Rat Small Intestinal Cell Line Research Articles

Study on the involvement of Zinc in homeostasis under intestinal Inflammation

Background & Aim: Zinc is one of the essential trace elements, and adequate amounts of zinc are crucial for good health. It is thought that zinc is absorbed primarily in the small intestine and also plays an important role in maintaining intestinal homeostasis. Small intestine is a key organ involved in digestion/absorption and is also sensitive to intestinal bacteria and easily inflamed. Alkaline phosphatase (ALP), which has zinc as its active center, detoxifies lipopolysaccharide and is thought to be involved in the suppression of small intestinal inflammation. In this study, we investigated the role of zinc in small intestinal inflammation using a mouse model, as well as the transporters ZIP4 and ZIP14, which are involved in ALP and zinc absorption. Materials & Methods: Male ICR mice were used. Indomethacin (IM, 12 mg/kg s.c.) was administered to mice to induce small intestinal inflammation. Four days prior to IM administration, zinc sulfate heptahydrate (Zn) was administered twice daily for 6 days. Forty-eight hours after IM administration, the animals were sacrificed and the lesion area was measured. Hematoxylin and eosin staining was performed. Myeloperoxidase (MPO) activity in the ileum was measured as an indicator of the degree of inflammation. ZIP4 and ZIP14 mRNA expression levels were examined by RT-PCR. ALP activity of the ileal was examined. IEC-6 cells, a rat small intestinal epithelial cell line, were seeded in 96-well plates, and Zn was applied 24 hours after seeding, and IM was added 24 hours after seeding. Cell viability was measured 24 hours after the addition of IM. Results: After administration of IM, bloody stools were observed from 24 hours after administration. At 48 hours after IM administration, disruption of villi structure and shortening of small intestine length were observed in the jejunum and ileum, and MPO activity was significantly increased. ZIP4 and ZIP14 mRNA expression levels were decreased in the small intestine 48 hours after IM treatment, and ALP activity was increased compared to the Vehicle group. On the other hand, pretreatment with Zn suppressed the occurrence of small intestinal inflammation, and the disruption of small intestinal villi structure was weak. The increase in MPO activity induced by IM tended to be suppressed by Zn pre-administration. ZIP4 and ZIP14 mRNA expression in Zn pre-administration group was same as in the IM alone group, and ALP activity was higher than in the IM alone group. IM treatment of IEC-6 also decreased cell viability in a concentration-dependent manner, which was inhibited by pretreatment with Zn in a concentration-dependent manner. Conclusion: These results suggest that zinc is effective in preventing the occurrence of NSAID-induced small intestinal inflammation. ZIP4 and ZIP14 are normally highly expressed in the intestinal epithelium and contribute greatly to zinc absorption, but the amount of zinc absorption may be decreased during inflammation induc University This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Nucleotide Supplements Alter Proliferation and Differentiation of Cultured Human (Caco-2) and Rat (IEC-6) Intestinal Epithelial Cells

The effect of exogenous nucleotides on the proliferation and differentiation of enterocytes was comparatively studied using a human colon tumor cell line (Caco-2) and a normal rat small intestinal crypt cell line (IEC-6). Caco-2 cells exhibited more active endogenous nucleic acid metabolism than did IEC-6 cells, as evidenced by greater cellular pools of nucleotides and their metabolites. To determine the supplemental nutritional effect of nucleotides, a mixture containing equal amounts (10 mg/L) of AMP, CMP, IMP, GMP and UMP was added to the culture medium. The results showed that a nucleotide supplement under normal culture conditions did not affect proliferation and differentiation of Caco-2 cells. In contrast, nucleotide supplements under normal culture conditions promoted proliferation of IEC-6 cells. The addition of nucleotides to the culture medium also enhanced differentiation of IEC-6 cells when grown on an extracellular matrix (Matrigel). Furthermore, when glutamine levels were less than optimal (nutritional stress conditions), nucleotide supplements enhanced growth and maturation of both cell lines. We conclude that the de novo biosynthesis of nucleotides is sufficient to support proliferation of tumor Caco-2 cells but not of the normal crypt IEC-6 cells. Thus, nucleotide supplements may enhance normal enterocyte growth and maturation as well as spare the need for exogenous glutamine in cell maintenance and development.

Beta-carotene regulates the biological activity of EGF in IEC6 cells by alleviating the inflammatory process

ABSTRACT Epidermal growth factor (EGF) has many important biological functions. It plays an important role in regulating the growth, survival, migration, apoptosis, proliferation, and differentiation of intestinal tissues and cells. However, until now, the effect of inflammation on the biological activity of EGF in intestinal cells or tissues is still unclear. For this reason, in the current research, we have conducted a detailed study on this issue. Using the rat small intestinal crypt epithelial cell line (IEC6) was used as an in vitro model, and Confocal laser scanning microscope (CLSM), Flow cytometry (FCM), Indirect immunofluorescence assay (IFA), Western-blotting (WB), and Quantitative real-time RT-PCR (QRT-PCR) methods were used to explore the effects of inflammation on EGF/EGFR biological activity and signal transduction profiles. We found that the EGF/EGFR nuclear signal almost disappeared in the inflammatory state, and the phosphorylation levels of EGFR, AKT, and STAT3 were all significantly down-regulated. In addition, we also studied the effect of β-carotene on the biological activity of EGF, and found that when cells were pretreated with β-carotene, the cellular behavior, biological activity, and nuclear signal of EGF/EGFR under inflammation stimulation were partially restored. In summary, the current study shows that inflammation can disrupt EGF/EGFR-mediated signaling in IEC6 cells, suggesting that inflammation negatively regulates the biological activity of EGF/EGFR. Furthermore, we found that β-carotene not only attenuated lipopolysaccharide (LPS)-induced inflammation but also partially restored the biological activity of EGF in IEC6 cells, laying a solid foundation for studying the biological functions of EGF and β-carotene.

Sesamin Protects against and Ameliorates Rat Intestinal Ischemia/Reperfusion Injury with Involvement of Activating Nrf2/HO-1/NQO1 Signaling Pathway.

Intestinal ischemia-reperfusion (I/R) may induce cell/tissue injuries, leading to multiple organ failure. Based on our preexperiments, we proposed that sesamin could protect against and ameliorate intestinal I/R injuries and related disorders with involvement of activating Nrf2 signaling pathway. This proposal was evaluated using SD intestinal I/R injury rats in vivo and hypoxia/reoxygenation- (H/R-) injured rat small intestinal crypt epithelial cell line (IEC-6 cells) in vitro. Sesamin significantly alleviated I/R-induced intestinal histopathological injuries and significantly reduced serum biochemical indicators ALT and AST, alleviating I/R-induced intestinal injury in rats. Sesamin also significantly reversed I/R-increased TNF-α, IL-6, IL-1β, and MPO activity in serum and MDA in tissues and I/R-decreased GSH in tissues and SOD in both tissues and IEC-6 cells, indicating its anti-inflammatory and antioxidative stress effects. Further, sesamin significantly decreased TUNEL-positive cells, downregulated the increased Bax and caspase-3 protein expression, upregulated the decreased protein expression of Bcl-2 in I/R-injured intestinal tissues, and significantly reversed H/R-reduced IEC-6 cell viability as well as reduced the number of apoptotic cells among H/R-injured IEC-6 cell, showing antiapoptotic effects. Activation of Nrf2 is known to ameliorate tissue/cell injuries. Consistent with sesamin-induced ameliorations of both intestinal I/R injuries and H/R injuries, transfection of Nrf2 cDNA significantly upregulated the expression of Nrf2, HO-1, and NQO1, respectively. On the contrary, either Nrf2 inhibitor (ML385) or Nrf2 siRNA transfection significantly decreased the expression of these proteins. Our results suggest that activation of the Nrf2/HO-1/NQO1 signaling pathway is involved in sesamin-induced anti-inflammatory, antioxidative, and antiapoptotic effects in protection against and amelioration of intestinal I/R injuries.

Betaine inhibits Toll-like receptor 4 responses and restores intestinal microbiota in acute liver failure mice

Previous research has revealed that the gut microbiome has a marked impact on acute liver failure (ALF). Here, we evaluated the impact of betaine on the gut microbiota composition in an ALF animal model. The potential protective effect of betaine by regulating Toll-like receptor 4 (TLR4) responses was explored as well. Both mouse and cell experiments included normal, model, and betaine groups. The rat small intestinal cell line IEC-18 was used for in vitro experiments. Betaine ameliorated the small intestine tissue and IEC-18 cell damage in the model group by reducing the high expression of TLR4 and MyD88. Furthermore, the intestinal permeability in the model group was improved by enhancing the expression of the (ZO)-1 and occludin tight junction proteins. There were 509 operational taxonomic units (OTUs) that were identified in mouse fecal samples, including 156 core microbiome taxa. Betaine significantly improved the microbial communities, depleted the gut microbiota constituents Coriobacteriaceae, Lachnospiraceae, Enterorhabdus and Coriobacteriales and markedly enriched the taxa Bacteroidaceae, Bacteroides, Parabacteroides and Prevotella in the model group. Betaine effectively improved intestinal injury in ALF by inhibiting the TLR4/MyD88 signaling pathway, improving the intestinal mucosal barrier and maintaining the gut microbiota composition.

Abstract 99: Phenotypic characterization of knockdown of CDKs in the intestinal epithelial cells

Abstract Cyclin-dependent kinases (CDKs) are serine/threonine kinases that bind to cyclins and together act as regulators of cell cycle progression. CDK inhibitors have various therapeutic potentials including cancer (e.g. CDK4/6 inhibitors). Intestinal epithelial cells are highly proliferative; however, the impact of individual CDK inhibition on intestinal cell proliferation has not been well studied. The IEC6 cell line, a non-transformed rat small intestinal epithelial cell line with characteristics of crypt epithelial cells, was utilized in the current study to understand the role of CDKs in the proliferation and survival of intestinal cells. In the initial experiment CDK4/6 inhibitor palbociclib treatment demonstrated a lack of potent toxicity in the IEC6 in comparison with MCF7 (breast cancer cell line), indicating the absence of intestinal cell reliance on CDK4 or CDK6 for cell cycle progression. To further illustrate the role of CDKs in intestinal cells, targeted gene knockdown experiments using CDK1, 2, 4, and 6 siRNA and lipofectamine-mediated transfection were conducted. Surprisingly, only CDK1 knockdown causes profound cell death. CDK2, 4, or 6 knockdowns, whether single, double or triple combinations, did not have significant impact on IEC6 cells, as measured by multiple endpoints including impedance, ATP viability, and caspase activity. To further understand the role of CDK1 in cell cycle, various seeding densities of IEC6 cells were used with CDK1 siRNA. Although the impact of CDK1 knockdown on IEC6 cells decreased at the initial proliferation phase with the higher seeding density, there was still a decrease of viability observed at the later time point (when cells reached proliferation plateau), indicating a non-canonical role of CDK1 that is not related to cell cycle. This research supports the concept that CDK1, but not CDKs 2, 4, or 6, is essential for intestinal cell cycle progression and explains the lack of GI toxicity observed with palbociclib. Citation Format: Shuyan Lu, Wenyue Hu, Tae Sung, Brad Hirakawa, Marina Amaro, Bart Jessen. Phenotypic characterization of knockdown of CDKs in the intestinal epithelial cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 99.

Cellular differentiation of non-transformed intestinal epithelial cells is regulated by Lactobacillus rhamnosus and L. casei strains.

The aim of this study was to characterize an in vitro modulating effect of three commensal Lactobacillus strains on cellular differentiation of non-transformed crypt-like rat small intestinal cell line IEC-18. IEC-18 was grown on extracellular matrix, with or without presence of Lactobacillus strains. Gene expression of IEC-18 bacterial detection system - such as Toll-like receptors TLR-2, TLR-4, signal adapter MyD88, cytoplasmic NOD2 receptor, inflammatory cytokines IL-18, IL-1beta, chemokine IL-8 and enzyme caspase-1 - was evaluated using real-time PCR. Expression and localization of TLR-2, TLR-4, IL-18 and caspase-1 proteins was demonstrated by Western blotting and immunofluorescent staining. Secretion of IL-18 to apical and basolateral surfaces was assayed by ELISA. Our results suggested that L. casei LOCK0919 accelerated differentiation of IEC-18 by stimulating TLR-2, TLR-4, MyD88, IL-18, caspase-1 mRNAs and proteins. L. casei LOCK0919 increased expression and transfer of villin and beta-catenin from cytoplasm to cell membrane. Presence of L. rhamnosus LOCK0900 resulted in detachment of IEC-18 layer from extracellular matrix leading to induction of IL-1beta, of TLR-2 and IL-8 mRNAs and stimulation of MyD88, caspase-1 and cytosolic receptor NOD2 mRNAs. L. rhamnosus LOCK0908 was not recognized by TLR-2 or TLR-4 receptors. Lactobacilli-IEC-18 crosstalk enhanced immune and barrier mucosal functions.

Interleukin-23 Increases Intestinal Epithelial Cell Permeability In Vitro.

Background Breast milk has a heterogeneous composition that differs between mothers and changes throughout the first weeks after birth. The proinflammatory cytokine IL-23 has a highly variable expression in human breast milk. We hypothesize that IL-23 found in human breast milk is biologically active and promotes epithelial barrier dysfunction. Methods The immature rat small intestinal epithelial cell line, IEC-18, was grown on cell inserts or standard cell culture plates. Confluent cultures were exposed to human breast milk with high or low levels of IL-23 and barrier function was measured using a flux of fluorescein isothiocyanate-dextran (FD-70). In addition, protein and mRNA expression of occludin and ZO-1 were measured and immunofluorescence used to stain occludin and ZO-1. Results Exposure to breast milk with high levels of IL-23 caused an increase flux of FD-70 compared with both controls and breast milk with low levels of IL-23. The protein expression of ZO-1 but not occludin was decreased by exposure to high levels of IL-23. These results correlate with immunofluorescent staining of ZO-1 and occludin which show decreased staining of occludin in both the groups exposed to breast milk with high and low IL-23. Conversely, cells exposed to high IL-23 breast milk had little peripheral staining of ZO-1 compared with controls and low IL-23 breast milk. Conclusion IL-23 in human breast milk is biologically active and negatively affects the barrier function of intestinal epithelial cells through the degradation of tight junction proteins.

CCR6 Regulation of the Actin Cytoskeleton Orchestrates Human Beta Defensin-2- and CCL20-mediated Restitution of Colonic Epithelial Cells

Intestinal inflammation is exacerbated by defects in the epithelial barrier and subsequent infiltration of microbes and toxins into the underlying mucosa. Production of chemokines and antimicrobial peptides by an intact epithelium provide the first line of defense against invading organisms. In addition to its antimicrobial actions, human beta defensin-2 (HBD2) may also stimulate the migration of dendritic cells through binding the chemokine receptor CCR6. As human colonic epithelium expresses CCR6, we investigated the potential of HBD2 to stimulate intestinal epithelial migration. Using polarized human intestinal Caco2 and T84 cells and non-transformed IEC6 cells, HBD2 was equipotent to CCL20 in stimulating migration. Neutralizing antibodies confirmed HBD2 and CCL20 engagement to CCR6 were sufficient to induce epithelial cell migration. Consistent with restitution, motogenic concentrations of HBD2 and CCL20 did not induce proliferation. Stimulation with those CCR6 ligands leads to calcium mobilization and elevated active RhoA, phosphorylated myosin light chain, and F-actin accumulation. HBD2 and CCL20 were unable to stimulate migration in the presence of either Rho-kinase or phosphoinositide 3-kinase inhibitors or an intracellular calcium chelator. Together, these data indicate that the canonical wound healing regulatory pathway, along with calcium mobilization, regulates CCR6-directed epithelial cell migration. These findings expand the mechanistic role for chemokines and HBD2 in mucosal inflammation to include immunocyte trafficking and killing of microbes with the concomitant activation of restitutive migration and barrier repair.

Lipopolysaccharides stimulate adrenomedullin synthesis in intestinal epithelial cells: Release kinetics and secretion polarity

Adrenomedullin (AM), a potent vasodilator peptide initially isolated from a human pheochromocytoma, functions as an antimicrobial peptide in host defense. In this study, we investigated changes in AM levels in intestinal epithelial cells and the mechanism of its secretion and cellular polarity after exposure to lipopolysaccharides (LPS). When a rat small intestinal cell line (IEC-18 cells) was exposed to LPS, enzyme-linked immunosorbent assay revealed a dose-dependent increase in AM together with an increase in AM mRNA expression, as determined by real-time polymerase chain reaction. Up-regulation of AM by LPS was dose-dependently inhibited by LY294002, PD98059, SP600125 and calphostin-C, suggesting the involvement of the phosphatidylinositol 3 kinase, extracellular signal-regulated kinase, c-Jun NH2-terminal kinase and protein kinase C pathways, respectively, in this process. When polarized IEC-18 cells in a Transwell chamber were stimulated with LPS, AM secretion was directed primarily toward the side of LPS administration (either the apical or basolateral side). In situ hybridization revealed that AM mRNA was expressed in epithelial cells and in the connective tissue in the lamina propria of the jejunum after intraperitoneal or oral administration of LPS. Higher levels of AM mRNA expression were observed in rats treated with LPS via the intraperitoneal route, compared with those treated via the oral route. These findings suggest that intestinal AM plays an important role in mucosal defense in the case of intestinal luminal infection, as well as in the modulation of hemodynamics in endotoxemia.

M1721 Role of Organic Cation Transporters (Oct1, Oct2 and Oct3) in the Uptake of the Duodenal Ulcerogen Cysteamine in Rat Duodenal Mucosa and in Rat Small Intestinal Epithelial Cell Line IEC-6: Use of siRNA Technology

SGLT1 siRNA treated cells, n=3). Kinetic studies demonstrated that the mechanism of SGLT1 siRNA mediated stimulation of Na:H exchange in IEC-18 cells was secondary to a significant increase in the Vmax (10.0 ±1.1 nmol/mg protein/30 sec in control cells and 31.0 ± 2.5 in SGLT1 siRNA treated cells, n=3, p<0.05) without an alteration in the affinity of the exchanger for Na. RTQ-PCR demonstrated significant decrease in SGLT1 mRNA while NHE3 mRNA was significantly increased with SGLT1 siRNA treatment of IEC-18 cells. Western blot studies demonstrated that whereas SGLT1 protein was nearly absent NHE3 protein increased 2 fold SGLT1 siRNA treated IEC 18 cells. Conclusions: A reduction in BBM SGLT1 protein levels directly stimulates BBMNHE3 in IEC-18 cells. Themechanism of this stimulation is secondary to an increase in NHE3 transporter numbers. Thus, these data indicate that BBM SGLT compensatorily regulates BBM NHE3 in intestinal epithelial cells to maintain intracellular Na homeostasis.

Regulation of expression of 1,25D3-MARRS/ERp57/PDIA3 in rat IEC-6 cells by TGFβ and 1,25(OH)2D3

We examined the transcriptional regulation of expression of the redox-sensitive Membrane-Associated-Rapid Response, Steroid-binding (1,25D3-MARRS) protein specific for 1,25(OH)2D3 in a rat small intestinal cell line, IEC-6, that demonstrates rapid responses to 1,25(OH)2D3. 1,25D3-MARRS binds and is activated by 1,25(OH)2D3, but is not itself up-regulated by treatment with 1,25(OH)2D3, nor is there a Vitamin D response element (VDRE) in its proximal promoter. We previously reported that transforming growth factor β (TGFβ) increased steady state levels of 1,25D3-MARRS transcript and protein approximately two-fold [Rohe B, Safford SE, Nemere I, Farach-Carson, MC. Identification and characterization of 1,25D3-membrane-associated rapid response, steroid (1,25D3-MARRS)-binding protein in rat IEC-6 cells. Steroids 2005;70:458–63]. To determine if this up-regulation could be attributed to the function of a highly conserved consensus smad 3 binding element present in the proximal promoter of the 1,25D3-MARRS gene, we created a promoter-reporter [SEAP] construct that was responsive to TGFβ (200pM). Deletion or mutation of the smad 3 element greatly reduced the response of the 1,25D3-MARRS promoter to TGFβ. Subsequent studies found that the smad 3 response element is bound by a protein found in the IEC-6 nuclear extract, most likely smad 3. Interestingly, although 1,25(OH)2D3 alone did not increase expression of the 1,25D3-MARRS promoter-reporter, co-treatment of transfected IEC-6 cells with 1,25(OH)2D3 and TGFβ shifted the dose–response curve to a lower effective concentration (100pM peptide). We conclude that TGFβ is a transcriptional regulator of 1,25D3-MARRS expression via a functional smad 3 element and that cross-talk with non-classical 1,25(OH)2D3-stimulated pathways occurs. The findings have broad implications for redox-sensitive signaling phenomena including those that regulate phosphate transport in the intestine.

Correlation of in vitro cytotoxicity with paracellular permeability in mortal rat intestinal cells

Introduction: The rat small intestinal cell line, IEC-18, was used as an in vitro model to differentiate between acute cytotoxicity (AC) and paracellular permeability (PP) of selected chemicals. Methods: This study compares the low resistance rat intestinal mortal cell line, IEC-18 (transepithelial electrical resistance, TEER = 160 ± 10 Ω cm 2) with the high resistance human intestinal cell line, Caco-2 (TEER = 900 ± 100 Ω cm 2). The two cell lines differ in state of differentiation, TEER and paracellular permeability characteristics. The IEC-18 cell line is originated from the ileum and resembles more closely the small intestine than the Caco-2. Cytotoxicity was carried out using MTT cell viability assay in 96-well plates for 24-h exposure time. PP was measured using TEER (membrane integrity indicator) and PP markers such as [ 3H]- d-mannitol, lucifer yellow (LY) and FITC-dextran (fluorescein-dextran) on cells grown on inserts. Results: The data showed that there is a high correlation ( R 2 = 0.99) between MTT and TEER using IEC-18 cell for 24-h exposure time. IEC-18 is as sensitive as Caco-2 for both MTT and TEER measurements. Decrease in TEER is inversely proportional with increase in PP of tight junction indicators. There is a good correlation between IC 50's MTT, TEER and Registry of Cytotoxicity (RC) data. Discussion: Based on the results from the experiments, IEC-18 can be used as an in vitro model to differentiate between concentrations needed for AC and those required for PP.

Ethanol-Induced CXC-Chemokine Synthesis and Barrier Dysfunction in Intestinal Epithelial Cells

Ethanol exposure contributes to infectious complications in burn and trauma patients through a process known as "bacterial translocation." Two major factors, 1) physical disruption of the intestinal mucosal barrier and 2) suppression of immune defense, explain this phenomenon. However, little information is available concerning the immune mechanisms of ethanol-induced bacterial translocation. In this study we investigated the effect of physiological concentrations of ethanol on immune function, especially on CXC-chemokine secretion, neutrophil migration, and barrier function in the small intestine A rat small intestinal intestinal cell line (IEC-18 cells) was exposed to 50-500 mM ethanol for 24 hr with or without IL-1 beta. Secretion of CXC chemokines (GRO/CINC-1 and MIP-2) was measured by ELISA assay, and barrier dysfunction was assessed by the apical-to-basolateral flux of HRP-dextran. Neutrophil transmigration was assessed by enzyme histochemistry (AS-D chloroesterase staining) Exposure to ethanol concentrations of 200 mM and over increased GRO/CINC-1 secretion, and MIP-2 secretion increased at 500 mM. Administration of ethanol in combination with IL-1 beta had no additive effect on the release of GRO/CINC-1 and MIP-2. Exposure of IEC-18 monolayers to ethanol resulted in a dose-dependent increase in permeability but IL-1 beta had no effect on barrier function. Ethanol had no effect on neutrophil migration in enzyme histochemistry analysis The above observations suggest that ethanol induced physical disruption of the intestine but not neutrophil transmigration is the main cause of the bacterial translocation that leads to bacteremia and endotoxemia in alcoholics.

Identification and characterization of 1,25D 3-membrane-associated rapid response, steroid (1,25D 3-MARRS)-binding protein in rat IEC-6 cells

We report the presence of a mammalian equivalent of the avian Membrane- Associated Rapid Response, Steroid (1,25D 3-MARRS)-binding protein specific for 1,25(OH) 2D 3 in a rat small intestinal cell line, IEC-6, that demonstrates rapid responses to the steroid hormone. Identification of transcript and protein was achieved using RT-PCR with several specific primer sets, Western blot analysis with two separate antibodies recognizing distinct regions of the protein, ribozyme knockdown and immunohistochemistry. Promoter analysis of the 1000-bp upstream region of the 1,25D 3-MARRS gene in several species revealed the presence of a conserved smad-3 element in the 5′ proximal promoter region, but no classical vitamin D response element (VDRE). Treatment of IEC-6 cells with transforming growth factor β1 (TGFβ1) increased steady-state levels of 1,25D 3-MARRS (mRNA and protein) approximately two-fold over a 24-h period. In contrast, treatment with 1,25(OH) 2D 3 failed to significantly change 1,25D 3-MARRS protein or mRNA levels. Localization studies showed rapid nuclear translocation of a pool of 1,25D 3-MARRS protein after 1,25(OH) 2D 3 treatment, suggesting that the protein is subject to membrane-initiated signal pathway activation. Together these data point to complex interactions between the two important 1,25(OH) 2D 3 sensitive response systems in intestinal cells, 1,25D 3-MARRS protein and the well-studied nVDR, that together work to fine tune intestinal Ca 2+ absorption in a variety of avian and mammalian species.

Oral bioavailability of glyphosate: Studies using two intestinal cell lines

Glyphosate is a commonly used nonselective herbicide that inhibits plant growth through interference with the production of essential aromatic amino acids. In vivo studies in mammals with radiolabeled glyphosate have shown that 34% of radioactivity was associated with intestinal tissue 2 h after oral administration. The aim of our research was to investigate the transport, binding, and toxicity of glyphosate to the cultured human intestinal epithelial cell line, Caco-2, and the rat small intestinal crypt-derived cell line, ileum epithelial cells-18 (IEC-18). An in vitro analysis of the transport kinetics of [14C]-glyphosate showed that 4 h after exposure, approximately 8% of radiolabeled glyphosate moved through the Caco-2 monolayer in a dose-dependent manner. Binding of glyphosate to cells was saturable and approximately 4 x 10(11) binding sites/cell were estimated from bound [14C]. Exposure of Caco-2 cells to > or =10 mg/ml glyphosate reduced transmembrane electrical resistance (TEER) by 82 to 96% and increased permeability to [3H]-mannitol, indicating that paracellular permeability increased in glyphosate-treated cells. At 10-mg/ml glyphosate, both IEC-18 and Caco-2 cells showed disruption in the actin cytoskeleton. In Caco-2 cells, significant lactate dehydrogenase leakage was observed when cells were exposed to 15 mg/ml of glyphosate. These data indicate that at doses >10 mg/ml, glyphosate significantly disrupts the barrier properties of cultured intestinal cells.

Endotoxin, but not platelet-activating factor, activates nuclear factor-kappaB and increases IkappaBalpha and IkappaBbeta turnover in enterocytes.

Bacterial endotoxin (lipopolysaccharide; LPS) and platelet-activating factor (PAF) are important triggers of bowel inflammation and injury. We have previously shown that LPS activates the transcription factor nuclear factor (NF)-kappaB in the intestine, which up-regulates many pro-inflammatory genes. This effect partly depends on neutrophils and endogenous PAF. However, whether LPS and PAF directly activate NF-kappaB in enterocytes remains controversial. In this study, we first investigated the effect of LPS and PAF on NF-kappaB activation in IEC-6 (a non-transformed rat small intestinal crypt cell line) cells, by electrophoresis mobility shift assay and supershift, and found that LPS, but not PAF, activates NF-kappaB mostly as p50-p65 heterodimers. The effect was slower than tumour necrosis factor (TNF). Both LPS and TNF induce the expression of the NF-kappaB-dependent gene inducible nitric oxide synthase (iNOS), which occurs subsequent to NF-kappaB activation. We then examined the effect of LPS and TNF on the inhibitory molecules IkappaBalpha and IkappaBbeta. We found that TNF causes rapid degradation of IkappaBalpha and IkappaBbeta. In contrast, LPS did not change the levels of IkappaBalpha and IkappaBbeta up to 4 hr (by Western blot). However, in the presence of cycloheximide, there was a slow reduction of IkappaBalpha and IkappaBbeta, which disappeared almost completely at 4 hr. These observations suggest that LPS causes slow degradation and synthesis of IkappaBalpha and IkappaBbeta and therefore activates NF-kappaBeta via at least two mechanisms: initially, through an IkappaB-independent mechanism, and later, via an increased turnover of the inhibitor IkappaB. NF-kappaBeta activation precedes the gene expression of iNOS (assayed by reverse transcription-polymerase chain reaction), suggesting that LPS up-regulates iNOS via this transcription factor.

Induction of Cyclooxygenase-2 and Invasiveness by Transforming Growth Factor-β 1 in Immortalized Mouse Colonocytes Expressing Oncogenic Ras

Cyclooxygenase-2 (COX-2) expression appears to be important in colorectal carcinogenesis. Elevated COX-2 expression and activity have been observed in several different transformed cell types. Prior studies implicating involvement of the Ras oncogene and growth factors on COX-2 expression were largely derived from rat small intestinal cell lines. We have investigated whether mouse colonocyte COX-2 levels are regulated by oncogenic Ras or transforming growth factor-β 1 (TGF-β 1), and whether these factors also serve to regulate cellular invasiveness. Young adult mouse colonocyte cells are colonocytes derived from the “Immortomouse” and immortalized by the SV40 large T antigen. Young adult mouse colonocyte Ras cells were derived by transfection of young adult mouse colonocyte cells with oncogenic Ha-Ras and are known to be tumorigenic. We found that the induction of COX-2 and eicosanoid release were augmented in the presence of activated Ras and that TGF-β 1 caused a further increase in COX-2 in the Ras-transformed mouse colonocytes. Increased COX-2 expression was correlated with increased release of prostaglandins E 2 and I 2. Activated Ras and TGF-β increased the invasiveness of the young adult mouse colonocyte cells, but treatment with a COX-2 inhibitor did not inhibit invasiveness. Thus we found that transforming growth factor-β collaborates to increase COX-2 expression, protaglandin release, and invasiveness in mouse colonocytes, but the increased COX-2 activity does not appear to contribute to the invasive response. ( J Gastrointest Surg 2002;6:304–309.)

Monolayers of IEC-18 cells as an in vitro model for screening the passive transcellular and paracellular transport across the intestinal barrier: comparison of active and passive transport with the human colon carcinoma Caco-2 cell line

Purpose: previous studies have shown that the rat small intestinal cell line IEC-18 provides a size-selective barrier for paracellularly transported hydrophilic macromolecules. In order to determine the utility of IEC-18 cells as an in vitro model to screen the passive paracellular and transcellular components of the intestinal transport of nutrients and drugs, we have now examined the transport of GlySar (H +-coupled di/tripeptide carrier), O-methyl- d-glucose (glucose carrier), vincristine and rhodamine 123 (P-glycoprotein), and calcein and DNPSG (MRPs) and the bidirectional transport of paracellularly transported compounds. Transport of these compounds across the filter grown IEC-18 cells was compared with transport across the human colon carcinoma Caco-2 cells. Results: in IEC-18 cells, transepithelial transport of GlySar and methylglucose was as fast as the transport of mannitol, which is transported passively via the paracellular route. Whereas in Caco-2 cells, mannitol transport was much slower than the transport of GlySar and methylglucose. In contrast to Caco-2 cells, no H +-coupled transport of GlySar could be measured in IEC-18 cells. P-Glycoprotein-mediated transport was characterised in Caco-2 cells by an enhanced transport of vincristine and rhodamine 123 in the basolateral to apical direction and by the inhibition of this transport by verapamil. In IEC-18 cells, permeability of vincristine and rhodamine 123 was similar in both directions and verapamil had no effect on the transport of these compounds. Both IEC-18 and Caco-2 cells efflux the organic anions calcein and DNPSG to the apical and basolateral compartments, and this efflux could be inhibited by probenecid. Conclusions: in conclusion, no carrier-mediated transport of GlySar, methylglucose, vincristine and rhodamine 123 could be determined in IEC-18 cells in contrast to Caco-2 cells. However, both IEC-18 and Caco-2 cells showed MRP-mediated eflux system(s) in the apical and basolateral membrane. Monolayers of IEC-18 cells appear to be more suitable than monolayers of Caco-2 cells as an in vitro system to screen the passive component of the intestinal transport in a deconvoluted screening regimen, where passive transport is represented by the IEC-18 monolayer permeability and active transport is represented by monolayers of cells expressing the transport proteins heterologously.

Mesalamine induces manganese superoxide dismutase in rat intestinal epithelial cell lines and in vivo.

Mesalamine (5-ASA) is effective in the treatment of inflammatory bowel diseases. However, the mechanisms of action of 5-ASA remain unclear. IEC-6 and IRD-98, nontransformed rat small intestinal epithelial cell lines, were used to examine the effect of 5-ASA on the expression of manganese superoxide dismutase (MnSOD). Rats were given 5-ASA enemas to determine the effect on colonic MnSOD expression. Treatment with 5-ASA at 0.02 or 2 mg/ml induced MnSOD mRNA levels 2.67-fold or 5.66-fold, respectively. Inhibition of 5-lipoxygenase activating protein with MK-886 or cyclooxygenase with indomethacin did not influence the level of MnSOD mRNA. Nuclear run-on experiments demonstrated an increase in de novo transcription following treatment with 5-ASA. MnSOD protein levels were induced 2-fold at 24 h and 4.23-fold at 48 h following treatment with 1 mg/ml 5-ASA. 5-ASA increased MnSOD 1.7-fold in vivo. Pretreatment with 5-ASA significantly protected IRD-98 cells from tumor necrosis factor-alpha cytotoxicity. This is the first example of transcriptional gene regulation by 5-ASA. The induction of MnSOD by 5-ASA may contribute to the therapeutic mechanism of 5-ASA.