Gene expression profiling of intestinal epithelial cell maturation along the crypt-villus axis

BACKGROUND & AIMS: To define the genetic reprogramming that drives intestinal epithelial cell maturation along the crypt-villus axis, enterocytes were sequentially isolated from the villus tip to the crypts of mouse small intestine. METHODS: Changes in gene expression were assessed using 27,405-element complementary DNA microarrays (14,685 unique genes) and specific changes validated by Western blotting. RESULTS: A total of 1113 genes differentially expressed between the crypt and villus were identified. Among these, established markers of absorptive and goblet cell differentiation were up-regulated in villus cells, whereas Paneth cell markers were maximally expressed in crypt cells. The 1113 differentially expressed genes were significantly enriched for genes involved in cell cycle progression, RNA processing, and translation (all predominantly down-regulated during maturation) and genes involved in cytoskeleton assembly and lipid uptake (predominantly up-regulated during maturation). No enrichment for apoptosis-regulating genes was observed. We confirmed that Wnt signaling was maximal in the proliferative compartment and observed a decrease in MYC and an increase in MAD and MAX expression during the maturation program. Consistent with these changes, the 1113 genes were enriched for MYC targets, establishing the importance of this network in intestinal cell maturation. CONCLUSIONS: This database serves as a resource for understanding the molecular mechanisms of intestinal cell maturation and for dissection of how perturbations in the maturation process can lead to changes in gastrointestinal physiology and pathology, particularly intestinal tumorigenesis.     

Mechanisms and regulation of intestinal iron absorption

Iron absorption from the small intestine is regulated according to the body's needs, increasing in iron deficiency and decreasing in iron overload. It has been proposed that the efficiency of absorption is determined by the amount of iron acquired by developing enterocytes when they are in the crypts of Lieberk?hn and that this regulates expression of iron transporters such as DMT1 in mature enterocytes of the intestinal villi. In the crypts the cells take up iron from plasma transferrin by receptor-mediated endocytosis, a process that is influenced by the hemochromatosis protein, HFE. Hence, the availability of plasma transferrin-bound iron and the expression and function of transferrin receptors (TfR1), HFE and DMT1 should all contribute to the absorptive capacity of villus enterocytes. These aspects of the regulation and mechanism of iron absorption were investigated in genetically normal rats and mice, and in Belgrade anemic (b/b) rats and HFE knockout mice. In most experiments the function of the TfR1 was assessed by the uptake of radiolabeled transferrin-bound iron given intravenously. Absorption of non-heme iron was measured using closed in situ duodenal loops. The expression and cellular distribution of DMT1 and TfR1 were determined by in situ hybridisation and immunohistochemistry. The uptake of transferrin-bound iron and expression of functional TfR1 was shown to occur mainly in crypt cells and to be proportional to the plasma concentration of iron. It was not impaired by the mutation of DMT1 that occurs in b/b rats but was impaired in HFE knockout mice. Iron absorption was increased in these mice but was still influenced by the level of iron stores, as in normal mice. These results are in accordance with the proposed regulation of iron absorption and suggest that DMT1 is not the only iron transporter operating within endosomes of crypt cells. This view was supported by the failure to detect DMT1 mRNA or protein in crypt cells. Expression of DMT1 mRNA and protein started at the crypt-villus junction and increased to reach highest levels in the mid-villus region. Greater expression was found in iron deficiency and less in iron loaded animals than in controls and in the iron deficient rats most of the protein was present on the brush border membrane. In normal rats the efficiency of iron absorption parallelled the level of DMT1 expression, but in b/b rats absorption was very low and independent of dietary iron content even though DMT1 was present in villus enterocytes. The results confirm the essential role of DMT1 in the uptake phase of non-heme iron absorption. When normal rats previously fed a low iron diet were given a bolus of iron by stomach tube, the subsequent absorption of iron from a test dose placed in the duodenum diminished in parallel with the expression of DMT1 mRNA and protein, commencing within 1hour and reaching low levels by 7 hours. The margination of DMT1 to the brush border membrane disappeared. These results show the level of expression and intracellular distribution and function of DMT1 respond very quickly to the iron content of the diet as well as being affected by storage iron levels.     

Effect of Fat Feeding on Pro-oxidant and Anti-oxidant Enzyme Systems in Rat Intestine: Possible Role in the Turnover of Enterocytes

Immature epithelial cells generated in the crypt base undergo differentiation while progressing to the villus tip, where the cells upon apoptosis are detached from the underlying muscular tissue. We previously reported that lipid peroxidation might be involved in the turnover of enterocytes across the crypt–villus axis in rat intestine (Dig Dis Sci 52:1840–1844, 2007). To examine whether long-term feeding of fat with different fatty-acid composition influences this process, in the present study we investigated the effect of feeding fish oil (n − 3) and corn oil (n − 6) polyunsaturated fatty acids on lipid per-oxidation and anti-oxidant systems in different epithelial cell fractions isolated in rat intestine. Feeding fish oil or corn oil markedly enhanced lipid per-oxidation levels of enterocytes throughout villus height compared with control, but there was no difference in the distribution profile of pro- and anti-oxidant enzyme systems and lipid per-oxidation across the crypt–villus axis under these conditions. Analysis of lipid peroxidation levels in different cell fractions revealed that the thiobarbituric acid reactive substance were 9- to 11-fold higher at the villus tip compared with at the crypt base. The activities of glutathione reductase and glutathione-S-transferase were 2- to 5-fold higher in villus tip compared to the crypt region. However, the activities of superoxide dismutase and catalase were 6- to 8-fold high at the crypt base compared with at villus tip cells. Immunocytolocalization of superoxide dismutase showed high staining in crypt base compared with that in villus, tip cells. These findings further suggest that generation of reactive oxygen species in enterocytes across the crypt–villus axis may be involved in turnover of enterocytes across the crypt–villus unit in rat intestine.     

Crypt/villus site of substrate-dependent regulation of mouse intestinal glucose transporters.

The intestinal epithelium is in a constant state of turnover, with cells differentiating at the crypts and then migrating toward the tips of the villi. Does substrate-dependent regulation of intestinal Na+/D-glucose cotransporters occur only in crypt cells, or can transport activity be subsequently reprogrammed in mature enterocytes? We used in situ, glucose-protectable specific phlorizin binding to determine site density of brush border glucose transporters in enterocytes fractionated along the crypt/villus axis of mice that were killed shortly after drastic changes in carbohydrate levels of their diets. Dietary carbohydrate-induced changes in site density of specific phlorizin binding initially appeared only in crypt cells before spreading, over the course of several days, to the villus tips. Thus, only crypt cells perceive the signal for glucose transporter regulation, and the observed time lag of diet-induced changes in intestinal glucose uptake is due mainly to cell migration times.     

Cholesterol synthesis and low density lipoprotein uptake are regulated independently in rat small intestinal epithelium.

We have compared the rates of low density lipoprotein (LDL) uptake and cholesterol synthesis in the rat intestine. By using a constant infusion technique, total and receptor-independent uptake was determined with homologous rat LDL (rLDL) and methylated human LDL (Me-hLDL), respectively. The absolute rates of sterol synthesis were measured with [1-(14) C]-octanoate and [3H]water. The rates of rLDL uptake in whole gut segments were similar along the length of the small intestine, whereas the rates of sterol synthesis varied over a 5-fold range and were highest in the duodenum and distal ileum. When the mucosal epithelium was fractionated along the villus/crypt axis, both rLDL and Me-hLDL clearance by the enterocytes increased approximately 3-fold in going from the upper villus to the crypt cell fractions, in both jejunum and ileum. In both the whole gut segments and isolated cells, approximately 60% of LDL uptake was receptor dependent. When the rates of rLDL cholesterol uptake were calculated and related to the absolute rates of sterol synthesis in the same cell fractions in vivo, both processes were found to be distributed similarly along the villus/crypt axis. Furthermore, the majority of mucosal cholesterol (64-86%) was derived from local synthesis rather than from rLDL uptake at all locations along the intestinal villus. Finally, when sterol synthesis in the epithelial cells was varied up to 7-fold by feeding cholesterol, triglyceride, cholestyramine, or surfomer, rLDL uptake was essentially unchanged. Thus, in intestinal epithelial cells in vivo, the rate of LDL uptake was constant under circumstances in which changing needs for cellular cholesterol were met by changes in the rates of sterol synthesis.     

Crypt-villus differentiation reflected by lectin and protein binding to rat small intestinal brush border membranes

To compare differentiation along the crypt-villus axis in adult rats with changes observed in postnatal maturation with respect to binding capacities for lectins and food proteins, crypts and villi were isolated byin vivo perfusion andin vitro incubation. Brush border membranes were prepared from adults and newborns, and binding of¹²⁵I-labeled lectins and food proteins was assessed by airfuge ultracentrifugation. Crypt and villus membrane protein patterns looked almost identical, unlike newborn membranes. Considerable shifts in lectin binding to membranes were observed during postnatal maturation, but not in crypt-villus differentiation. For instance, fucose-specific lectin binding patterns in both preparations resembled the general adult mode. Contrary to differences in food protein binding between newborn and adult membranes, food protein binding did not show a consistent significant difference between membranes of crypt and villus origin in adult animals. In conclusion, membrane differentiation along the crypt-villus axis was found to follow a pattern dissimilar from neonatal maturation as far as protein and carbohydrate composition and food protein binding were concerned.Supported by Deutsche Forschungsgemeinschaft DFG Ste 305.     

The Profile of Antioxidant Systems and Lipid Peroxidation Across the Crypt-Villus Axis in Rat Intestine

The distribution of lipid peroxiation and profile of antioxidant-pro-oxidant enzyme systems have been studied in rat intestinal enterocyte across the length of villi. The MDA levels estimated as a measure of lipid peroxidation, under induced or uninduced in vitro conditions, indicated markedly high levels at villus tip cells compared to that in the crypt base. The activities of glutathione-S-transferase and glutathione reductase were three- to sixfold higher in villus tip cells compared to that in the crypt base. However the levels of catalase and superoxide dismutase showed a reverse pattern, being high in the crypt base and lowest in the villus tip region. Feeding coconut oil, sunflower oil, or groundnut oil did not modify the distribution pattern of these systems across crypt-villus unit in rat intestine. These findings suggest that the large amount of free radicals generated in villus tip cells may be responsible for the release of enterocytes from the villus tip as a consequence of apoptosis.     

Regulation of transferrin-mediated iron uptake by HFE, the protein defective in hereditary hemochromatosis

The protein defective in hereditary hemochromatosis, called HFE, is similar to MHC class I-type proteins and associates with beta2-microglobulin (beta2M). Its association with beta2M was previously shown to be necessary for its stability, normal intracellular processing, and cell surface expression in transfected COS cells. Here we use stably transfected Chinese hamster ovary cell lines expressing both HFE and beta2M or HFE alone to study the effects of beta2M on the stability and maturation of the HFE protein and on the role of HFE in transferrin receptor 1 (TfR1)-mediated iron uptake. In agreement with prior studies on other cell lines, we found that overexpression of HFE, without overexpressing beta2M, resulted in a decrease in TfR1dependent iron uptake and in lower iron levels in the cells, as evidenced by ferritin and TfR1 levels measured at steady state. However, overexpression of both HFE and beta2M had the reverse effect and resulted in an increase in TfR1-dependent iron uptake and increased iron levels in the cells. The HFE-beta2M complex did not affect the affinity of TfR1 for transferrin or the internalization rate of transferrin-bound TfR1. Instead, HFE-beta2M enhanced the rate of recycling of TfR1 and resulted in an increase in the steady-state level of TfR1 at the cell surface of stably transfected cells. We propose that Chinese hamster ovary cells provide a model to explain the effect of the HFE-beta2M complex in duodenal crypt cells, where the HFE-beta2M complex appears to facilitate the uptake of transferrin-bound iron to sense the level of body iron stores. Impairment of this process in duodenal crypt cells leads them to be iron poor and to signal the differentiating enterocytes to take up iron excessively after they mature into villus cells in the duodenum of hereditary hemochromatosis patients.     

Induction of apoptosis before shedding of human intestinal epithelial cells

OBJECTIVES: Human intestinal epithelial cells (IECs) derive from stem cells at the crypt base and migrate along the so-called crypt-villus axis toward the intestinal lumen. As they reach the luminal surface in the colon or the villus tip in the small intestine, IECs are shed and their life cycle is terminated. The role of apoptosis during IEC migration along the crypt-villus axis has been subject to studies with conflicting results. In this study we use a novel approach to identify the initiation of apoptosis within normal human IECs. METHODS: Normal mucosa from the large and small human intestine was analyzed employing a novel antibody directed against activated caspase-3--an early marker of apoptosis. RESULTS: IECs initiate the apoptotic cascade as they approach the area of shedding before displaying evident morphological features of apoptosis. IECs of the small bowel also show caspase-3 activation in the small intestinal crypt base, whereas IECs of the colononic crypt base rarely show evidence of ongoing apoptosis. CONCLUSIONS: These findings indicate that apoptosis is initiated in human IECs as they reach the luminal surface/villus tip and before shedding. Furthermore, they show that different sections of the intestinal tract vary significantly in the rate of IEC apoptosis, possibly reflecting their difference in susceptibility to epithelial cell neoplasia.     

Regulation of enterocyte apoptosis by acyl-CoA synthetase 5 splicing

BACKGROUND AND AIMS: The constant renewal of enterocytes along the crypt-villus axis (CVA) of human small intestine is due to cell-inherent changes resulting in the apoptotic cell death of senescent enterocytes. The aim of the present study was to examine underlying molecular mechanisms of the cell death at the villus tip. METHODS: Characterization of human acyl-coenzyme A (CoA) synthetase 5 (ACSL5) was performed by cloning, recombinant protein expression, biochemical approaches, and several functional and in situ analyses. RESULTS: Our data show that different amounts of acyl-CoA synthetase 5-full length (ACSL5-fl) and a so far unknown splice variant lacking exon 20 (ACSL5-Delta 20) are found in human enterocytes. In contrast with the splice variant ACSL5-Delta 20, recombinant and purified ACSL5-fl protein is active at a highly alkaline pH. Over expression of ACSL5-fl protein is associated with a decrease of the anti-apoptotic FLIP protein in a ceramide-dependent manner and an increased cell-surface expression of the death receptor TRAIL-R1. Expression analyses revealed that the ACSL5-fl/ACSL5-Delta 20 ratio increases along the CVA, thereby sensitizing ACSL5-fl-dominated cells at the villus tip to the death ligand TRAIL, which is corroborated by functional studies with human small intestinal mucosal samples and an immortalized human small intestinal cell line. CONCLUSIONS: Our results suggest an ACSL5-dependent regulatory mechanism that contributes to the cellular renewal along the CVA in human small intestine. Deregulation of the ACSL5-fl/ACSL5-Delta 20 homeostasis in the maturation and shedding of cells along the CVA might also be of relevance for the development of intestinal neoplasia.     

Ileal epithelial cell migration after 40% small-bowel resection

Adaptation in the small bowel after resection is associated with increases in crypt cell proliferation and villus height. This paper gives the results of an autoradiographic investigation with [³H]thymidine of epithelial cell migration 60 days after 40% small-bowel resection in the rat. The mean number of cell positions between the crypt-villus junction and the leading labeled cell 30 hr after injection was increased by 19.4% in the resected group (P<0.02). The mean total number of cells per villus column was increased by 27.8% (P<0.002). Migration rate estimated in cell positions per hour was accelerated by 18.9% (P<0.001) after resection. The 8.1% lengthened life span of villus cells was not statistically significant. The increased number of cells per villus column and unaltered life span of villus cells would facilitate functional adaptation. The causal relationship between the larger villus cell population and accelerated migration after resection and increased crypt cell proliferation is unknown.This investigation was supported by the National Health and Medical Research Council.     

Alterations in quantitative distribution of Na,K-ATPase activity along crypt-villus axis in animal model of malabsorption characterized by hyperproliferative crypt cytokinetics

The distribution of sodium- and potassium-stimulated ATPase (Na, K-ATPase) along the crypt-villus axis and crypt cytokinetics were examined in an infective model of celiac disease produced by infection of the rat with the nematodeNippostrongylus brasiliensis. In controls, levels of enzyme activity remained stable during enterocyte migration to the villous apex. In the jejunum of infected rats, the structural lesion of villous atrophy and crypt hyperplasia, observed at day 10 of infection, was associated with a three-dimensional expansion of the crypts. Cell cycle time was shortened and this resulted in a markedly increased crypt cell production rate. Enterocytes emerged from the crypts at a faster rate, and this functional immaturity was paralleled by decreased Na, K-ATPase activity. Further decreases in enzym levels were observed during enterocyte migration along the villi. This may reflect enterocyte damage or increased enzyme turnover. In the ileum of these animals, enterocyte maturation was prolonged and enzyme activity was increased at the level of the crypt villus junction with further increases noted during enterocyte transit. These changes in ileal Na, K-ATPase appear to be adaptive.This study was presented in part at the Annual Meeting of the American Gastroenterological Association, Chicago, May 12, 1987) and published in abstract from (Gastroenterology 92:1695, 1987).These studies were supported by a grant from the Medical Research Council of Canada.     

Study protocol: population screening for colorectal cancer by colonoscopy or CT colonography: a randomized controlled trial

Background

Maturation of enterocytes along the small intestinal crypt-villus axis is associated with significant changes in gene expression profiles. fls485 coding a putative chaperone protein has been recently suggested as a gene involved in this process. The aim of the present study was to analyze fls485 expression in human small intestinal mucosa.

Methods

fls485 expression in purified normal or intestinal mucosa affected with celiac disease was investigated with a molecular approach including qRT-PCR, Western blotting, and expression strategies. Molecular data were corroborated with several in situ techniques and usage of newly synthesized mouse monoclonal antibodies.

Results

fls485 mRNA expression was preferentially found in enterocytes and chromaffine cells of human intestinal mucosa as well as in several cell lines including Rko, Lovo, and CaCo2 cells. Western blot analysis with our new anti-fls485 antibodies revealed at least two fls485 proteins. In a functional CaCo2 model, an increase in fls485 expression was paralleled by cellular maturation stage. Immunohistochemistry demonstrated fls485 as a cytosolic protein with a slightly increasing expression gradient along the crypt-villus axis which was impaired in celiac disease Marsh IIIa-c.

Conclusions

Expression and synthesis of fls485 are found in surface lining epithelia of normal human intestinal mucosa and deriving epithelial cell lines. An interdependence of enterocyte differentiation along the crypt-villus axis and fls485 chaperone activity might be possible.     

Active absorption of vitamin B12 and conjugated bile salts by guinea pig ileum occurs in villous and not crypt cells

We isolated highly enriched fractions of villous and crypt cells from guinea pig intestine to determine whether this preparation provided a suitable model for comparing the transport of cobalamin and conjugated bile salts by these cell populations. The uptake of [⁵⁷Co] cyanocobalamin by ileal villous cells was 30-fold greater when incubated with cobalamin bound to intrinsic factor than with free cobalamin. Intrinsic factor-mediated uptake of cobalamin could not be demonstrated using ileal crypt or jejunal villous or crypt cells. When incubated with [³H] taurocholate, the uptake by ileal villous cells was significantly greater than by ileal crypt or jejunal villous cells. These results indicate the suitability of using isolated guinea pig villous and crypt cells to examine transport processess of molecules that involve specialized mechanisms. The results also demonstrate that the undifferentiated crypt cell lacks specific transport processes necessary for the active absorption of cobalamin and taurocholate.     

Duodenal mucosal reductase in wild-type and Hfe knockout mice on iron adequate,iron deficient,and iron rich feeding

BACKGROUND: Genetic haemochromatosis is a common hereditary iron loading disorder in humans. The disease is associated with loss of function mutations in the HFE gene. This is thought to change iron stores via increased iron absorption. AIMS: In this study we investigated how adaptation of mucosal reductase activity is engaged in this process and how the changes compare with adaptation seen when an iron deficient diet is fed. METHODS: Duodenal mucosal surface reductase was measured with nitroblue tetrazolium in age matched groups of male Hfe knockout mice (Hfe) and wild- type mice fed a purified diet containing normal (iron adequate), high (iron rich), or low (iron deficient) iron concentrations. RESULTS: Reductase activity increased when mice were fed an iron deficient diet and decreased when they were fed an iron rich diet. Total villus activity, as measured by the average area under the activity curve along the crypt-villus axis, was increased 2.8-2.9-fold by iron deficiency in both genotypes. Approximately half of this difference was attributable to the significantly increased length of the villi in mice on an iron deficient diet (p<0.05). Hfe knockout did not affect villus length but increased mucosal reductase activity near the villus tips. Similar increases (1.3-1.6-fold) were seen on all diets but the increase was significant for iron deficient and iron loaded diets only (p<0.05). CONCLUSION: Hfe gene product and dietary iron downregulate villus reductase activity in mice.     

Developmental characteristics of adapting mouse small intestine crypt cells

BACKGROUND & AIMS: Following massive small bowel resection (SBR), the remnant intestine undergoes an adaptive process characterized by increases in a number of physiologic and morphologic parameters. These changes are the result of a stimulus that increases crypt cell mitosis and augments cellular progression along the villus axis. To better define this process, we identified patterns of gene expression specifically within adapting intestinal crypt cells following SBR. METHODS: Laser capture microdissection was used to isolate mouse intestinal crypt cells following SBR or sham operation. Multiple biological and technical complementary DNA microarray replicates allowed rigorous statistical analyses for identification of important expression profiles. Major groups of genes were classified as to site of action, functional pathway, and possible regulatory groups. RESULTS: A total of 300 genes differentially expressed at significant levels within adapting crypt enterocytes were analyzed. Comparison of this list of differentially expressed adapting crypt cell genes with a generalized mouse gene expression database (from 82 developing and adult mouse tissues) showed the greatest overlap with developing and immature intestinal tissues. We identified prominent groups of genes involved with cell growth, signal transduction, and nucleic acid binding. Genes not previously shown to be involved with adaptation or development and maturation were identified. CONCLUSIONS: Identification of similar genes coordinately regulated during both adaptation and development, processes that share key morphologic features, provides a basis for new mechanistic insights into these shared characteristics.     

Delayed hepcidin response explains the lag period in iron absorption following a stimulus to increase erythropoiesis

INTRODUCTION: The delay of several days between an erythropoietic stimulus and the subsequent increase in intestinal iron absorption is commonly believed to represent the time required for body signals to programme the immature crypt enterocytes and for these cells to migrate to the villus. Recent data however suggest that signals from the body to alter absorption are mediated by circulating hepcidin and that this peptide exerts its effect on mature villus enterocytes. METHODS: We have examined the delay in the absorptive response following stimulated erythropoiesis using phenylhydrazine induced haemolysis and correlated this with expression of hepcidin in the liver and iron transporters in the duodenum. RESULTS: There was a delay of four days following haemolysis before a significant increase in iron absorption was observed. Hepatic hepcidin expression did not decrease until day 3, reaching almost undetectable levels by days 4 and 5. This coincided with the increase in duodenal expression of divalent metal transporter 1, duodenal cytochrome b, and Ireg1. CONCLUSION: These results suggest that the delayed increase in iron absorption following stimulated erythropoiesis is attributable to a lag in the hepcidin response rather than crypt programming, and are consistent with a direct effect of the hepcidin pathway on mature villus enterocytes.