Effects of TGF-alpha gene knockout on epithelial cell kinetics and repair of methotrexate-induced damage in mouse small intestine

While previous studies have indicated that exogenous TGF-alpha stimulates epithelial growth, maintenance, and repair of the gut, roles of endogenous TGF-alpha are less well-defined particularly in the small bowel. The current study examined effects of TGF-alpha knockout on adult small intestinal epithelial cell proliferation, migration, apoptosis, and damage/repair response after methotrexate treatment. Compared to normal mice, TGF-alpha gene knockout did not affect crypt cell production, mitosis position, migration, and apoptosis in non-injured intestine. RT-PCR gene expression analysis revealed presence of four out of six TGF-alpha related EGF family ligands in the normal intestine, suggesting a possible functional redundancy of the EGF family in maintenance of the intestine. Although TGF-alpha gene knockout did not significantly impair the overall mucosal repair in methotrexate-induced acute damage in the small intestine, it resulted in a higher apoptotic response in the early hours following methotrexate challenge, and a delayed and reduced crypt cell proliferation during repair. Consistently, after methotrexate challenge, intestinal TGF-alpha mRNA was found to be markedly upregulated in the early hours and during repair in the wild type, and there were similar profiles in the increased expression of all other ligands (except EGF) between the wild type and knockout intestines. Therefore, despite a possible functional redundancy among the EGF family ligands in the normal small intestine, TGF-alpha may play a role in modulating the early apoptotic events and in enhancing the subsequent reparative proliferative response in the methotrexate-damaged intestine.     

Distribution of i.v. administered epidermal growth factor in the rat

The distribution of i.v. injected ¹²⁵I-labeled epidermal growth factor (EGF) was examined in the rat. The uptake of radioactivity was examined for the following tissues: liver, kidney, skin, stomach, small intestine, colon, brain, submandibular gland, lung, spleen, and testis. ¹²⁵I-EGF was cleared from the circulation within minutes. At 2.5 min after the injection only 7% of the label was left in the blood. Most of the label was found in the liver (52%), the kidneys (14%), the small intestine (11%) and the skin (7%). The other organs examined contained 1% or less of the radioactivity. The uptake of ¹²⁵I-EGF per g tissue was markedly higher for the liver and kidneys than for the rest of the organs. By autoradiography ¹²⁵I-EGF was found in the peripheral parts of the classical liver lobule, in the proximal tubules of the kidneys, in the surface epithelium of the stomach, and in the surface epithelium of the villi in the small intestine. In conclusion the present study showed that small doses of homologous EGF was cleared from the circulation of rats within minutes, mainly by the liver, the kidneys, and the small intestine.     

Selective sparing of goblet cells and paneth cells in the intestine of methotrexate-treated rats

Proliferation, differentiation, and cell death were studied in small intestinal and colonic epithelia of rats after treatment with methotrexate. Days 1-2 after treatment were characterized by decreased proliferation, increased apoptosis, and decreased numbers and depths of small intestinal crypts in a proximal-to-distal decreasing gradient along the small intestine. The remaining crypt epithelium appeared flattened, except for Paneth cells, in which lysozyme protein and mRNA expression was increased. Regeneration through increased proliferation during days 3-4 coincided with villus atrophy, showing decreased numbers of villus enterocytes and decreased expression of the enterocyte-specific genes sucrase-isomaltase and carbamoyl phosphate synthase I. Remarkably, goblet cells were spared at villus tips and remained functional, displaying Muc2 and trefoil factor 3 expression. On days 8-10, all parameters had returned to normal in the whole small intestine. No methotrexate-induced changes were seen in epithelial morphology, proliferation, apoptosis, Muc2, and TFF3 immunostaining in the colon. The observed small intestinal sparing of Paneth cells and goblet cells following exposure to methotrexate is likely to contribute to epithelial defense during increased vulnerability of the intestinal epithelium.     

Role of epidermal growth factor and its receptor in chemotherapy-induced intestinal injury

Several growth factors are trophic for the gastrointestinal tract and able to reduce the degree of intestinal damage caused by cytotoxic agents. However, studies of epidermal growth factor (EGF) for chemotherapy-induced intestinal injury are conflicting. The development of a transgenic mouse that specifically overexpresses EGF in the small intestine provided a unique opportunity to assess the contribution of EGF in mucositis. After a course of fluorouracil, transgenic mice fared no better than control mice. Weight recovery was inferior, and mucosal architecture was not preserved. Apoptosis was not decreased and proliferation was not increased in the crypts. To corroborate the findings in transgenic mice, ICR mice were treated with exogenous EGF after receiving fluorouracil. Despite ileal upregulation of native and activated EGF receptor, the mice were not protected from intestinal damage. No benefits were observed with different EGF doses or schedules or routes of EGF administration. Finally, mucositis was induced in mutant mice with specific defects of the EGF signaling axis. Compared with control mice, clinical and histological parameters of intestinal injury after fluorouracil were no different in waved-2 mice, which have functionally diminished EGF receptors, or waved-1 mice, which lack transforming growth factor-alpha, another major ligand for the EGF receptor. These findings do not support a critical role for EGF or its receptor in chemotherapy-induced intestinal injury.     

Regulation of the biosynthesis of two distinct fatty acid-binding proteins in rat liver and intestine. Influences of sex difference and of clofibrate

Two distinct fatty acid-binding proteins (FABPs) have been identified in rat intestine, gFABP (15,063 Da) which is confined to intestinal epithelium and hFABP (14,184 Da) which is found in both liver and intestine. We have examined the influence of sex difference and the effect of clofibrate, both of which affect cellular fatty acid metabolism and hFABP levels, on the concentration, and mRNA levels of both hepatic and intestinal FABPs. In the liver, hFABP concentration was approximately 2-fold greater in females and in clofibrate-treated males than in untreated male rats. These differences were not accompanied by changes in the fractional turnover of the polypeptide but rather by parallel increases in hFABP mRNA. In the intestine, the two FABPs exhibited different regulatory responses. Intestinal hFABP turnover was 33% greater in females than in males, whereas mRNA concentration was 50% greater. Thus, unlike hFABP in liver, there was no sex-related difference in the steady-state level of hFABP in intestine. However, clofibrate treatment, similar to its effects in the liver, doubled intestinal hFABP protein and mRNA concentration. In contrast to hFABP, neither gFABP protein nor mRNA concentration were sex dependent, whereas clofibrate produced only a modest increase in gFABP concentration without significantly changing gFABP mRNA levels. The results indicate that the influence of sex difference and the effect of clofibrate on hepatic fatty acid metabolism are both associated with changes in hFABP synthesis mediated pretranslationally. The differential response of hFABP and gFABP in intestine suggests that these proteins play distinct roles in the cellular metabolism of fatty acids.     

黑麂肝、小肠和大肠的组织学结构及Ghrelin的分布

研究了成年雌性黑麂(Muntiacus crinifrons)的肝、小肠和大肠的组织学结构及Ghrelin的分布。采用H.E染色法观察组织学结构,免疫组化PV-9000两步法并结合DAB显色技术确定Ghrelin的分布。结果表明,黑麂的肝组织分为被膜、肝小叶、肝中央静脉、门管区等结构。被膜为浆膜结构,肝小叶不明显。肝细胞以中央静脉为中心,呈放射状排列。肝血窦的形状不规则。肠黏膜上皮为单层柱状上皮,小肠、盲肠和结肠的黏膜肌层很薄,管壁皱襞与肠绒毛等形态在消化道各部也存在差异。免疫组化结果显示肝细胞中有Ghrelin阳性细胞的表达;在肠道,免疫阳性细胞在十二指肠、空肠、回肠、盲肠和直肠的黏膜、黏膜下层和肌层均有分布,尤其在肠绒毛上皮和黏膜下层分布较多。黑麂肝、小肠和大肠结构与哺乳动物基本相似,但无十二指肠腺;Ghrelin阳性细胞在肝、小肠和大肠均有分布,这表明Ghrelin可能对消化有一定的调节作用。     

EphB receptors coordinate migration and proliferation in the intestinal stem cell niche

More than 10(10) cells are generated every day in the human intestine. Wnt proteins are key regulators of proliferation and are known endogenous mitogens for intestinal progenitor cells. The positioning of cells within the stem cell niche in the intestinal epithelium is controlled by B subclass ephrins through their interaction with EphB receptors. We report that EphB receptors, in addition to directing cell migration, regulate proliferation in the intestine. EphB signaling promotes cell-cycle reentry of progenitor cells and accounts for approximately 50% of the mitogenic activity in the adult mouse small intestine and colon. These data establish EphB receptors as key coordinators of migration and proliferation in the intestinal stem cell niche.     

Effect of anthracyclines on the intestine and its lymphoid organs (literature review)]

Anthracycline drugs after their administration first of all affect the cells with active proliferation and differentiation resulting not only in inhibition of the processes but also in induction of apoptosis, that precisely explains the pathological shifts in the intestinal epithelium, i.e. in the tissue with many dividing and maturating cells. The damaged epithelium after the use of anthracycline drugs becomes permeable for many substance of the antigenic nature from the intestine lumen that stimulate the response of the lymphoid organs.     

The Anatomy of the Oesophagus,Stomach and Intestine in Common Wolffish (Anarhichas lupus L.): A Basis for Diagnostic Work and Research

The alimentary canal of laboratory-reared common wolffish (Anarhichas lupus L.) was studied using light and electron microscopy. In the oesophagus, a simple columnar microvillous epithelium with transport characteristics was observed in addition to the main striated squamous epithelium. An osmoregulatory function is proposed for the simple columnar epithelium, which was supported by wide, thin-walled vessels. In the stomach, a separate type of neck cells was observed leading into the acinar gastric glands, which morphologically consist of one cell type: chief cells. The intestine was divided into a proximal and distal segment by an intestinal valve. Pyloric caeca were not present. We propose that shallow, crypt-like structures in the intestinal mucosa are the sites of epithelial-cell proliferation in juveniles and adults. The length of microvilli decreased from approximately 4 μm in the cranial part of the proximal intestine, to 1.5 μm in the distal intestine. In the distal intestine, rod-shaped bacteria were observed between microvilli. An extensive system of thin-walled vessels was observed in the submucosa of juvenile and adult wolffish stomach and intestine. Eosinophilic granular cells were numerous in the perivascular connective tissue in the gastric and intestinal submucosa of adults and juveniles, but were not observed in larvae.     

Dietary betaine accumulates in the liver and intestinal tissue and stabilizes the intestinal epithelial structure in healthy and coccidia-infected broiler chicks

The aim of this experiment was to study the patterns of betaine accumulation into intestinal tissue, liver and plasma of broiler chicks with or without coccidial infection. The chicks were raised on a corn-based, low-betaine diet with or without 1000 ppm betaine supplementation and with or without intestinal microparasite (Eimeria maxima) challenge to the age of 21 days. Plasma, liver, intestinal tissue and digesta of non-challenged (NC) birds and plasma and intestinal tissue of coccidiosis challenged (CC) birds were analysed for betaine content. NC birds were also analyzed for homocysteine in plasma and S-adenosylmethionine (S-AM) in liver. The jejunal epithelium was histologically examined for the presence of coccidia and the crypt-villus ratio was measured. Dietary betaine supplementation decreased the plasma homocysteine concentration but had no effect on liver S-AM of NC birds. The data suggest that chicks on a low-betaine diet accumulate betaine into the intestinal tissue. When the diet was supplemented with betaine, betaine accumulated heavily into liver and to a lesser degree into intestinal tissue. The concentration of betaine in jejunal and ileal digesta was low suggesting that dietary betaine was mainly absorbed from the proximal small intestine. The coccidial challenge decreased the concentration of betaine in the liver, but greatly increased that in the intestinal tissue. The crypt-villus ratio was decreased by the dietary betaine supplementation in healthy and challenged chicks, suggesting that dietary betaine both protects the jejunal villi against coccidial infection and also stabilizes the mucosal structure in healthy broiler chicks. These results support our earlier findings suggesting that betaine is likely to act as an important intestinal osmolyte in broiler chicks.     

Immunolocalization of EGF receptor (EGFr) in intestinal epithelium: recognition of apoptotic cells

The EGF-like family of growth factors are known to be involved in the control of the intestinal epithelium. The intracellular events are mediated by the EGF receptor (EGFr), a transmembrane glycoprotein which is overexpressed in many malignancies and also in many radiosensitive cell types. The precise mode of action of the receptor in controlling proliferation and whether the factor is also involved in controlling apoptosis in this tissue is not clear. Using polyclonal antibodies raised against a cytoplasmic region of the receptor distant to the phosphorylation site and one raised against the peptide sequence DVVDADEYLIPQ, which is present in the cytoplasmic tail phosphorylation site of the EGFr, we have examined the immunostaining in normal and irradiated murine intestine. The former antibody labelled the basolateral membranes of the epithelial cells in the proliferative zones of both the small intestine and colon, in both control and irradiated tissue. The latter antibody however, strongly labelled the Goblet cells and the microvilli of the enterocyte apical membrane in control tissue. Following irradiation\ the apical labelling redistributed and was localized in the apical cytoplasm and in a paranuclear region. Furthermore, strong labelling was now seen in many of the apoptotic cells of the small intestinal epithelium. The greatly differing results with the two antibodies indicates that interpretation of such immunostaining must be viewed with caution and may relate to the availability of each particular epitope. These results also suggest that antibodies to DVVDADEYLIPQ may be a useful marker of apoptotic calls and could imply a correlation between high levels of epitope availability, the radiosensitive (frequently p53 expressing) cells of the crypt epithelium and the induction of apoptosis.This work was supported by the Cancer Research Campaign.     

Comparison of the tissue-specific expression and developmental regulation of two closely linked rodent genes encoding cytosolic retinol-binding proteins

Cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBP II) are two highly homologous cytoplasmic proteins that bind all-trans-retinol. We have recently demonstrated that the mouse genes encoding CRBP and CRBP II are closely linked on chromosome 9 and that both human genes are located on chromosome 3 (Demmer, L.A., Birkenmeier, E.H., Sweetser, D.A., Levin, M.S., Zollman, S., Sparkes, R.S., Mohandas, T., Lusis, A.J., and Gordon, J.I. (1987) J. Biol. Chem. 262, 2458-2467). We have now used RNA blot hybridization analysis to assess the degree to which these genes are coordinately expressed in fetal, suckling, weaning, and adult rat tissues. Both genes exhibit different developmental patterns of expression in liver, intestine, lung, kidney, testes, and placenta. In the intestine, CRBP mRNA was detected during the 16th day of gestation--prior to the development of a well-differentiated absorptive epithelium--and remained essentially unchanged throughout the peri- and postpartum periods. By contrast, the pattern of intestinal CRBP II mRNA accumulation closely parallels the times of first appearance, and subsequent proliferation, of the intestinal absorptive columnar epithelium, supporting the hypothesis that CRBP II is involved in the intestinal uptake or intracellular trafficking of this hydrophobic vitamin. In the fetal liver, both genes were expressed by gestational day 16. Whereas the concentration of hepatic CRBP mRNA increased markedly during the suckling and early weaning periods, CRBP II mRNA levels fell abruptly immediately after birth. These peripartum changes were not paralleled by remarkable alterations in the steady state levels of hepatic retinol. Marked changes in the expression of CRBP in the liver and of CRBP II in the intestine were also documented in pregnant and lactating female rats. These differences in CRBP/CRBP II gene expression strongly suggest that their proteins serve different physiological functions. The peripartum liver may provide a useful model for dissecting the relative roles played by these homologous proteins in retinoid metabolism as well as the factors which modulate activation and repression their genes.     

Modulatory effect of rat small intestinal epithelial cell-conditioned medium on lymphocyte proliferation

Summary The small intestinal epithelium plays an important role in the mucosal host defense. Intestinal epithelial cells have been known to release substances that suppress lymphocyte proliferation, suggesting an immunoregulatory function. We investigated how intestinal epithelial cells affect lymphocyte proliferation. Serum-free medium that was conditioned by incubating epithelial cells, particularly crypt cells, of the rat small intestine affected proliferation of allogeneic spleen lymphocytes stimulated with concanavalin A, as assessed by measuring cellular [³H]thymidine incorporation. Less than 1% and greater than 2% of the conditioned medium enhanced and suppressed, respectively, lymphocyte proliferation. The causative substances found in the conditioned medium were dialyzable and heat-stable. Suppression was not due to toxicity to splenocytes. Exposure of splenocytes to a suppressive concentration of the conditioned medium beginning at 30 min before an onset of lectin stimulation decreased the suppression of lymphocyte proliferation. Splenocyte exposure to the suppressive concentration of the conditioned medium beginning at 30 min to 4 h after the onset of the stimulation inversely strengthened the suppression. A brief exposure of splenocytes to the conditioned medium for the last 4 h during a total 72-h culture period still suppressed lymphocyte proliferation. Thus, intestinal epithelial cells produce low-molecular-weight lymphocyte proliferation-modulating substances that suppress the proliferation of lectin-activated lymphocytes, but not resting ones, by affecting earlier intracellular events and the following DNA synthesis when incubated in culture medium.     

Effects of growth factors on an intestinal epithelial cell line: transforming growth factor beta inhibits proliferation and stimulates differentiation

The effects of epidermal growth factor transforming growth factor beta (TGF beta) and other growth factors on the proliferation and differentiation of a cell line derived from rat intestinal crypt epithelium (IEC-6) were defined. Incorporation of [3H]-thymidine was stimulated 1.4-2.4 fold by insulin, insulin like growth factor (IGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF) and 2% fetal calf serum (FCS) respectively. Additive stimulation was observed when FCS was supplemented by insulin,IGF-I or PDGF but not EGF. Incorporation of [3H]-thymidine by IEC-6 was strongly inhibited by TGF beta with greater than 80% inhibition of incorporation at concentration approximately equal to 2.0 pM. IEC-6 cells bound 4.1 +/- 0.15 X 10(4) molecules TGF beta/cell and appeared to have only a single class of high affinity receptors (Kd approximately equal to 0.5 pM). TGF beta inhibition was unaffected by the presence of insulin or IGF-I suggesting it inhibits proliferation at a step subsequent to that at which these growth factors stimulate [3H]-thymidine incorporation. TGF beta also reduced the stimulation induced by FCS by 65%. In contrast EGF reduced TGF beta inhibition by 60%. IEC-6 cells demonstrated the appearance of sucrase activity after greater than 18 hours treatment with TGF beta. These findings suggest that TGF beta may inhibit proliferative activity and promote the development of differentiated function in intestinal epithelial cells.     

Hyaluronic acid regulates normal intestinal and colonic growth in mice

Hyaluronic acid (HA), a component of the extracellular matrix, affects gastrointestinal epithelial proliferation in injury models, but its role in normal growth is unknown. We sought to determine the effects of exogenous HA on intestinal and colonic growth by intraperitoneal injection of HA twice a week into C57BL/6 mice from 3 to 8 wk of age. Similarly, to determine the effects of endogenous HA on intestinal and colonic growth, we administered PEP-1, a peptide that blocks the binding of HA to its receptors, on the same schedule. In mice treated with exogenous HA, villus height and crypt depth in the intestine, crypt depth in the colon, and epithelial proliferation in the intestine and colon were increased. In mice treated with PEP-1, intestinal and colonic length were markedly decreased and crypt depth and villus height in the intestine, crypt depth in the colon, and epithelial proliferation in the intestine and colon were decreased. Administration of HA was associated with increased levels of EGF (intestine) and IGF-I (colon), whereas administration of PEP-1 was associated with decreased levels of IGF-I (intestine) and epiregulin (colon). Exogenous HA increases intestinal and colonic epithelial proliferation, resulting in hyperplasia. Blocking the binding of endogenous HA to its receptors results in decreased intestinal and colonic length and a mucosal picture of hypoplasia, suggesting that endogenous HA contributes to the regulation of normal intestinal and colonic growth.     

Comitogenic effects of vasoactive intestinal polypeptide on rat hepatocytes

The primary mitogens such as epidermal growth factor and transforming growth factor-α are known to stimulate DNA synthesis in primary cultures of adult rat hepatocytes. Vasoactive intestinal polypeptide (VIP) was found to amplify DNA synthesis induced by the primary mitogens and thus acted as a comitogen. The comitogenic effect of VIP was specific for the culture medium, suggesting that minor components in the medium were required for hepatocytes to fully respond to VIP. Glutamic acid is probably one of these minor components, although other components present in the nutrient-rich medium were also necessary for the full comitogenic effect. Other comitogens such as insulin, vasopressin, and angiotensin II interacted additively with low concentrations of VIP. The comitogenic effect of VIP was also found in hepatocytes cultured from regenerating rat liver after a partial hepatectomy. In the regenerating hepatocyte cultures, VIP can act as a mitogen even in the absence of the primary mitogen EGF. VIP mRNA was found in several organs including brain, intestine, and liver, and its expression was slightly induced in liver 24 h after a partial hepatectomy. These results suggest that VIP can act as a hepatic comitogen and may play a role in liver cell proliferation. © 1996 Wiley-Liss, Inc.     

RNA-binding protein HuR promotes growth of small intestinal mucosa by activating the Wnt signaling pathway

Inhibition of growth of the intestinal epithelium, a rapidly self-renewing tissue, is commonly found in various critical disorders. The RNA-binding protein HuR is highly expressed in the gut mucosa and modulates the stability and translation of target mRNAs, but its exact biological function in the intestinal epithelium remains unclear. Here, we investigated the role of HuR in intestinal homeostasis using a genetic model and further defined its target mRNAs. Targeted deletion of HuR in intestinal epithelial cells caused significant mucosal atrophy in the small intestine, as indicated by decreased cell proliferation within the crypts and subsequent shrinkages of crypts and villi. In addition, the HuR-deficient intestinal epithelium also displayed decreased regenerative potential of crypt progenitors after exposure to irradiation. HuR deficiency decreased expression of the Wnt coreceptor LDL receptor–related protein 6 (LRP6) in the mucosal tissues. At the molecular level, HuR was found to bind the Lrp6 mRNA via its 3′-untranslated region and enhanced LRP6 expression by stabilizing Lrp6 mRNA and stimulating its translation. These results indicate that HuR is essential for normal mucosal growth in the small intestine by altering Wnt signals through up-regulation of LRP6 expression and highlight a novel role of HuR deficiency in the pathogenesis of intestinal mucosal atrophy under pathological conditions.     

Perchloric acid-soluble protein is expressed in enterocytes and goblet cells in the intestine and upregulated by dietary lipid

We previously identified perchloric acid-soluble protein (PSP) in the rat liver, kidney, brain and lung, and reported that it appeared to be related to repression of cell proliferation. In the present study, we clarified that PSP was expressed in the intestine, and found that the amino acid sequence of the intestinal PSP was consistent with those of other PSPs present in other tissues. An immunohistochemical study revealed that PSP was expressed in enterocytes and goblet cells, but not in other cell types among the lamina propria epithelial cells. A comparison of the expressions of PSP and proliferating cell nuclear antigen demonstrated that the proliferating cells did not express PSP. Intestinal PSP expression was induced by approximately 3-fold by oral administration of dietary fat. These findings indicate that the proliferation repression activity may be related to renewal of the intestinal epithelium, and that PSP is one of the fatty acid-inducible proteins.     

Epidermal growth factor promotes the chemotactic migration of cultured rat intestinal epithelial cells

The RIE-1 cell line is an untransformed, epithelial cell line derived from the rat small intestine. We report that epidermal growth factor (EGF), which regulates the proliferation of RIE-1 cells, also directs their movement. We measured cell migration through gelatin-coated filters in blind-well Boyden chambers. The migration of RIE-1 cells was stimulated up to approximately 100-fold by EGF, with a half-maximal response at 1-2 ng/ml and a maximal effect at 10 ng/ml. Further analysis showed that the RIE-1 cells responded directionally to a gradient of EGF in solution. Other growth factors tested did not stimulate RIE-1 cell migration, and EGF did not stimulate the migration of fibroblasts in this assay. We conclude that EGF is a potent and specific chemo-attractant for RIE-1 intestinal epithelial cells and suggest that EGF might influence epithelial cell migration in vivo.