Small Intestinal Crypt Cell Line Research Articles

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.

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.

In vitro and in vivo effects of exogenous nucleotides on the proliferation and maturation of intestinal epithelial cells.

To determine the nutritional role of nucleotides, the in vitro and in vivo effects of exogenous nucleotides on the development of intestine were investigated. First, the in vitro effects of nucleotides on the proliferation and maturation of enterocytes were studied by using a human colon tumor cell line (Caco-2) and a rat normal small intestinal crypt cell line (IEC-6). Second, the in vivo effects of nucleotides were also studied in early weaned rats fed nucleotide-unsupplemented or high-nucleotide-supplemented diet. Nucleotide composition resembled that of human milk (CMP:UMP:AMP:IMP:GMP = 10:1:1:1:1, in weight). Nucleotide supplement did not enhance Caco-2 cells proliferation; however, it significantly enhanced maltase and sucrase activities. In contrast, nucleotides supplement enhanced ICE-6 cells proliferation and maltase activity. CMP, predominantly contained in the mixture, enhanced most effectively the proliferation and maturation of cells. In the in vivo experiment, nucleotides significantly enhanced sucrase activity in the intestinal mucosa of early weaned rats. The results presented here suggest that a nucleotide supplement may enhance enterocyte proliferation and/or maturation in vivo and in vitro. Therefore exogenous nucleotides may play an important role in the development of the intestine.

Vitamin A Up-Regulates Expression of Bone-Type Alkaline Phosphatase in Rat Small Intestinal Crypt Cell Line and Fetal Rat Small Intestine

Vitamin A is a potent inducer for liver/bone/kidney alkaline phosphatase (L/B/K ALP) in a variety of tissues. However, the evidence for induction of L/B/K ALP by vitamin A in small intestine is limited. In this study, we investigated the influence of vitamin A on L/B/K ALP expression in rat small intestinal crypt IEC-6 cells and fetal rat small intestine. Treatment of IEC-6 cells with all-trans retinoic acid (RA) increased the levels of activity, protein and mRNA of L/B/K ALP, whereas enterocyte-specific proteins, including intestinal ALP, sucrase-isomaltase and glucose transporter-2, were not induced. The reverse transcription-polymerase chain reaction technique revealed that this L/B/K ALP transcript had the bone-type but not the liver-type leader exon. IEC-6 cells constitutively expressed mRNAs of all subtypes of retinoic acid receptor (RAR) and retinoid X receptor (RXR) at varied concentrations. Among these receptor mRNAs, RARβ mRNA quickly responded to RA treatment, and the level was doubled within 4 h. Gel mobility shift assay showed that RA induced an RXRE-binding activity in IEC-6 cells. The L/B/K ALP transcript, expressed in fetal rat small intestine, also contained the bone-type leader exon. Intragastric administration of 10 mg retinyl acetate to pregnant rats from gestational d 7 to 15 increased the levels of this transcript and enzyme in 15-d fetal rat small intestine. Our results suggest that vitamin A may be an important regulator for L/B/K ALP expression in fetal rat small intestine as well as in IEC-6 cells.

Polyamines are important for attachment of IEC-6 cells to extracellular matrix.

The inhibition of ornithine decarboxylase, a rate-limiting enzyme of polyamine biosynthesis, with alpha-difluoromethylornithine in IEC-6 cells (small intestinal crypt cell line) reduces cell migration by 70%, inhibits protein cross-linking, and affects the cytoskeletal assembly. The current study examines the effects of intracellular polyamine depletion on attachment of IEC-6 cells to different matrices. Polyamine deficiency inhibited cell attachment to plastic, laminin, fibronectin, collagen IV, and Matrigel by different extents. Intracellular putrescine restored attachment to all matrices. The presence of a specific inhibitor of protein cross-linking also inhibited attachment to laminin in a dose-dependent manner. The inhibition of cell attachment to plastic and Matrigel was correlated with the inhibition of cell migration. Immunofluorescence studies showed that polyamines are essential for the correct expression of the integrin subunit alpha 2 but not for the expression of the alpha 1-subunit. This study demonstrates that polyamines are important for cell attachment and expression of the integrin alpha 2 beta 1, a putative receptor for collagen and laminin. The impairment of protein cross-linking and the inhibition of the expression of cell surface receptors that bind extracellular matrix (ECM) proteins may be part of the mechanism by which polyamine deficiency retards cell migration in the small intestine.

Transforming growth factor-α increases tyrosine phosphorylation of microtubule-associated protein kinase in a small intestinal crypt cell line (IEC-6)

The small intestinal crypt cell line (IEC-6) is an undifferentiated, untransformed, mitotically active cell used in this study to determine the effect of transforming growth factor-alpha (TGF-alpha) on tyrosine phosphorylation levels of cellular proteins. Thymidine incorporation increased maximally after addition of 2 ng/ml TGF-alpha for 24 h. At the same dose, TGF-alpha induced the tyrosine phosphorylation of proteins with approximate molecular masses of 42, 44, 52, 80, 150 and 175 kDa as shown by Western blots treated with anti-phosphotyrosine antibody. The most intense phosphorylation was seen in the 42 kDa (p42) and 44 kDa (p44) proteins, which were identified as two isoforms of microtubule-associated protein kinase (MAPK). This phosphorylation was seen as early as 5 min post stimulation and was dose dependent. Both p42 and p44 were found in the nucleus after stimulation, although a basal level of unphosphorylated protein was present before stimulation. The observed tyrosine phosphorylation of p42 and p44 was inhibited by genistein, a tyrosine kinase inhibitor, and tyrphostin 23, an epidermal growth factor receptor tyrosine kinase inhibitor. We conclude that MAPK is tyrosine phosphorylated in response to TGF-alpha stimulation of IEC-6 cells.

Glutamine is essential for epidermal growth factor-stimulated intestinal cell proliferation.

In this study, Ko et al determined whether L‐glutamine (GLN) is essential for epidermal growth factor (EGF)‐stimulated enterocyte proliferation in an in vitro cell culture system by using the nontransformed rat small intestinal crypt cell line IEC‐6. In addition, specific mitogenic actions of EGF that require GLN were investigated.IEC‐6 cells were maintained in monolayer culture by incubation in standard culture medium (Dulbecco's minimum essential medium) containing 5% bovine serum, grown to quiescence, and then incubated in serum‐free Dulbecco's minimum essential medium containing 0.1 to 0.2 mmol/L GLN for 36 hours (as in almost all other cell culture systems, GLN is required to prevent IEC‐6 cell death in serum‐free medium). Cells were then stimulated with a previously determined maximal trophic dose of EGF (20 ng/mL) plus varying concentrations of L‐GLN (0 to 10 mmol/L) added to the culture medium. Under these conditions, DNA, RNA, and protein synthesis were quantitated over time (0 to 30 hours) by using tritiated thymidine, tritiated uridine, and 14C‐leucine, respectively. Cell number was determined after 72 hours of treatment with varying amounts of added GLN with or without EGF. Total RNA was isolated from the EGF/GLN‐treated cells in other experiments for determination of messenger RNA (mRNA) levels of the “early response” proto‐oncogene transcription factors zif268, jun‐B, and c‐myc by Northern blotting.GLN was required for EGF‐stimulated DNA synthesis in IEC‐6 cells. Thus, EGF (20 ng/mL) added to quiescent cells without additional GLN (ie, not more than the 0.1 mmol/L required to prevent cell death) did not stimulate DNA synthesis at any time from 0 to 30 hours of incubation. However, addition of a larger amount of GLN (1.0 mmol/L) to EGF significantly stimulated DNA synthesis beginning 9 hours after incubation, and this effect on DNA synthesis steadily increased over time. The increased DNA synthesis in cells treated with EGF was related to L‐GLN dose; stimulation occurred only after 0.3 mmol/L GLN. In contrast, DNA synthesis was not stimulated significantly by addition of increasing amounts of GLN alone to the medium and was maximal at 1 to 3 mmol/L GLN. The requirement for GLN under these conditions was amino acid specific and stereospecific, inasmuch as D‐GLN, L‐arginine, and L‐alanine were not effective in enhancing EGF‐stimulated DNA synthesis.The addition of 1 mmol/L GLN to EGF also significantly increased RNA and protein synthesis beginning 3 hours after incubation. Without the addition of L‐GLN to standard media, EGF‐induced protein synthesis was reduced 80% and RNA synthesis 70%. Cell growth studies revealed that GLN added at a dose of either 1.0 or 10 mmol/L significantly increased (~fourfold) cell number compared with control cells (without EGF in standard medium containing 0.1 mmol/L GLN) and cells treated with EGF alone in standard medium.Northern analysis for mRNA levels of the proto‐oncogene transcription factors zif268, jun‐B, and c‐myc showed significantly increased mRNA expression of all three genes after 30 minutes of exposure to EGF; however, these responses were similar with or without supplemental L‐GLN. Interestingly, mRNA levels for lf268, jun‐B, and c‐myc remained persistently elevated at 2 hours after treatment with EGF alone, but levels decreased to a variable degree with EGF plus GLN at this time point.

Polyamines are necessary for cell migration by a small intestinal crypt cell line.

Studies from our laboratory have shown that polyamines are essential for the normal repair of duodenal erosions induced in vivo in a rat stress-ulcer model. In that model, the inhibition of ornithine decarboxylase, a rate-limiting enzyme of polyamine biosynthesis, with alpha-difluoromethylornithine (DFMO) almost entirely prevented healing. Healing could be restored by oral polyamines. In this paper, we have investigated whether the polyamines are required for the early stages of epithelial restitution using an IEC-6 cell culture model of cell migration. Treatment of the cells with DFMO for 4 days reduced cell migration 80%. Migration could be restored to normal by concomitant treatment with putrescine (PUT), spermidine (SPD), or spermine (SPM), but not by their addition during the migration period (6 h) only. If DFMO treatment was not begun until the migration period, it still reduced cell migration 20%, and this deficit could not be restored by concomitant addition of the polyamines. Intracellular polyamine levels at these times, i.e., 6 h or 4 days, were an important factor in these results. Only PUT was undetectable after 6 h of DFMO. SPD and SPM were still at normal levels at 6 h. SPD was undetectable at 4 days, but SPM was still at 40% of normal. These data give added importance to PUT because its absence reduced cell migration after only 6 h, while SPD and SPM were still present in normal amounts. Perhaps exogenous SPD and SPM restored cell migration when present with DFMO for 4 days treatment primarily because they contributed to intracellular PUT through the acetyltransferases.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of putrescine on S-adenosylmethionine decarboxylase in a small intestinal crypt cell line.

Two key enzymes in polyamine biosynthesis are ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC). SAMDC decarboxylates S-adenosylmethionine, which then donates aminopropyl groups for spermidine and spermine synthesis. The purpose of our study was to determine whether putrescine, taken up from medium or synthesized endogenously by ODC, alters SAMDC activity. Studies were conducted in the IEC-6 cell line derived from rat small intestinal crypt cells. Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum (dFBS). They were deprived of serum for 24 h before experiments. Basal SAMDC activity was increased significantly by > or = 10(-4) M of putrescine. Lower doses had no significant effect. The same doses of putrescine decreased ODC activity to near zero. Asparagine at 10 mM or 5% dFBS not only stimulated ODC activity and the intracellular putrescine levels but also increased significantly SAMDC activity as well. ODC activity peaked at 3 h, and the maximum level of SAMDC occurred 3-4 h after exposure to asparagine or serum. Treatment with DL-alpha-difluoromethylornithine (DFMO), a specific ODC inhibitor, prevented the increases in both cellular putrescine levels and SAMDC activity in asparagine- and serum-treated cells. In the presence of DFMO, exogenous putrescine returned SAMDC activity toward control levels but had no effect on ODC. A very slight increase of SAMDC half-life in IEC-6 cells grown in the presence of putrescine was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+/calmodulin regulation of putrescine uptake in cultured gastrointestinal epithelial cells.

Regulation of putrescine uptake in a small intestinal crypt cell line, IEC-6 cells, was examined. Uptake of [14C]putrescine was measured throughout a normal growth curve and was found to be inversely related to growth. Kinetic analysis at low and high cell density revealed the inhibition of uptake in confluent cells was due to a five-fold reduction in Vmax of uptake, 199.5 vs. 43.1 pmol.10(5) cells-1.h-1, respectively. Three gastrointestinal hormones, gastrin, secretin, and cholecystokinin, produced partial inhibition of [14C]putrescine uptake. Conversely, treatment of quiescent cells with 5% fetal bovine serum to stimulate growth did not affect uptake. Influence of putrescine uptake on free ionized intracellular Ca2+ ([Ca2+]i) was measured by microspectrofluorometry using the Ca(2+)-sensitive fluoroprobe fura-2. Basal [Ca2+]i was calculated to be 112 nM and increased rapidly to 313 nM upon addition of 10 microM putrescine. Preventing the rise in [Ca2+]i using an intracellular Ca2+ buffer, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, decreased [14C]putrescine uptake to 29.5 +/- 5.3% of control values. 45Ca2+ flux experiments and measurement of transport in 0 Ca2+ and 0.5 mM EDTA suggested an intracellular source of calcium was mobilized during putrescine uptake. Finally, use of the putative calmodulin antagonist N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide caused a dose-dependent inhibition of [14C]putrescine uptake with 50% inhibitory concentration of approximately 7 microM. These data suggest that putrescine uptake in IEC-6 cells may be regulated by a Ca2+/calmodulin-dependent mechanism.

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.