Departments of 1Surgery and 2Pathology, University of Maryland School of Medicine, and 3Baltimore Department of Veterans Affairs Medical Center, Baltimore, Maryland 21201
Submitted 13 January 2003 ; accepted in final form 7 July 2003
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ABSTRACT |
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ornithine decarboxylase; tight junction; catenins; intracellular calcium; normal intestinal epithelial cells
The cadherins are a group of Ca2+-dependent adhesion molecules essential for the induction and maintenance of intercellular contacts (4, 14, 50). E-cadherin is primarily found at the adherens junctions and is the major cadherin expressed in epithelial cells, including those of the gastrointestinal tract (14, 16, 46, 50). As a classic member of the adhesion molecules, E-cadherin is a single transmembrane protein containing an extracellular domain with Ca2+-binding sequence repeat motifs and a carboxy-terminal cytoplasmic domain (4, 50). In epithelial cells, E-cadherin dimers bind homo-typically to similar dimers on neighboring cells, and its conserved cytoplasmic domain interacts with the cytoskeleton through actin binding proteins, leading to strengthened cell-cell adhesion (45, 50, 51, 62). The strong E-cadherin-mediated cell-cell adhesions are thought to be necessary for epithelial barrier function, and signals that are transmitted through the adherens junctions may also regulate tight junctions (4, 29, 50, 53). It is likely that adherens junctions bring two plasma membranes together physically and permit or enhance tight junction assembly. In support of this possibility, anti-cadherin antibodies that block the formation of adherens junctions also inhibit the formation of tight junctions, thus resulting in an increase in epithelial paracellular permeability (4, 29, 50).
The natural polyamines, including spermidine, spermine, and their precursor putrescine, are organic cations found in all eukaryotic cells (49). Polyamines have been implicated in a wide variety of biological functions, and regulation of cellular polyamines has been recognized to be the central convergence point for the multiple signaling pathways driving different epithelial cell functions including cell motility (30, 56, 58), proliferation (24, 26, 35-37), and apoptosis (25, 44). We (41, 43, 59) recently found that polyamines regulate intracellular free Ca2+ concentration ([Ca2+]cyt) primarily by governing membrane potential (Em) through control of voltage-gated K+ (Kv) channel expression and that elevated [Ca2+]cyt is a major mediator for the stimulation of distinct intestinal epithelial functions after increase in polyamines. Because intestinal epithelial cells do not express L-type voltage-dependent Ca2+ channels (VDCC), polyamines stimulate Kv channel expression, resulting in membrane hyperpolarization, and increase [Ca2+]cyt by enhancing the driving force for Ca2+ influx. Using an in vitro cell migration model mimicking the early cell division-independent stage of mucosal restitution, we (15) further demonstrated that polyamines regulate -catenin tyrosine phosphorylation through [Ca2+]cyt, implicating these compounds in the modulation of cell-cell adhesions and barrier function in the intestinal epithelium.
The aim of current study was to test the hypothesis that polyamines are involved in regulation of intestinal epithelial barrier function by altering E-cadherin expression through Ca2+. First, we examined effects of polyamine depletion on expression and cellular distribution of the adherens junction proteins E-cadherin, -catenin, and
-catenin and the tight junction proteins zona occludens (ZO)-1, ZO-2, and junctional adhesion molecule (JAM)-1 in normal intestinal epithelial cells (IEC-6 line). Second, we determined whether manipulation of [Ca2+]cyt, either increase or decrease, alters E-cadherin expression in the presence or absence of polyamines. Third, we investigated whether the observed changes in E-cadherin expression affect intestinal epithelial paracellular permeability after polyamine depletion.
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MATERIALS AND METHODS |
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Cell cultures and general experimental protocols. The IEC-6 cell line was purchased from the American Type Culture Collection (ATCC) at passage 13. The cell line was derived from normal rat intestine and was developed and characterized by Quaroni et al. (39). IEC-6 cells originated from intestinal crypt cells, as judged by morphological and immunologic criteria. They are nontumorigenic and retain the undifferentiated character of epithelial stem cells. Stock cells were maintained in T-150 flasks in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% heat-inactivated FBS, 10 µg/ml insulin, and 50 µg/ml gentamicin sulfate. Flasks were incubated at 37°C in a humidified atmosphere of 90% air-10% CO2. Stock cells were subcultured once a week at 1:20; medium was changed three times weekly. The cells were restarted from original frozen stock every seven passages. Tests for mycoplasma were routinely negative, and passages 15-20 were used in the experiments.
The stable Cdx2-transfected-IEC-6 cell line was developed and characterized by Suh and Traber (48) and was a kind gift from Dr. Peter G. Traber (University of Pennsylvania, Philadelphia, PA). The expression vector, the LacSwitch System (Stratagene, La Jolla, CA), was used for directing the conditional expression of Cdx2, and isopropyl--D-thiogalactopyranoside (IPTG) served as the inducer for gene expression. IEC-6 cells were transfected with pOPRSVCdx2 by an electroporation technique and clones resistant to selection medium containing 0.6 mg/ml G418 and 0.3 mg/ml hygromycin B were isolated and screened for Cdx2 expression by Northern blot, RNase protection assays, and electrophoretic mobility shift assay. Stock stable Cdx2-transfected IEC-6 cells were grown in the DMEM used in parental nontransfected IEC-6 cells. Before experiments, cells were grown in DMEM containing 4 mM IPTG for 16 days to induce cell differentiation.
In the first series of studies, we examined the effects of polyamine depletion on expression and cellular distribution of E-cadherin, -catenin,
-catenin, ZO-1, ZO-2, and JAM-1 in IEC-6 cells. Cells were grown in control cultures or cultures containing 5 mM DFMO or DFMO plus 5 µM spermidine for 4, 6, or 8 days, and the monolayers were washed three times with ice-cold Dulbecco's PBS (DPBS). Different solutions were added according to the assays to be conducted.
In the second series of studies, we investigated whether manipulation of [Ca2+]cyt, either increase or decrease, alters E-cadherin expression in the presence or absence of polyamines. The Ca2+ ionophore ionomycin (1 µM) was used to increase [Ca2+]cyt, whereas Ca2+-free medium was used to decrease [Ca2+]cyt. In the Ca2+-free medium, 1.8 mM CaCl2 was replaced by 1.8 mM MgCl2 and an additional 0.1 mM EGTA was added to chelate the residual Ca2+. Free Ca2+ in the Ca2+-free medium was <0.002 mM. Levels of E-cadherin mRNA and protein were measured at various times after treatment with ionomycin or Ca2+-free medium in normal (without DFMO) and polyamine-deficient (with DFMO) IEC-6 cells.
In the third series of studies, we investigated whether observed changes in E-cadherin expression after polyamine depletion alter paracellular permeability in parental IEC-6 cells and differentiated IEC-Cdx2L1 cells. Permeability was measured by paracellular tracer flux assays, and membrane-impermeable molecules, [14C]mannitol and [3H]inulin, served as tracers.
Reverse transcription and PCR. Total RNA was isolated by using an RNeasy Mini Kit (Qiagen, Valencia, CA). Equal amounts of total RNA (2 µg) were transcribed to synthesize single-strand cDNA with a reverse transcription (RT)-PCR kit (Invitrogen Life Technologies, Carlsbad, CA). Specific primers for E-cadherin, -catenin,
-catenin, ZO-1, and ZO-2 were designed from the cDNA sequences of the coding regions corresponding to these genes (Table 1). These particular sequences were chosen on the basis of specificity established by previous publications from others (8, 21, 64). RTPCR was performed as described in our earlier publications (43, 60). To quantify the PCR products (the amounts of mRNA) of E-cadherin,
-catenin,
-catenin, ZO-1, and ZO-2, an invariant mRNA of
-actin was used as an internal control. The optical density (OD) values for each band on the gel were measured by a Gel Documentation System (UVP, Upland, CA), and their signals were normalized to the OD values in the
-actin signals.
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Western blot analysis. Cell samples dissolved in ice-cold RIPA buffer (50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1 mM DTT, 0.5 mM EDTA, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 2 µg/ml leupeptin, and 2 mM sodium orthovanadate) were sonicated and centrifuged at 14,000 rpm for 15 min at 4°C. The protein concentration of the supernatant was measured by the methods described by Bradford (5), and each lane was loaded with 20 µg of protein equivalent. The supernatant was boiled for 5 min and then subjected to electrophoresis on 7.5% acrylamide gels according to Laemmli (23). Briefly, after the transfer of protein onto nitrocellulose filters, the filters were incubated for 1 h in 5% nonfat dry milk in 1x TBS-T buffer (Tris-buffered saline, pH 7.4, with 0.1% Tween 20). Immunologic evaluation was then performed overnight at 4°C in 5% nonfat dry milk-TBS-T buffer containing specific antibodies against E-cadherin, -catenin,
-catenin, ZO-1, ZO-2, or JAM-1 proteins. The filters were subsequently washed with 1x TBS-T and incubated with the secondary antibodies conjugated with horseradish peroxidase (HRP) for 1 h at room temperature. The immunocomplexes on the filters were reacted for 1 min with chemiluminescence reagent (NEL-100, DuPont NEN).
Pulse-chase analysis and immunoprecipitation of E-cadherin. Pulse-chase experiments were performed as described by Varlamov et al. (53) with a minor change. Briefly, cells were grown in the presence or absence of 5 mM DFMO for 6 days and then incubated with the pulse medium containing 200 µCi/ml L-[35S]methionine-cysteine (Perkin-Elmer Life Sciences, Boston, MA) at 37°C for 60 min. Cells were harvested in radioimmune precipitation assay buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 20 mM pyrophosphate, 1% Triton X-100, 100 mM NaF, 2 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 20 µg/ml leupeptin, and 2 mM sodium orthovanadate) at different time points after incubation with the methionine- and cysteine-supplemented medium. Cell samples were sonicated and centrifuged at 4°C, and the supernatants were collected for immunoprecipitation. Equal amounts of proteins (300 µg) for each sample were incubated with the specific antibody against E-cadherin (2 µg) at 4°C for 3 h, and protein G-PLUS-Agarose was added and incubated overnight at 4°C. The precipitates were washed with ice-cold D-PBS, and the beads were resuspended in SDS sample buffer for subsequent gel electrophoresis (SDS-PAGE, 7.5% ready gel). The gels were dried and exposed to X-ray film with an intensifying screen.
Immunofluorescence staining. The immunofluorescence procedure was carried out according to the method of Vielkind and Swierenga (55) with minor changes (15, 58). Cells were incubated with the primary antibody against E-cadherin, -catenin,
-catenin, ZO-1, or ZO-2 at 4°C overnight and then incubated with secondary antibody conjugated with FITC for 2 h at room temperature. After the slides were rinsed three times, they were mounted and viewed through a Zeiss confocal microscope (model LSM410). Images were processed with Photoshop software (Adobe, San Jose, CA).
Measurement of [Ca2+]cyt. Details of the digital imaging methods used for measuring [Ca2+]cyt are described in our previous publications (41, 60). Briefly, cells were plated on 25-mm coverslips and were incubated in culture medium containing 3.3 µM fura 2-AM for 30-40 min at room temperature (22-24°C) under an atmosphere of 10% CO2 in air. Fluorescent images were obtained with a microchannel plate image intensifier (Amperex XX1381; Opelco, Washington, DC) coupled by fiber optics to a Pulnix charge-coupled device video camera (Stanford Photonics, Stanford, CA). Image acquisition and analysis were performed with a Metamorph Imaging System (Universal Imaging). [Ca2+]cyt was calculated from fura 2 fluorescence emission excited at 380 and 360 nm by the ratio method (34).
Paracellular tracer flux assay. Flux assays were performed with the 12-mm Transwell as described by Wong and Gumbiner (63) with minor modification. Briefly, IEC-6 or differentiated IEC-Cdx2L1 cells were grown in control cultures or cultures containing DFMO or DFMO plus spermidine for 4 days and then trypsinized, plated at a confluent density of 4 x 105 cells/cm2 on the insert, and maintained under the same culture conditions for an additional 48 h to establish tight monolayers. Two different membrane-impermeable molecules, [14C]mannitol (mol wet 184) and [3H]inulin (mol wt 5,200), served as paracellular tracers in this experiment. At the beginning of the flux assay, both sides of the bathing wells of Transwell filters were replaced with fresh medium containing either 5 mM unlabeled mannitol or 0.5 mM unlabeled inulin. Each of the tracers was added to a final concentration of 3.6 nM for [14C]mannitol and 0.36 nM for [3H]inulin to the apical bathing wells that contained 0.5 ml of medium. The basal bathing well had no added tracers and contained 1.5 ml of the same flux assay medium as in the apical compartment. All flux assays were performed at 37°C, and the basal medium was collected 2 h after addition of [14C]mannitol or [3H]inulin for counting in a Beckman liquid scintillation counter. The results were expressed as percentage of total count values of each tracer.
Statistics. All data are expressed as means ± SE from six or eight samples. Autoradiographic and immunofluorescence labeling experiments were repeated three times. The significance of the difference between means was determined by analysis of variance. The level of significance was determined with the Duncan's multiple-range test (17).
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RESULTS |
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As shown in Fig. 1, depletion of cellular polyamines by DFMO selectively and significantly inhibited mRNA expression of E-cadherin but negligibly affected levels of -catenin,
-catenin, ZO-1, and ZO-2 mRNAs. The levels of E-cadherin mRNA in the cells treated with DFMO for 4, 6, and 8 days were decreased by
40% (Fig. 1B). The changes in E-cadherin mRNA were paralleled by those of E-cadherin proteins (Fig. 2). The levels of E-cadherin protein in the cells exposed to DFMO for 4, 6, and 8 days were decreased by
70% (Fig. 2B, a). Although there was no significant inhibition of
-catenin and
-catenin mRNA expression in DFMO-treated cells, protein levels of these two adherens junction proteins decreased significantly at 6 and 8 days after polyamine depletion (Fig. 2, A and B, b and c). The levels of
-catenin and
-catenin proteins were decreased by
30% and
20%, respectively. On the other hand, polyamine depletion had no significant effects on expression of ZO-1, ZO-2, and JAM-1 proteins (Fig. 2A). In the presence of DFMO, decreased levels of E-cadherin mRNA and protein and
-catenin and
-catenin proteins were completely prevented by addition of exogenous spermidine (5 µM). Levels of E-cadherin,
-catenin, and
-catenin expression in cells grown in the presence of DFMO plus spermidine were indistinguishable from those of control cells. We also examined changes in claudin-1, claudin-4, and occludin proteins in the presence or absence of DFMO for 4, 6, and 8 days and demonstrated that basal levels of these proteins were very low and undetectable by Western blot analysis in IEC-6 cells (data not shown). To verify the sensitivity of the assay system we used in these studies, our methods clearly detected claudin-1, claudin-4, and occludin proteins in positive control samples (data not shown).
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Effects of polyamine deficiency on cellular distribution of E-cadherin, -catenin,
-catenin, ZO-1, and ZO-2 proteins. To extend these positive findings of decreased adherens junction proteins after polyamine depletion, immunofluorescence staining was performed to determine cellular distribution of E-cadherin and
-catenin in IEC-6 cells. In control cells (Fig. 3A), immunoreactivities for E-cadherin and
-catenin were mainly located along the entire cell-cell contact region of adjacent cells. These results were consistent with our data from Western blot analysis that revealed high basal levels of E-cadherin and
-catenin proteins in normal IEC-6 cells. In DFMO-treated cells (Fig. 3B), these membrane immunoreactivities for E-cadherin and
-catenin markedly decreased and were hardly detected, as expected. Exogenous spermidine given together with DFMO reversed the inhibitory effect of polyamine depletion on E-cadherin and
-catenin formation. The appearances of E-cadherin and
-catenin in cells treated with DFMO plus spermidine were the same as in control cells (Fig. 3, A vs. C). In contrast, immunostaining levels for
-catenin were not affected by polyamine depletion (data not shown).
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We also examined the effect of polyamine depletion on cellular distribution of tight junction proteins in IEC-6 cells. The immunoreactivities for ZO-1 protein were primarily observed along the entire cell-cell contact region of adjacent cells in IEC-6 cells (Fig. 3, bottom). Polyamine depletion did not alter cellular organization of ZO-1 protein. There were no significant differences in cellular distribution and immunostaining levels of ZO-1 protein between control cells and cells treated with DFMO in the presence or absence of spermidine. Similarly, polyamine depletion also did not affect cellular distribution of ZO-2 protein in IEC-6 cells (data not shown).
Effect of [Ca2+]cyt on E-cadherin expression. We recently demonstrated that polyamines modulate [Ca2+]cyt by governing the driving force for Ca2+ influx via controlling activity of Kv channels (41, 43, 60) and that Ca2+ mediates distinct actions of intestinal epithelial cells after polyamine increase (15, 40). In this study, we tested the possibility that polyamines regulate E-cadherin expression by altering [Ca2+]cyt. Because IEC-6 cells do not express L-type VDCC (43, 60), the inhibition of Kv channel expression and resultant membrane depolarization through polyamine depletion decrease the driving force for Ca2+ influx and reduce [Ca2+]cyt. In fact, [Ca2+]cyt in cells exposed to DFMO for 6 days decreased significantly (from 115 ± 6.4 to 60.3 ± 3.2 nM; n = 12, P < 0.05). Spermidine given together with DFMO restored [Ca2+]cyt to normal levels.
In the first study, we examined whether the decreased [Ca2+]cyt caused by removal of extracellular Ca2+ alters expression of E-cadherin mRNA and protein in control cells (without DFMO). As shown in Figs. 4A and 5A, removal of extracellular Ca2+ not only decreased E-cadherin mRNA but also inhibited E-cadherin protein expression. This inhibitory effect on E-cadherin expression occurred at 4 h and peaked at 6 h after exposure to the Ca2+-free medium. Levels of E-cadherin mRNA and protein were decreased by 60% at 4 h and by
90% at 6 h, respectively, after incubation with the Ca2+-free medium. Some small protein fragmentations were regularly detected in cells exposed to the Ca2+-free medium for 4 and 6 h, probably resulting from the stimulation of E-cadherin degradation. The regulatory effect of Ca2+ on E-cadherin is specific because expression of
-catenin protein was not affected after removal of extracellular Ca2+ (Fig. 5A, a). There were no significant differences in levels of
-catenin protein between control cells and cells exposed to Ca2+-free medium for 2, 4, and 6 h.
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In the second study, we determined whether increased [Ca2+]cyt caused by the Ca2+ ionophore ionomycin prevents the inhibition of E-cadherin expression in polyamine-deficient cells. Consistent with our previous findings (15, 41, 43), exposure to 1 µM ionomycin dramatically increased [Ca2+]cyt in DFMO-treated cells (data not shown). Increased [Ca2+]cyt caused by ionomycin also increased levels of E-cadherin mRNA (Fig. 4B) and protein (Fig. 5B) in the absence of polyamines. Treatment of polyamine-deficient cells with ionomycin for 6 h restored E-cadherin mRNA to 85% of control value and its protein to
70%. Neither the Ca2+-free medium nor ionomycin affected cell attachment and cell viability in control and DFMO-treated cells as measured by Trypan blue staining (data not shown). These results indicate that polyamines regulate E-cadherin expression, at least partially, through [Ca2+]cyt in intestinal epithelial cells.
Effects of polyamine depletion and [Ca2+]cyt on E-cadherin protein stability. To keep this study focused, we specifically examined changes in E-cadherin protein stability in the presence or absence of polyamines. Although E-cadherin is relatively stable, the levels of E-cadherin protein in control cells declined gradually after protein synthesis was inhibited by administration of cycloheximide, with a half-life of 900 min as calculated by linear regression analysis (Fig. 6, A, a, and B). In DFMO-treated cells, the stability of E-cadherin protein decreased significantly compared with that observed in control cells (Fig. 6A, a vs. b). The half-life of E-cadherin protein in polyamine-deficient cells was
330 min (Fig. 6B). Spermidine given together with DFMO completely prevented the decreased stability of E-cadherin protein, and levels of E-cadherin in cells exposed to DFMO plus spermidine decreased at a rate similar to that observed in controls.
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To validate the methods used in this study and confirm these interesting findings, we also examined the time course for the degradation of E-cadherin in control and polyamine-deficient cells by pulse-chase analysis. As shown in Fig. 7, after the 60-min pulse, E-cadherin protein decreased at a relatively slow rate, with 65% still remaining after 6 h. This result is similar to that obtained from the cycloheximide method described in Fig. 6. In contrast to that observed in control cells, the level of E-cadherin protein in polyamine-deficient cells decreased quickly, with a half-life of
200 min. These results clearly indicate that polyamine depletion destabilizes E-cadherin protein in intestinal epithelial cells.
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Because polyamine depletion decreased [Ca2+]cyt, we further determined the involvement of [Ca2+]cyt in E-cadherin protein stability in IEC-6 cells. As shown in Fig. 8, removal of extracellular Ca2+ from the culture medium dramatically decreased E-cadherin stability when protein synthesis was inhibited by cycloheximide, with a half-life of 80 min. Some small fragmentations of E-cadherin protein were regularly detected in cells exposed to Ca2+-free medium for 2, 3, 4, and 6 h (Fig. 8A, b). Together, these results indicate that polyamines and [Ca2+]cyt are necessary for maintenance of E-cadherin protein stability in intestinal epithelial cells, suggesting that the reduction of E-cadherin by polyamine depletion results, at least partially, from instability of E-cadherin protein after decrease in [Ca2+]cyt.
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Effects of decrease in E-cadherin expression by polyamine depletion on paracellular permeability in IEC-6 cells. To study the possible role of decreased E-cadherin in intestinal epithelial paracellular barrier function, we assessed the paracellular flux of membrane-impermeable tracers across the confluent monolayer in the presence or absence of polyamines. Cells were grown in control cultures or cultures containing DFMO or DFMO plus spermidine for 4 days and then plated at confluent density on the insert and maintained for an additional 48 h to establish a tight monolayer. Two widely accepted hydrophilic paracellular tracer molecules, [14C]mannitol and [3H]inulin, were used in this study. To verify the system used for paracellular permeability assays, the effects of decreased [Ca2+]cyt on the paracellular flux of [14C]mannitol and [3H]inulin were tested and served as a positive control. As expected, exposure to Ca2+-free medium for 2 h markedly increased paracellular permeability in normal cells (without DFMO) (Fig. 9B, left). Consistent with the effect on E-cadherin expression, polyamine depletion also significantly increased paracellular permeability (Fig. 9B, right). In DFMO-treated cells, levels of paracellular flux of [14C]mannitol and [3H]inulin were increased by 30% and
25%, respectively. Spermidine given together with DFMO restored the paracellular permeability to normal levels. Levels of paracellular permeability in cells exposed to DFMO plus spermidine were similar to those observed in control cells.
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Changes in E-cadherin expression and paracellular permeability after polyamine depletion in differentiated IEC-Cdx2L1 cells. To determine whether the inhibition of E-cadherin expression and resultant dysfunction of intestinal epithelial barrier by polyamine depletion occur in differentiated intestinal epithelial cells, we examined changes in levels of E-cadherin protein expression and paracellular permeability in differentiated IEC-Cdx2L1 cells. Our previous studies (42) and others (48) demonstrated that forced expression of the Cdx2 gene in undifferentiated parental IEC-6 cells induces the development of a differentiated phenotype. After treatment with the gene expression inducer IPTG for 16 days, these stable IEC-Cdx2L1 cells were polarized, showed multiple morphological characteristics of villus-type enterocytes, and highly expressed brush border enzymes such as sucrase-isomaltase. Consistent with parental IEC-6 cells, depletion of cellular polyamines by DFMO also inhibited expression of E-cadherin protein (Fig. 10A) and increased paracellular permeability (Fig. 10C) in differentiated IECCdx2L1 cells. E-cadherin protein in cells exposed to DFMO for 4, 6, and 8 days was decreased by 45% (Fig. 10B), and levels of paracellular permeability were increased by
20% in [14C]mannitol and
80% in [3H]inulin (Fig. 10C). E-cadherin expression and paracellular permeability were returned to normal levels when DFMO was given together with spermidine. These results indicate that polyamines are essential for the E-cadherin expression that is absolutely required for integrity of intestinal epithelial paracellular barrier function.
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DISCUSSION |
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It has been recognized that cell-cell adhesion, cytoskeleton, and signaling transduction functions overlap at the adherens junction to integrate a variety of cellular information and regulate intestinal epithelial barrier function. The cytoplasmic domain of E-cadherin directly associates -catenin in intestinal epithelial cells, and this cadherin-catenin complex is linked through
-catenin either directly or indirectly to the actin cytoskeleton (4, 50). This association of the cadherin-catenin complex with the actin filament network is crucial for strong cell-cell adhesions and has a functional role in forming the intestinal epithelial barrier. During the assembly of different intercellular junctions, cadherin-catenin complexes are stabilized at sites of cell-cell contact and subsequently develop into mature adherens junctions (1, 3, 19, 46). Inhibition of adherens junctions by either treatment with the specific antibody against E-cadherin (4, 29) or ectopic expression of a dominant-negative mutant E-cadherin (54, 61) retards the assembly of tight junctions and disrupts epithelial paracellular barrier functions. In contrast, stimulation of E-cadherin expression by exposure of gastric epithelium to a low-pH environment enhances adherens junction formation and stabilizes the epithelial barrier against acid back-diffusion (32). Consistent with observations in epithelial cells, over-expression of a mutated nonfunctional N-cadherin inhibits endothelial adhesions, impairs tight junction assembly, and decreases vascular endothelial barrier function (2, 11). More recently, it was found that over-expression of a mutant VE-cadherin lacking an adhesive extracellular domain disrupts endothelial barrier function and also inhibits endothelial growth in dermal microvascular endothelial cells (54).
The findings reported here indicate that expression of adherens junction proteins in normal intestinal epithelial cells requires polyamines and that polyamine depletion by DFMO dramatically decreases levels of both E-cadherin mRNA and protein and, to a lesser degree, also inhibits expression of -catenin and
-catenin proteins, suggesting that polyamines have distinct roles in expression of different adherens junction proteins. This result is not surprising, because polyamines are implicated in multiple signaling pathways and involved at different levels in the expression of various genes in intestinal epithelial cells. For example, polyamines stimulate the transcription of c-myc and c-jun but have no effect on these two genes post-transcriptionally (36). On the other hand, polyamines destabilize the mRNAs of transforming growth factor (TGF)-
(35), p53 (26), and junD (24) genes without affecting their transcriptional rates. In addition, the regulatory effects of polyamines on expression of adherens junction proteins are specific because there are no significant differences in tight junction proteins ZO-1 and ZO-2 between control cells and polyamine-deficient cells.
It is interesting and of biological significance that polyamine depletion decreases expression of adherens junction proteins, particularly E-cadherin, leading to dysfunction of the intestinal epithelial barrier. On one hand, intestinal epithelial cells contain high levels of polyamines, and the intracellular polyamine pool is critically controlled by polyamine synthesis, uptake, and degradation (49, 56, 57). Cellular polyamines increase rapidly in response to various physiological and pathogenic stimuli and affect distinct epithelial functions. On the other hand, adherens junctions integrate various intracellular and extracellular signals in regulation of the epithelial barrier and are vulnerable under various pathological conditions. It has been shown that E-cadherin is the target for cadmium toxicity in intestinal epithelial cells and that exposure to cadmium chloride damages E-cadherin-mediated cell-cell adhesions and decreases epithelial barrier function (10, 38). Expression of adherens junctions in patients with inflammatory bowel disease (IBD) is generally more affected by active inflammation than that of tight junctions and desmosomes (12, 18, 20, 22). Levels of E-cadherin and -catenin in actively inflamed IBD tissues are dramatically reduced in epithelial cells, whereas several tight junction- and desmosome-associated proteins remain to express at normal levels. This decreased expression of adherens junctions is associated with a significant change in intestinal epithelial paracellular permeability in these patients (12, 22).
The data from the current studies also show that polyamines regulate expression of the E-cadherin gene, at least partially, through Ca2+. Polyamine depletion by DFMO reduced [Ca2+]cyt and decreased levels of E-cadherin mRNA and protein. As noted in Fig. 4, elevation of [Ca2+]cyt by the Ca2+ ionophore ionomycin restored E-cadherin mRNA to near normal in polyamine-deficient cells, whereas reduction of [Ca2+]cyt through the removal of extracellular Ca2+ decreased the level of E-cadherin mRNA in normal cells (without DFMO), suggesting that polyamine-regulated [Ca2+]cyt is implicated in the regulation of either E-cadherin mRNA synthesis or mRNA degradation or both. The current studies further show that polyamine-regulated [Ca2+]cyt is also implicated in the posttranslational regulation of E-cadherin. Reduction of [Ca2+]cyt by polyamine depletion or exposure to Ca2+-free medium significantly decreased the stability of E-cadherin protein, as indicated by a decrease in its protein half-life (Figs. 6, 7, 8). This finding that polyamines are involved in the maintenance of E-cadherin protein stability via Ca2+ is consistent with our previous studies (41) that demonstrate that stabilization of the RhoA protein by polyamines is mediated by Ca2+ in intestinal epithelial cells.
The results presented in Figs. 9 and 10 indicate that decreased expression of adherens junction after polyamine depletion increases intestinal epithelial paracellular permeability. Our results are consistent with those of others who have demonstrated that disruption of adherens junctions impairs epithelial barrier function (12, 29, 51, 54). However, most of the studies that used an in vitro system that mimicked the intestinal epithelial barrier used T84 and Caco-2 cells (27, 33, 66). Although these cells are associated with the differentiated phenotype and have well-characterized tight junctions, they are cells of carcinoma origin. In this study, we used IEC-6 cells as a model, which are derived from normal intestinal epithelial cells and highly express adherens junction proteins E-cadherin, -catenin, and
-catenin as well as tight junction proteins ZO-1 and ZO-2. Because expression of tight junction proteins ZO-1 and ZO-2 was not affected in DFMO-treated cells, increased paracellular permeability after polyamine depletion results primarily from inhibition of expression of adherens junction proteins. Considering the fact that IEC-6 cells are undifferentiated epithelial cells and lack some tight junction proteins such as occludin (data not shown), differentiated IEC-Cdx2L1 cells were also used in the present experiments. Consistent with the observation in parental IEC-6 cells, polyamine depletion by DFMO also decreased E-cadherin expression and increased epithelial paracellular permeability in differentiated intestinal epithelial cells.
The mechanism by which intestinal epithelial paracellular permeability is increased after inhibition of adherens junction protein expression in polyamine-deficient cells is unknown. Because tight junctions seal epithelial cells together and prevent leaking between cells, it is possible that disruption of adherens junctions by polyamine depletion inhibits tight junction assembly in intestinal epithelial cells. Numerous intracellular signals have been shown to regulate tight junction assembly and function, and some of these signaling transduction proteins are associated with the adherens junctions (4, 65). Two models are proposed to delineate how adherens junctions regulate tight junction assembly, although they are not mutually exclusive. The first model hypothesizes that the cadherincatenin complex in the adherens junctions physically nucleates tight junction assembly. In support of this hypothesis, inhibition of cadherin adhesions prevents tight junction assembly (13, 61). The second model suggests that adherens junctions activate specific intracellular signaling pathways such as protein kinase C and Rho-family GTPases, which then activate tight junction assembly (4, 7). Impairment of adherens junctions could alter the balance of these signaling systems, thus leading to disruption of tight junction function. In addition, a third possibility also exists, suggesting that the effects of polyamines and Ca2+ could act independently on the adherens junctions and tight junctions in intestinal epithelial cells. It is not clear at present whether tight junction assembly is inhibited after decreased expression of adherens junctions in polyamine-deficient cells and whether other signaling transduction proteins are also involved in this process. Clearly, further studies are necessary to determine the exact roles of polyamines and polyamine-mediated expression of adherens junctions in the regulation of tight junction assembly and intestinal epithelial paracellular barrier function.
In summary, these results indicate that polyamines are required for expression of adherens junction proteins and involved in the regulation of intestinal epithelial barrier function. Depletion of cellular polyamines by DFMO decreases expression of the adherens junction proteins E-cadherin, -catenin, and
-catenin but has no effect on the tight junction proteins ZO-1, ZO-2, and JAM-1. This decreased expression of adherens junctions is associated with a significant increase in intestinal epithelial paracellular permeability. These results also show that polyamine depletion reduces [Ca2+]cyt in intestinal epithelial cells and that decreased [Ca2+]cyt caused by either polyamine depletion or exposure to Ca2+-free medium significantly inhibits expression of E-cadherin mRNA and decreases the half-life of E-cadherin proteins. It is indicated that decreased levels of E-cadherin in polyamine-deficient cells result from both decrease in mRNA expression and increase in the rate of its protein degradation. These findings suggest that polyamines regulate intestinal epithelial barrier function by altering expression of adherens junctions, at least partially through [Ca2+]cyt.
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