and growth of adult crypt stem cells, and the selection of totipotent embryonic stem cells capable of differentiating into intestinal tissues should facilitate studies of the genomic basis of endodermal tissue differentiation.     

Human intestinal epithelial cells express a novel receptor for IgA

Binding and transport of polymeric Igs (pIgA and IgM) across epithelia is mediated by the polymeric Ig receptor (pIgR), which is expressed on the basolateral surface of secretory epithelial cells. Although an Fc receptor for IgA (FcalphaR) has been identified on myeloid cells and some cultured mesangial cells, the expression of an FcalphaR on epithelial cells has not been described. In this study, binding of IgA to a human epithelial line, HT-29/19A, with features of differentiated colonic epithelial cells, was examined. Radiolabeled monomeric IgA (mIgA) showed a dose-dependent, saturable, and cation-independent binding to confluent monolayers of HT-29/19A cells. Excess of unlabeled mIgA, but not IgG or IgM, competed for the mIgA binding, indicating that the binding was IgA isotype-specific and was not mediated by the pIgR. The lack of competition by asialoorosomucoid and the lack of requirement for divalent cations excluded the possibility that IgA binding to HT-29/19A cells was due to the asialoglycoprotein receptor or beta-1, 4-galactosyltransferase, previously described on HT-29 cells. Moreover, the FcalphaR (CD89) protein and message were undetectable in HT-29/19A cells. FACS analysis of IgA binding demonstrated two discrete populations of HT-29/19 cells, which bound different amounts of mIgA. IgA binding to other colon carcinoma cell lines was also demonstrated by FACS analysis, suggesting that an IgA receptor, distinct from the pIgR, asialoglycoprotein receptor, galactosyltransferase, and CD89 is constitutively expressed on cultured human enterocytes. The function of this novel IgA receptor in mucosal immunity remains to be elucidated.     

Protein traffic in intestinal epithelial cells.

Cell culture systems, in particular Caco-2, and sucrase-isomaltase deficiency in humans are attractive models to study exocytic protein traffic in absorptive intestinal epithelial cells. Transport from ER to and through the Golgi is asynchronous and may depend on protein folding rather than oligomerization. Apical and basolateral proteins are sorted both intracellularly and from the basolateral membrane. A model is presented for the sorting of apical and basolateral proteins. Brush border proteins in lysosomes mainly originate from the Golgi and may reflect a regulatory or quality control mechanism. Apical transport and transcytosis but not basolateral transport are facilitated by microtubules.     

Vasopressin-mediated mitogenic signaling in intestinal epithelial cells

The role of G protein-coupled receptorsand their ligands in intestinal epithelial cell signaling andproliferation is poorly understood. Here, we demonstrate that argininevasopressin (AVP) induces multiple intracellular signal transductionpathways in rat intestinal epithelial IEC-18 cells via aV_1A receptor. Addition of AVP to these cells induces arapid and transient increase in cytosolic Ca²⁺concentration and promotes protein kinase D (PKD) activation through aprotein kinase C (PKC)-dependent pathway, as revealed by in vitrokinase assays and immunoblotting with an antibody that recognizesautophosphorylated PKD at Ser⁹¹⁶. AVP also stimulates thetyrosine phosphorylation of the nonreceptor tyrosine kinaseproline-rich tyrosine kinase 2 (Pyk2) and promotes Src family kinasephosphorylation at Tyr⁴¹⁸, indicative of Src activation.AVP induces extracellular signal-related kinase (ERK)-1(p44^mapk) and ERK-2 (p42^mapk) activation, aresponse prevented by treatment with mitogen-activated protein kinasekinase (MEK) inhibitors (PD-98059 and U-0126), specific PKC inhibitors(GF-I and Ro-31-8220), depletion of Ca²⁺ (EGTA andthapsigargin), selective epidermal growth factor receptor (EGFR)tyrosine kinase inhibitors (tyrphostin AG-1478, compound 56), or theselective Src family kinase inhibitor PP-2. Furthermore, AVP acts as apotent growth factor for IEC-18 cells, inducing DNA synthesis and cellproliferation through ERK-, Ca²⁺-, PKC-, EGFR tyrosinekinase-, and Src-dependent pathways.

     

Solute transport process in intestinal epithelial cells

In rat small intestine, the active transport of organic solutes results in significant depolarization of the membrane potential measured in an epithelial cell with respect to a grounded mucosal solution and in an increase in the transepithelial potential difference. According to the analysis with an equivalent circuit model for the epithelium, the changes in emf's of mucosal and serosal membranes induced by active solute transport were calculated using the measured conductive parameters. The result indicates that the mucosal cell membrane depolarizes while the serosal cell membrane remarkably hyperpolarizes on the active solute transport. Corresponding results are derived from the calculations of emf's in a variety of intestines, using the data that have hitherto been reported. The hyperpolarization of serosal membrane induced by the active solute transport might be ascribed to activation of the serosal electrogenic sodium pump. In an attempt to determine the causative factors in mucosal membrane depolarization during active solute transport, cell water contents and ion concentrations were measured. The cell water content remarkably increased and, at the same time, intracellular monovalent ion concentrations significantly decreased with glucose transport. Net gain of glucose within the cell was estimated from the restraint of osmotic balance between intracellular and extracellular fluids. In contrast to the apparent decreases in intracellular Na+ and K+ concentrations, significant gains of Na+ and K+ occurred with glucose transport. The quantitative relationships among net gains of Na+, K+ and glucose during active glucose transport suggest that the coupling ratio between glucose and Na+ entry by the carrier mechanism on the mucosal membrane is approximately 1:1 and the coupling ratio between Na+-efflux and K+-influx of the serosal electrogenic sodium pump is approximately 4:3 in rat small intestine. In addition to the electrogenic ternary complex inflow across the mucosal cell membrane, the decreases in intracellular monovalent ion concentrations, the temporary formation of an osmotic pressure gradient across the cell membrane and the streaming potential induced by water inflow through negatively charged pores of the cell membrane in the course of an active solute transport in intestinal epithelial cells are apparently all possible causes of mucosal membrane depolarization.     

mRNA localization in polarized intestinal epithelial cells

An important feature of enterocyte maturation is the asymmetrical distribution of cellular functions including protein localization. mRNA sorting is one mechanism for establishment and maintenance of this process in other systems, and we have previously demonstrated differential localization of mRNAs in human enterocytes. To study regulation of mRNA sorting, we established a model in polarized Caco-2 cells. Proxy cDNA constructs containing beta-galactosidase (beta-gal)/green fluorescence protein (GFP) and the 3'-untranslated region (3'-UTR) of either human sucrase-isomaltase or villin were transfected transiently or stably. A control construct contained poly-A sequence in place of 3'-UTR. Expression of GFP was observed by confocal microscopy; intracellular location of the construct mRNA was imaged by in situ hybridization. The sucrase-isomaltase mRNA proxy localized to an apical position in Caco-2 cells as in native enterocytes; the villin mRNA proxy did not show significant localization. The control construct was not localized and was found diffusely throughout the cell. Proxy GFP proteins tended to localize with their mRNA proxies, but with less precision. This study establishes a valuable model for the investigation of mRNA localization in intestinal epithelial cells. Mechanisms controlling asymmetrical distribution of intestinal mRNAs can be now be elucidated.     

Unique subset of thymic epithelial cells defined by the expression of a novel neuroendocrine antigen

Murine monoclonal antibodies (MoAbs) were produced against a rat medullary thyroid carcinoma to identify neuroendocrine differentiation antigens. One of these antibodies (1D4) identified a novel 62- to 69-kDa antigen expressed by subsets of immune system epithelial and neuroendocrine cells. This antigen is expressed by distinct subsets of thymic epithelial and splenic reticular cells and is shared by discrete subsets of anterior pituitary and thyroid neuroendocrine cells. In the thymus, 1D4 expression identified a unique subset of stellate-shaped Ia+ medullary epithelial cells which did not react with thymosin alpha-1 antisera nor with the MoAb A2B5 specific for a GQ ganglioside expressed by thymic hormone-producing cells. The availability of the 1D4 MoAb should facilitate further characterization of 1D4+ immune system epithelial cells and may advance our understanding of neuroendocrine-immune system interactions.     

Cellular internalization of lactoferrin in intestinal epithelial cells

We studied the cellular internalization of lactoferrin (Lf) in an intestinal epithelial cell line, Caco-2, to investigate the mechanism of biological actions of ingested Lf. RT-PCR and Western blotting analyses revealed that differentiated Caco-2 cells express LfR mRNA and its protein with a 34 kD molecular weight under reducing conditions. Biotin-labeled Lf showed specific binding to the cellular membrane of differentiated Caco-2 cells with a dissociation constant (Kd) of 0.16 microM. The cellular internalization of Lf was studied in differentiated Caco-2 cells grown as monolayers on Transwell inserts, and compared to that of human transferrin (Tf). After labeling with fluorescent dye, either Lf or Tf was added to Caco-2 cells from the apical side or the basolateral one. Laser scanning confocal microscopy showed that labeled Lf was internalized only from the apical side and localized to the nuclei. On the other hand, labeled Tf was internalized from the basolateral side, not from the apical side, and localized in the cytoplasm. The internalization of labeled Lf was inhibited by excess of unlabeled Lf, but not of Tf. The internalization of labeled Lf, but not of labeled Tf, was also suppressed by heparin. This indicates that a heparin-binding site in the N-terminal region of Lf could be important for the internalization of Lf. These findings suggest that ingested Lf might be internalized by the intestinal epithelium in a manner different from Tf and might function in the nucleus.     

Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells

Homeostasis of the gastrointestinal epithelium is dependent upon a balance between cell proliferation and apoptosis. Cyclin-dependent kinases (Cdks) are well known for their role in cell proliferation. Previous studies from our group have shown that polyamine-depletion of intestinal epithelial cells (IEC-6) decreases cyclin-dependent kinase 2 (Cdk2) activity, increases p53 and p21Cip1 protein levels, induces G1 arrest, and protects cells from camptothecin (CPT)-induced apoptosis. Although emerging evidence suggests that members of the Cdk family are involved in the regulation of apoptosis, their roles directing apoptosis of IEC-6 cells are not known. In this study, we report that inhibition of Cdk1, 2, and 9 (with the broad range Cdk inhibitor, AZD5438) in proliferating IEC-6 cells triggered DNA damage, activated p53 signaling, inhibited proliferation, and induced apoptosis. By contrast, inhibition of Cdk2 (with NU6140) increased p53 protein and activity, inhibited proliferation, but had no effect on apoptosis. Notably, AZD5438 sensitized, whereas, NU6140 rescued proliferating IEC-6 cells from CPT-induced apoptosis. However, in colon carcinoma (Caco-2) cells with mutant p53, treatment with either AZD5438 or NU6140 blocked proliferation, albeit more robustly with AZD5438. Both Cdk inhibitors induced apoptosis in Caco-2 cells in a p53-independent manner. In serum starved quiescent IEC-6 cells, both AZD5438 and NU6140 decreased TNF-α/CPT-induced activation of p53 and, consequently, rescued cells from apoptosis, indicating that sustained Cdk activity is required for apoptosis of quiescent cells. Furthermore, AZD5438 partially reversed the protective effect of polyamine depletion whereas NU6140 had no effect. Together, these results demonstrate that Cdks possess opposing roles in the control of apoptosis in quiescent and proliferating cells. In addition, Cdk inhibitors uncouple proliferation from apoptosis in a p53-dependent manner.     

Biochemical and functional characterization of a molecule expressed by a subset of thymic medullary epithelial cells.

A monoclonal antibody (mAb), named TE-4F 10, was produced by fusing P3X-Ag8 myeloma cells with splenocytes of BALB/c mice immunized with a rat medullary thymic epithelial cell (TEC) line, (TE-R 2.5), previously established in our Institute. Flow cytometry showed that 85-95% TE-R 2.5 cells expressed the TE-4F10 antigen. The mAb immunoprecipitated a 29 kDa molecule from the TE-R2.5 cell lysate. Immunohistochemical analysis using single and double staining of the thymus with anti-cytokeratin (CK) mAb, showed that TE- 4F10 mAb selectively stains a subpopulation of medullary TEC. Hematopoietic and lymphoid cells were negative. The expression of the TE-4F10 antigen on TE-R 2.5 cells in vitro was significantly upregulated by interleukin 1 (IL-1) and tumor necrosis factor (TNFalpha). Other cytokines IL-4, IL-6, IL-10 and granulocyte - macrophage colony stimulating factor (GM-CSF) showed lesser stimulation on its expression, whereas interferon gamma (IFN) and dexamethasone were without significant effect. The TE-R 2.5 cell line strongly bound and induced apoptosis of a rat / mouse thymocyte heterohybridoma (BWRT8), phenotypically alphabetaTCRhiCD4hiCD8lo. TE-4F10 mAb significantly inhibited binding (40-50%) of both BWRT8 cells and the BWRT8 - MDP.1 subclone to TE-R 2.5 cells. The inhibition was enhanced when TEC were stimulated with IL-1 + TNFalpha. The mAb also significantly blocked apoptosis of BWRT8 but did not modulate cell death of the BWRT8 - MDP.1 subclone, which was resistant to TEC-induced apoptosis. These findings indicate that the TE-4F10 antigen might be selectively involved in adhesion and selection processes in the medullary thymic microenvironment. The mAb of the same characteristics has not been described so far.     

The gut microflora and intestinal epithelial cells: a continuing dialogue

The mammalian intestinal epithelium effectively performs its physiological functions in a microbe-rich environment, while the prokaryotic population thrives amidst efficient cellular defenses. Recent delineation of the mechanisms by which bacteria communicate with their eukaryotic hosts and the cellular sites that microbial signals act in may shed light on these complex interactions.     

Interaction of vasoactive intestinal peptide with rat small intestinal epithelial cells after intestinal resection

Specific binding of vasoactive intestinal peptide (VIP) and VIP-stimulated c y c l i c AMP accumulation were studied in small intestinal epithelial cells (both of crypt and villous levels) 3, 7 and 14 d after a 60% resection of the small intestine . The affinity, but not the binding capacity, of VIP receptors decreased during the adaptive hyperplastic response. Basal cyclic AMP levels were similar in cells of both control and resected rats. Resection induced a decrease of potency, but not of efficiency, of VIP on the stimulation of cyclic AMP accumulation.