Peroxisome proliferator-activated receptor gamma is induced during differentiation of colon epithelium cells.

Peroxisome proliferator-activated receptor gamma (PPARgamma), a fatty acid-activated nuclear receptor, is implicated in adipocyte differentiation and insulin sensitisation. In view of the association of dietary fat intake and bowel disease, the expression of PPARgamma in rodent and human intestine was studied. Expression of PPARgamma mRNA was examined by Northern blot hybridisation, RNase protection, and/or competitive RT-PCR assays, whereas PPARgamma protein levels were evaluated by immunoblotting and immunohistochemistry. PPARgamma mRNA and protein were abundantly expressed in colon relative to the small intestine both in rodents and in man. Interestingly, expression of PPARgamma was primarily localised in the more differentiated epithelial cells in the colon. The level of expression of PPARgamma in colon was similar to the levels seen in adipose tissue. Expression of PPARgamma increased from proximal to distal segments of the colon in man. In Caco-2 and HT-29 human adenocarcinoma cells, PPARgamma expression increased upon differentiation, consistent with PPARgamma being associated with a differentiated epithelial phenotype. High-level expression of PPARgamma was observed in the colon, but not in the small intestine, suggesting a potential role of this nuclear receptor in the colon.     

Epidermal growth factor receptors during postnatal development of the mouse colon

The present study was undertaken to establish the postnatal profile of specific epidermal growth factor (EGF) binding in the maturing mouse colon, with particular emphasis on possible regional differences between both proximal and distal colonic binding patterns vs. those of the small intestine. Binding studies using [125I]EGF were performed on isolated epithelial cells obtained from 2-, 5-, 9-, 16-, and 22-day-old mice as well as adults. At 2 days, cells isolated from the entire colon bound 4 times more [125I]EGF than did corresponding intestinal cells, whereas between the ages of 5 days to adult, colonic cells bound between 1.7-2.5 times more labeled EGF than their intestinal counterparts. The immature colon already exhibited maximal binding after birth as opposed to the small intestine where binding only reached maximal values by the third week of life. Comparison between the proximal and distal colon in 9-, 16-, and 22-day-old mice revealed a further 2-fold increase in EGF binding in the distal colon compared to that in the proximal colon. Scatchard plots of [125I]EGF displacement by native EGF in both proximal and distal colonic segments also revealed the presence of two classes of binding sites, with high affinity constants (K1) significantly greater in the distal colon. These results demonstrate for the first time not only the continued presence of EGF receptors in mouse colonic epithelium, but also significant regional differences in EGF-binding capacity within the digestive tract throughout the postnatal period.     

Adaptive regulation of the ileal apical sodium dependent bile acid transporter (ASBT) in patients with obstructive cholestasis

BACKGROUND/AIMS: The apical sodium dependent bile acid transporter ASBT (SLC10A2) contributes substantially to the enterohepatic circulation of bile acids by their reabsorption from the intestine. In the rat, its adaptive regulation was observed in the kidneys, cholangiocytes, and terminal ileum after bile duct ligation. Whether adaptive regulation of the human intestinal ASBT exists during obstructive cholestasis is not known. METHODS: Human ASBT mRNA expression along the intestinal tract was analysed by real time polymerase chain reaction in biopsies of 14 control subjects undergoing both gastroscopy and colonoscopy. Their duodenal ASBT mRNA expression was compared with 20 patients with obstructive cholestasis. Additionally, in four patients with obstructive cholestasis, duodenal ASBT mRNA expression was measured after reconstitution of bile flow. RESULTS: Normalised ASBT expression in control subjects was highest (mean arbitrary units (SEM)) in the terminal ileum (1010 (330)). Low ASBT expression was found in colonic segments (8.3 (5), 4.9 (0.9), 4.8 (1.7), and 1.1 (0.2) in the ascending, transverse, descending, and sigmoid colon, respectively). Duodenal ASBT expression in control subjects (171.8 (20.3)) was found to be approximately fourfold higher compared with patients with obstructive cholestasis (37.9 (6.5); p<0.0001). Individual ASBT mRNA expression was inversely correlated with bile acid and bilirubin plasma concentrations. In four cholestatic patients, average ASBT mRNA increased from 76 (18) before to 113 (18) after relief of cholestasis (NS). Immunohistochemical assessment indicated that ASBT protein was expressed on the apical surface of duodenal epithelial cells. CONCLUSION: Obstructive cholestasis in humans leads to downregulation of ASBT mRNA expression in the distal part of the human duodenum.     

Ephrin-B2 is differentially expressed in the intestinal epithelium in Crohn＇s disease and contributes to accelerated epithelial wound healing in vitro

AIM: Eph receptor tyrosine kinases and their membrane bound receptor-like ligands, the ephrins, represent a bi-directional cell-cell contact signaling system that directs epithelial movements in development. The meaning of this system in the adult human gut is unknown. We investigated the Eph/ephrin mRNA expression in the intestinal epithelium of healthy controls and patients with inflammatory bowel disease (IBD). METHODS: mRNA expression profiles of all Eph/ephrin family members in normal small intestine and colon were established by real-time RT-PCR. In addition, differential expression in IBD was investigated by cDNA array technology, and validated by both real-time RT-PCR and immunohistochemistry. Potential effects of enhanced EphB/ephrin-B signaling were analyzed in an in vitro IEC-6 cell scratch wound model. RESULTS: Human adult intestinal mucosa exhibits a complex pattern of Eph receptors and ephrins. Beside the known prominent co-expression of EphA2 and ephrinAl, we found abundantly co-expressed EphB2 and ephrin-B1/2. Interestingly, cDNA array data, validated by real-time PCR and immunohistochemistry, showed upregulation of ephrin-B2 in both perilesional and lesional intestinal epithelial cells of IBD patients, suggesting a role in epithelial homeostasis. Stimulation of ephrin-B signaling in ephrin- B1/2 expressing rat IEC-6-cells with recombinant EphB1-Fc resulted in a significant dose-dependent acceleration of wound closure. Furthermore, fluorescence microscopy showed that EphB1-Fc induced coordinated migration of wound edge cells is associated with enhanced formation of lamellipodial protrusions into the wound, increased actin stress fiber assembly and production of laminin at the wound edge. CONCLUSION: EphB/ephrin-B signaling might represent a novel protective mechanism that promotes intestinal epithelial wound healing, with potential impact on epithelial restitution in IBD.     

Degradation of intestinal mRNA:A matter of treatment

AIM: To characterize the influence of location, species and treatment upon RNA degradation in tissue samples from the gastrointestinal tract. METHODS: The intestinal samples were stored in different medium for different times under varyingconditions: different species(human and rat), varying temperature(storage on crushed ice or room temperature), time point of dissection of the submucous-mucous layer from the smooth muscle(before or after storage), different rinsing methods(rinsing with Medium, PBS, RNALater or without rinsing at all) and different regions of the gut(proximal and distal small intestine, caecum, colon and rectum). The total RNA from different parts of the gut(rat: proximal and distal small intestine, caecum, colon and rectum, human: colon and rectum) and individual gut layers(muscle and submucosal/mucosal) was extracted. The quality of the RNA was assessed by micro capillary electrophoresis. The RNA quality was expressed by the RNA integrity number which is calculated from the relative height and area of the 18 S and 28 S RNA peaks. From rat distal small intestine q PCR was performed for neuronal and glial markers.RESULTS: RNA obtained from smooth muscle tissue is much longer stable than those from submucosal/mucosal tissue. At RT muscle RNA degrades after one day, on ice it is stable at least three days. Cleaning and separation of gut layers before storage and use of RNALater, maintains the stability of muscle RNA at RT for much longer periods. Different parts of the gut show varying degradation periods. RNA obtained from the submucosal/mucosal layer always showed a much worse amplification rate than RNA from muscle tissue. In general RNA harvested from rat tissue, either smooth muscle layer or submucosal/mucosal layer is much longer stable than RNA from human gut tissue, and RNA obtained from smooth muscle tissue shows an increased stability compared to RNA from submucosal/mucosal tissue. At RT muscle RNA degrades after one day, while the stability on ice lasts at least three days. Cleaning and separation of gut layers before storage and use of RNALater, maintains the stability of muscle RNA at RT for much longer periods. Different parts of the gut show varying degradation periods. The RNA from muscle and submucosal/mucosal tissue of the proximal small intestine degrades much faster than the RNA of distal small intestine, caecum or colonwith rectum. RNA obtained from the submucosal/mucosal layer always showed a much more reduced amplification rate than RNA from muscle tissue [β-Tubulin Ⅲ for muscle quantification cycle(Cp): 22.07 ± 0.25, for β-Tubulin Ⅲ submucosal/mucosal Cp: 27.42 ± 0.19].CONCLUSION: Degradation of intestinal m RNA depends on preparation and storage conditions of the tissue. Cooling, rinsing and separating of intestinal tissue reduce the degradation of m RNA.     

Intestinal transferrin receptors and iron absorption in the neonatal rat

The transferrin receptor is a major protein found on the basolateral membranes of intestinal epithelial cells, yet its possible role in intestinal iron metabolism and also in iron absorption is unclear. We have studied intestinal transferrin receptor expression during the peri- and postnatal development of the small intestine of the rat using immunohistochemistry with a monoclonal antibody to the rat receptor. Two major changes in transferrin receptor expression in the developing small intestine were found, a decrease in receptor expression associated with birth, and an increase at the time of weaning. Around the time of weaning there was a large decrease in iron absorption, but there was no direct correlation between absorption and transferrin receptor expression. However, at both birth and weaning there were major changes in intestinal cell kinetics, and the distribution of receptor correlated well with the distribution of proliferating cell populations. In addition, as the intestinal epithelial cells differentiated and stopped dividing, there was a redistribution of transferrin receptors from the cell surface to intracellular sites. These data suggest that the most likely role of the transferrin receptor in the neonatal intestine is in the supply of iron to the developing epithelial cells in the crypts, and that the receptor does not play a direct role in iron transit across the intestinal epithelium.     

Expression of iron absorption genes in mouse large intestine

OBJECTIVE: The large intestine has been reported to have a capacity for iron absorption and expresses genes for iron absorption normally found in the duodenum. The importance and function of these genes in the large intestine are not understood. We therefore investigated the cellular localization and regulation of expression of these genes in mouse caecum and colon. MATERIAL AND METHODS: Gene expression was measured by real-time PCR using RNA extracted from iron-deficient and hypoxic mouse large intestine, compared to controls. Protein localization and regulation were measured by immunohistochemistry using frozen sections of the large intestine from the same mice. RESULTS: Dcytb (duodenal ferric reductase) was expressed at very low levels in the large intestine, compared to the duodenum, while Ireg1 and DMT1 were expressed at significant levels in the large intestine and were increased in iron-deficient caecum, proximal and distal colon, with the most significant increases seen in the distal colon. Hypoxia increased Ireg1 expression in the proximal colon. Immunohistochemistry detected significant levels of only IREG1, which was localized to the basolateral membrane of colonic epithelial cells. CONCLUSIONS: Iron absorption genes were expressed at lower levels in mouse caecum and colon than in the duodenum. They are regulated by body iron requirements. Colonic epithelial cells express basolateral IREG1in the same fashion as in the duodenum and this protein could regulate colonic epithelial cell iron levels.     

Epithelial proliferation,cell death,and gene expression in experimental colitis: alterations in carbonic anhydrase I,mucin MUC2, and trefoil factor 3 expression

BACKGROUND AND AIMS: To gain insight in intestinal epithelial proliferation, cell death, and gene expression during experimental colitis rats were treated with dextran sulfate sodium (DSS) for 7 days. MATERIALS AND METHODS: Proximal and distal colonic segments were excised on days 2, 5, 7, and 28. Epithelial proliferation, cell death, enterocyte gene expression (carbonic anhydrase I (CA I) and goblet cell gene expression (mucin, MUC2; trefoil factor 3, TFF3) were studied immunohistochemically and biochemically. RESULTS: Proliferative activity was decreased in the proximal and distal colon at the onset of disease (day 2). However, during active disease (days 5-7) epithelial proliferation was increased in the entire proximal colon and in the proximity of ulcerations in the distal colon. During DSS treatment the number of apoptotic cells in the epithelium of both colonic segments was increased. In the entire colon surface enterocytes became flattened and CA I negative during active disease (day 5-7). Additionally, CA I levels in the distal colon significantly decreased during this phase. In contrast, during the regenerative phase (day 28) CA I levels were restored in the distal colon and up-regulated in the proximal colon. During all disease phases increased numbers of goblet cells were observed in the surface epithelium of the entire colon. In the distal colon TFF3 expression extended to the bottom of the crypts during active disease. Finally, MUC2 and TFF3 expression was increased in the proximal colon during disease. CONCLUSION: DSS affected the epithelium by inhibiting proliferation and inducing apoptosis. DSS-induced inhibition of CA I expression indicates down-regulation of specific enterocyte functions. Accumulation of goblet cells in the surface epithelium and up-regulation of MUC2 and TFF3 expression in the proximal colon underline the importance of goblet cells in epithelial protection and repair, respectively.     

Localization of peptide YY (PYY) in gastrointestinal endocrine cells and effects on intestinal blood flow and motility.

In immunohistochemical studies using antisera to peptide YY (PYY), a 36 amino acid polypeptide isolated from porcine duodenum, it was found that PYY-like immunoreactivity occurred mainly in endocrine cells of the gastrointestinal mucosa. PYY-immunoreactive cells were particularly abundant in the distal intestine and have been observed in five species, including man. By radioimmunoassay it was found that, in the rat, the amount of PYY immunoreactivity was about 100-fold higher in the colon than in the duodenum. The chromatographic profiles of PYY immunoreactivity from the rat colon and porcine PYY on a SP-Sephadex ion exchanger were similar. Furthermore, serial dilutions of extracts from the rat colon and porcine PYY had parallel displacement curves in radioimmunoassay. Close intraarterial administration of PYY in cats caused an intestinal vasoconstriction and an inhibition of jejunal and colonic motility. Simultaneously there was a rise in systemic arterial blood pressure. These effects of PYY were also observed after pretreatment with adrenergic blocking agents. It is concluded that PYY is a gastrointestinal peptide that is present mainly in endocrine cells of distal intestine and that has effect on both intestinal motility and the cardiovascular system.     

Fetal characteristics of small intestinal crypt cells.

Nine monoclonal antibodies were prepared against luminal membranes purified from rat intestinal cells at day 19 of gestation, and seven of them were found to define antigens common to adult crypt cells and fetal or embryonic intestinal epithelial cells. The FBB 2/29 antigen was first detected over the entire intestinal epithelial population at days 14-15 of gestation, a period of development characterized by formation of a stratified intestinal epithelium and differentiation of the surrounding mesenchyme. This antigen, identified as a set of high molecular mass proteins, became restricted to the crypt and lower villus cells after birth and was exclusively expressed by the crypt cells in adult intestine. It also was found to be expressed by the epithelial cells of the distal tubuli in the kidney of adult rats and by cultured human tumor colonic cells. The FBB 1/54/1, FBB 3/46, and FBB 3/78/9 antibodies stained only the epithelial cells present at the base of the villi in fetal intestine, starting at days 20-21 of gestation (about 1-2 days before birth), and stained the crypt and lower villus cells in newborn and adult intestine; these antigens may be regarded as specific markers for the developing crypt cells in fetal intestine shortly before birth. The FBB 1/20 and FBB 4/2 antigens were first detected on the fetal intestinal cells at day 18 of gestation; they were located over the entire epithelium in newborn rats and became restricted to the crypts after weaning. The FBB 2/28 antigen was expressed by the entire intestinal epithelium at all stages of development, starting from days 18-19 of gestation in the fetus. Two antibodies, FBB 3/4 and FBB 3/24, were found to be specific for lactase. These results have demonstrated the expression of cell- and tissue-specific components in rat intestine during early embryonic development and revealed a marked similarity in surface membrane antigens between fetal intestinal epithelial cells and adult crypt cells.     

Mucosal pathobiology and molecular signature of epithelial barrier dysfunction in the small intestine in irritable bowel syndrome

Irritable bowel syndrome (IBS) is one of the most prevalent gastrointestinal disorders in developed countries. Its etiology remains unknown; however, a common finding, regardless of IBS subtype, is the presence of altered intestinal barrier. In fact, signaling and location of cell‐to‐cell adhesion proteins, in connection with increased immune activity, seem abnormal in the intestinal epithelium of IBS patients. Despite that most research is performed on distal segments of the intestine, altered permeability has been reported in both, the small and the large bowel of all IBS subtypes. The small intestine carries out digestion and nutrient absorption and is also the site where the majority of immune responses to luminal antigens takes place. In fact, the upper intestine is more exposed to environmental antigens than the colon and is also a site of symptom generation. Recent studies have revealed small intestinal structural alterations of the epithelial barrier and mucosal immune activation in association with intestinal dysfunction, suggesting the commitment of the intestine as a whole in the pathogenesis of IBS. This review summarizes the most recent findings on mucosal barrier alterations and its relationship to symptoms arising from the small intestine in IBS, including epithelial structural abnormalities, mucosal immune activation, and microbial dysbiosis, further supporting the hypothesis of an organic origin of IBS.