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At least 19 recordsLinked to original sources
PubMed2000

Dietary nucleotides increase the proportion of a TCR gammadelta+ subset of intraepithelial lymphocytes (IEL) and IL-7 production by intestinal epithelial cells (IEC); implications for modification of cellular and molecular cross-talk between IEL and IEC by dietary nucleotides.

We have investigated the effects of dietary nucleotides on intraepithelial lymphocytes (IEL) and intestinal epithelial cells (IEC) in weanling mice. The proportion of T-cell receptor (TCR) gammadelta+ IEL in BALB/c mice fed a diet supplemented with nucleotides (NT(+) diet) was significantly higher than that in mice fed the nucleotide-free diet, while the proportion of TCR alphabeta+ IEL in NT(+) diet-fed mice was significantly decreased. The change of the TCR alphabeta+/TCR gammadelta+ ratio was mainly observed in a CD8 alphaalpha+ subset of IEL. IEC from NT(+) diet-fed mice produced a higher level of IL-7, which is important in the development of TCR gammadelta+ IEL, than those from control diet-fed mice. The expression levels of IL-7 and IL-2 receptors on IEL were not different between the two dietary groups. Our findings suggest that the increased population of a TCR gammadelta+ IEL subset by feeding nucleotides may be caused by the increased production of IL-7 by IEC.

Animals↗
PubMed1992

Secretion of insulin-like growth factor II (IGF-II) and IGF-binding protein-2 by intestinal epithelial (IEC-6) cells: implications for autocrine growth regulation.

To identify the factors regulating the proliferation of intestinal epithelium, we examined the effects of various growth factors on [3H] thymidine incorporation into the DNA of IEC-6 cells, an intestinal epithelial cell line derived from rat jejunal crypts. Insulin-like growth factor-I (IGF-I), IGF-II, and insulin stimulated the DNA and protein synthesis of IEC-6 cells in serum-free medium supplemented with transferrin, dexamethasone, and BSA (basal medium). Concentration-response experiments demonstrated that IGF-I is approximately 10 times more potent than IGF-II or insulin in producing 2- to 3-fold stimulations of DNA and protein synthesis by IEC-6 cells. In addition, IEC-6 cells proliferated slowly in the basal medium without any added growth factors. Analysis of medium conditioned by IEC-6 cells by gel filtration chromatography, RIA, HPLC, and N-terminal sequencing revealed that IEC-6 cells synthesize and secrete mature, 7,500 mo wt (M(r)) IGF-II as well as high M(r) forms of IGF-II. In addition, ligand blot, immunoblot, and N-terminal sequence analyses showed that IEC-6 cells produce the 34,000 M(r) IGF-binding protein-2 (IGFBP-2). To determine if IGFBP-2 modulates IGF responses in IEC-6 cells, the IGF-I analogs, Des-(1-3)-IGF-I and [Gln3,Ala4,Tyr15,Leu16]IGF-I, both of which have a reduced affinity for IGFBPs, were tested for their effects on IEC-6 cell proliferation. Both analogs exhibited 10-fold greater potency than IGF-I, presumably because endogenously secreted IGFBPs depress IGF-I binding to cell surface receptors. Finally, purified IGFBP-2 attenuated the DNA synthesis of IEC-6 cells in a dose-dependent manner. We conclude that IGFBP-2 secreted by intestinal epithelial cells is capable of limiting the mitogenic activity of both exogenous and endogenous IGFs by blocking the association of the growth factors with cell surface binding sites. These results further suggest that the growth of IEC-6 cells is modulated by autocrine mechanisms involving IGF-II and IGFBP-2.

Amino Acid Sequence↗
PubMed2004

Preservation of folate transport activity with a low-pH optimum in rat IEC-6 intestinal epithelial cell lines that lack reduced folate carrier function.

Intestinal folate transport has been well characterized, and rat small intestinal epithelial (IEC-6) cells have been used as a model system for the study of this process on the cellular level. The major intestinal folate transport activity has a low-pH optimum, and the current paradigm is that this process is mediated by the reduced folate carrier (RFC), despite the fact that this carrier has a neutral pH optimum in leukemia cells. The current study addressed the question of whether constitutive low-pH folate transport activity in IEC-6 cells is mediated by RFC. Two independent IEC-6 sublines, IEC-6/A4 and IEC-6/PT1, were generated by chemical mutagenesis followed by selective pressure with antifolates. In IEC-6/A4 cells, a premature stop resulted in truncation of RFC at Gln(420). A green fluorescent protein (GFP) fusion with the truncated protein was not stable. In IEC-6/PT1 cells, Ser(135) was deleted, and this alteration resulted in the failure of localization of the GFP fusion protein in the plasma membrane. In both cell lines, methotrexate (MTX) influx at neutral pH was markedly decreased compared with wild-type IEC-6 cells, but MTX influx at pH 5.5 was not depressed. Transient transfection of the GFP-mutated RFC constructs into RFC-null HeLa cells confirmed their lack of transport function. These results indicate that in IEC-6 cells, folate transport at neutral pH is mediated predominantly by RFC; however, the folate transport activity at pH 5.5 is RFC independent. Hence, constitutive folate transport activity with a low-pH optimum in this intestinal cell model is mediated by a process entirely distinct from that of RFC.

Animals↗
PubMed1993

Low-conductance chloride channels in IEC-6 and CF nasal cells expressing CFTR.

The properties of the cystic fibrosis gene product (CFTR) were studied by expression of cloned cDNA in different cell systems. Infection of both simian fibroblast (Vero) cells and immortalized CF nasal polyp cells (NCF3A) with a vaccinia virus encoding CFTR induced forskolin-induced Cl- permeability and low-conductance (8 pS) Cl- channels. By stable transfection of the rat intestinal crypt-derived cell line IEC-6 we have isolated a clone, IEC-CF7, which expresses CFTR mRNA and antigen. IEC-CF7 cells, but not IEC-6, display forskolin-induced Cl- permeability and multiple linear low-conductance (+/- 8 pS) Cl- channels in cell-attached membrane patches. In excised patches of IEC-CF7 cells, low-conductance Cl- channels could be activated by addition of the catalytic subunit of the adenosine 3',5'-cyclic monophosphate-dependent protein kinase A (PKA) plus ATP. During bath fluid replacement studies, the activated low-conductance channel remained active in the absence of ATP at room temperature and showed saturation kinetics. Rectifying (32 pS) Cl- channels were not observed in either IEC-6 cells or IEC-CF7 cells, indicating that there is no relation between CFTR expression and the incidence of this channel. Our data strongly support the conclusion that CFTR can act as a low-conductance Cl- channel, gated by PKA. The IEC-6-derived cell line IEC-CF7 may prove to be a useful model in the study of CFTR function because of the absence of 32-pS Cl- channel activity and its potential for differentiation.

Adenosine Triphosphate↗
PubMed2000

Functional characterization of cultured cells derived from an intraepidermal carcinoma of the skin (IEC-1).

We have successfully isolated a cell line (IEC-1) from an intraepidermal carcinoma of the skin of a patient and compared its behavior, in vitro, to normal human epidermal keratinocytes (HEK) and squamous cell carcinoma cell lines (SCCs). HEK differentiation comprises an initial growth arrest followed by an induction of squamous differentiation-specific genes such as transglutaminase type 1 (TG-1). Using thymidine uptake and TG-1 induction as markers of proliferation and differentiation, respectively, we were able to show that HEKs and the IEC-1 cells undergo growth arrest and induce TG-1 mRNA expression in response to various differentiation-inducing stimuli, while neoplastic SCC cell lines did not. However, differentiation in HEKs was an irreversible process whereas differentiation of the IEC-1 cells was reversible. Furthermore, growth of IEC-1 cells in organotypic raft cultures revealed differences in their ability to complete a squamous differentiation program compared with that of normal HEKs. The IEC-1 cells also exhibited a transitional phenotype with respect to replicative lifespan; HEKs had a lifespan of 4-6 passages, IEC-1 cells of 15-17 passages, and SCC cells were immortal. These alterations in IEC-1 cell behavior were not associated with functional inactivation or mutations of the p53 gene. These data indicate that the IEC-1 cells, derived from a preneoplastic skin tumor, exhibit differences in their ability to undergo terminal differentiation and have an extended replicative lifespan.

Aged↗
PubMedDate not supplied

Lead transport in IEC-6 intestinal epithelial cells.

This study evaluated the use of IEC-6 cells as a model for studying lead (Pb) transport by intestinal epithelial cells (IECs) and examined potential transport mechanisms for Pb uptake and extrusion. Pb accumulation in IEC-6 cells exposed to 5 and 10 microM Pb for up to 60 min was time- and dose-dependent. Reduction of incubation temperature significantly reduced the total cellular Pb content of IEC-6 cells. Simultaneous exposed of cells to zinc (Zn) and Pb resulted in decreased total cellular Pb contents compared to total cellular Pb contents of cells exposed to Pb only. IEC-6 cells treated with ouabain (1 mM) or sodium azide (1 mM) and 5 microM Pb accumulated more Pb than cells exposed to Pb only. Cells treated with p-chloromercuribenzensulfonic acid (50 microM), p-chloromercuribenzoic acid (50 microM), or iodoacetimide (50 microM) accumulated less Pb than cells treated with Pb only. We conclude that Pb uptake by IEC-6 cells depends on the extracellular Pb concentration. Our data suggest that the mechanism of Pb uptake by IECs is complex, and that Pb transport in IEC-6 cells is time- and temperature-dependent, involves sulfhydryl groups, and is decreased by the presence of Zn. Extrusion of Pb is at least partially dependent on metabolic energy.

Animals↗
PubMed2003

Non-steroidal anti-inflammatory drugs affect the methotrexate transport in IEC-6 cells.

Methotrexate (MTX) is used not only for the cancer chemotherapy but also for the treatment of rheumatic disease, often together with non-steroidal anti-inflammatory drugs (NSAIDs). MTX is actively cotransported with H(+) in the small intestine, mediated by a reduced folate carrier (RFC). The coadministration of some NSAIDs with MTX to rats caused a decrease of MTX absorption through the small intestine. This may be due to the uncoupling effect of oxidative phosphorylation of the NSAIDs. The present study investigated whether flufenamic acid, diclofenac and indomethacin, NSAIDs, decreased ATP content of rat-derived intestinal epithelial cell line IEC-6 cells and affected the MTX transport in IEC-6 cells. The MTX uptake in IEC-6 cells was dependent on medium pH and maximum around pH 4.5-5.5. The MTX uptake was composed of a transport inhibited by 4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS) and a non-saturable one. The DIDS-sensitive component in the MTX uptake showed a saturation kinetics (Michaelis-Menten constant (Km): 3.91 +/- 0.52 microM, Maximum velocity (Vmax): 94.66 +/- 6.56 pmol/mg protein/5 min). The cellular ATP content in IEC-6 cells decreased significantly at 30 min after the cells were started to incubate with the NSAIDs (250 microM flufenamic acid, 500 microM diclofenac and 500 microM indomethacin). The MTX uptake in IEC-6 cells in the presence of the NSAIDs decreased with the reduction of cellular ATP content and showed a good correlation with the ATP content (correlation coefficient: 0.982). Thus it seems likely that the ATP content in IEC-6 cells with the NSAIDs decreased due to the uncoupling effect of oxidative phosphorylation of the NSAIDs, resulting in the inhibition of the secondary active transport of MTX in IEC-6 cells. The present results also suggest that IEC-6 cells are useful to evaluate the drug interaction relating to this carrier system.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1996

IL-4 enhances IEC-6 intestinal epithelial cell proliferation yet has no effect on IL-6 secretion.

Intestinal epithelial cells (IEC) form an important line of defence at the intestinal mucosa by providing a barrier to lumenal contents and also by their ability to secrete various inflammatory cytokines. Recently, several T cell-derived cytokines have been shown to regulate specific IEC functions. In this study, the effect of IL-4 on IEC proliferation and secretion of the inflammatory cytokine IL-6 was investigated using the non-transformed rat IEC-6 intestinal epithelial cell line. Recombinant rat (rr)IL-4 was found to enhance IEC-6 cell proliferation over 4 days of culture, and this enhancement was dose-dependent. Further studies using specific antibodies confirmed that IL-4 induced the effect and that the effect was not mediated by autocrine-produced transforming growth factor-alpha. However, IL-4 did not induce IL-6 secretion by the IEC-6 cells, nor did it alter IL-1 beta-induced IL-6 secretion. These results indicate that T cells may be capable of regulating IEC proliferation via the secretion of IL-4 without altering the capacity of the IEC to function in the inflammatory response by secreting IL-6.

Animals↗
PubMedDate not supplied

Transforming growth factor-beta1 enhances the secretion of monocyte chemoattractant protein-1 by the IEC-18 intestinal epithelial cell line.

Intestinal epithelial cells (IEC) are known to produce monocyte chemoattractant protein-1 (MCP-1). However, MCP-1 production, as with many other cytokines, can be regulated by a network of cytokines present in the environment of the IEC. Both IEC and inflammatory cells have been shown to produce transforming growth factor-beta (TGF-beta), and the regulatory effect of this cytokine on MCP-1 secretion by IEC has not been determined. Using the IEC-18 cell line, we have found that TGF-beta1 alone induced the secretion of high levels of MCP-1. Treatment with TGF-beta1 also enhanced the levels of MCP-1 messenger ribonucleic acid. However, costimulation of the cells with TGF-beta1 and interleukin-1beta (IL-1beta) resulted in significant, but less than additive, increases in MCP-1 secretion. Finally, the enhancing effect of TGF-beta1 on MCP-1 secretion was not due to IL-6. These results suggest that TGF-beta1 from IEC or inflammatory cells may significantly enhance the secretion of MCP-1 by IEC and play an important role in inflamed mucosal tissues.

Animals↗
PubMed2001

Short-chain fatty acids induce intestinal epithelial heat shock protein 25 expression in rats and IEC 18 cells.

BACKGROUND & AIMS: Because short-chain fatty acids (SCFAs) and heat shock proteins (hsps) confer protection to intestinal epithelia cells (IECs), we studied whether SCFAs modulate IEC hsp expression. METHODS: Hsp 25, hsp72, and hsc73 protein expression in rat intestinal tissues and IEC-18 cells were determined by Western blot and immunohistochemistry. Cell survival under conditions of oxidant stress (monochloramine) was determined using (51)Cr release in hsp25 cDNA anti-sense and sense-transfected cells expressing minimal and increased hsp25, respectively. RESULTS: Butyrate induces a time- and concentration-dependent increase in hsp25, but not hsp72 or hsc73, protein expression in rat IEC-18 cells but not 3T3 fibroblasts. Other SCFAs, including the poorly metabolized isobutyate, also induced selective expression of hsp25. Butyrate treatment significantly improved the ability of IEC-18 cells to withstand oxidant (monochloramine) injury. This effect could be blocked in cells in which hsp25 induction by butyrate was blocked by stable hsp25 antisense transfection. Additionally, hsp25-transfected overexpressing IEC-18 cells showed increased resistance to monochloramine. In vivo, increasing dietary fiber increased colonic, but not proximal, ileal hsp25 while having no effect on hsp72 or hsc73 expression. CONCLUSIONS: SCFAs, the predominant anions of colonic fluid derived from bacterial flora metabolism of luminal carbohydrates, protect IECs against oxidant injury, an effect mediated in part by cell-specific hsp25 induction.

3T3 Cells↗
PubMed2006

Vascular endothelial growth factor (VEGF164) ameliorates intestinal epithelial injury in vitro in IEC-18 and Caco-2 monolayers via induction of TGF-beta release from epithelial cells.

OBJECTIVE: VEGF is a glycoprotein with various (e.g. angiogenic) activities. So far, research has focused on its angiogenic properties. VEGF receptors are localized on epithelial cells of patients with inflammatory bowel disease (IBD) and also on Caco-2 and IEC-18 cells. Our aim was to evaluate the role of VEGF on intestinal epithelial cell (IEC) migration and proliferation by utilizing an established in vitro model. METHODS: IEC-18 and Caco-2 monolayers were wounded with a razor blade as described previously. Cells were incubated in medium w/o rat VEGF(164). After 24 h, migration was assessed by counting cells across the wound edge. Migration was blocked with neutralizing TGF-beta(1) antibodies. IEC proliferation was assessed using the MTT (3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide) test. Semi-quantitative changes of the TGF-beta(1) mRNA expression were evaluated before and after stimulation of the cells with VEGF(164) by RT-PCR. Statistical analysis was performed with ANOVA and the Wilcoxon test. RESULTS: VEGF(164) significantly induced epithelial cell migration in Caco-2 and IEC-18 cells compared to control. TGF-beta(1) antibodies completely abolished this VEGF-induced cell migration. TGF-beta(1) mRNA significantly increased in IEC-18 and Caco-2 cells after stimulation with VEGF. VEGF significantly inhibited epithelial cell proliferation in IEC-18 and in Caco-2 cells, indicating that the observed effects on cell migration were not due to any proliferate effects. CONCLUSION: VEGF effects on epithelial cell migration play an important part in epithelial cell restitution by maintaining mucosal homeostasis after mucosal injury. This effect is mediated by TGF-beta(1). Our results obtain another possible role for increased VEGF levels in the intestinal mucosa of patients with IBD as reported recently by others.

Animals↗
PubMed1998

Comparison of intestinal folate carrier clone expressed in IEC-6 cells and in Xenopus oocytes.

We recently identified a cDNA clone from mouse small intestine, which appears to be involved in folate transport when expressed in Xenopus oocytes. The open reading frame of this clone is identical to that of the reduced folate carrier (RFC) (K. H. Dixon, B. C. Lanpher, J. Chiu, K. Kelley, and K. H. Cowan. J. Biol. Chem. 269: 17-20, 1994). The characteristics of this cDNA clone [previously referred to as intestinal folate carrier 1 (IFC-1)] expressed in Xenopus oocytes, however, were found to be different from the characteristics of folate transport in native small intestinal epithelial cells. To further study these differences, we determined the characteristics of RFC when expressed in an intestinal epithelial cell line, IEC-6, and compared the findings to its characteristics when expressed in Xenopus oocytes. RFC was stably transfected into IEC-6 cells by electroporation; its cRNA was microinjected into Xenopus oocytes. Northern blot analysis of poly(A)+ RNA from IEC-6 cells stably transfected with RFC cDNA (IEC-6/RFC) showed a twofold increase in RFC mRNA levels over controls. Similarly, uptake of folic acid and 5-methyltetrahydrofolate (5-MTHF) by IEC-6/RFC was found to be fourfold higher than uptake in control sublines. This increase in folic acid and 5-MTHF uptake was inhibited by treating IEC-6/RFC cells with cholesterol-modified antisense DNA oligonucleotides. The increase in uptake was found to be mainly mediated through an increase in the maximal velocity (Vmax) of the uptake process [the apparent Michaelis-Menten constant (K(m)) also changed (range was 0.31 to 1.56 microM), but no specific trend was seen]. In both IEC-6/RFC and control sublines, the uptake of both folic acid and 5-MTHF displayed 1) pH dependency, with a higher uptake at acidic pH 5.5 compared with pH 7.5, and 2) inhibition to the same extent by both reduced and oxidized folate derivatives. These characteristics are very similar to those seen in native intestinal epithelial cells. In contrast, RFC expressed in Xenopus oocytes showed 1) higher uptake at neutral and alkaline pH 7.5 compared with acidic pH 5.5 and 2) higher sensitivity to reduced compared with oxidized folate derivatives. Results of these studies demonstrate that the characteristics of RFC vary depending on the cell system in which it is expressed. Furthermore, the results may suggest the involvement of cell-or tissue-specific posttranslational modification(s) and/or the existence of an auxiliary protein that may account for the differences in the characteristics of the intestinal RFC when expressed in Xenopus oocytes compared with when expressed in intestinal epithelial cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Growth-regulatory effects of sensory neuropeptides, epidermal growth factor, insulin, and somatostatin on the non-transformed intestinal epithelial cell line IEC-6 and the colon cancer cell line HT 29.

A non-transformed small-intestinal cell line from the rat (IEC-6) and a human colon cancer cell line (HT 29) were examined for their trophic response to sensory neuropeptides. Substance P, neurokinin A (NKA), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), and peptide YY (PYY) were tested. Epidermal growth factor (EGF), insulin, and somatostatin-14 were also used. Interaction studies were performed on IEC-6 cells by combining EGF or insulin with somatostatin-14. The sensory neuropeptides had no effect either on IEC-6 cell growth and DNA synthesis or on HT29 cell growth. EGF and insulin stimulated cell growth and DNA synthesis in IEC-6 cells and cell growth in HT 29 cells in a dose-dependent fashion. Somatostatin-14 had no effect either alone or in combination with EGF or insulin on IEC-6 cell growth and DNA synthesis. HT 29 cell growth was inhibited by somatostatin-14 only in the presence of serum with a maximal and significant response at 10(-7) M. Our observations suggest that the sensory neuropeptides do not exert a direct growth-regulatory effect either on IEC-6 cells or on HT 29 cells. Somatostatin, however, inhibits serum-induced HT 29 cell growth but does not interfere directly with the proliferative effect of serum, EGF, or insulin on IEC-6 cells in this model.

Animals↗
PubMed1987

The effects of gastrin, epidermal growth factor, and somatostatin on DNA synthesis in a small intestinal crypt cell line (IEC-6).

Exposure of IEC-6 cells for 24 hr to either gastrin (50-500 ng/ml) or EGF (100-500 ng/ml) significantly stimulated (100-165%) the rate of [3H]thymidine incorporation into DNA (referred to as DNA synthesis) when compared with the corresponding basal levels. Somatostatin (10-500 ng/ml) produced no apparent change in DNA synthesis in IEC cells. On the other hand, somatostatin completely inhibited the EGF-induced rise in DNA synthesis. The gastrin-mediated stimulation in DNA synthesis was not affected by somatostatin. The rate of DNA synthesis in IEC cells in the presence of both gastrin and EGF was found to be greater (additive) than that caused by either of the peptides alone. A similar but less dramatic change in the actual number of cells (assessment of cell replication) was observed when the IEC cells were exposed for 24 hr to gastrin, EGF, and somatostatin, either alone or in combination. Whereas gastrin (250 ng/ml) and EGF (250 ng/ml) by themselves increased the number of cells significantly by 29 and 37%, respectively, together they caused a 72% stimulation, when compared with the basal levels. Somatostatin by itself caused no apparent change in IEC cell population, but it significantly inhibited the EGF- but not the gastrin-induced stimulation in IEC cell replication. It is concluded that both gastrin and EGF exert a direct proliferative effect on IEC cells, and the EGF action is regulated by somatostatin.

Animals↗