|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
A dynamic ratio of the α+ and α− isoforms of the tight junction protein ZO-1 is characteristic of Caco-2 cells and correlates with their degree of differentiation
Annarita Ciana*1, Katharina Meier†1, Nicole Daum†, Stefan Gerbes‡, Michael Veith‡, Claus‑Michael Lehr† and Giampaolo Minetti*2
*Department of Biochemistry, University of Pavia, via Bassi 21, 27100 Pavia, Italy, †Biopharmaceutics and Pharmaceutical Technology, Saarland University, Campus A4 1, 66123 Saarbrcken, Germany, and ‡INMLeibniz Institute for New Materials, Campus D2 2, 66123 Saarbrcken, Germany
ZO-1 is a peripheral protein that plays a central role in the macromolecular assembly of tight junctions by interacting with integral proteins (occludin, claudins, JAMs) of the membrane of adjoining cells, with the actin cytoskeleton, and with nuclear factors. Human ZO-1 is expressed in all epithelia and some specialized endothelia as variable amounts of two related isoforms, which originate from the alternatively spliced mRNA transcripts α+ and α− and whose specific differential role is still unknown. Moreover, little is known about the timing of expression of ZO-1 isoforms at the protein and mRNA level. This study shows that during growth of freshly plated Caco-2 cells, the α+/α− ratio increased as a result of simultaneous increase of α+ and decrease of α−. Differences in the isoform ratio also correlated with differences in epithelium differentiation. This was determined by aminopeptidase N measurements of cells grown on conventional substrates and on modified, micro/nano-patterned surfaces. A comparable shift of ZO-1 isoforms was not observed in other tumour cell lines of non-intestinal origin (A549, Calu-3). Pancreatic stem cells, propagated without exogenous differentiation stimuli, displayed a slight, stable prevalence of the α− isoform. Of the intestinal cell lines examined (Caco-2 and T84), only Caco-2 cells displayed a dramatic shift in isoform expression. This suggests that this tumour cell line retains to a higher degree a developmental programme related to the dynamic of enterocytic differentiation in vivo.
Key words: Caco-2, endothelial cell line, epithelial cell line, pancreatic stem cell, zonula occludens-1 (ZO-1) isoform
Abbreviations: APN, aminopeptidase N, BrdU, 5′-bromo-2′-deoxy-uridine, DMEM, Dulbecco’s modified Eagle’s medium, FBS, fetal bovine serum, GPTS, (3-glycidyloxypropyl)trimethoxysilane, MAGUKs, membrane-associated guanylate kinase homologues, PET, polyethylene therephtalate, PSC, pancreatic stem cells, RT, reverse transcription, SiO2, silica, TEER, transepithelial electrical resistance, TJ, tight junctions, ZO-1, zonula occludens-1
1Annarita Ciana and Katharina Meier contributed equally to this work.
2To whom correspondence should be addressed (email email@example.com).
Of the approximately 200 different cell types of the human body, more than 60% are epithelial. Epithelial cells interconnect through a series of junctional complexes [TJ (tight junctions), adherens junctions and gap junctions, desmosomes] that regulate barrier function and differentiation of epithelium. TJ are located at the apex of the cell and are composed of more than 40 different types of proteins. ZO-1 (zonula occludens-1), a peripheral protein of approximately 220 kDa, is found at the centre of the macromolecular assembly of the TJ (Feldman et al., 2005). It is localized at the cytoplasmic side of the TJ and belongs to the family of multidomain proteins known as MAGUKs (membrane-associated guanylate kinase homologues). All these have in common the presence of an SH3 domain, a guanylate kinase homology domain, GK (Anderson et al., 1995) and at least one PDZ domain. These are 80–90 residue domains originally identified in the postsynaptic density protein PSD95/SAP90 (Cho et al., 1992), in the Drosophila tumour-suppressor protein dlg-A and in ZO-1 (hence the acronym PDZ). They are frequently found in membrane-associated proteins that play a role as adapters and molecular organizers in apical and basolateral junctional complexes. Thus, ZO-1 appears to have both a structural role by binding to the integral membrane proteins of adjoining cells [occludin, claudins, JAMs (junction adhesion molecules)] and to the cell cytoskeleton (actin) (Fanning et al., 1998) and a regulatory role by modulating cell proliferation through association with the transcription factor ZONAB (Balda et al., 2003). The gene coding for ZO-1 (TJP1) in the human genome maps in chromosome 15 at 15q13 and its primary transcript is subject to alternative splicing. The two protein isoforms translated from the mature mRNAs differ in the presence of an 80-residue insert in the internal region of the sequence in ZO-1 α+ and its absence in ZO-1 α− (Willott et al., 1992).
Although, to date, there is no information on possibly different functions of the two isoforms, their relative abundance appears to be tissue-specific and related to the junctional ‘plasticity’, i.e. the ability of the junction to open and reseal in response to certain stimuli. There is no correlation, however, between the ratio of expression of the two forms and the TEER (transepithelial electrical resistance) of the epithelium, since a wide range of electrical resistance is recorded in epithelia that express predominantly either isoform (Balda and Anderson, 1993).
Caco-2 is a cell line established in the 1970s from a human colon adenocarcinoma. The peculiar property of Caco-2 cells is their ability to undergo spontaneous differentiation during long-term culture in vitro, forming a functional monolayer that expresses a wide range of biochemical and morphological features of enterocytes of adult small intestine (Pinto et al., 1983). Therefore, Caco-2 cells are widely used as a biotechnological tool in many areas from pharmacology to toxicology to nutrition research (Artursson et al., 2001; Chen et al., 2002; Sambuy et al., 2005).
In most studies, the expression of ZO-1 isoforms has been characterized at fixed time points for a given cell type or tissue, usually at relatively late stages of growth and differentiation. As a result of this static representation, the ratio α+/α− has been proposed as a characteristic, invariant parameter for the cell type under study (Willott et al., 1992). This work, however, evaluated the dynamic expression of ZO-1 isoforms, at both the protein and mRNA levels, during in vitro proliferation of cells. Besides Caco-2 cells and other tumour epithelial cell lines of intestinal (T84) and pulmonary origin (A549 and Calu-3), also PSCs (pancreatic stem cells) and the endothelial cell line ECV304 were investigated. Caco-2 cells displayed a pronounced shift in isoform expression from ZO-1 α− to ZO-1 α+ during the exponential phase of cell growth that translated into an increasing α+/α− ratio, levelling off around cell confluence. This dynamic isoform expression was also evident at the mRNA level, suggesting that the process is transcriptionally regulated. The shift in expression was delayed when cells were cultured on modified surfaces. These surfaces partially inhibited spontaneous differentiation, as determined by measuring the APN (aminopeptidase N) brush border enzyme activity. None of the other examined cell types displayed such a dynamic isoform expression as Caco-2. This suggests that Caco-2, although being a tumour cell line, retains an articulated developmental program that is related to the dynamics of enterocytic differentiation in vivo.
2. Materials and methods
2.1. Cell culture and experimental setup
Caco-2 cells (ATCC-No: HTB-37; p. 36–52) were maintained in RPMI-1640 medium with stable l-glutamine (Euroclone-Celbio), 10% FBS (fetal bovine serum; Cambrex Bioscience) and were routinely subcultured twice a week with a seeding density of 2.5×104 cells/cm2. A549 cells (ATCC-No: CCL-185; p. 88–109) were maintained in RPMI-1640–high glucose with l-glutamine (GIBCO Invitrogen) supplemented with 10% FBS and were routinely subcultured once a week in a ratio of 1:50. Calu-3 cells (ATCC-No: HTB-55; p. 31–50) were maintained in RPMI-1640–high glucose with l-glutamine (PAA) supplemented with 10% FBS GOLD (PAA) and 1 mM sodium pyruvate (Lonzy), and were subcultured once a week in a ratio of 1:7.5. T84 cells (ATCC-No: CCL-248; p. 60–66) were maintained in DMEM (Dulbecco’s modified Eagle’s medium)/F12 (GIBCO Invitrogen) supplemented with 5% FBS (PAA), and were routinely subcultured once a week in a ratio of 1:5. The adult human PSC, isolation Cepan 3b (p. 17 and 18), were a gift from Fraunhofer Institut St. Ingbert, Germany. Cell isolation was performed as described earlier (Kruse et al., 2004). PSC were maintained in DMEM supplemented with 10% FBS GOLD, 100 units/ml penicillin and 100 μg/ml streptomycin (all from PAA). PSC were subcultured once a week and seeded at a density of 6.5×103 cells/cm2. ECV304 cells (ATCC-No: CRL-1998; p. 133–150) were maintained as described for Caco-2 and subcultivated twice a week at a ratio of 1:10. All cell cultures were maintained at 37°C and 5% CO2 and were regularly tested for mycoplasm infection. For setup of experiments, all cells were seeded at a starting density of 3×104 cells/cm2, except for ECV304, which were seeded at 8×103.
For investigating the substrate influence on Caco-2, the surfaces of polystyrene Petri dishes (Corning-Celbio), of standard microscopy glass slides and of glass slides coated with a patterned nanocomposite layer (GPTS04p) (see below), were divided among eight culture chambers of 0.9 cm2 by a reusable silicone insert (FlexiPERM®-slide, Sigma–Aldrich). Alternatively, Caco-2 cells were seeded on 24-well polystyrene plates or on PET (polyethylene therephtalate) 12-well permeable inserts (ThinCert™, cod. 665641, Greiner Bio-One GmbH). For comparative study of ZO-1 expression in different cell lines and stem cells, experiments were carried out in 12-well plates, or for determination of TEER, in Transwell® 12-well permeable supports (all from Corning).
2.2. GPTS04p slides
GPTS04 was developed by INM for use in cell cultures as a non-fluorinated material for patterned coatings in which SiO2 (silica) particles are incorporated for enhancing scratch resistance. First, the SiO2 particles were synthesized according to the Stöber method (Stöber and Fink, 1968). Subsequently, the particles [BET: 200 m2/g, diameter (d50): 7.7 nm] were modified with GPTS [(3-glycidyloxypropyl)trimethoxysilane, Brenntag] in order to obtain epoxidic functions on the particle surface, which ensure the incorporation into the coating. In the next step, a suspension of the SiO2 particles in n-propanol, GPTS and MTEOS (methyl-triethoxysilane, Fluka) and water were mixed and stirred for 18 h at 85°C. After cooling down to room temperature, Polypox R20 (trimethylolpropantriglycidylether, UPPC) was added, and the mixture was stirred for another 15 min. Next, the photoinitiator Cyracure UV-I 6976 (Dow Chemicals) was added, and after mixing at room temperature, in the absence of UV light, the coating material was ready for application.
In order to obtain patterned surfaces (samples GPTS04p), a cleaned glass slide was flooded with the GPTS04 varnish. Then, a stamp made from silicone rubber (Elastosil RT 604, Wacker Chemie) was pressed on to the wet coating on the slides. The coating with the stamp on top of it was dried for 3 min at 90°C followed by photocuring by UV light for 5 min from underneath. After removing the stamp, the slides were heated at 130°C for 4 min in order to evaporate the remaining solvent. The patterns on the samples consist of eight fields of parallel grooves with pitches of 1, 2, 4 and 6 μm; the corresponding widths of the grooves were 0.5, 1, 2 and 4 μm.
GPTS04p slides and control, uncoated glass slides were sterilized by immersion in 70% ethanol for 20 min and allowed to dry in a laminar flow hood. FlexiPERM®-slide inserts were then mounted on top of the slides making sure that each of the eight patterned fields of the GPTS04p slides fitted with each of the eight FlexiPERM®-slide chambers.
2.3. Assays for cell number and proliferation
At each time point of cell growth, a number of wells of the cell culture chambers were dedicated to the accurate determination of cell number for the various samples. Cell number was measured by two assays, based on dye fluorescence enhancement upon binding to cellular nucleic acids. In both assays, cells were at first treated with an identical lysis buffer before the fluorescent dye was added. For the Hoechst 33258 assay (used with Caco-2 and EVC304 cells), adherent cells were washed once with PBS and lysed in 75 μl of 50% (v/v) CelLytic™-M (Sigma–Aldrich) solution in water, at 37°C for 45 min. Cell lysates were resuspended in 9 vol. of 5 μg/ml Hoechst 33258 dye (Sigma–Aldrich) solution by repeated pipetting and read within 25 min at 360/465 nm in a fluorescence microplate reader (GENios Plus, Tecan). The dye solution was freshly prepared by stepwise addition of 1 mg/ml dye stock solution to prewarmed 2×TNE (20 mM Tris/HCl, 4 M NaCl, 2 mM EDTA, pH 7.4) and subsequently diluting TNE with water to the final 1× concentration. The CyQuant assay (used with all other cell types) was slightly adapted from the manufacturer’s manual (CyQuant Cell Proliferation Assay Kit, Invitrogen). Instead of repeated freeze and thaw cycles for inducing cell lysis, cells were incubated with the same lysis buffer used for the Hoechst assay. For both assays, a calibration was performed by including standard DNA in the same microplate as the unknown samples. For the Hoechst assay, a calf thymus standard DNA (Sigma–Aldrich) was used, while the DNA standard supplied by the manufacturer was applied for CyQuant. A conversion factor between DNA amount and cell number was obtained by comparing the fluorescence of standard DNA with that of cell lysates obtained from cell suspensions of accurately determined cell number.
Cell proliferation was followed by measuring the incorporation of BrdU (5′-bromo-2′-deoxy-uridine) into newly synthesized DNA of replicating cells, by using the Cell Proliferation ELISA, BrdU (colorimetric) assay by Roche, according to the manufacturer’s manual. In short, cells were incubated for 24 h with BrdU labelling solution, fixed, incubated with anti-BrdU–peroxidase solution and then with the substrate solution for 15 min. The reaction was stopped by addition of a stopping solution and absorbance at 450 nm was measured.
2.4. Assay for Caco-2 cell differentiation
Caco-2 differentiation was evaluated by APN activity assay (Stefanovic et al., 1992). At the indicated times, cells adherent on different types of surfaces in Flexiperm-slide inserts were washed once in PBS and incubated with 1.5 mM Ala-p-nitroanilide substrate in Hepes buffer (20 mM Hepes, 150 mM NaCl, pH 7.4) at 37°C for 2 h (0.45 ml of substrate solution/cm2). Aliquots (100 μl) of the incubated solution in triplicate were aspirated from two wells for each type of surface and transferred in a microplate reader, where the p-nitroanilide released by APN was measured by reading the absorbance at 405 nm, in parallel with a standard curve of p-nitroanilide (0.05–0.8 mM). APN activity is expressed as mUnits/cm2. One unit is defined as the activity that releases one micromole per minute of p-nitroanilide from Ala-p-nitroanilide, at 37°C in Hepes buffer pH 7.4.
TEER was measured with an epithelial volt–ohmmeter (EVOM, World Precision Instruments). TEER of plain filters was subtracted as background.
2.6. Western blotting
Adherent cells were preincubated with 5 mM di-isopropylfluorophosphate in PBS (0.3 ml/cm2) for 5 min and subsequently dissolved in 0.5 vol. of SDS/PAGE sample buffer [50 mM Tris/HCl, 5% (w/v) SDS, 35% (w/v) sucrose, 5 mM EDTA, 200 mM dithiothreitol and 0.01% Bromophenol Blue, pH 6.8] by repeated pipetting. Samples were incubated at 60°C for 15 min and subjected to SDS/PAGE (Laemmli, 1970) in 8% acrylamide gels. Sample volumes corresponding to 2.0×104 cells were loaded in each lane, according to the quantification of cell number carried out on at least two culture wells for each substrate, as described in section 2.3. In order to improve the separation of the two ZO-1 isoforms, the electrophoretic run was prolonged for 1 h after the front dye had reached the bottom of the gel. Samples were electrotransferred to a 0.2-μm PVDF membrane, taking the gels as a whole (Towbin et al., 1979) or performing a multistrip Western blotting (Aksamitiene et al., 2007). PVDF membranes were blocked with 20 mM Tris/HCl, 150 mM NaCl, 0.05% Tween-20, and 5% skimmed milk (w/v), pH 7.4, incubated overnight at 4°C with primary antibody, and revealed by chemiluminescent detection (ECL kit Amersham, GE Healthcare) after incubation with the proper secondary antibody. Primary antibodies used were mouse monoclonal anti-ZO-1 (Becton Dickinson Italia) and rabbit polyclonal anti-occludin (Zymed Laboratories, Invitrogen). The ZO-1 isoform ratio was calculated after densitometric quantification of the protein bands on a digital image of the films, using the software Scion Image (Scion Corporation).
2.7. RT (reverse transcription)-PCR
In parallel with cell sampling for Western blotting, mRNA was isolated by use of RNeasy Mini Kit (Qiagen). Alternatively, total RNA was extracted from Caco-2 and ECV304 cells with 1 ml of TRIzol® (Invitrogen) for not more than 2×106 cells. First-strand cDNA was synthesized using Omniscript RT Kit (Qiagen) or by the High-Capacity cDNA Archive Kit (Applied Biosystems), according to the supplier’s recommendations.
PCR was carried out with Taq PCR Master Mix Kit (Qiagen) in a final volume of 50 μl containing 25 pmol of each nucleotide primer. For specific amplification of ZO-1 and β-actin, Taq PCR Master Mix Kit was supplemented with 25 mM MgCl2 to yield a final concentration of 2.5 mM MgCl2 in the reaction mixture. PCR of ZO-1 isoforms was performed with primers flanking the α motif, as in Willott et al. (1992). Amplification products of 474 and 234 bp correspond to ZO-1 α+ and ZO-1 α− mRNA, respectively (Table 1). One PCR cycle was 94°C for denaturation, 60°C for annealing and 72°C for extension, with a total of 40 cycles for ZO-1 and 30 cycles for β-actin. For occludin, the PCR cycle was 95°C for denaturation, 52°C for annealing and 72°C for extension in a total of 35 cycles.
Table 1 Specific primers used for RT-PCR of ZO-1 α− (234 bp) and α+ (474 bp) isoforms, occludin and β-actin
2.8. Statistical analysis
Statistical comparison of means was performed using unpaired two-tailed Student’s t tests. Significant differences between samples are indicated by one (P<0.05) or two (P<0.005) asterisks.
3.1. Shift in the expression of ZO-1 isoforms from α− to α+ during Caco-2 cell growth on solid and permeable substrates
Caco-2 cells seeded on two different solid surfaces, standard cell culture polystyrene and borosilicate glass, displayed superimposable logistic curves of cell growth (Figure 1A) over a period of 20 days. At different time points, cells were subjected to Western blotting for immunodetection of ZO-1 with an antibody that recognizes both α+ and α− isoform of the protein. A progressive decrease in the signal associated with the α− isoform and a parallel increase in the α+ signal was observed. This change in relative amounts of the isoform was comparable for Caco-2 cells cultured on polystyrene and glass (Figure 1B).
Transport in Caco-2 cells grown on non-porous substrates does not take place to the same extent as transepithelial transport does when cells are cultured on permeable supports, which allow for the formation of a correctly polarized cell layer. In order to determine whether the observed isoform shift was due to the culture conditions, Caco-2 cells were grown for 24 days on PET permeable inserts, which divide the culture dish into an apical and a basolateral compartment. The shift in expression of ZO-1 isoforms was observed also for Caco-2 cells grown under these conditions (Figure 1C). As a second key component of TJ, also occludin was examined, and its amount was shown to be relatively constant starting from day 9 on (Figure 1C).
3.2. Dynamic expression of ZO-1 protein isoforms in Caco-2 is paralleled by a corresponding shift at the mRNA level
In order to determine whether the gradual transition in the expression of the two protein isoforms was paralleled by a corresponding shift at the mRNA level, RT-PCR was carried out over a period of 16 days with primers discriminating between the two isoforms. At each time point, both isoforms were present even though the α− isoform was prevailing initially. As cells proliferated, the expression of α− decreased and that of α+ increased, so that the latter became the most expressed form in differentiated cells. This development took place in parallel at both the protein (Figure 1D, WB) and the mRNA levels (Figure 1D, mRNA).
3.3. Dynamics of expression of ZO-1 isoforms in other human epithelial tumour cell lines, PSC and in the endothelial cell line ECV304
For investigating whether the differential expression of ZO-1 isoforms was a peculiar feature of Caco-2 cells or whether it took place also in other tumour cell lines, of colonic and non-colonic origin, the following human cell types were compared with Caco-2: the tumour cell line T84, derived from the metastatic site (lung) of a colon adenocarcinoma; A549 cells, derived from an adenocarcinoma of the lung and displaying characters of the alveolar epithelium; Calu-3, derived from the pleural effusion of a lung adenocarcinoma and corresponding to a more bronchiolar cell type; PSC, as a model of undifferentiated human adult stem cells and ECV304 derived from a human umbilical cord transformed endothelium.
Cells were grown on standard polystyrene surfaces and analysed at intervals, starting from day 2 to day 16 postseeding, by means of Western blotting and RT-PCR for analysis of the content of the isoforms at protein (Figure 2A) and mRNA levels (Figure 2B). To evaluate whether other TJ proteins showed changes in expression levels during epithelial differentiation, the mRNA for occludin was also examined.
In all cell lines, except for Caco-2, the ZO-1 isoforms were expressed in relatively constant amounts, both at the protein and the mRNA levels (Figures 2A, 2B). For the other colon-derived cell line, T84, a slight decrease of α− at later time points was detected, while the expression of α+ was already stable at a high level from day 2. In cell lines of lung origin (A549 and Calu-3) ZO-1 α+ was the prevailing isoform throughout the whole period examined, with both isoforms being expressed at constant levels. Interestingly, PSC displayed a prevalence of ZO-1 α−, both at protein and mRNA levels, which remained constant during the observation period (Figures 2A and 2B).
All epithelial cell lines were found to express mRNA encoding for occludin without an apparent relationship to the amount of ZO-1 isoforms. While occludin mRNA was constant for most cell lines, a distinct increase was shown for Calu-3, and no band was detected in PSC at all (Figure 2B).
In order to investigate whether expression of isoforms was correlated with cell density, proliferating activity or tightness of cell layer, assays for determination of cell number and proliferation and measurements of TEER were performed.
For the epithelial cell lines, the number of cells increased strongly in the beginning of the growth period and levelled off from day 7 for A549 and Calu-3 and from day 11 for Caco-2 and T84 (Figure 2C). Although total proliferation as determined by BrdU assay increased permanently throughout cell growth, proliferation related to number of cells decreased strongly after the first sampling point (Figure 2D). TEER values for Caco-2 increased up to day 11 and weakened slightly thereafter (Figure 2E). Calu-3 showed a very strong and constant increase from day 7 on. A549 and T84 displayed no significant increase. PSC showed no significant change in cell number, proliferation or TEER values (Figures 2C, 2D and 2E).
Endothelia in mouse tissues are reported to express the α− isoform only or predominantly (Balda and Anderson, 1993). Therefore, this study investigated ZO-1 isoform expression in ECV304 cells as a model of endothelium. It was found that ECV304 do in fact express the α− isoform only at both the protein and the mRNA levels even after 16 days of culture (Figures 2F, 2G and 2H).
3.4. ZO-1 isoform ratio α+/α− in Caco-2 correlates with cell differentiation and is influenced by substrate micropatterning
It is well known that the composition of the substrate used for cell culture can influence cell differentiation (Piana et al., 2007; Piana et al., 2008). This study investigated whether a coating with embossed line patterns of increasing nano/micrometric width and pitch (GPTS04p) had an impact on differentiation of Caco-2 cells and whether this influence correlated with the expression of ZO-1 isoforms. Caco-2 grown on polystyrene and glass substrates served as controls. Cell number was determined by the Hoechst assay. Differentiation was evaluated by measuring the activity of APN, an enterocyte brush border hydrolase whose activity is commonly used as a marker of Caco-2 cell differentiation (Pinto et al., 1983). Finally, expression of ZO-1 isoforms was determined by Western blotting.
Caco-2 cells grew to a comparable extent on all substrates for at least 14 days (Figure 3A), while their differentiation was significantly lower on microstructured substrates independent on the pattern size (Figure 3B). At all time points, ZO-1 Western blotting was also performed to quantify by densitometry the α+/α− ratios, which are displayed in the graph of Figure 3(D). A representative Western blot for day 14 is shown in Figure 3(C). It is clearly visible that expression of α+ in cells grown on GPTS04p patterned substrates was much lower than in cells grown on control substrates. Statistically significant lower α+/α− ratios at days 11, 14 and 17 were determined for GPTS04p substrates compared with controls, thus demonstrating that a correlation exists between the differentiation level and the ZO-1 α+/α− ratio in Caco-2 cells.
In the present work, the dynamics of expression of the two isoforms, α+ and α−, of TJ protein ZO-1, in a number of human cell lines of intestinal and non-intestinal origin and in human PSC were examined. A transition in the expression of ZO-1 isoforms from α− to α+ was particularly evident in Caco-2 cells. The strongest shift was visible at relatively early stages of growth in vitro before reaching full cell differentiation. The transition occurred to a similar extent in cells grown on permeable membranes and on solid polystyrene or glass surfaces. The change in isoform expression was detected by means of Western blotting and RT-PCR at the protein and mRNA level, respectively. This proves that the shift was related to protein turnover regulated at the level of transcription and not the result of post-translational events. Despite the large amount of information available on Caco-2 cells and on ZO-1 isoform expression in various tissues and cell lines, the evidence of a time-dependent shift in isoform expression was not reported previously. So far, only a tissue specificity with prevalence of either ZO-1 isoform has been described for a given type of endothelial or epithelial cells (Willott et al., 1992; Balda and Anderson, 1993). ZO-1 α+ was found to be present in most epithelial TJs and ZO-1 α− as the prevailing form in various types of endothelia.
ZO-1 has the role of a junctional adaptor within TJ, where it establishes contacts between integral membrane proteins on the one side, and cytoplasmic proteins and the actin cytoskeleton, on the other. However, a previous study of the mouse epithelial cell line Eph4 revealed that suppression of ZO-1 biosynthesis did not significantly affect the formation of TJs (Umeda et al., 2004). This was ascribed to a certain functional redundancy of MAGUK proteins in epithelial cells. But more recent evidence with the same cell line has confirmed the essential role of ZO proteins in the assembly of the TJ once more. In particular, it proved to be pivotal for recruitment and polymerization of claudins, as the simultaneous suppression of ZO-1, ZO-2 and ZO-3 expression resulted in the complete absence of TJs (Umeda et al., 2006). Furthermore, ZO-1 was found to be located in the nucleus (Willott et al., 1992), although not all investigators detected a nuclear pool (Matter and Balda, 2007). Studies aimed at defining the role for conserved protein-binding domains in ZO-1 revealed their important role in the binding to cytoskeletal proteins (Fanning et al., 2002), for the targeting of ZO-1 to the TJ and for controlling the location of other TJ and membrane proteins (Fanning et al., 2007; Ikenouchi et al., 2007). Focusing on the differential expression of the two isoforms, they were found to co-localize at the level of the membrane (Willott et al., 1992). Further investigation showed, in accordance with a hypothesis of the Anderson group (Balda and Anderson, 1993), a possible co-localization of ZO-1 α+ with F-actin and of ZO-1 α− with G-actin in guinea pig testis (Pelletier et al., 1997). For non-epithelial cells, where ZO-1 participates in the cadherin-mediated cell adhesion, it is not known yet whether it is expressed as one prevailing isoform (Itoh et al., 1993). However, no evidence about the role of the ZO-1 α-motif has emerged from these important studies.
The expression of relatively high levels of the α− isoform, as occurring in endothelia, was related to the structural plasticity of the junction. This is the “capacity to actively open and reseal the intercellular space or to move within the plane of the plasma membrane during normal physiological activity” (Balda and Anderson, 1993). No correlation, however, was found between the ratio of expression of the two ZO-1 isoforms and the TEER of the various epithelia examined, since a wide range of electrical resistance is recorded in epithelia that express predominantly either isoform. Vascular endothelium and epithelial glomerular podocytes of the kidney are characterized by a high, although finely regulated, permeability and thus lower TEER. In these tissues, ZO-1α− prevails. This is also true for the endothelium of some brain capillaries, which is, however, characterized by higher resistance (Balda and Anderson, 1993). A more recent study has shown that, in response to treatment with vascular endothelial growth factor, the expression of ZO-1 α+ and of ZO-1 α− is down-regulated in vascular endothelial cells, while it is up-regulated in retinal pigmented epithelial cells. In both cell types, the changes in ZO-1 levels positively correlated with changes in TEER values. However, again, no indication of a differential role of the two isoforms was obtained, since their levels altered simultaneously and in parallel in response to the treatment (Ghassemifar et al., 2006).
In order to investigate whether the dynamic ZO-1 isoform expression is unique for Caco-2, the present work evaluated the ZO-1 α+/α− ratio in different epithelial tumour cell lines and in undifferentiated stem cells with respect to cell density, cell proliferation, cell confluence, TEER and transcriptional expression of occludin, as one of the physiologic ligands for ZO-1 in the plasma membrane. However, none of these parameters was found to correlate with ZO-1 isoform expression, confirming previous reports (Balda and Anderson, 1993). While a marked increase in ZO-1 α+/α− ratio was observed during Caco-2 growth, other tumour cell lines of intestinal and lung origin displayed a constant ZO-1 α+/α− ratio and a prevalence of the α+ isoform starting from the first days of culture. PSC also displayed a constant ZO-1 isoform ratio, but with α− as the prevailing form. The question as to the differential role of the two ZO-1 isoforms is thus still open.
The endothelial cell line ECV304, originating from human umbilical vein endothelial cells by spontaneous transformation, expresses endothelial markers and is considered a good model of endothelium (Suda et al., 2001). However, their genotype was shown to be identical with urinary bladder carcinoma cell line T24 (Brown et al., 2000). Thus, this cell line is also discussed as a derivative of T24 cells. In this study, ECV304 expressed a constant amount of α− isoform, both at the mRNA and the protein level, during 16 days of culture. The permanent and exclusive expression of this isoform supports the definition of ECV304 as endothelial cells.
A number of works (reviewed in Eckert and Fleming, 2008) studied ZO-1 isoforms in ontogenesis. The temporal and spatial isoform expression was investigated at different stages of preimplantation in the mouse embryo. ZO-1 α− was present in oocytes and all preimplantation stages, while α+ transcripts were detected in co-localization with occludin in embryos not before the late morula stage. Therefore, ZO-1 α+ expression was understood as a relatively late and essential step in the maturation of fully functional TJs that ensures a proper paracellular seal (Sheth et al., 1997; Eckert and Fleming, 2008). The prevailing expression of the α− isoform in the investigated PSC underlines these findings. All in all, these results give the impression that the α− isoform is expressed at relatively early stages of embryogenesis, in stem cells and in some tumour cells. Furthermore, it remains the most expressed isoform in those cells whose final differentiated state belongs to the group of endothelia (ECV304).
Furthermore, the presence of both isoforms in PSC can also be interpreted in a different way. As shown earlier, PSC express already in their undifferentiated state epithelial and endothelial markers at mRNA level and even show spontaneous tube formation on a semisolid matrix as a typical characteristic for endothelial cells (Meier et al., 2009). The presence of the α+ isoform is therefore consistent with the epithelial characteristics of PSC, while the dominance of the α− isoform hints at their endothelial predisposition.
Caco-2 cells are a tumour cell line of adult colonic origin, which spontaneously differentiate into a phenotype of ileal epithelium, with microvilli, polarized barrier and expression of some markers of adult enterocyte. Compared with other colon carcinoma cell lines, Caco-2 exhibit a better morphological and functional enterocytic differentiation. However, it has been suggested that this process reflects the shift from a tumour/adult colonic phenotype to a fetal colonic one that expresses many small intestinal functions. Their distinctions to the mature enterocyte may represent an incomplete conversion from the proliferative (crypt) to the differentiated (villous) phenotype (Engle et al., 1998; Sambuy et al., 2005). Recently, the patterns of gene expression during growth of Caco-2 cells have been analysed by cDNA microarrays. These investigations showed that the transition from proliferating, non-polarized cells to postmitotic polarizing cells involves a switch in gene expression programs upon formation of cell–cell contacts. The gene pattern in proliferating, non-polarized cells was highly comparable with patterns seen in human colon cancer in vivo. In polarized Caco-2 cells, however, it switched to a pattern more closely resembling that in normal colon tissue. The temporal program of gene expression involved changes in signalling pathways in patterns similar to those occurring during migration and differentiation of intestinal epithelial cells in vivo. Most remarkably, such transition in gene expression occurs despite the absence of morphogen gradients and the interaction with stromal cells, which is characteristic of enterocyte differentiation in situ along the crypt–villus axis. This highly co-ordinated transition involves genes responsible for the formation of structural and functional characteristics of polarized epithelial cells like the formation of brush border and the apical junction complex, the decrease in paracellular permeability and the expression of brush border hydrolases. The increase in APN transcripts has, in fact, also been detected during Caco-2 differentiation (Halbleib et al., 2007). The correlation found between the increase in APN activity and the increase in ZO-1 α+/α− ratio in differentiating Caco-2 cells (Figure 3) is thus reasonably explained. The dynamic ZO-1 expression described in the present work is, at the level of a single TJ protein, a further indication of the peculiar switch in gene expression program that appears to be under control of an innate timing in Caco-2 cells (Sääf et al., 2007; Halbleib et al., 2007). Moreover, for future studies, a thorough analysis of the localization of the two isoforms during enterocyte differentiation in situ is of major interest.
The question of whether the ZO-1 isoform shift was specific for intestinal cell lines was addressed here by examining the T84 line. These cells did not show a transition comparable with levels observed in Caco-2 cells. On the other hand, the dynamic isoform expression was also observed in Caco-2 cells from different subclones (not shown), making it an invariant property of this cell line. Despite the instability and the heterogeneity that in general characterize tumour cells, Caco-2 cells apparently retain to a higher degree a more articulated and autonomous differentiation program than other lines. This could be related to the level of aneuploidy of Caco-2 cells and their attainment of a particularly ‘stably unstable’ karyotype, among the human cancer cell lines (Roschke et al., 2002; Duesberg et al., 2005).
In conclusion, this is the first study to report a dynamic ZO-1 isoform expression as a time-dependent process during Caco-2 cell growth and consistent with progressing differentiation. This shift was unique for Caco-2 cells and could not be found in other epithelial cell lines of intestinal or lung origin, in endothelial cells or stem cells investigated. Caco-2 cells may therefore prove useful as an in vitro cell model for better understanding the function of ZO-1 α-motif, in terms of the central role played by this scaffold protein in the assembly of TJs.
Annarita Ciana made the original experimental observation on Caco-2 cells, designed and performed the study of ZO-1 isoforms at the protein and mRNA levels for Caco-2 cells, and at the level of protein for all other cell types, contributed to the writing of the methods, results and discussion sections. Katharina Meier performed the study of ZO-1 isoform expression at the level of mRNA for all cell types, measured the transepithelial electrical resistance, the proliferation and differentiation parameters for all cell types, contributed to the interpretation of results and in the writing of the manuscript. Nicole Daum contributed in designing and executing some of the experiments and in the interpretation of results and writing the manuscript. Stefan Gerbes prepared the micro/nanopatterned substrates used in the study and provided the material in several batches in co-ordination with the biologists. He also contributed in the revision of the manuscript. Michael Veith co-ordinated the preparation of patterned materials and contributed to the revision of the manuscript. Claus-Michael Lehr co-ordinated the experimental work carried out at his institution, contributed to the interpretation of results and writing of the manuscript. Giampaolo Minetti designed the original experiments on Caco-2 cells, performed some of the experiments, co-ordinated the work of all collaborators, interpreted the results and wrote the manuscript.
This publication was partly generated in the context of the CellPROM project, funded under the
Anderson, JM, Fanning, AS, Lapierre, L and Van Itallie, CM (1995) Zonula occludens (ZO)-1 and ZO-2: membrane-associated guanylate kinase homologues (MAGuKs) of the tight junction. Biochem Soc Trans 23, 470-5
Balda, MS, Garrett, MD and Matter, K (2003) The ZO-1-associated Y-box factor ZONAB regulates epithelial cell proliferation and cell density. J Cell Biol 60, 423-32
Brown, J, Reading, SJ, Jones, S, Fitchett, CJ, Howl, J and Martin, A (2000) Critical evaluation of ECV304 as a human endothelial cell model defined by genetic analysis and functional responses: a comparison with the human bladder cancer derived epithelial cell line T24/83. Lab Invest 80, 37-45
Chen, W, Tang, F, Horie, K and Borchardt, RT (2002) Caco-2 cell monolayers as a model for studies of drug transport across human intestinal epithelium. In Cell culture models of biological barriers, in-vitro test systems for drug absorption and delivery, In:. (Lehr CM, ed.), pp. 143-63, London, Taylor and Francis
Cho, KO, Hunt, CA and Kennedy, MB (1992) The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs–large tumor suppressor protein. Neuron 9, 929-42
Fanning, AS, Jameson, BJ, Jesaitis, LA and Anderson, JM (1998) The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton. J Biol Chem 273, 29745-53
Fanning, AS, Little, BP, Rahner, C, Utepbergenov, D, Walther, Z and Anderson, JM (2007) The unique-5 and -6 motifs of ZO-1 regulate tight junction strand localization and scaffolding properties. Mol Biol Cell 18, 721-31
Ghassemifar, R, Lai, CM and Rakoczy, PE (2006) VEGF differentially regulates transcription and translation of ZO-1alpha+ and ZO-1alpha- and mediates trans-epithelial resistance in cultured endothelial and epithelial cells. Cell Tissue Res 323, 117-25
Halbleib, JM, Sääf, AM, Brown, PO and Nelson, WJ (2007) Transcriptional modulation of genes encoding structural characteristics of differentiating enterocytes during development of a polarized epithelium in vitro. Mol Biol Cell 18, 4261-78
Ikenouchi, J, Umeda, K, Tsukita, S, Furuse, M and Tsukita, S (2007) Requirement of ZO-1 for the formation of belt-like adherens junctions during epithelial cell polarization. J Cell Biol 176, 779-86
Itoh, M, Nagafuchi, A, Yonemura, S, Kitani-Yasuda, T, Tsukita, S and Tsukita, S (1993) The 220-kD protein colocalizing with cadherins in non-epithelial cells is identical to ZO-1, a tight junction-associated protein in epithelial cells: cDNA cloning and immunoelectron microscopy. J Cell Biol 121, 491-502
Kruse, C, Birth, M, Rohwedel, J, Assmuth, K, Goepel, A and Wedel, T (2004) Pluripotency of adult stem cells derived from human and rat pancreas. Appl Phys A-Mater 79, 1617-24
Pelletier, RM, Okawara, Y, Vitale, ML and Anderson, JM (1997) Differential distribution of the tight-junction-associated protein ZO-1 isoforms α+ and α− in guinea pig Sertoli cells: a possible association with F-actin and G-actin. Biol Reprod 57, 367-76
Piana, C, Güll, I, Gerbes, S, Gerdes, R, Mills, C and Samitier, J (2007) Influence of surface modification on vitality and differentiation of Caco-2 cells. Differentiation 75, 308-17
Piana, C, Toegel, S, Guell, I, Gerbes, S, Viernstein, H and Wirth, M (2008) Growth surface-induced gene and protein expression patterns in Caco-2 cells. Acta Biomater 4, 1819-26
Pinto, M, Robine-Leon, S, Appay, MD, Kedinger, M, Triadou, N and Dussaulx, E (1983) Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol Cell 47, 323-30
Roschke, AV, Stover, K, Tonon, G, Schäffer, AA and Kirsch, IR (2002) Stable karyotypes in epithelial cancer cell lines despite high rates of ongoing structural and numerical chromosomal instability. Neoplasia 4, 19-31
Sääf, AM, Halbleib, JM, Chen, X, Yuen, ST, Leung, SY and Nelson, WJ (2007) Parallels between global transcriptional programs of polarizing Caco-2 intestinal epithelial cells in vitro and gene expression programs in normal colon and colon cancer. Mol Biol Cell 18, 4245-60
Sambuy, Y, De Angelis, I, Ranaldi, G, Scarino, ML, Stammati, A and Zucco, F (2005) The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biol Toxicol 21, 1-26
Sheth, B, Fesenko, I, Collins, JE, Moran, B, Wild, AE and Anderson, JM (1997) Tight junction assembly during mouse blastocyst formation is regulated by late expression of ZO-1 alpha+ isoform. Development 124, 2027-37
Stefanovic, V, Vlahovic, P, Ardaillou, N, Ronco, P and Ardaillou, R (1992) Cell surface aminopeptidase A and N activities in human glomerular epithelial cells. Kidney Int 41, 1571-80
Suda, K, Rothen-Rutishauser, B, Günthert, M and Wunderli-Allenspach, H (2001) Phenotypic characterization of human umbilical vein endothelial (ECV304) and urinary carcinoma (T24) cells: endothelial versus epithelial features. In Vitro Cell Dev Biol Anim 37, 505-14
Towbin, H, Staehelin, T and Gordon, J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76, 4350-4
Umeda, K, Ikenouchi, J, Katahira-Tayama, S, Furuse, K, Sasaki, H and Nakayama, M (2006) ZO-1 and ZO-2 independently determine where claudins are polymerized in tight-junction strand formation. Cell 126, 741-54
Umeda, K, Matsui, T, Nakayama, M, Furuse, K, Sasaki, H and Furuse, M (2004) Establishment and characterization of cultured epithelial cells lacking expression of ZO-1. J Biol Chem 279, 44785-94
Willott, E, Balda, MS, Heintzelman, M, Jameson, B and Anderson, JM (1992) Localization and differential expression of two isoforms of the tight junction protein ZO-1. Am J Physiol 262, C1119-24
Received 6 May 2009/22 March 2010; accepted 26 March 2010
Published as Cell Biology International Immediate Publication 26 March 2010, doi:10.1042/CBI20090067
© The Author(s) Journal compilation © 2010 Portland Press Limited
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)
Figure 2 ZO-1 isoforms expression, cell growth and proliferation, and TEER in Caco-2 compared with the tumour cell lines T84, A549 and Calu-3 and to the PSC over a period of 16 days