Asymmetrical targeting of type II Na-Pi cotransporters in renal and intestinal epithelial cell lines

N. Hernando, S. Sheikh, Z. Karim-Jimenez, H. Galliker, J. Forgo, J. Biber, and H. Murer

Institute of Physiology, University of Zürich, Zürich CH-8057, Switzerland


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Targeting of newly synthesized transporters to either the apical or basolateral domains of polarized cells is crucial for the function of epithelia, such as in the renal proximal tubule or in the small intestine. Recently, different sodium-phosphate cotransporters have been identified. Type II cotransporters can be subdivided into two groups: type IIa and type IIb. Type IIa is predominantly expressed in renal proximal tubules, whereas type IIb is located on the intestinal and lung epithelia. To gain some insights into the polarized targeting of the type II cotransporters, we have transiently expressed type IIa and type IIb cotransporters in several epithelial cell lines: two lines derived from renal proximal cells (opossum kidney and LLC-PK1), one from renal distal cells (Madin-Darby canine kidney), and one from colonic epithelium (CaCo-2). We studied the expression of the transporters fused to the enhanced green fluorescent protein. Our data indicate that the polarized targeting is dependent on molecular determinants most probably located at the COOH terminus of the cotransporters as well as on the cellular context.

polarized cells; sorting


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SODIUM-PHOSPHATE (Na-Pi) cotransporters located at the apical membranes (brush border) control the direction and rate of transepithelial Pi movements in renal proximal tubules and in small intestine. Over the last several years, three different cotransporters, type I, type II, and type III, have been identified (for review, see Ref. 22). The type I transporter is found in renal proximal tubular brush-border membranes and mediates, in addition to Na-Pi cotransport, a multispecific anion conductance. The type II family can be subdivided into two groups, type IIa and type IIb, with an overall homology of ~60%, the major differences between both groups being at the COOH terminus (11). Type IIa is expressed in the apical membrane of the renal proximal epithelium (6) and is largely responsible for renal Pi reabsorption, as indicated by recent knockout experiments (3). On the other hand, type IIb, which it is not detected in mammalian renal tissues, is located on the apical membrane of the intestinal and lung epithelia (11, 32). Finally, the retroviral receptors Glvr-1 and Ram-1, which are expressed in both epithelial and nonepithelial cells, constitute the type III cotransporters (13).

The ability of the epithelial cells to mediate vectorial transport of ions and solutes against their concentration gradients depends on the asymmetrical distribution of transporters at the cellular surface. Thus polarized expression of Na-Pi cotransporters at the apical membrane of epithelial cells allows the reabsorption of Pi from the luminal compartment (of either renal proximal tubules or small intestine) to the blood. It is not clear how the newly synthesized proteins are asymmetrically targeted, but on the basis of studies in different cell lines, sorting seems to involve at least two intracellular compartments: the trans-Golgi network (TGN) and the endosomes. In Madin-Darby canine kidney (MDCK) cells, sorting takes place mostly in the TGN, and proteins are directly targeted to their final location, whereas in CaCo-2 cells and hepatocytes sorting is a function of the endosomes, and proteins reach their final location after endocytosis and transcytosis (2, 12, 20, 34). Because all types of cells undergo continuous endocytosis at both membrane domains, sorting at the endosomal level is essential to maintain the cell polarity. In addition, the so-called subapical compartment (SAC), which is distally located to the early endosomes (where plasma membrane proteins are first sorted), seems also to be involved in transcytosis and recycling of proteins and lipids (1, 33). Similar signals seem to control sorting in both TGN and endosomes (18). At least two types of sorting signals are involved in basolateral targeting: cytoplasmic tyrosine- and dileucine-based motifs (17, 19). The identity of the apical signal is not well understood. It has been postulated that it resides in the extracellular (or luminal) domain of the protein, and several groups have proposed that the N-linked glycosylation may function as an apical sorting motif (29). However, not all the apically targeted proteins are glycosylated, ruling out this as a universal signal. Some proteins may achieve apical targeting due to their ability to segregate from the trans-Golgi in clusters of sphingolipids-rich vesicles (30). According to this model, the ability of a protein to associate with sphingolipid rafts would determine the final destination of the protein, therefore focusing attention on those hydrophobic portions of the polypeptides that may interact with the lipid bilayer. Indeed, a sorting signal within a predicted transmembrane domain has been reported (5). Finally, the presence of critical apical sorting information in the COOH-terminal cytoplasmic domain has also been recently found (23).

To obtain information about the specific targeting of the type II Na-Pi cotransporters, we have transfected several cell lines derived from renal and intestinal epithelia with three cotransporters: NaPi2, NaPi5, and NaPi2b. NaPi2 is the rat renal cotransporter that was the first type IIa protein cloned (16). It is expressed exclusively in the apical membrane of proximal tubules (6). NaPi5 is a flounder cotransporter detected in both kidney and intestine (14, 35). Because of its presence in kidney, it was initially considered as the fish homolog of the mammalian type IIa; however, its COOH-terminal tail shows a higher homology with the recently reported type IIb subfamily than with the type IIa. NaPi2b, isolated from mouse small intestine, was the first type IIb cotransporter described (11). It is detected in the apical domain of intestinal and lung epithelia but not in kidney (32). Our data suggest that sorting depends on both molecular determinants within the targeted protein and the cellular context in which targeting takes place.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fusion of Na-Pi cotransporters to the enhanced green fluorescent protein (EGFP). The cDNAs encoding for the NaPi2, NaPi5, and NaPi2b cotransporters were fused to the COOH terminus of the cDNA encoding EGFP, as described elsewhere (N. Hernando, J. Forgo, J. Biber, and H. Murer, unpublished observations). Briefly, the cotransporter cDNAs were fused, through PCR and subcloning, in a mammalian expression vector that contains the EGFP cDNA under the control of the CMV promoter (pEGFP-C1; Clontech). The generated fusion protein contained the EGFP at the NH2 terminus of the transporter.

Cell culture and transfections. Opossum kidney cells (0K cells; clone 3B/2) were maintained in DMEM: Ham's F-12 medium (1:1), the porcine kidney cell line LLC-PK1 in alpha minimal essential salts medium (alpha MEM), MDCK cells (strain II) in DMEM containing 1% nonessential amino acids, and human colon adenocarcinoma CaCo-2 cells in DMEM also supplemented with nonessential amino acids as previously described (9, 21 28). All media contained 10% (vol/vol) fetal calf serum (20% for CaCo), 2 mM glutamine, 22 mM bicarbonate, 50 IU/ml penicillin, and 50 µg/ml streptomycin. Cells were plated on coverslips in 35-mm dishes (Nunc), and cultures were transfected with either the empty pEGFP plasmid or with the pEGFP constructs containing the different cotransporters. Transfections of subconfluent cultures were carried out over night (ON), using 1 µg of DNA and 3 µl of FuGene (Boehringer Mannheim)/dish, according to manufacture's procedure. After cultures reached confluency (2-3 days after transfection), we studied the subcellular location of the transfected cotransporters by confocal microscopy. With this experimental approach we analyzed the targeting in a "steady-state" situation; therefore, this information reflects the final location of the proteins but not the intermediary steps.

Subcellular location: confocal microscopy. Cells grown on coverslips were fixed for 10 min in 3% paraformaldehyde in PBS, washed with PBS, and incubated for 10 additional min with 20 mM L-glycine in PBS. After 30-min permeabilization in 0.1% saponine, cells were incubated either with polyclonal antibodies against NaPi2 (6) or NaPi2b (11), followed by incubation with a goat anti-rabbit rhodamine-conjugated IgG, or with phalloidine-rhodamine (Calbiochem) for actin detection. After extensive washings in PBS, the coverslips were mounted by using Dako-Glycergel (Dakopatts) containing 2.5% 1,4-diazabicyclo-[2.2.2]octane (Sigma Chemical) as a fading retardant. Confocal images were taken by using a Leica laser scan microscope equipped with a ×63 oil-immersion objective.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The efficiency of transfection 24 h after addition of the different pEGFP constructs was ~10%, in all cell types analyzed. The cultures reached confluence, required for proper differentiation, 2 days after transfection. Therefore, by the time the confocal analysis was performed, <10% of the cells were transfected. This is the reason why each picture contains only one or two transfected cells. We performed at least three independent transfections for each experiment, and from each transfection at least 10 cells were analyzed. In almost all the cases the final sorting behavior of the given isoform was consistent. The only exception was the targeting of the NaPi 2b cotransporter in LLC-PK1, as indicated below.

Confocal microscopy of confluent OK, LLC-PK1, MDCK, and CaCo-2 cultures transfected with the empty plasmid showed that the native EGFP exhibited a diffused distribution throughout the cell (Fig. 1). In cross sections (Fig. 1, small rectangles) the fluorescence was homogeneously detected in the cytoplasm and nucleus. By contrast, the fluorescence of cells transfected with the EGFP-fused cotransporters was located at the plasma membrane. To prove that the EGFP fluorescence of the fused cotransporters reflected the pattern of expression of the native cotransporters, we stained OK cells transfected with NaPi2 and NaPi2b with antibodies against specific peptides of both proteins. As shown in Fig. 2, there was a total overlapping between the intrinsic fluorescence of the cotransporters (EGFP signal) and the NaPi2 or NaPi2b antibody immunostaining. Similar data were obtained with the other three cell lines (data not shown). Therefore, the intrinsic fluorescence of the EGFP-fused NaPi2 and NaPi2b indeed reflected the presence of the cotransporter and mimics the pattern of expression detected after antibody staining.


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Fig. 1.   Native enhanced green fluorescent protein (EGFP) is expressed intracellularly. Cultures of opossum kidney (OK; A), LLC-PK1 (B), Madin-Darby canine kidney (MDCK; C), and CaCo-2 (D) cells transfected with empty pEGFP plasmid were processed for immunofluorescence as described in MATERIALS AND METHODS. Actin staining is shown in red, and endogenous EGFP fluorescence in green. For each type of cell, an apical focal plane (squares) and a confocal cross section along planes depicted by white lines (rectangles) are shown. In cross sections, apical membranes are on top and basolateral on the bottom.



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Fig. 2.   Intrinsic fluorescence of EGFP-fused NaPi reflects presence of cotransporter. OK cells transfected with EGFP-fused NaPi2 (A-C) or NaPi2b (D-F) were stained with corresponding specific antibodies as described in text. Endogenous EGFP fluorescence is shown in green (A and D), NaPi2 and NaPi2b antibody staining in red (B and E), and merge of both signals in yellow (C and F). As indicated before for each set of images, an apical focal plane (squares) and a confocal cross section along planes depicted by white lines (rectangles) are shown.

We first studied the expression of the cotransporters in two proximal cells lines derived from opossum and pig kidney, OK cells and LLC-PK1 cells, respectively. OK cells express an endogenous type IIa cotransporter (NaPi4) at the apical membrane that shows many of the characteristics of proximal tubular Na-Pi cotransport (31). In contrast, LLC-PK1 cells do not express an intrinsic type II transporter, although they exhibit Na-Pi cotransport activity not yet molecularly defined (4). The experiments performed with both proximal cell lines provided very similar information. Tranfection of the EGFP-fused constructs in OK cells lead to a "patchy" pattern of fluorescence (Fig. 3). We have previously shown that the NaPi4 apical "patches" represent clusters of the cotransporter within the OK cells microvilli (25). Cross sections of the transfected cells confirmed that the fluorescent patches reflected the presence of the EGFP-fused cotransporters in the apical membrane (Fig. 3, small rectangles). Although very little, some basolateral fluorescence was also detected. The expression of the EGFP-fused constructs in the other proximal cell type, the LLC-PK1 cells, did not lead to fluorescent patches but instead to a homogeneously distributed fluorescence. The absence of patches is probably due to a less differentiated brush border than in the OK line (8). Confocal microscope sections show that, like the OK cells, the NaPi2 cotransporter was targeted mostly to the apical domain of the LLC-PK1 cells, although some basolateral signal was also detected (Fig. 4A). However, the NaPi5 signal was exclusively apical (Fig. 4C) whereas NaPi2b appeared either exclusively or mostly apical (Fig. 4, B1 and B2). We cannot rule out that this last observation may be due to some heterogeneity within the LLC-PK1 cells.


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Fig. 3.   Expression of Na-Pi cotransporters in OK cells. OK cells were transfected with the NaPi2 (A), NaPi2b (B), and NaPi5 (C) cotransporters and processed for immunofluorescence as described in MATERIALS AND METHODS. Actin staining is shown in red and endogenous EGFP fluorescence in green. Both an apical focal plane (squares) and a confocal cross-section along planes depicted by white lines (rectangles) are shown.



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Fig. 4.   Expression of Na-Pi cotransporters in LLC-PK1 cells. LLC-PK1 cells transfected with NaPi2 (A), NaPi2b (B1 and B2), and NaPi5 (C) cotransporters were processed for confocal analysis as indicated in Fig. 3.

We next analyzed the expression of the different NaPi constructs in MDCK cells, a well-characterized cell line derived from canine kidney. MDCK cells do not express type II cotransporters endogenously. The transfection experiments performed in this cell line showed some differences from those described for the proximal tubule models. First, the NaPi2 fluorescence was detected at a high intensity both apically and basolaterally (Fig. 5A), in contrast to the expression in the proximal cell types, where the basolateral signal was very faint. Moreover, NaPi5 and NaPi2b appeared to be exclusively apical (Fig. 5, B and C).


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Fig. 5.   Expression of Na-Pi cotransporters in MDCK cells. MDCK cells were transfected with NaPi2 (A), NaPi2b (B,) and NaPi5 (C) cotransporters. Confocal images were processed as indicated in Fig 3.

Finally, we studied the pattern of expression of the different cotransporters in CaCo-2 cells, a cell line derived from human colon carcinoma. As mentioned above, whereas NaPi2 has not been detected in extrarenal tissues (16), expression of NaPi5 has been reported in both kidney and intestine (14), and NaPi2b is detected in intestine but not in kidney (11). Our data indicate that CaCo-2 cells target the NaPi5 and NaPi2b cotransporters almost exclusively to the apical membrane (Fig. 6, B and C). However, most of the cells transfected with the NaPi2 construct contained numerous vacuole-like structures, and in general these cells were unable to target the NaPi2 cotransporter to either membrane (Fig. 6A).


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Fig. 6.   Expression of Na-Pi cotransporters in CaCo-2 cells. CaCo-2 cells were transfected with NaPi2 (A), NaPi2b (B,) and NaPi5 (C) cotransporters. Confocal images were processed as indicated in Fig 3.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To achieve transcellular transport, transporter proteins must be asymmetrically expressed at either cell surface of epithelial cells. Thus in the renal proximal tubules and small intestine of mammals the type II (a and b, respectively) Na-Pi cotransporters are expressed at the brush-border membrane (6, 11). In contrast, in nonmammalian vertebrates (e.g., flounder) the type II transporter (NaPi5) can be expressed apically or basolaterally depending on the specific epithelial function (14). Sorting of proteins takes place in the TGN as well as in endosomes, and there is evidence suggesting that the same sorting signals operate both in the TGN and in endosomes (18). Partial truncations as well as chimeras of isoforms of proteins that are differentially targeted have been performed, and they have proved useful in defining the sorting signals (7, 15, 17, 19, 23). Although the sorting pathway seems to be protein and cell type specific, three models have been postulated: 1) a direct pathway whereby apical and basolateral-resident proteins are separated from each other before their delivery to the plasma membrane, in the trans-Golgi network; 2) an indirect pathway whereby all newly synthesized proteins are first delivered together to one cell surface, and subsequently those proteins that should appear in the opposite compartment are transcytosed; and 3) a random pathway whereby apical and basolateral proteins are first delivered randomly to both surfaces, and subsequently transcytosed to their final location. The last two pathways involve sorting in endosomes.

The aim of our work was to gain some information about the specific targeting of the Na-Pi cotransportsers. For that aim we have transfected several cell lines derived from renal (OK, LLC-PK1 and MDCK) and intestinal (CaCo-2) epithelia with three cotransporters: NaPi2, NaPi5, and NaPi2b. A summary of these data is shown in Table 1. Our data suggest, first, that the three transporters are not targeted in a similar way within the same cell line and, second, that the same cotransporter is differently targeted in different cell lines.

                              
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Table 1.   Summary of the polarized expression of Na-Pi type II cotransporters in epithelial cells

Regarding the first observation, we found that targeting of NaPi2 is different from that of NaPi5 and NaPi2b, the latter two being very similar to each other. This pattern of expression was especially clear in MDCK cells, where NaPi2 was detected at a high intensity in the both apical and basolateral membranes whereas NaPi5 and NaPi2b were exclusively apical, and in CaCo-2 cells, where no specific targeting of NaPi2 seems to take place whereas NaPi5 and NaPi2b were apically located. Both cells types could specifically sort the NaPi5 and NaPi2b cotransporters to the apical membrane. Therefore, the lack of specific apical targeting of the NaPi2 isoform cannot be explained by the inability of the transfected cells to properly keep their polarity. Interestingly, the COOH-terminal tail of the NaPi5 cotransporter displays a higher homology to the type IIb (NaPi2b) than to the type IIa (NaPi2) cotransporters. As shown in Fig. 7, although the type IIa and type IIb cotransporters show a high degree of homology, their COOH-terminal tails are very different, and NaPi5 shares some of the most remarkable features of the NaPi2b such as C-rich stretches. The last three amino acids of the cotransporters resemble the PDZ binding motif (S/T)X Z, where X can be any amino acid and Z is preferentially a hydrophobic residue. Although this motif has been involved in the apical targeting of some transporters (23), it does not seem to be essential for the apical targeting of the Na-Pi cotransporters studied in this paper (12a).


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Fig. 7.   Comparison of COOH-terminal tails of NaPi2b, NaPi5, and NaPi2 cotransporters. Identical amino acids are shown in black and conserved substitutions in gray. Two horizontal lines mark the position of the last 2 transmembrane domains. The beginning of the COOH-terminal tails is marked with vertical arrow.

On the other hand, we found that the same cotransporter can be targeted differently in different cell lines. Thus NaPi2 was mostly apical in OK and LLC-PK1, both apical and basolateral in MDCK, and basically not targeted in CaCo-2 cells. Although the faint basolateral expression detected in OK and LLC-PK1 cells could be considered as some degree of mistargeting due to the overexpression of the protein, this is clearly not the case in MDCK cells, where both apical and basolateral signals had a high intensity. The dual expression of NaPi2 in MDCK cells is in agreement with a previous report from our group in which we described that stably transfected MDCK cells grown on permeable supports showed Na-Pi cotransport activity both at the apical and basolateral surfaces, and dual location of the cotransporter was also detected by immunohistochemistry (26). Therefore, the lack of asymmetry of the NaPi2 cotransporter can neither be explained by a disturbed polarity of the MDCK cells plated on coverslips. Although speculative, it seems that the apical signal contained in NaPi2 (a type IIa cotransporter) is decoded with higher efficiency in cells from renal proximal epithelium than in cells from more distal segments, whereas such a signal is not identified in an extrarenal environment.

During the last few years it became clear that the basolateral targeting of proteins containing Y-based and LL-based sorting motifs requires the interaction of these motifs with the adaptor complex machinery (24, 27). Although very little is known about the apical sorting signals, most probably they also require interaction with specific components of the sorting machinery. Therefore, the lack of specific apical targeting of NaPi2 in MDCK as well as in CaCo cells could reflect the absence of specific protein(s) required for the proper decoding of the apical sorting signal.

When all our data are taken together, they strongly suggest that targeting of polarized proteins is a highly specific process that depends not only on the particular protein of interest but also on the specific cell line in which targeting is studied. For the renal-specific type IIa cotransporter in particular, both a correct cellular context as well as molecular determinants probably located at the COOH terminus seem to be involved in apical sorting.


    ACKNOWLEDGEMENTS

We thank C. Gasser for assistance in preparing the figures.


    FOOTNOTES

This work was supported by Swiss National Science Foundation Grant 31.46523 (to H. Murer), the Hartmann Müller-Stiftung (Zürich, Switzerland), the Olga Mayenflsch-Stiftung, and the Schwerzerische Bank-Gesellschaft (Zürich; Bu 70417-1).

N. Hernando and S. Sheikh contributed equally to this study.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: N. Hernando, Physiologisches Institut der Universität Zürich, Winterthurerstrasse 190, Zürich, CH-8057, Switzerland (E-mail: hernando{at}physiol.unizh.ch).

Received 26 April 1999; accepted in final form 6 October 1999.


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DISCUSSION
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