sPAR-3, a splicing variant of PAR-3, shows cellular localization and an expression pattern different from that of PAR-3 during enterocyte polarization

Takako Yoshii,1 Keiko Mizuno,2 Tomonori Hirose,2 Atsushi Nakajima,1 Hisahiko Sekihara,1 and Shigeo Ohno2

1The Third Department of Internal Medicine and 2Department of Molecular Biology, Yokohama City University School of Medicine, Yokohama, Japan

Submitted 29 September 2003 ; accepted in final form 2 September 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PAR-3 (partitioning-defective) is a scaffold-like PDZ (postsynaptic density-95/discs large/zonula occludens-1) domain-containing protein that forms a complex with PAR-6 and atypical PKC, localizes to tight junctions, and contributes to the formation of functional tight junctions. There are several alternatively spliced isoforms of PAR-3, although their physiological significance remains unknown. In this study, we show that one of the major isoforms of PAR-3, sPAR-3, is predominantly expressed in the Caco-2 cells derived from colon carcinoma and is used as a model to investigate the events involved in the epithelial cell differentiation and cell polarity development. During the polarization of Caco-2 cells, the expression of PAR-3 increases as do those of other cell-cell junction proteins, whereas the expression of sPAR-3 decreases. Biochemical characterization revealed that sPAR-3 associates with atypical PKC, as does PAR-3. On the other hand, immunofluorescence microscopy revealed that sPAR-3 does not concentrate at the cell-cell contact region in fully polarized cells, whereas it concentrates at premature cell-cell junctions. This makes a contrast to PAR-3, which concentrates at tight junctions in fully polarized cells. These results provide evidence suggesting the difference in the role between sPAR-3 and PAR-3 in epithelial cells.

Caco-2 cells; intestinal epithelial cells; tight junction; differentiation; atypical protein kinase C


IN MAMMALIAN SMALL intestine, enterocytes arise from a proliferative zone of undifferentiated stem cells within crypts, migrate into the intestinal lumen at the villus tip, and differentiate to the functional component during this process (2). The human colon cancer cell line Caco-2 is a well-characterized model for the study of intestinal epithelial cell differentiation. When Caco-2 cells reach confluence in vitro, they spontaneously differentiate and exhibit many characteristics of a fully differentiated small intestine epithelium, namely, growth retardation and secretion of the digestive enzymes, including alkaline phosphatase. Structural development is also observed in monolayers of Caco-2 cells in postconfluent cultures. The cells become more compact, the dome is formed, and microvilli develop on the apical side of the cells (9–11, 18, 19, 26–28, 35). These processes accompany the development of epithelium-specific cell-cell junctional structures such as tight junctions (TJs) and adherence junctions (AJs) (11, 26) and the increases in expression of junctional components, such as E-cadherin and {beta}-catenin (1, 18). Thus the differentiation of Caco-2 cells is tightly coupled to the development of apical-basal polarity of the epithelial monolayer.

A recent study (24) has identified a set of conserved proteins required for the establishment of cell polarity in a variety of biological contexts. One example is PAR-3, a scaffold-like PDZ-containing protein that forms a complex with PAR-6 and atypical PKC (aPKC). These proteins localize at TJ and are required for the maturation of epithelium-specific junctional structures (14, 15, 17, 31, 34). PAR-3 has three conserved regions (CRs), namely CR1, -2, and -3 (14). CR3 provides the binding site for aPKC (22). CR2 contains three PDZ domains, and the interaction between the PDZ domain of PAR-3 and the COOH terminus of the junctional adhesion molecule (JAM), one of the membrane proteins of TJ, is required for recruitment of PAR-3 to premature cell-cell contact regions of epithelial cells (3, 13). CR1 contributes to the self-association required for localization of PAR-3 at cell-cell contact regions during the polarization of epithelial cells (21). Mammalian PAR-3 has several alternatively spliced isoforms, and two of them, 180-kDa PAR-3 and 150-kDa sPAR-3, are expressed in a variety of cells and tissues, although the physiological significance of the variation remains unknown (4, 7, 12, 17). In this study, we show that sPAR-3, a predominant isoform expressed in Caco-2 cells, associates with aPKC as PAR-3 does. During the polarization of Caco-2 cells, however, the expression levels of PAR-3 and other junctional component proteins increase, while that of sPAR-3 decreases. Furthermore, sPAR-3 is not concentrated to TJ in polarized Caco-2 cells, whereas it is concentrated at the cell-cell contact regions during the initial phase of cell polarization, making a clear contrast to PAR-3 concentrated at TJs in fully polarized cells. These results suggest a novel role of sPAR-3 different from PAR-3, one of the components of TJs in polarized epithelial cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials. Rabbit anti-PAR-3 polyclonal antibodies were previously described (12, 14) (see also Fig. 1A). C2 was raised against GST-PAR-3-(712–936) and recognizes PAR-3 and sPAR-3 equally. PAR-3 isoform-specific antibody A2 was raised against GST-PAR-3- (1137–1337), whereas Yap, sPAR-3-specific antibody, was raised against a synthetic peptide identical to sPAR-3-specific COOH-terminal sequence. Rabbit anti-PAR-6 antibody BC42 was described previously (31). Mouse anti-aPKC-{lambda}, anti-E-cadherin, and anti-{beta}-catenin monoclonal antibodies were purchased from Transduction Laboratories. Anti-zonula occludens-1 (ZO-1) monoclonal (for immunofluorescence study) and polyclonal (for Western blot analysis) antibodies were obtained from Zymed Laboratories (South San Francisco, CA). Rabbit anti-actin antibody and mouse anti-His-tag monoclonal antibody were purchased from Biomedial Tec and Santa Cruz Biotechnology (Santa Cruz, CA), respectively.



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Fig. 1. A: domain structure of PAR-3 and a splicing variant of PAR-3 (sPAR-3). Epitopes for C2, A2, and Yap antibodies are indicated. CR, evolutionally conserved region; a.a., amino acid. B: Western blot analysis of cell lysates prepared from near-confluent Madin-Darby canine kidney (MDCK; lane 1) and Caco-2 (lane 2) cells were performed by using the indicated anti-PAR-3 antibodies. His-tagged PAR-3 (lane 3) and sPAR-3 (lane 4) expressed in COS1 cells were used as positive controls. PAR-3 isoform-specific antibodies, A2 and Yap, specifically recognized PAR-3 and sPAR-3, respectively.

 
Cell culture. Caco-2 cells were maintained at 37°C in high-glucose (25 mM) DMEM (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum under a humidified atmosphere containing 5% CO2. For the enterocytic differentiation experiments, cells were seeded at 4 x 103 cells/cm2 and the culture medium was exchanged every day after cells reached confluence. Whole cell lysates were prepared at the indicated times, and alkaline phosphatase activity was measured according to previously reported methods (26). Two epithelium-like cell lines derived from kidney cancer (DRY) and from colon cancer (CoMet) were kind gifts from Dr. Nagashima (School of Medicine, Yokohama City University, Yokohama, Japan) and were maintained in the RPMI medium (Invitrogen) containing 10% calf serum. Madin-Darby canine kidney (MDCK), HPB-ALL, and Jurkat cells were maintained as previously described (20, 31). For preparation of nonpolarized MDCK cells, confluent MDCK cells were maintained in low calcium medium (3 µM CaCl2) at 37°C for 20 h, as previously described (31). Polarized MDCK cells were maintained for at least 2 days after reaching confluence .

Western blot analysis and immunoprecipitation. Whole cell lysates of Caco-2 cells were prepared by using Laemmli's SDS-sample buffer 2 and 13 days after seeding and were analyzed by Western blot analyses as previously described (31). Proteins were visualized by an enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ) and analyzed using a luminescent image analyzer, LAS-1000 (Fuji film, Tokyo, Japan).

Cells grown in 10-cm dishes were suspended in 800 µl of lysis buffer containing (in mM) 20 HEPES (pH 7.2), 150 NaCl, 1 EDTA, 2 Na3VO4, 50 NaF, and 2 phenylmethylsulfonyl fluoride, plus 10 µg/ml leupeptin, 1 µg/ml aprotinin, and 1% (wt/vol) Triton X-100. After incubation for 30 min on ice, the Triton X-100-soluble fractions were prepared by centrifugation at 14,000 rpm for 30 min. Insoluble materials were suspended in 400 µl of the lysis buffer and solubilized by addition of the same volume of 2x Laemmli's SDS-sample buffer. The lysates (soluble fractions) were incubated with anti-PAR-3 antibodies preabsorbed on protein A-Sepharose (Amersham Biosciences) for 2 h at 4°C. After being washed five times with the lysis buffer, the immunoprecipitants were eluted with Laemmli's SDS-sample buffer.

Immunofluorescence microscopy. Caco-2 cells grown on coverslips were fixed with 2% paraformaldehyde as described previously (31). After blocking with 10% calf serum, the cells were incubated with the appropriate antibodies at 37°C for 45 min in 10 mM Tris·HCl buffer (pH 7.5) containing 150 mM NaCl, 0.01% Tween 20, and 0.1% bovine serum albumin. The secondary antibodies used were Alexa Flour 488-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR) and Cy3-conjugated goat anti-mouse IgG (Amersham Biosciences). For negative control, the Yap antibody was preabsorbed with 100 µg/ml GST-sPAR-3-(956–1034). The cells were observed under a fluorescence microscope (model BX60; Olympus, Tokyo, Japan) and a confocal microscope system (Bio-Rad, Hercules, CA).

For analysis of the localization of ectopically expressed sPAR-3, cells were seeded on coverslips at 3 and 10 x 104 cells/24-well plate 20 h before transfection. T7-His-tagged PAR-3 and sPAR-3 (14, 21) were transiently transfected by using the Lipofectamine PLUS reagent (Invitrogen) according to the manufacturer's instructions. After 48 h of culture, the cells were fixed with 2% paraformaldehyde, permeabilized with 0.5% Triton X-100 in phosphate-buffered saline, and double-stained with anti-His-tag and the anti-ZO-1 antibody.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
sPAR-3, a major splicing isoform of PAR-3, is predominantly expressed in Caco-2 cells. Western blot analysis using the anti-PAR-3 antibody C2 demonstrated that exponentially growing Caco-2 cells express at least two PAR-3 isoforms, a scarce isoform with a molecular mass of 180 kDa and an abundant isoform of 150 kDa (Fig. 1B). The C2 antibody raised against GST-PAR-3-(712–936), can recognize the two isoforms 180-kDa PAR-3 and 150-kDa sPAR-3, which contain a specific 10 amino acid sequence at the COOH terminus (4, 7, 12, 17) (Fig. 1A). To further characterize these isoforms, we employed two antibodies, A2 and Yap, which were raised against the isoform-specific COOH-terminal sequences (Fig. 1A). As expected from previous observations, the 180-kDa band was recognized by PAR-3-specific A2 antibody, and the abundant band migrating at ~150 kDa was recognized by the Yap antibody specific to sPAR-3 (Fig. 1B).

Expression level of PAR-3 increases, but that of sPAR-3 decreases during the polarization of Caco-2 cells. Electron microscopic analysis and the measurement of transepithelial electrical resistance have established that Caco-2 cells develop epithelial-specific junctional structures (11, 26). We have followed the previous method to seed and culture the cells (11, 26). Under a phase-contrast microscope, the cells reached confluence 5 days after seeding. After confluence, the cells became more compact and columnar in shape, cell thickness increased, and the dome was formed (data not shown). We also confirmed retardation of cell growth and the secretion of a differentiation marker, alkaline phosphatase (Fig. 2A). These phenotypic changes reached the plateau until day 13.



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Fig. 2. A: cell growth and alkaline phosphatase (ALP) activity were measured during the culture of Caco-2 cells. Data shown are means ± SD of 3 determinations and are representative of 3 different passages. Polarization and differentiation of Caco-2 cells 13 days after seeding (day 13) were confirmed. BF: total cell lysates were prepared from nonpolarized (day 2) and polarized (day 13) Caco-2 cells, and total proteins were estimated by protein assay. Aliquots were analyzed by Western blot analysis using antibodies against the indicated proteins. Data shown are representative of 3–5 experiments. The quantity of each band was estimated by LAS-1000 analysis and was normalized by counter-immunostaining with anti-actin antibody (data not shown). The quantity in nonpolarized cells was standardized to 1.00. Data shown are the means ± SD of 3–5 experiments. Relative amounts of PAR-3 and sPAR-3 at days 2 and 13 were presented in E. The total expression level of both isoforms on day 2 was estimated to be 100% and was slightly decreased during the polarization of Caco-2 cells. ZO-1, zonula occludens-1.

 
To evaluate the changes in expression of PAR-3, sPAR-3, and other junctional proteins during the process, we prepared total protein extracts on 2 days (as the nonpolarized phase) and on 13 days (as the polarized phase). Consistent with a previous report (18), the amounts of E-cadherin and {beta}-catenin, components of AJ, increased up to 1.5- and 2.1-fold, respectively (Fig. 2B). The amounts of occludin and claudin-1, structural components of TJ (5, 6), also increased (Fig. 2C). Interestingly, ZO-1, a peripheral protein of TJ, showed no change in the total amount, but showed a shift in migration to a higher molecular weight. This may suggest either a change in the splicing or in the phosphorylation states (1, 29). The amount of PAR-3 increased up to 1.6-fold during polarization, consistent with the notion that PAR-3 is a component of TJs in polarized epithelial cells. On the other hand, the amount of sPAR-3 decreased (Fig. 2D). Total amounts of PAR-3 and sPAR-3 slightly decreased during the period (Fig. 2E). These results suggest that sPAR-3 plays a role different from that of PAR-3 during the differentiation and polarization of Caco-2 cells. Interestingly, the amounts of aPKC and PAR-6, binding partners of PAR-3, slightly decreased during these processes (Fig. 2F).

sPAR-3 associates with aPKC in Caco-2 cells. Consistent with previous observations (14), aPKC was coimmunoprecipitated by the anti-PAR-3 antibody C2 from nonpolarized and polarized Caco-2 cell extracts (Fig. 3A). Isoform-specific antibodies, A2 and Yap, immunoprecipitated PAR-3 and sPAR-3, respectively, from both nonpolarized and polarized cells (Fig. 3A). These results suggest that sPAR-3 associates with aPKC independent of PAR-3 in nonpolarized and polarized cells. Interestingly, most of sPAR-3 was recovered in the Triton X-100-soluble fraction while a significant portion of PAR-3 was recovered in the Triton X-100-insoluble fraction (data not shown), suggesting that sPAR-3 is more soluble than PAR-3. To confirm this, we compared the solubility of PAR-3 and sPAR-3 using nonpolarized and polarized MDCK cells, because MDCK cells expresses comparable amounts of PAR-3 to sPAR-3 (Fig. 1B, lane 1). Incubation under low calcium condition to release the cells from calcium-dependent cell-cell adhesions make cells nonpolarized in which C2 antibody shows cytoplasmic staining (14, 31). Consistent with these observations, <10% of PAR-3 was fractionated in the insoluble fraction of nonpolarized cells (Fig. 3, B and C). On the other hand, when cells were polarized under normal calcium condition, insoluble PAR-3 increased up to 30% (Fig. 3, B and C). In contrast to PAR-3, only 12% of sPAR-3 was fractionated into the insoluble fractions even under polarized condition. These results support the difference in the solubility between PAR-3 and sPAR-3 and imply the difference in localization between sPAR-3 and PAR-3 in polarized cells.



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Fig. 3. A: Triton X-100-soluble fraction was prepared from nonpolarized (day 2) and polarized (day 13) Caco-2 cells and immunoprecipitation (IP) was performed by using the indicated anti-PAR-3 antibodies. Immunoprecipitants were analyzed by Western blot analysis using C2 and anti-aPKC antibodies. B and C: nonpolarized (low calcium treatment) and polarized (postconfluent) MDCK cells were fractionated into the Triton X-100-soluble and insoluble fractions, as described in MATERIALS AND METHODS. Aliquots of the soluble fractions and double amounts of the insoluble fractions were analyzed by Western blot (WB) analysis using the C2 antibody. The data shown in B are representative of 4 experiments. The amounts of PAR-3 and sPAR-3 were analyzed by using LAS-1000 (Fuji film), and the percent PAR-3 or sPAR-3 in the insoluble fractions under both conditions was determined (C). Data shown are the means ± SD of 4 separate experiments. *P value of <0.002; **P value of <0.01 in Student's t-test.

 
sPAR-3 dose not concentrate at TJs in polarized Caco-2 cells. We then analyzed the localization of PAR-3 and sPAR-3 in nonpolarized (day 2) and polarized (day 13) Caco-2 cells (Fig. 4). C2 and A2 antibodies showed very similar stainings. Both antibodies strongly stained the cell-cell junctions and the staining overlaps with that of ZO-1 in polarized cells, supporting that PAR-3 localizes at premature and mature cell-cell junctions in Caco-2 cells. In addition to the cell-cell contact region, both antibodies faintly stained the cytoplasm and the nucleus only for nonpolarized cells, although the nature of the staining remains unclear (day 2).



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Fig. 4. Nonpolarized (day 2) and polarized (day 13) Caco-2 cells were fixed, double-stained with the indicated anti-PAR-3 antibodies (green) and anti-ZO-1 antibody (red, cell-cell junction marker), and analyzed under a confocal microscope. Data shown are representative of 3 experiments.

 
In contrast, the staining with Yap antibody was different. Importantly, the staining with Yap antibody seemed to change during cell polarization. The clear staining at the cell-cell contact region in nonpolarized cells became faint and unclear in most of the polarized cells. Yap antibody also shows punctuate cytoplasmic staining in both nonpolarized and polarized cells. Cytoplasmic staining as well as the staining at the cell-cell contact region in nonpolarized cells disappeared when it was incubated with GST-sPAR-3-(956–1034) (data not shown). Furthermore, similar staining was also observed for C2 antibody, suggesting that these stainings reflect the distribution of endogenous sPAR-3, although the nature of the punctuate cytoplasmic staining remains to be clarified. The punctuate staining with C2 or Yap in the apical part of the cytosol in polarized cells partially overlaps with that of anti-{beta}-tubulin antibody (data not shown), which showed a dense network staining at the apical part of the cells as previously described (8). On the other hand, the strong nuclear staining obtained with Yap antibody only in nonpolarized cells disappears after adsorption with GST-sPAR-3-(956–1034) but was not reproducible (data not shown). Although the nature of the punctuate cytoplasmic staining remains unclear, these results suggest that sPAR-3 shows a difference in its localization with PAR-3, especially in polarized cells.

To confirm the change in cellular distribution of sPAR-3, we compared the localization of exogenously expressed PAR-3 and sPAR-3. Caco-2 cells expressing ectopic proteins were either sparsely or densely (near confluent) seeded, cultured for 48 additional hours, and stained with anti-tag (His) antibody. The sparsely seeded cells represent nonpolarized cells and the densely seeded cells represent partially polarized cells. We could classify the cells on the basis of the distribution of ectopic proteins. In type 1 cells, ectopic protein distributes to the cell-cell contact region as well as the cytoplasm. In type 2 cells, the ectopic protein distributes to the cytosol (Fig. 5A). The percentage of type 2 cells expressing sPAR-3 was similar to that for PAR-3 under nonpolarized conditions. In partially polarized cells, however, the percentage of type 2 cells expressing sPAR-3 markedly increased (Fig. 5B). These results support the notion that sPAR-3 changes its localization from the cell-cell contact regions to the cytosol during epithelial cell polarization.



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Fig. 5. A: His-tagged PAR-3 (or sPAR-3) is transiently overexpressed in sparse (for nonpolarized) and near-confluent (for partially polarized) Caco-2 cells. After being cultured for 48 h, cells were double stained with anti-His-tag (green) and the anti-ZO-1 antibody (data not shown). In type 1 cells, the colocalization of ectopic proteins with ZO-1 at the cell-cell contacts can be clearly observed, but in type 2 cells, the staining by anti-His-tag antibody is observed mainly in cytosol, and the colocalization of PAR-3 with ZO-1 is unclear. B: percentage of type 2 cells among the cells expressing PAR-3 or sPAR-3 (n = 200). Data shown are the means ± SD of 3 separate experiments. *P value of <0.002; **P value of <0.01 in Student's t-test.

 
Nonadhesive cells predominantly express sPAR-3. Western blot analysis previously demonstrated that not only PAR-3 but also sPAR-3 is expressed in various tissues and that sPAR-3 is predominantly expressed in intestinal epithelial cells, kidney, and heart (12). Therefore, we analyzed the expression of sPAR-3 in various cell lines (Fig. 6). Two human epithelial cell lines, DRY and CoMet, express both PAR-3 and sPAR-3 similar to Caco-2 and MDCK cells. Interestingly, blood cell lines, HPB-ALL and Jurkat, which have no cell-cell junctions, predominantly expressed sPAR-3 compared with PAR-3, further supporting the notion that sPAR-3 has a role other than formation of cell-cell junctions.



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Fig. 6. Total cell lysates were prepared from various human cell lines, and analyzed by Western blot analysis using the C2 antibody. The epithelial cell lines Dry and CoMet derive from kidney and colon cancer, respectively.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Because of its evolutionarily conserved structure and functions, PAR-3 has been the focus of attention, and its critical roles in the regulation of mammalian epithelial cell polarity have been demonstrated (12, 21, 22, 24). Although several alternatively spliced isoforms of PAR-3 including sPAR-3 have been reported (4, 7, 12, 17), they have hardly attracted attention. In this study, we characterized sPAR-3 predominantly expressed in Caco-2 cells.

During the early phase of polarization of mammalian epithelial cells, PAR-3 is recruited to premature cell-cell junctions for their formation in cooperation with E-cadherin and ZO-1, and it plays a scaffolding role in the recruitment of aPKC and PAR-6 to the premature cell-cell junctions (3, 21, 30). The self-multimerization of PAR-3 mediated by CR1 domain and the binding to JAM through PDZ1 of PAR-3 are required for PAR-3 localization at the premature cell-cell junctions, because the overexpression of CR1 domain of PAR-3 or a deletion mutant of JAM lacking the cytoplasmic domain results in the mislocalization of aPKC and PAR-6 as well as PAR-3 (3, 21). As shown in Fig. 1A, because sPAR-3 contains three conserved domains (CR1–3), sPAR-3 is expected to contribute to the recruitment of aPKC and PAR-6 to the premature cell-cell junctions, similar to PAR-3.

aPKC binds to PAR-3 recognizing the 26 amino acid sequence 816–841 including the CR3 domain, and this binding is regulated by the phosphorylation of Ser-827 in this sequence (22). A part of CR3 is encoded in exon 17 of par-3 gene and PAR-3-spliced variants lacking exon 17 were reported (4, 22) to be unable to bind to aPKC. RT-PCR experiments revealed that sPAR-3 in Caco-2 cells has both variants with or without exon 17 (data not shown), and we showed that aPKC is immunoprecipitated with sPAR-3 by using the sPAR-3-specific antibody (Fig. 3A), indicating that sPAR-3 can form a complex with aPKC, similar to PAR-3. On the other hand, we previously demonstrated that His-sPAR-3 overexpressed in MDCK cells colocalized with ZO-1 at the cell-cell junctions (21). In this study, immunofluorescence experiments using isoform-specific antibodies demonstrated that endogenous sPAR-3 is recruited to the premature cell-cell junction during early polarization of Caco-2 cells (Fig. 4). Furthermore, ectopic His-sPAR-3 concentrates at the premature cell-cell junctions as does His-PAR-3 (Fig. 5B). Taken together, not only PAR-3, but also sPAR-3 contributes to the recruitment of aPKC and PAR-6 to the premature cell-cell junctions in the early phase of epithelial cell polarization.

In polarized cells, however, the role of both isoforms remains unknown, because the overexpression of dominant-negative mutants of PAR-3 or aPKC in polarized epithelial cells shows no effects (3, 21, 30, 31). In Drosophila epithelial cells, Bazooka, a homologue of PAR-3, localizes on the apical side of AJ and is required for the regulation of cell polarity (25, 33). We demonstrated the localization of PAR-3 at TJs in polarized Caco-2 cells (Figs. 4 and 5), consistent with previous reports (12, 14, 30). It was demonstrated that the amount of PAR-3 increased in regenerating rat liver after hepatectomy, along with TJ formation and cell polarization (32). We found an increase in the amount of PAR-3, as well as other junctional proteins, during polarization of Caco-2 cells (Fig. 2, BF). These results suggest the role of PAR-3 not only as a component of TJ but also in maintaining cell polarity. Furthermore, our results clearly indicate the importance of PAR-3-specific COOH-terminal sequence (1025–1337) for its localization at TJs in polarized epithelial cells.

In contrast to PAR-3, endogenous and ectopic sPAR-3 do not clearly concentrate at TJs in polarized cells (Figs. 4 and 5). Furthermore, sPAR-3 was more preferably fractionated into detergent-soluble fractions, suggesting a lower interaction of sPAR-3 than PAR-3 with junctional components (Fig. 3B). During the polarization of Caco-2 cells, the amount of sPAR-3 decreased, making a contrast to TJ proteins such as occludin, claudin-1, and PAR-3 (Fig. 2). These results suggest that sPAR-3 plays a different role from PAR-3 in polarized Caco-2 cells. Interestingly, the cell lines that lack cell adhesion, namely, HPB-ALL and Jurkat, express sPAR-3 predominantly (Fig. 6). The expression of sPAR-3 in the kidney and prostate was also reported (12), suggesting the role of sPAR-3 in these organs. Further studies are required to clarify the role of PAR-3 and sPAR-3 after the establishment of epithelial cell polarity or in nonepithelial cells and the involvement of aPKC in these cells.

In conclusion, we found that sPAR-3 may have different functions according to the phase of intestinal cell polarization. It was reported that aberrant cell polarity is related to pathogenesis of some diseases, particularly cancer (16, 23). The clarification of the role of each isoform of PAR-3 may, in turn, clarify the pathogenesis of these diseases.


    ACKNOWLEDGMENTS
 
This work was supported, in part, by grants from the Japan Society for the Promotion of Science and from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. Ohno, Dept. of Molecular Biology, Yokohama City Univ., School of Medicine, Fuku-ura 3-9, Kanazawa-ku, Yokohama 236-0004, Japan (E-mail; ohnos{at}med.yokohama-cu.ac.jp)

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.


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