Polyamines are necessary for synthesis and stability of occludin protein in intestinal epithelial cells
Xin Guo,1,3,*
Jaladanki N. Rao,1,3,*
Lan Liu,1,3
Tongtong Zou,1,3
Kaspar M. Keledjian,1,3
Dessy Boneva,1,3
Bernard S. Marasa,2,3 and
Jian-Ying Wang1,2,3
Departments of 1Surgery and 2Pathology, University of Maryland School of Medicine and 3Baltimore Veterans Affairs Medical Center, Baltimore, Maryland
Submitted 8 September 2004
; accepted in final form 31 January 2005
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ABSTRACT
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Occludin is an integral membrane protein that forms the sealing element of tight junctions and is critical for epithelial barrier function. Polyamines are implicated in multiple signaling pathways driving different biological functions of intestinal epithelial cells (IEC). The present study determined whether polyamines are involved in expression of occludin and play a role in intestinal epithelial barrier function. Studies were conducted in stable Cdx2-transfected IEC-6 cells (IEC-Cdx2L1) associated with a highly differentiated phenotype. Polyamine depletion by
-difluoromethylornithine (DFMO) decreased levels of occludin protein but failed to affect expression of its mRNA. Other tight junction proteins, zonula occludens (ZO)-1, ZO-2, claudin-2, and claudin-3, were also decreased in polyamine-deficient cells. Decreased levels of tight junction proteins in DFMO-treated cells were associated with dysfunction of the epithelial barrier, which was overcome by exogenous polyamine spermidine. Decreased levels of occludin in polyamine-deficient cells was not due to the reduction of intracellular-free Ca2+ concentration ([Ca2+]cyt), because either increased or decreased [Ca2+]cyt did not alter levels of occludin in the presence or absence of polyamines. The level of newly synthesized occludin protein was decreased by
70% following polyamine depletion, whereas its protein half-life was reduced from
120 min in control cells to
75 min in polyamine-deficient cells. These findings indicate that polyamines are necessary for the synthesis and stability of occludin protein and that polyamine depletion disrupts the epithelial barrier function, at least partially, by decreasing occludin.
epithelial barrier; paracellular permeability; cdx2 gene; claudin; zonula occludens-1; zonula occludens-2; ornithine decarboxylase; protein stability
EPITHELIAL CELLS LINING THE gastrointestinal mucosa form an important barrier to a wide array of noxious substances in the lumen. Integrity of normal function of the intestinal epithelial barrier depends on specialized structures involved in cell-cell contacts known as tight junctions and adherens junctions. The tight junction located at the apical region of epithelial lateral membrane provides the barrier that is selectively permeable to certain hydrophilic molecules, ions, and nutrients, whereas the adherens junction mediates strong cell-to-cell adhesions between adjacent epithelial cells and regulates the tight junction assembly and function (9, 32, 38, 50). The tight junction seals epithelial cells together in a way that prevents even small molecules from leaking between cells and also functionally separates the plasma membrane into an apical and a basolateral domain (9, 38, 41). Although the understanding about the architecture and function of the tight junction is far from complete, there is no doubt that tight junction is a multiprotein complex that is highly dynamic and tightly regulated by numerous factors.
Major transmembrane and cytosolic tight junction proteins in the mammalian epithelium include occludin, claudins, zonula occludens (ZO)-1, and ZO-2 (8, 12, 41, 54, 66). Occludin is an integral membrane protein specifically localized at tight junction complexes and required for normal tight junction physiology (14, 25, 64). Occludin has a tetraspanning membrane topology with two extracellular loops and three cytoplasmic domains, among which the extracellular loops are important for occludin localization (2, 11, 13, 27, 39, 64). Increasing evidence indicates that occludin is the protein that forms the actual sealing element of tight junctions and is involved in fence functions of the epithelial barrier (4, 10, 27, 34, 64, 65). For example, ectopic expression of wild-type occludin in Madin-Darby canine kidney cells increases the number of tight junction strands and promotes the epithelial barrier function (34). In contrast, inhibition of occludin activity by a dominant negative occludin mutant disrupts tight junction structures and results in dysfunction of the epithelial barrier (4). Claudins are another family of integral membrane proteins of tight junctions and can interact with occludin in a collaborating way to achieve the full function of tight junctions (8, 9, 13, 24). On the other hand, ZO-1 and ZO-2 are the cytoplasmic face of tight junctions and directly bind to the COOH terminus of intracellular domain of occludin, and the interaction between occludin and ZO-1 or ZO-2 protein is crucial for maintaining normal structure of the tight junctions and epithelial barrier function (10, 15, 24, 63).
The natural polyamines, spermidine and spermine and their precursor putrescine, are organic cations found in all eukaryotic cells and have distinct regulatory roles in intestinal epithelial cells (IECs) (35, 56). Polyamines modulate expression of various genes involved in mucosal growth, repair, and apoptosis (30, 36, 51, 5961), and the control of cellular polyamines is thought to be a central convergence point for the multiple signaling pathways driving different epithelial cell functions. We (17, 18) have recently demonstrated that polyamines modulate intercellular junctions in normal IECs (IEC-6 line) and that depletion of cellular polyamines decreases adherens junction proteins such as E-cadherin,
-catenin, and
-catenin. Because the IEC-6 cells are undifferentiated crypt-type epithelial cells lacking expression of some tight junctions (18, 45), an in vitro model using normal differentiated IECs is necessary for identifying the fundamental mechanisms by which epithelial barrier function is regulated under biological conditions. Our previous studies (47, 49) and others (55) have shown that forced expression of the Cdx2 gene in IEC-6 cells induces the development of a differentiated phenotype. These differentiated Cdx2-transfected IEC-6 cells (IEC-Cdx2L1 cell line) exhibit multiple morphological features of villus-type enterocytes with well-developed tight junctions and appear to provide an excellent in vitro system for investigating intestinal epithelial barrier function. The present study was designed to test the hypothesis that polyamines are involved in expression of occludin in differentiated IEC-Cdx2L1 cells. Some of these data have been published previously in abstract form (19).
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MATERIALS AND METHODS
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Chemicals and supplies.
Disposable culture ware was purchased from Corning Glass Works (Corning, NY). Tissue culture media and dialyzed FBS were obtained from GIBCO-BRL (Gaithersburg, MD), and biochemicals were from Sigma (St. Louis, MO). The affinity-purified rabbit polyclonal antibodies against occludin, claudin-2, claudin-3, ZO-1, and ZO-2 were purchased from Zymed Laboratories (San Francisco, CA). The monoclonal antibody against E-cadherin was purchased from Transduction Laboratories (Lexington, KY).
-Difluoromethylornithine (DFMO) was purchased from Ilex Oncology (San Antonio, TX). The 12-mm Transwell filters (0.4 µm pore size, clear polyester) were obtained from Costar (Cambridge, MA). Fluorescein-conjugated goat anti-mouse and goat anti-rabbit antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). L-35S-labeled methionine, 14C-labeled mannitol and 3H-labeled inulin were obtained from Amersham Pharmacia Biotech (Piscataway, NJ).
Cell cultures and general experimental protocols.
The IEC-6 cell line at passage 13 was purchased from the American Type Culture Collection. The cell line was derived from normal rat intestine and was developed and characterized by Quaroni et al. (45). IEC-6 cells originated from intestinal crypt cells, as judged by morphological and immunological criteria. They are nontumorigenic and retain the undifferentiated character of epithelial stem cells. The stable IEC-Cdx2L1 cells were developed and characterized by Suh and Traber (55) and were kind gifts from Dr. Peter G. Traber (Baylor College of Medicine, Houston, TX). 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 an inducer for the gene expression. Stock stable IEC-Cdx2L1 cells were grown in DMEM as described in our previous publications (47, 49). 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 determined the effect of polyamine depletion on expression and cellular distribution of occludin and epithelial barrier function in differentiated IEC-Cdx2L1 cells. Cells were grown in the control cultures or cultures containing 5 mM DFMO or DFMO plus 5 µM spermidine for 4, 6, and 8 days, and the monolayers were washed three times with ice-cold Dulbecco's PBS. Different solutions were added according to the assays to be conducted. Expression of occludin mRNAs and protein was measured by semiquantitative RT-PCR and Western blot analysis, whereas barrier function of the cell monolayers was examined by measurements of transepithelial electrical resistance (TEER) and paracellular permeability.
In the second series of studies, we determined whether manipulation of [Ca2+]cyt, either increased or decreased, altered occludin expression in the presence or absence of polyamines. Based on our previous studies (48, 62), the Ca2+ ionophore ionomycin (1 µM) was used to increase [Ca2+]cyt, whereas the Ca2+-free medium was employed to decrease [Ca2+]cyt. In the Ca2+-free medium, 1.8 mM CaCl2 was replaced by 1.8 mM MgCl2, and additional 0.1 mM EGTA was added to chelate the residual Ca2+. Free Ca2+ concentration in the Ca2+-free medium was <0.002 µM. Levels of mRNAs and proteins of occludin, ZO-1, and ZO-2 were measured at various times after treatment with the Ca2+-free medium or ionomycin in normal (without DFMO) and polyamine-deficient (with DFMO) differentiated IEC-Cdx2L1-cells.
In the third series of studies, we focused on experiments to investigate the effect of cellular polyamines on the protein synthesis and stability of occludin in differentiated IEC-Cdx2L1 cells. The level of newly synthesized occludin protein was measured by using 35S-methionine-labeling technique, and the occludin stability was examined by determination of the protein half-life. Cells were grown in control cultures and cultures containing DFMO alone or DFMO plus spermidine for 6 days and then pulse-labeled with 35S-methionine. To determine the half-life of occludin protein, cycloheximide (50 µg/ml) was added to cultures, and levels of occludin protein were assayed at different times after treatment with cycloheximide by Western blot analysis.
RT-PCR.
Total cellular RNA was isolated by using RNeasy Mini Kit (Qiagen, Valencia, CA). Equal amounts of total RNA (2 µg) were transcribed to synthesize single-strand cDNA with a RT-PCR kit (Invitrogen Life Technologies, Carlsbad, CA). The specific sense and antisense primers for occludin included 5'-TTG GGA CAG AGG CTA TGG-3' and 5'-ACC CAC TCT TCA ACA TTG GG-3' and the expected size of occludin fragments was 623 bp. The specific sense and antisense primers for ZO-1 included 5'-GCCTCTGCAGTTAAGCAT-3' and 5'-AAGAGCTGGCTGTTTTAA-3', and the expected size of ZO-1 fragments was 249 bp. The specific sense and antisense primers for ZO-2 included 5'-CGCTGAAGACCGCATGTCCT-3' and 5'-GAGTAGAAGGCTTCAGGATGGAT-3', and the expected size of ZO-2 fragments was 461 bp. The specific sense and antisense primers for claudin-2 included 5'-TTCAACTGGTGGGCTACATCC-3' and 5'-GTGTGTCGCACAC-TCCATCC-3', and the expected size of claudin-2 was 154 bp. The specific sense and antisense primers for claudin-3 included 5'-CATCCTGCTGGCCGCCTTCG-3' and 5'-CCTGATGATGGTGTTGGCCGAC-3', and the expected size of claudin-3 was 174 bp. These particular sequences are chosen based on specificity established by previous publications (18, 53). RT-PCR was performed as described in our earlier publications (18, 49). To ensure that we were working within the linear phase of each amplication reaction, aliquots of individual PCR reactions were removed at two- to three-cycle intervals, electrophoresed on 1% agarose gels, and stained with ethidium bromide. To quantify the PCR products (the amounts of mRNA) of occludin, ZO-1, ZO-2, claudin-2, and claudin-3, 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.
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 phenylmethyl-sulfonyl 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 (7), 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. 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 occludin, ZO-1, ZO-2, claudin-2, and claudin-3 proteins. The filters were subsequently washed with 1x TBS-T and incubated with the secondary antibodies conjugated with horseradish peroxidase for 1 h at room temperature. The immunocomplexes on the filters were reacted for 1 min with chemiluminiscence reagent (cat. no. NEL-100; DuPont New England Nuclear).
Immunofluorescence staining.
The immunofluorescence staining procedure was carried out according to the method of Vielkind and Swierenga (58) with minor changes (17). After the monolayers of IEC-Cdx2L1 cells were fixed and rehydrated, they were incubated with the primary antibody against occludin and ZO-1 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 using Photoshop software (Adobe, San Jose, CA).
Measurement of occludin protein synthesis.
Occludin protein synthesis was examined by using 35S-methionine-labeling technique (30). After cells were grown in control culture medium and in culture medium containing 5 mM DFMO alone or DFMO plus spermidine for 6 days, they were washed with the methionine-free medium and incubated with the medium containing 35S-methionine (100 µCi/ml) for 2 h. The cells were rinsed with cold Dulbecco's PBS containing 2 mM methionine and harvested by scraping. Cell samples were disrupted by passing through a 21-gauge syringe needle, and then the suspension was centrifuged at 4°C for 15 min. The supernatant (cell lysate) was collected and incubated with a normal rabbit IgG together with the protein G PLUS-agarose for 30 min on a rocker platform with a rotating device at 4°C. Beads were isolated by centrifugation, and the preclear cell lysate was transferred into a new tube. The cell lysate (400 µg) was incubated with the anti-occludin antibody (3 µg) for 2 h at 4°C. The protein G PLUS-agarose was added, and the samples were incubated overnight. Immunoprecipitates were carefully collected after centrifugation at 3,500 rpm for 5 min, and pellets were washed with cold PBS and resuspended in 30 µl of 1x electrophoresis sample buffer. The supernatant was analyzed by SDS-PAGE followed by autoradiography.
Measurement of TEER.
TEER across the cell monolayers was measured by a method described previously (3, 31). Cells were grown in control cultures or cultures containing 5 mM DFMO or DFMO plus 5 µM spermidine for 4 days and plated in 12-mm Transwell filters for an additional 48 h to establish tight cell monolayers under the same culture conditions. Transwell inserts containing the cell monolayers were placed inside the Endohm-12 chamber (World Precision Instruments, Sarasota, FL) and TEER was measured across the monolayers using an Epithelial Tissue Voltohmmeter (World Precision Instruments, Sarasota, FL) and was expressed as ohms per centimeter square.
Paracellular tracer flux assay.
Flux assays were performed on the 12-mm Transwell filters as described in detail in our previous publication (18). Briefly, cells were grown in control cultures or cultures containing 5 mM DFMO or DFMO plus 5 µM spermidine for 4 days and then trypsinized and plated at confluent density of 4 x 104 cells/cm2 on the insert, and maintained at the same culture conditions for an additional 48 h to establish tight monolayers. Two different membrane-impermeable molecules, 14C-mannitol (mol wt 184) and 3H-inulin (mol wt 5,200), were 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 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 a Beckman liquid scintillation counter. The results were expressed as percentage of total count values of each tracer.
Statistical analysis.
All data are expressed as means ± SE from six samples. Autoradiographic and immunofluorescence labeling results were repeated three times. The significance of the difference between means was determined by ANOVA. The level of significance was determined using Dunnett's multiple range test (20).
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RESULTS
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Tight junction expression and epithelial barrier function in differentiated IECs.
As reported in our previous studies (47, 49) and others (55), the stable IEC-Cdx2L1 cells grown in the presence of 4 mM IPTG for 16 days were associated with a significant development of differentiated phenotype. These differentiated IEC-Cdx2L1 cells were polarized, showed lateral membrane interdigitations and microvilli at the apical pole, and also expressed brush-border enzymes such as sucrase-isomaltase (data not shown). These differentiated IEC-Cdx2L1 cells highly expressed tight junction proteins occludin, ZO-1, ZO-2, claudin-2, and claudin-3 (Fig. 1A). Basal levels of claudin-1 and claudin-4 proteins were too low to be detectable by Western blot analysis in differentiated Cdx2L1 cells (data not shown). In contrast, undifferentiated parental IEC-6 cells did not express occludin and claudins, although they highly expressed ZO-1 and ZO-2 proteins (Fig. 1C).

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Fig. 1. Changes in expression of occludin, zonula occludens (ZO)-1, ZO-2, claudin-2, and claudin-3 proteins in control differentiated Cdx2-transfected IEC-6 cells (IEC-Cdx2L1) and cells treated with either -difluoromethylornithine (DFMO) alone or DFMO + spermidine (SPD) are shown. Before experiments, stable IEC-Cdx2L1 cells were grown in DMEM containing 5% FBS and 4 mM isopropyl -D-thiogalactopyranoside (IPTG), the inducer for expression of the Cdx2 gene, for 16 days to induce cell differentiation. These differentiated IEC-Cdx2L1 cells were grown in DMEM containing 5% dialyzed FBS in the presence or absence of DFMO (5 mM) or DFMO plus SPD (5 µM) for 4, 6, and 8 days. Whole cell lysates were harvested for Western blot analysis. A: representative immunoblots of Western analysis. Twenty micrograms of total protein were applied to each lane and subjected to electrophoresis on 7.5% acrylamide gel. Immunoblots were hybridized with the antibodies specific for occludin ( 65 kDa), ZO-1 ( 225 kDa), ZO-2 ( 160 kDa), claudin-2 ( 22 kDa), and claudin-3 ( 24 kDa) as described in MATERIALS AND METHODS. After the blot was stripped, actin ( 42 kDa) immunoblotting was performed as an internal control for equal loading. B: quantitative analysis derived from densitometric analysis of immunoblots of occludin from cells described in A. Values are means ± SE of data from 3 separate experiments; relative levels of proteins were corrected for loading as measured by densitometry of actin. *P < 0.05 compared with the corresponding control and DFMO + SPD. C: representative immunoblots of Western analysis for ZO-1 and ZO-2 proteins in parental IEC-6 cells exposed to DFMO or DFMO plus SPD for 4, 6, and 8 days. Three experiments were performed that showed similar results.
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Consistently, barrier function in differentiated IEC-Cdx2L1 cells was improved significantly as indicated by TEER across the cell monolayer and paracellular tracer flux. Differentiated IEC-Cdx2L1 cells exhibited a higher steady-state level of TEER and lower basal paracellular permeability than parental IEC-6 cells. The value of TEER in differentiated IEC-Cdx2L1 cells was
1.8-fold greater than that of parental IEC-6 cells (from 23.1 ± 0.6 to 41.4 ± 1.2
·cm2, n = 18, P < 0.05), whereas levels of paracellular flux of two widely accepted membrane impermeable tracers, 3H-inulin and 14C-mannitol, were decreased by
80% and
30% in differentiated IEC-Cdx2L1 cells, respectively. In addition, enhanced barrier function in differentiated IEC-Cdx2L1 cells is not due simply to clonal variation, because identical results were observed when another independently transfected clone, IEC-Cdx2L2, was analyzed (data not shown). Increased barrier function in differentiated IEC-Cdx2L1 cells also is not due to the effects of IPTG. There were no significant differences in levels of TEER and paracellular permeability between nontransfected parental IEC-6 cells and cells transfected with the empty vector containing no Cdx2 cDNA but maintained in the medium containing IPTG for 16 days (data not shown). Treatment with IPTG for 16 days also did not affect levels of tight junction proteins nor epithelial barrier function in nontransfected parental IEC-6 cells. Furthermore, levels of various tight junction proteins and barrier function in Cdx2-transfected IEC-6 cells before treatment with IPTG to induce differentiation were identical to those of nontransfected parental IEC-6 cells (data not shown). These results suggest that stable IEC-Cdx2L1 cells with a differentiated phenotype are a better system to characterize epithelial barrier events in vitro.
Changes in expression of tight junctions following polyamine depletion.
Our previous studies (18) have demonstrated that polyamines are necessary for expression of adherens junction proteins in undifferentiated parental IEC-6 cells. Because tight junctions are not well developed in parental IEC-6 cells, differentiated IEC-Cdx2L1 cells were used in the present study. To determine the role of cellular polyamines in the regulation of tight junction expression, differentiated IEC-Cdx2L1 cells were cultured in the DMEM containing DFMO, a specific inhibitor of polyamine synthesis, for 4, 6, and 8 days. Consistent with our previous publications (47, 49), exposure to 5 mM DFMO completely depleted putrescine within 48 h, but it took 4 days to totally deplete spermidine and significantly deplete spermine (by
60%) (data not shown).
Results presented in Fig. 1 show that depletion of cellular polyamines by treatment with DFMO decreased protein levels of tight junctions occludin, ZO-1, ZO-2, claudin-2, and claudin-3 in differentiated IEC-Cdx2L1 cells. The levels of occludin protein in the cells exposed to DFMO for 4, 6, and 8 days were decreased by >80% (Fig. 1A, top, and B). Although there was no inhibition of ZO-1 and ZO-2 expression in undifferentiated parental IEC-6 cells treated with DFMO (Fig. 1C), levels of ZO-1 and ZO-2 proteins in differentiated IEC-Cdx2L1 cells decreased significantly after polyamine depletion. Levels of ZO-1 and ZO-2 proteins in differentiated IEC-Cdx2L1 cells exposed to DFMO for 4, 6, and 8 days were decreased by
55% and
40%, respectively. Treatment with DFMO for 4 days did not alter expression of claudin-2, but its levels were decreased by
50% on day 6 and by
80% on day 8, respectively. Changes in claudin-3 expression were similar to those observed in cladudin-2 following polyamine depletion, and its protein levels were decreased by
50% in cells exposed to DFMO for 6 and 8 days. In the presence of DFMO, decreased levels of occludin, ZO-1, ZO-2, claudin-2 and claudin-3 proteins were completely abolished by addition of exogenous spermidine (5 µM). Putrescine (10 µM) had an effect equal to spermidine on levels of tight junctions when it was added to cultures that contained DFMO (data not shown). On the other hand, the steady-state levels of occludin, ZO-1, and ZO-2 proteins were not affected by the addition of exogenous spermidine (5 µM) in cells grown without DFMO (data not shown); and the similar effect of spermidine on basal levels of other proteins, such as p53 and c-Myc, has been reported in our previous publications (30, 42).
To extend these positive findings that polyamine depletion decreased levels of intercellular junction proteins, immunofluorescence staining was performed to determine the cellular distribution of occludin, ZO-1, and E-cadherin in differentiated IEC-Cdx2L1 cells. In control cells (Fig. 2A), immunoreactivities for occludin, ZO-1, and E-cadherin proteins were primarily located along the entire cell-to-cell contact regions of adjacent cells. Consistent with our data from Western blot analysis, these membrane immunoreactivities for occludin, ZO-1, and E-cadherin proteins markedly decreased and were hardly detected in polyamine-deficient cells (Fig. 2B), as expected. Spermidine given together with DFMO prevented the decreased immunostaining levels for occludin, ZO-1, and E-cadherin (Fig. 2C). The cellular distribution of occludin, ZO-1, and E-cadherin in the cells exposed to DFMO plus spermidine was indistinguishable from those observed in control cells (Fig. 2, A vs. C).

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Fig. 2. Cellular distribution of occludin, ZO-1, and E-cadherin (E-cad) proteins in differentiated IEC-Cdx2L1 cells described in Fig. 1. Cells were plated in a 4-well chamber slide, grown in DMEM containing 5% dialyzed FBS in the presence or absence of DFMO or DFMO + SPD for 6 days, and then fixed for immunostaining. Cells were permeabilized and incubated with the specific antibody against occludin, ZO-1, or E-cad, and then with anti-IgG conjugated with FITC. Slides were viewed through a Zeiss confocal microscope. A: control. B: cells treated with DFMO alone for 6 days. C: cells treated with DFMO + SPD for 6 days. Original magnification: x1,000. Three experiments were performed that showed similar results.
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Decreased expression of tight junctions associated with dysfunction of epithelial barrier following polyamine depletion.
To determine the role of decreased tight junctions in intestinal epithelial barrier function, TEER and paracellular permeability were assessed in differentiated IEC-Cdx2L1 cells in the presence or absence of polyamines. Cells were grown in control cultures or cultures containing DFMO or DFMO plus spermidine for 4 days, then plated at confluent density on the insert, and maintained for an additional 48 h to establish a tight monolayer. As shown in Fig. 3, polyamine depletion resulted in dysfunction of the epithelial barrier as indicated by a decrease in TEER and increase in paracellular permeability. Values of TEER were decreased by
35% in cells exposed to DFMO for 6 days (from 46.2 ± 1 to 30.2 ± 0.7
·cm2, n = 16, P < 0.05), while levels of paracellular flux of 3H-inulin and 14C-mannitol were increased by
50 and
20%, respectively. Spermidine given together with DFMO restored TEER and paracellular permeability to normal levels. Levels of TEER and paracellular flux of 3H-inulin and 14C-mannitol in cells exposed to DFMO plus spermidine were similar to those observed in control cells. These results suggest that polyamine depletion downregulates expression of tight junctions, which is associated with a disruption of the intestinal epithelial barrier function.

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Fig. 3. Changes in epithelial barrier function in differentiated IEC-Cdx2L1 cells described in Fig. 1. A: changes in transepithelial electrical resistance (TEER) in cells exposed to DFMO or DFMO + SPD. Cells were grown in control cultures or cultures containing 5 mM DFMO alone or DFMO plus 5 µM SPD for 6 days. TEER assays were performed on 12-mm Transwell filters as described in MATERIALS AND METHODS. Values are means ± SE of data from 8 samples. B: 3H-inulin permeability. C: 14C-mannitol permeability after cells were grown in control cultures or cultures containing DFMO or DFMO plus SPD for 4 days and were then trypsinized, plated at confluent density on the insert, and maintained under the same culture conditions for additional 48 h. Membrane-impermeable tracer molecules, 3H-inulin and 14C-mannitol, were added to the insert medium, and the entire basal medium was collected 2 h thereafter for paracellular trace flux assays. Values are means ± SE of data from 8 samples. *P < 0.05 compared with control and DFMO + SPD.
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Effect of polyamine depletion on expression of tight junction mRNAs.
To define levels where polyamines are implicated in expression of tight junctions, changes in mRNAs of occludin, ZO-1, ZO-2, claudin-2, and claudin-3 were examined in the presence or absence of cellular polyamines. As shown in Fig. 4, depletion of cellular polyamines by DFMO did not inhibit expression of occludin, ZO-1, and ZO-2 mRNAs, although it significantly decreased levels of their proteins (Fig. 1). There were no significant changes in mRNA levels of occludin, ZO-1, and ZO-2, regardless of treatment with DFMO alone or DFMO plus spermidine for 4 and 6 days (Fig. 4). On the other hand, polyamine depletion inhibited expression of claudin-2 and claudin-3 mRNAs, which was completely prevented by exogenous spermidine (Fig. 4, bottom). These findings suggest that polyamines are involved in expression of various tight junction proteins through different signaling pathways in IECs.

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Fig. 4. Changes in expression of occludin, ZO-1, ZO-2, claudin-2, and claudin-3 mRNAs in differentiated IEC-Cdx2L1 cells described in Fig. 1. Cells were grown in control cultures or cultures containing DFMO or DFMO plus SPD for 4 and 6 days, and total cellular RNA was harvested for RT-PCR analysis. A: PCR-amplified products displayed in agarose gels for occludin (623 bp), ZO-1 (249 bp), ZO-2 (461 bp), claudin-2 (154 bp), claudin-3 (174 bp), and -actin (244 bp). The first-strand cDNAs, synthesized from total RNA, were amplified with the specific sense and antisense primers and displayed in agarose gels stained with ethidium bromide. B: quantitative analysis of RT-PCR results of occludin mRNA by densitometry from cells described in A. Data were normalized to the amount of -actin (optical density of the occludin mRNA/optical density of the -actin mRNA) and are expressed as means ± SE of data from 3 separate experiments.
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Effect of [Ca2+]cyt on occludin expression.
To keep this study focused, we specifically elucidated the mechanism by which polyamines modulate occludin in IECs. Our previous studies have demonstrated that cellular polyamines regulate [Ca2+]cyt concentration by governing the driving force for Ca2+ influx via controlling activity of voltage-gated K+ (KV) channels in IECs and that depletion of cellular polyamines inhibited KV channel expression and resulted in membrane depolarization, which was associated with a decrease in [Ca2+]cyt (17, 48, 49, 62). In fact, [Ca2+]cyt in differentiated IEC-Cdx2L1 cells exposed to DFMO for 6 days was decreased by
35% (from 139 ± 6 nM in control cells to 91 ± 3 nM in DFMO-treated cells; n = 28, P < 0.05). Spermidine, given together with DFMO, restored [Ca2+]cyt to normal levels.
To test the possibility that polyamines modulate occludin expression by altering [Ca2+]cyt, the following two studies were performed in differentiated IEC-Cdx2L1 cells. First, we determined whether the decreased [Ca2+]cyt caused by removal of extracellular Ca2+ inhibits expression of occludin in control cells (without DFMO). As shown in Fig. 5, exposure to the Ca2+-free medium for 6 h did not alter expression of occludin. There were no significant differences in levels of occludin protein and mRNA between control cells and cells exposed to the Ca2+-free medium for 2, 4, and 6 h. In addition, decreased [Ca2+]cyt by exposure to the Ca2+-free medium also had no effect on expression of ZO-1 and ZO-2 proteins. However, expression of E-cadherin protein, which is a calcium-dependent protein, was dramatically decreased after exposure to the Ca2+-free medium (Fig. 5A, bottom, and B). Levels of E-cadherin protein were decreased by
60% at 2 h, by
85% at 4 h, and by
90% at 6 h after exposure to the Ca2+-free medium.

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Fig. 5. Effect of decreased intracellular Ca2+ concentration by exposure to the Ca2+-free medium on expression of occludin, ZO-1, ZO-2, and E-cadherin in normal differentiated IEC-Cdx2L1 cells. A: representative immunoblots of Western analysis for occludin, ZO-1, ZO-2, and E-cadherin ( 120 kDa) proteins. Cells were grown in the standard DMEM for 6 days and then incubated with the Ca2+-free medium. Whole cell lysates were harvested at indicated times after incubation with Ca2+-free medium, and protein levels of occludin, ZO-1, ZO-2, and E-cadherin were measured by Western blotting analysis using various specific antibodies. After the blot was stripped, actin immunoblotting was performed as an internal control for equal loading. B: quantitative analysis derived from densitometric analysis of immunoblots from cells described in A. Values are means ± SE of data from 3 separate experiments. C: changes in occludin mRNA in cells described in A. Total cellular RNA was harvested at various times after exposure to Ca2+-free medium, and occludin mRNA levels were assayed by RT-PCR analysis. Equal loading was monitored by measurement of -actin mRNA. Three separate experiments were performed that showed similar results.
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Second, we examined whether increased [Ca2+]cyt by the Ca2+ ionophore, ionomycin, induced expression of occludin in control and polyamine-deficient IEC-Cdx2L1 cells. Consistent with our previous studies (48, 49), exposure to 1 µM ionomycin dramatically increased [Ca2+]cyt in control and DFMO-treated cells (data not shown). Results presented in Fig. 6 show that increased [Ca2+]cyt by ionomycin did not increase levels of occludin protein in control and polyamine-deficient cells. Consistently, there were no significant changes in ZO-1 and ZO-2 proteins after exposure to ionomycin. In addition, neither the Ca2+-free medium nor ionomycin affected cell attachment and cell viability nor induced apoptosis in control and DFMO-treated cells as measured by Trypan blue staining and Annexin-V staining for apoptotic cells (data not shown). These results indicate that polyamines regulate expression of occludin protein through a mechanism that is independent of [Ca2+]cyt in differentiated IEC-Cdx2L1 cells.

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Fig. 6. Effect of increased intracellular Ca2+ concentration by treatment with the Ca2+ ionophore, ionomycin (Iono), on expression of occludin, ZO-1, and ZO-2 proteins in normal and polyamine-deficient IEC-Cdx2L1 cells. A: representative immunoblots of Western analysis for occludin, ZO-1, and ZO-2 proteins. Differentiated IEC-Cdx2L1 cells were grown in control cultures or cultures containing DFMO (5 mM) for 6 days and then exposed to 1 µM ionomycin for 6 h. Whole cell lysates were harvested and levels of occludin, ZO-1, and ZO-2 proteins were assayed by Western blot analysis. Actin immunoblotting was performed as internal control for equal loading. B: quantitative analysis derived from densitometric analysis of immunoblots from cells described in A. Values are means ± SE of data from 3 separate experiments. *P < 0.05 compared with the corresponding control groups.
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Effect of polyamine depletion on occludin protein synthesis and stability.
To determine whether polyamines regulate occludin at the translation level, the level of newly synthesized occludin protein was examined in this study. As shown in Fig. 7, polyamine depletion by DFMO significantly decreased the occludin protein synthesis in differentiated IEC-Cdx-2L1 cells. The level of newly synthesized occludin protein was decreased by
70% in cells exposed to DFMO for 6 days. To determine the effect of polyamines on the stability of occludin, the half-life of occludin protein was examined in the presence or absence of cellular polyamines. The method used in this study was validated by pulse-chase analysis as described in our previous work (18). Results presented in Fig. 8 show that an increase in occludin protein degradation also contributes to the decreased levels of occludin in polyamine-deficient cells. In control cells (without DFMO), the levels of occludin protein declined gradually after protein synthesis was inhibited by administration of cycloheximide, with a half life of
120 min (Fig. 8, Aa and B). In DFMO-treated cells, the stability of occludin protein decreased significantly compared with that observed in control cells (Fig. 8A, a vs. b). The half-life of occludin protein in polyamine-deficient cells was
75 min (Fig. 8B). Spermidine given together with DFMO not only totally overcame the decrease in occludin protein synthesis but also completely prevented the instability of occludin protein as well. The level of newly synthesized occludin protein and its half-life in cells treated with DFMO plus spermidine was similar to those observed in controls (Fig. 8A, a vs. c). These results clearly indicate that polyamines are implicated in regulation of occludin protein synthesis and stability in IECs.

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Fig. 7. Incorporation of 35S-labeled methionine into newly synthesized occludin protein in differentiated IEC-Cdx2L1 cells grown in control cultures, cultures in which polyamine synthesis was inhibited with 5 mM DFMO, and cultures inhibited with DFMO and supplemented with exogenous SPD (5 µM). A: representative autoradiograms of occludin protein synthesis as measured by 35S-methionine incorporation. Cells were grown in control cultures or cultures containing DFMO or DFMO + SPD for 6 days and then pulse labeled with 35S-methionine (100 µCi/ml) for 2 h, followed by washing with Dulbecco's PBS. Cells were harvested, and cytosolic extracts were prepared as described in MATERIALS AND METHODS. The cytosolic lysates (400 µg) from each of the groups were processed for immunoprecipitation, SDS-PAGE analysis, and autoradiography. B: quantitative analysis of autoradiograms by densitometry from cells described in A. Values are means ± SE of data from 3 separate experiments. *P < 0.05 compared with control and DFMO + SPD.
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Fig. 8. Studies of occludin protein stability in differentiated IEC-Cdx2L1 cells described in Fig. 7. After cells were grown in the presence or absence of DFMO with or without SPD for 6 days, cycloheximide (CHX) at the concentration of 50 µg/ml was added to cultures and whole cell lysates were harvested at indicated times. Occludin protein levels were assayed by Western blot analysis, and loading of proteins was monitored by actin. A: representative immunoblots of Western analysis in cell extracts from control cells (a) and cells exposed to DFMO alone (b) or DFMO + SPD (c). B: quantitative analysis of Western immunoblots by densitometry from cells described in A. Values are means ± SE of data from 3 separate experiments, and the relative levels of occludin protein were corrected for protein loading as measured by densitometry of actin.
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DISCUSSION
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An increasing body of evidence indicates that cellular polyamines play a critical role in maintenance of the intestinal epithelial integrity, but few specific functions of polyamines at cellular and molecular levels are defined. We (18) have recently reported that polyamines are implicated in regulation of the intestinal epithelial barrier function and that depletion of cellular polyamines increases epithelial paracellular permeability at least partially by inhibiting expression of adherens junctions in undifferentiated parental IEC-6 cells. The present studies further confirm our previous observations by demonstrating that polyamines are crucial for expression of adherens junctions in differentiated IEC-Cdx2L1 cells. The most significant of the new findings reported in this study, however, is that polyamines are required for normal function of tight junctions and that polyamines regulate expression of various tight junction proteins through different mechanisms. Polyamines regulate occludin primarily by altering its protein synthesis and stability rather than its mRNA. These findings provide, for the first time, new evidence showing that cellular polyamines are necessary for normal function of the intestinal epithelial barrier in association with their ability to regulate levels of tight junction proteins, especially occludin.
The requirement of polyamines for expression of tight junction proteins is specific in differentiated IECs, because depletion of cellular polyamines fails to inhibit levels of ZO-1 and ZO-2 proteins in parental IEC-6 cells (Fig. 1C). Although the exact reasons for the different responses of ZO-1 and ZO-2 expression to polyamines in parental IEC-6 cells and differentiated IEC-Cdx2L1 cells remain unclear, it may be related to the following facts and possibilities. First, parental IEC-6 cells originate from intestinal crypts and retain the undifferentiated character of epithelial crypt cells (45). In contrast, stable IEC-Cdx2L1 cells have multiple morphological and molecular characteristics of differentiated phenotype and represent villus-type enterocytes (47, 49, 55). Second, polyamines may have different regulatory effects when these compounds are presented in the villus in general. Our previous studies (47, 60) and others (35) have shown that polyamines in the crypt are absolutely required for epithelial cell proliferation, but roles of induced polyamines in the villus are still unknown. Third, tight junctions are not well developed in undifferentiated parental IEC-6 cells. For example, expression of occludin and claudin-2 and -3 was observed only in differentiated IEC-Cdx2L1 cells, although both parental IEC-6 cells and differentiated IEC-Cdx2L1 cells expressed ZO-1 and ZO-2 proteins. It is possible that expression of the premature tight junctions in parental IEC-6 cells is regulated by a distinct mechanism insensitive to cellular polyamines. On the other hand, polyamines are necessary for expression of adherens junctions in both parental IEC-6 cells and differentiated IEC-Cdx2L1 cells and depletion of cellular polyamines decreases levels of E-cadherin,
-catenin, and
-catenin proteins.
The findings reported here indicate that polyamines are implicated in different levels in regulation of various tight junctions. Polyamine depletion decreased levels of occludin, ZO-1, and ZO-2 proteins without affecting their mRNAs, but inhibited expression of both mRNAs and proteins of claudin-2 and claudin-3 (Figs. 1 and 4). In addition, we have recently reported that polyamines regulate expression of the adherens junction protein E-cadherin at the transcriptional level and that depletion of cellular polyamines decreases E-cadherin mRNA and protein primarily through inhibition of transcription of the E-cadherin gene (18). These different mechanisms involved in regulation of adherens junctions and tight junctions by polyamines are not surprising, because polyamines have been involved in multiple signaling pathways in the expression of various genes in IECs. It has been shown that polyamines modulate transcription, but not posttranscription, of c-myc and c-jun genes in IEC-6 cells (42). In contrast, polyamines regulate the stability of mRNAs and proteins of p53 (30) and JunD (29) without affecting the transcriptional rates of these two genes.
It is of physiological significance that cellular polyamines regulate expression of tight junctions in IECs. Results presented in Fig. 1 show that polyamine depletion dramatically decreased levels of occludin, ZO-1, and ZO-2 proteins, but the epithelial barrier function was only inhibited by
30% in DFMO-treated cells as indicated by a decrease in TEER and increase in paracellular permeability (Fig. 3). Although the exact reasons causing the differences are unclear, these findings suggest that 1) normal epithelial barrier function depends on multiple tight junction proteins; and 2) decreased levels of occludin, ZO-1, and ZO-2 proteins following polyamine depletion are associated with functional compensation of other tight junction or adherens junction proteins. Under normal conditions, the epithelial cells contain high levels of polyamines, which is dynamically regulated by polyamine biosynthesis, uptake, and degradation (35, 56). Cellular levels of polyamines are changed rapidly, either increased or decreased, in response to various physiological and pathogenic stimuli, leading to the activation or inactivation of different cellular signaling pathways. On the other hand, tight junctions form a physical fence to the diffusion of macromolecules through the paracellular space and also are involved in various physiological processes, such as neutrophil transmigration across an endothelium (37), epithelial cell division (1), and extrusion (5). Disruption of tight junction function occurs commonly in various pathological conditions such as inflammatory bowel disease, intestinal infections, cancers, and critical surgical stresses (3, 16, 2123, 26, 46). To date, many signaling pathways, including tyrosine kinases, Ca2+, protein kinase C, and phospholipase C-
, have been implicated in the regulation of tight junction permeability in epithelial cells (28, 33, 40, 50, 52, 57). The present studies provide a strong evidence for a role of cellular polyamines in the control of intestinal epithelial tight junctions.
The regulatory effect of polyamines on occludin is not due to [Ca2+]cyt, because either increasing or decreasing [Ca2+]cyt did not alter levels of occludin protein in the presence or absence of polyamines. It has been reported that polyamines regulate [Ca2+]cyt concentration by governing membrane potential through control of KV channels and that the elevated [Ca2+]cyt is a major mediator for distinct biological functions of polyamines (48, 49, 62). Polyamine depletion inhibits KV channel expression and causes membrane depolarization, leading to a decrease in [Ca2+]cyt (48, 62). We have recently found that polyamines are essential for E-cadherin gene expression, acting at least partially through [Ca2+]cyt (18). Therefore, it was logical and reasonable to consider the possibility that polyamines regulate occludin by altering [Ca2+]cyt in this study. However, as noted in Figs. 5 and 6, polyamines are necessary for occludin protein expression through a mechanism that is independent of [Ca2+]cyt in differentiated IEC-Cdx2L1 cells.
Results presented in Figs. 7 and 8 clearly show that polyamines regulate expression of occludin primarily by controlling its protein synthesis and stability. Depletion of cellular polyamines by DFMO not only inhibited the level of newly synthesized occludin protein but also decreased its protein stability. Because the decreases in both occludin protein synthesis and its half-life in DFMO-treated cells are completely prevented by exogenous spermidine, the decrease in occludin expression at translation and posttranslation levels must be related to polyamine depletion rather than to the nonspecific effect of DFMO. In addition, the possibility that polyamines regulate occludin expression at the translational level is further supported by the results showing that cycloheximide decreases the spermidine-mediated prevention of DFMO effect on occludin protein synthesis (Fig. 8). Although the exact mechanisms by which polyamines regulate translation and posttranslation of occludin remain unknown, they are possibly related to the specific molecular structure of polyamines. At physiological pH, putrescine, spermidine, and spermine possess two, three, and four positive charges, respectively (56). These compounds are shown to bind to negatively charged macromolecules such as DNA, RNA, and proteins to influence the sequence-specific DNA-, RNA- or protein-protein interactions, which alter gene transcription and translation and the stability of mRNAs and proteins (6, 43, 44, 56). Clearly, further studies are needed to define and characterize the specific regions or domains of occludin, which mediate or are involved in the regulatory effects of polyamines.
In summary, these results indicate that polyamines are required for expression of tight junctions in differentiated IECs. Polyamines regulate expression of various tight junction proteins through distinct cellular signaling pathways. Although the inhibitory effect of polyamine depletion on expression of occludin protein is independent of intracellular Ca2+, results presented here clearly indicate that reduced levels of occludin in polyamine-deficient cells result primarily from decreases in its protein synthesis and stability. These findings suggest that cellular polyamines are the biological regulators for tight junction expression and play an important role in the maintenance of intestinal epithelial barrier integrity under physiological conditions.
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GRANTS
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This work was supported by Merit Review Grant from the Department of Veterans Affairs and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-57819, DK-61972, and DK-68491.
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ACKNOWLEDGMENTS
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J-Y. Wang is a Research Career Scientist, Medical Research Service, US Department of Veterans Affairs.
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FOOTNOTES
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Address for reprint requests and other correspondence: J.-Y. Wang, Dept. of Surgery, Baltimore Veterans Affairs Medical Center, 10 North Greene St., Baltimore, MD 21201 (E-mail:jwang{at}smail.umaryland.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* X. Guo and J. N. Rao contributed equally to this work. 
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REFERENCES
|
---|
- Baker J and Garrod D. Epithelial cells retain junctions during mitosis. J Cell Sci 104: 415425, 1993.[Abstract/Free Full Text]
- Balda MS, Flores-Maldonado C, Cereijido M, and Matter K. Multiple domains of occludin are involved in the regulation of paracellular permeability. J Cell Biochem 78: 8596, 2000.[CrossRef][ISI][Medline]
- Balda MS, Whitney JA, Flores C, Gonzalez S, Cereijido M, and Matter K. Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein. J Cell Biol 134: 10311049, 1996.[Abstract]
- Bamforth SD, Kniesel U, Wolburg H, Engelhardt B, and Risau W. A dominant mutant of occludin disrupts tight junction structure and function. J Cell Sci 112: 18791888, 1999.[Abstract/Free Full Text]
- Baron DA and Miller DH. Extrusion of colonic epithelial cells in vitro. J Electron Microsc (Tokyo) 16: 1524, 1990.
- Basu HS, Smirnov IV, Peng HF, Tiffany K, and Jackson V. Effects of spermine and its cytotoxic analogs on nucleosome formation on topologically stressed DNA in vitro. Eur J Biochem 243: 247258, 1997.[Abstract]
- Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248254, 1976.[CrossRef][ISI][Medline]
- Cereijido M, Shoshani L, and Contreras RG. Molecular physiology and pathophysiology of tight junctions. I. Biogenesis of tight junctions and epithelial polarity. Am J Physiol Gastrointest Liver Physiol 279: G477G482, 2000.[Abstract/Free Full Text]
- Cereijido M, Valdes J, Shoshani L, and Contreras RG. Role of tight junctions in establishing and maintaining cell polarity. Annu Rev Physiol 60: 161177, 1998.[CrossRef][ISI][Medline]
- Chen Y, Merzdorf C, Paul DL, and Goodenough DA. COOH terminus of occludin is required for tight junction barrier function in early Xenopus embryos. J Cell Biol 138: 891899, 1997.[Abstract/Free Full Text]
- Fanning AS, Mitic LL, and Anderson JM. Transmembrane proteins in the tight junction barrier. J Am Soc Nephrol 10: 13371345, 1999.[Abstract/Free Full Text]
- Fujimoto K. Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins: application to the immunogold labeling of intercellular junctional complexes. J Cell Sci 108: 34433449, 1995.[Abstract/Free Full Text]
- Furuse M, Fujita K, Hiiragi T, Fujimoto K, and Tsukita S. Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol 141: 15391550, 1998.[Abstract/Free Full Text]
- Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, Tsukita S, and Tsukita S. Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol 123: 17771788, 1993.[Abstract]
- Furuse M, Itoh M, Hirase T, Nagafuchi A, Yonemura S, Tsukita S, and Tsukita S. Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions. J Cell Biol 127: 16171626, 1994.[Abstract]
- Gassler N, Rohr C, Schneider A, Kartenbeck J, Bach A, Obermuller N, Otto HF, and Autschbach F. Inflammatory bowel disease is associated with changes of enterocytic junctions. Am J Physiol Gastrointest Liver Physiol 281: G216G228, 2001.[Abstract/Free Full Text]
- Guo X, Rao JN, Liu L, Rizvi M, Turner DJ, and Wang JY. Polyamines regulate beta-catenin tyrosine phosphorylation via Ca2+ during intestinal epithelial cell migration. Am J Physiol Cell Physiol 283: C722C734, 2002.[Abstract/Free Full Text]
- Guo X, Rao JN, Liu L, Zou TT, Turner DJ, Bass BL, and Wang JY. Regulation of adherens junctions and epithelial paracellular permeability: a novel function for polyamines. Am J Physiol Cell Physiol 285: C1174C1187, 2003.[Abstract/Free Full Text]
- Guo X, Rao JN, Liu L, Zou T, Zhang HM, Turner DJ, and Wang JY. Involvement of cellular polyamines regulation of occludin expression and intestinal epithelial barrier function (Abstract). FASEB J 18: A710, 2004.
- Harter JL. Critical values for Duncan's new multiple range tests. Biometrics 16: 671685, 1960.[ISI]
- Hermiston ML and Gordon JI. Inflammatory bowel disease and adenomas in mice expressing a dominant negative N-cadherin. Science 270: 12031207, 1995.[Abstract]
- Hollander D. Crohn's diseasea permeability disorder of the tight junction? Gut 29: 16211624, 1988.[ISI][Medline]
- Huber D, Balda MS, and Matter K. Occludin modulates transepithelial migration of neutrophils. J Biol Chem 275: 57735778, 2000.[Abstract/Free Full Text]
- Itoh M, Furuse M, Morita K, Kubota K, Saitou M, and Tsukita S. Direct binding of tight junction-associated MAGUKs, ZO-1, ZO-2 and ZO-3, with the COOH termini of claudins. J Cell Biol 147: 13511363, 1999.[Abstract/Free Full Text]
- Kimura Y, Shiozaki H, Hirao M, Maeno Y, Doki Y, Inoue M, Monden T, Ando-Akatsuka Y, Furuse M, Tsukita S, and Monden M. Expression of occludin, tight-junction-associated protein, in human digestive tract. Am J Pathol 151: 4554, 1997.[Abstract]
- Kucharzik T, Walsh SV, Chen J, Parkos CA, and Nusrat A. Neutrophil transmigration in inflammatory bowel disease is associated with differential expression of epithelial intercellular junction proteins. Am J Pathol 159: 20012009, 2001.[Abstract/Free Full Text]
- Lacaz-Vieira F, Jaeger MM, Farshori P, and Kachar B. Small synthetic peptides homologous to segments of the first external loop of occludin impair tight junction resealing. J Membr Biol 168: 289297, 1999.[CrossRef][ISI][Medline]
- Li D and Mrsny RJ. Oncogenic Raf-1 disrupts epithelial tight junctions via downregulation of occludin. J Cell Biol 148: 791800, 2000.[Abstract/Free Full Text]
- Li L, Liu L, Rao JN, Esmaili A, Strauch ED, Bass BL, and Wang JY. JunD stabilization results in inhibition of normal intestinal epithelial cell growth through p21 after polyamine depletion. Gastroenterology 123: 764779, 2002.[CrossRef][ISI][Medline]
- Li L, Rao JN, Guo X, Liu L, Santora R, Bass BL, and Wang JY. Polyamine depletion stabilizes p53 resulting in inhibition of normal intestinal epithelial cell proliferation. Am J Physiol Cell Physiol 281: C941C953, 2001.[Abstract/Free Full Text]
- Liu TS, Musch MW, Sugi K, Walsh-Reitz MM, Ropeleski MJ, Hendrickson BA, Pothoulakis C, Lamont JT, and Chang EB. Protective role of HSP72 against Clostridium difficile toxin A-induced intestinal epithelial cell dysfunction. Am J Physiol Cell Physiol 284: C1073C1082, 2003.[Abstract/Free Full Text]
- Liu Y, Nusrat A, Schnell FJ, Reaves TA, Walsh S, Pochet M, and Parkos CA. Human junction adhesion molecule regulates tight junction resealing in epithelia. J Cell Sci 113: 23632374, 2000.[Abstract/Free Full Text]
- Mankertz J, Tavalali S, Schmitz H, Mankertz A, Riecken EO, Fromm M, and Schulzke JD. Expression from the human occludin promoter is affected by tumor necrosis factor alpha and interferon gamma. J Cell Sci 113: 20852090, 2000.[Abstract/Free Full Text]
- McCarthy KM, Skare IB, Stankewich MC, Furuse M, Tsukita S, Rogers RA, Lynch RD, and Schneeberger EE. Occludin is a functional component of the tight junction. J Cell Sci 109: 22872298, 1996.[Abstract/Free Full Text]
- McCormack SA and Johnson LR. Role of polyamines in gastrointestinal mucosal growth. Am J Physiol Gastrointest Liver Physiol 260: G795G806, 1991.[Abstract/Free Full Text]
- McCormack SA, Wang JY, and Johnson LR. Polyamine deficiency causes reorganization of F-actin and tropomyosin in IEC-6 cells. Am J Physiol Cell Physiol 267: C715C722, 1994.[Abstract/Free Full Text]
- Milks LC, Conyers GP, and Cramer EB. The effect of neutrophil migration on epithelial permeability. J Cell Biol 103: 27292738, 1986.[Abstract]
- Mitic LL and Anderson JM. Molecular architecture of tight junctions. Annu Rev Physiol 60: 121142, 1998.[CrossRef][ISI][Medline]
- Nusrat A, Chen JA, Foley CS, Liang TW, Tom J, Cromwell M, Quan C, and Mrsny RJ. The coiled-coil domain of occludin can act to organize structural and functional elements of the epithelial tight junction. J Biol Chem 275: 2981629822, 2000.[Abstract/Free Full Text]
- Nusrat A, Eichel-Streiber C, Turner JR, Verkade P, Madara JL, and Parkos CA. Clostridium difficile toxins disrupt epithelial barrier function by altering membrane microdomain localization of tight junction proteins. Infect Immun 69: 13291336, 2001.[Abstract/Free Full Text]
- Nusrat A, Parkos CA, Verkade P, Foley CS, Liang TW, Innis-Whitehouse W, Eastburn KK, and Madara JL. Tight junctions are membrane microdomains. J Cell Sci 113: 17711781, 2000.[Abstract/Free Full Text]
- Patel AR and Wang JY. Polyamines modulate transcription but not posttranscription of c-myc and c-jun in IEC-6 cells. Am J Physiol Cell Physiol 273: C1020C1029, 1997.[Abstract/Free Full Text]
- Peng HF and Jackson V. In vitro studies on the maintenance of transcription-induced stress by histones and polyamines. J Biol Chem 275: 657668, 2000.[Abstract/Free Full Text]
- Pollard KJ, Samuels ML, Crowley KA, Hansen JC, and Peterson CL. Functional interaction between GCN5 and polyamines: a new role for core histone acetylation. EMBO J 18: 56225633, 1999.[Abstract/Free Full Text]
- Quaroni A, Wands J, Trelstad RL, and Isselbacher KJ. Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria. J Cell Biol 80: 248265, 1979.[Abstract]
- Ranaldi G, Marigliano I, Vespignani I, Perozzi G, and Sambuy Y. The effect of chitosan and other polycations on tight junction permeability in the human intestinal Caco-2 cell line. J Nutr Biochem 13: 157167, 2002.[CrossRef][ISI][Medline]
- Rao JN, Li J, Li L, Bass BL, and Wang JY. Differentiated intestinal epithelial cells exhibit increased migration through polyamines and myosin II. Am J Physiol Gastrointest Liver Physiol 277: G1149G1158, 1999.[Abstract/Free Full Text]
- Rao JN, Li L, Golovina VA, Platoshyn O, Strauch ED, Yuan JX, and Wang JY. Ca2+-RhoA signaling pathway required for polyamine-dependent intestinal epithelial cell migration. Am J Physiol Cell Physiol 280: C993C1007, 2001.[Abstract/Free Full Text]
- Rao JN, Platoshyn O, Li L, Guo X, Golovina VA, Yuan JX, and Wang JY. Activation of K+ channels and increased migration of differentiated intestinal epithelial cells after wounding. Am J Physiol Cell Physiol 282: C885C898, 2002.[Abstract/Free Full Text]
- Rao RK, Basuroy S, Rao VU, Karnaky KJ Jr, and Gupta A. Tyrosine phosphorylation and dissociation of occludin-ZO-1 and E-cadherin-
-catenin complexes from the cytoskeleton by oxidative stress. Biochem J 368: 471481, 2002.[CrossRef][ISI][Medline]
- Ray RM, Viar MJ, Yuan Q, and Johnson LR. Polyamine depletion delays apoptosis of rat intestinal epithelial cells. Am J Physiol Cell Physiol 278: C480C489, 2000.[Abstract/Free Full Text]
- Rothen-Rutishauser B, Riesen FK, Braun A, Gunthert M, and Wunderli-Allenspach H. Dynamics of tight and adherens junctions under EGTA treatment. J Membr Biol 188: 151162, 2002.[CrossRef][ISI][Medline]
- Saitou M, Fujimoto K, Doi Y, Itoh M, Fujimoto T, Furuse M, Takano H, Noda T, and Tsukita S. Occludin-deficient embryonic stem cells can differentiate into polarized epithelial cells bearing tight junctions. J Cell Biol 141: 397408, 1998.[Abstract/Free Full Text]
- Sakakibara A, Furuse M, Saitou M, Ando-Akatsuka Y, and Tsukita S. Possible involvement of phosphorylation of occludin in tight junction formation. J Cell Biol 137: 13931401, 1997.[Abstract/Free Full Text]
- Suh E and Traber PG. An intestine-specific homeobox gene regulates proliferation and differentiation. Mol Cell Biol 16: 619625, 1996.[Abstract]
- Tabor CW and Tabor H. Polyamines. Annu Rev Biochem 53: 749790, 1984.[CrossRef][ISI][Medline]
- Tsukamoto T and Nigam SK. Role of tyrosine phosphorylation in the reassembly of occludin and other tight junction proteins. Am J Physiol Renal Physiol 276: F737F750, 1999.[Abstract/Free Full Text]
- Vielkind U and Swierenga SH. A simple fixation procedure for immunofluorescent detection of different cytoskeletal components within the same cell. Histochemistry 91: 8188, 1989.[CrossRef][ISI][Medline]
- Wang JY and Johnson LR. Luminal polyamines stimulate repair of gastric mucosal stress ulcers. Am J Physiol Gastrointest Liver Physiol 259: G584G592, 1990.[Abstract/Free Full Text]
- Wang JY and Johnson LR. Polyamines and ornithine decarboxylase during repair of duodenal mucosa after stress in rats. Gastroenterology 100: 333343, 1991.[ISI][Medline]
- Wang JY, McCormack SA, and Johnson LR. Role of nonmuscle myosin II in polyamine-dependent intestinal epithelial cell migration. Am J Physiol Gastrointest Liver Physiol 270: G355G362, 1996.[Abstract/Free Full Text]
- Wang JY, Wang J, Golovina VA, Li L, Platoshyn O, and Yuan JX. Role of K+ channel expression in polyamine-dependent intestinal epithelial cell migration. Am J Physiol Cell Physiol 278: C303C314, 2000.[Abstract/Free Full Text]
- Wittchen ES, Haskins J, and Stevenson BR. Protein interactions at the tight junction: actin has multiple binding partners, and ZO-1 forms independent complexes with ZO-2 and ZO-3. J Biol Chem 149: 3517935185, 1999.[CrossRef]
- Wong V and Gumbiner BM. A synthetic peptide corresponding to the extracellular domain of occludin perturbs the tight junction permeability barrier. J Cell Biol 136: 399409, 1997.[Abstract/Free Full Text]
- Wu Z, Nybom P, and Magnusson KE. Distinct effects of Vibrio cholerae haemagglutinin/protease on the structure and localization of the tight junction-associated proteins occludin and ZO-1. Cell Microbiol 2: 1117, 2000.[CrossRef][ISI][Medline]
- Youakim A and Ahdieh M. Interferon-
decreases barrier function in T84 cells by reducing ZO-1 levels and disrupting apical actin. Am J Physiol Gastrointest Liver Physiol 276: G1279G1288, 1999.[Abstract/Free Full Text]
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