Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5120
Submitted 15 January 2003 ; accepted in final form 30 June 2003
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ABSTRACT |
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Madin-Darby canine kidney cells; fibroblasts; enhanced green fluorescent protein; fluorescence; transfection; laser scanning confocal microscopy
BGT1 transport activity is low in MDCK cells maintained in isotonic medium but is strongly induced in response to prolonged (24 h) hypertonic stress (23, 35, 51). Research on the cellular regulation of BGT1 during hypertonic stress has focused on the mechanisms that trigger increased transcription of the BGT1 gene. Transcription plays an essential role in upregulation of BGT1 transport activity. There is a marked increase in BGT1 gene transcription when cells are exposed to hypertonic medium (48). A 13-bp tonicity-responsive enhancer (TonE) sequence in the 5'-flanking region of the BGT1 gene confers responsiveness to hypertonicity (22). A TonE binding protein (TonEBP) specifically binds to TonE and is presumably a transcription factor. The abundance of TonEBP in the nucleus is dramatically increased by hypertonicity, a step that occurs before the increase in BGT1 mRNA (33, 50). TonEBP is also increased in the nucleus in response to amino acid deprivation under isotonic conditions, another situation that leads to a decrease in cell volume (12), suggesting that TonEBP may play a key role in the regulation of cell volume under both isosmotic and anisosmotic conditions. Several different kinase signaling pathways may play an important role in initiating the cellular responses to osmotic challenge (12, 34, 45, 49), although they may not be required to maintain the adaptation (2, 9, 27).
Our previous results (3) revealed the presence of intracellular BGT1 protein in MDCK cells that had been maintained in isotonic medium. This observation raises the possibility that insertion of preexisting BGT1 into the plasma membrane may be an important posttranscriptional step in the upregulation of the BGT1 transport capacity of the cell. There is ample evidence for posttranslational regulation and recycling of plasma membrane transporters in kidney and other tissues, such as the NaPi2 phosphate transporter (17, 25), GLUT1 and GLUT4 glucose transporters (18, 37), and the AQP2 aquaporin water channel (16). Little attention has been paid to posttranscriptional regulation of the BGT1 transporter during adaptation to hypertonic stress. We have investigated the subcellular distribution of BGT1 protein in live cells by transfection with BGT1 tagged with enhanced green fluorescent protein (EGFP). The functional properties of the fusion proteins and their response to hypertonic stress reveal that posttranscriptional regulation of BGT1 protein is a crucial component in cellular adaptation to hypertonic stress.
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METHODS |
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Cells were transfected 2 h after plating in isotonic medium in six-well plates by using 1 µg of plasmid DNA/well and Fugene 6 (Boehringer Mannheim), according to the manufacturer's instructions. Cells were used 24 h later. Transfection efficiency, typically 20%, was monitored by direct visualization of fluorescence.
Hypertonic stress was induced by replacing normal growth medium with growth medium made hypertonic by the addition of sucrose to achieve a final osmolarity of 500 mosmol/l, as in previous studies (3). The osmolarity of all solutions used for transport was matched to the osmolarity of the growth medium. The transport function of endogenous and transfected BGT1 in cell monolayers was determined by measuring cell uptake of [3H]GABA, as described previously in detail (3, 23). Briefly, [3H]GABA uptake was determined both in medium containing Na+ and also in medium in which Na+ was replaced by methyl-D-glucamine-HCl. The difference, which is referred to as the Na+-dependent component, represents transport specifically via BGT1.
EGFP-tagged BGT1 fusion proteins. The 1.87-kb coding sequence of the renal BGT1 gene was amplified from the pSPORT-BGT1 plasmid (51) by polymerase chain reaction by using Taq polymerase (Takara Biomedicals). The primers were 5'CTCCCAGTCCATCTCGAGGCTATGG3' and 5'CACAGGATCCAAGTGGGTCTCCTTCTCC3' and were designed to introduce XhoI and BamHI sites and to eliminate the BGT1 stop codon. The restricted product was cloned into the XhoI and BamHI sites of the mammalian expression vector pEGFPC3 (Clontech, Palo Alto, CA) so that the resulting fusion protein (EGFP-BGT) would have the NH2 terminus of BGT1 fused to the COOH terminus of EGFP, with an intermediate spacer of six amino acids. The BGT1 coding sequence in this construct was verified through double-strand primer walk sequencing (SeqWright, Houston, TX). The 1.87-kb full-length BGT1 coding sequence was excised from the construct by double digestion with XhoI and BamHI and cloned into the XhoI and BamHI sites of the mammalian expression vector pEGFPN1 (Clontech) to make a plasmid encoding a fusion protein (BGT-EGFP) in which the COOH terminus of BGT1 was fused to the NH2 terminus of EGFP with a spacer of six amino acids.
Visualization of BGT1 protein in cultured cells. Endogenous BGT1 in MDCK cell monolayers on glass coverslips was detected by immunofluorescence after fixation in cold methanol, as described previously (3). After being blocked with 2% gelatin for 30 min, the cells were incubated for 2 h with affinity-purified anti-dog BGT1 polyclonal antibody, diluted 1:100 in 1% gelatin. The primary antibody was detected by a 1-h incubation with affinity-purified goat anti-rabbit IgG conjugated to Cy5 (Jackson ImmunoResearch, West Grove, PA), diluted 1:100 in 1% gelatin. All solutions contained Tris-buffered saline and all incubations were at 37°C. Cells were mounted in 9% Mowiol solution, and fluorescent images were acquired with a LSM 510 laser scanning confocal microscope (Carl Zeiss Microimaging, Thornwood, NY) by using a water immersion x40 lens with 1.2 numerical aperture and 220 µm working distance. The optical section thickness was 1 µm, and the instrument gain and offset were the same for all samples. The excitation for Cy5 was at 633 nm, and emission was collected at >650 nm.
EGFP-tagged BGT1 proteins were detected in live cells on glass coverslips mounted in a chamber on the stage of the confocal microscope. The chamber contained a solution of 25 mM glucose in buffered physiological saline, pH 7.4 (15, 29). For detection of EGFP fluorescence, the excitation was at 488 nm and emission was collected at 500-550 nm.
Online studies of EGFP-BGT distribution in live cells at various times during hypertonic stress were conducted on cells grown on glass coverslips that were mounted across a 1-cm hole in the bottom of a standard 35-mm dish. At 24 h posttransfection, the dish was clamped to the stage of the confocal microscope by using a x40 water-immersion lens with silicone oil (Dow Corning 200) that has the same refractive properties as water. An appropriate group of cells was located, and the normal cell culture medium in the dish was carefully replaced by 5 ml of hypertonic medium (500 mosmol/l). The hypertonic medium was a 1:1 mixture of DMEM:Hams F12 lacking bicarbonate and phenol red (Sigma), buffered to pH 7.4 with 30 mM HEPES, and contained 10% bovine calf serum and sucrose. Preliminary tests showed that the pH was stable within 0.1 pH unit for 24 h, and the cells showed no adverse reaction (rounding, vacuolization, blebbing, etc.). A timed series (30-min intervals) of z-section images was begun immediately. Each z-stack was six images at 2 µm intervals.
All images were stored on CD-RW by using Zeiss LSM software and imported into Adobe Photoshop 5.0 (Adobe Systems, San Jose, CA).
Biotinylation and Western blotting of cell lysates. Surface biotinylation of MDCK cells transfected with EGFP-BGT was performed exactly as described previously in detail (7). An aliquot of the Triton lysate was removed before treatment with streptavidin beads. Both this fraction and the fraction recovered on the beads were analyzed for EGFP-BGT protein by Western blotting.
Cell monolayers in six-well plates were placed on ice and, if necessary, were washed twice with isotonic or hypertonic (as appropriate) Tris-buffered saline. The cells were lysed by the addition of 1% Triton X-100 solution containing 150 mM NaCl, 5 mM EDTA, 10% glycerol, 50 mM Tris, pH 7.5, and protease inhibitors (Halt cocktail; Pierce, Rockford, IL). Cells were collected by scraping, sonicated for 5 s, incubated for 15 min at 4°C, and centrifuged at 13,000 g for 15 min. The supernatant was removed and stored at -80°C. Aliquots were assayed for protein content by the BCA method (Pierce). Before being loaded (5-10 µg/lane) on a 10% polyacrylamide/SDS gel, samples of the supernatant were incubated in SDS sample buffer, containing 100 mM dithiothreitol, for 10 min at 65°C. Prestained molecular weight markers (Bio-Rad, Hercules, CA) were run in parallel. The separated proteins were electrotransferred to nitrocellulose membrane overnight using the Bio-Rad minigel system, as in previous studies (3, 24). The membranes were blocked with 5% milk powder and blotted for 2 h with affinity-purified rabbit polyclonal antibody to GFP (Abcam, Cambridge, UK), diluted 1:20,000 in 5% bovine serum albumin. This antibody recognizes all variants of GFP, including EGFP. The GFP antibody was detected by blotting for 45 min with goat anti-rabbit IgG coupled to horseradish peroxidase (Jackson ImmunoResearch), diluted 1:2,500 in 5% milk powder, and enhanced chemiluminescence (ECL system; Amersham Pharmacia Biotech, Piscataway, NJ). Short exposures to BioMax MR film (Eastman Kodak, Rochester, NY) that were not saturating were used for quantitation. The blots were stripped and reprobed with mouse monoclonal antibody to -actin (1:1,000, AC-40; Sigma, St. Louis, MO) and HRP-conjugated donkey anti-mouse IgG (1:5,000, Jackson ImmunoResearch), as an internal control for sample loading. In biotinylation experiments, E-cadherin was used as the internal control, as described previously (7). Films were scanned with a Bio-Rad GS-670 imaging densitometer.
Where appropriate, the data from different groups were analyzed by the Student t-test or by ANOVA for multiple comparisons.
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RESULTS |
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The normal hypertonic upregulation of endogenous BGT1 transport in MDCK cells was not altered by expression of foreign proteins such as EGFP (Fig. 2), suggesting there were no nonspecific effects on transport. Redistribution of the fusion proteins to the plasma membrane during hypertonic stress was correlated with an increase in hypertonic upregulation of GABA transport, at least in the case of EGFP-BGT (Fig. 2), indicating that the fusion proteins may retain normal transport functions.
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To avoid the complication due to endogenous BGT1 transporters, the transport functions of the fusion proteins were tested more rigorously after transient transfection into mouse embryo fibroblasts (C3H/10T). Nontransfected fibroblasts exhibited little endogenous Na+-dependent uptake of GABA under isotonic conditions, and GABA uptake was not activated by hypertonic stress for 24 h (Fig. 3, nontransfected). Expression of EGFP alone produced no change in GABA uptake. Expression of either EGFP-BGT or BGT-EGFP caused an increase in Na+-dependent GABA transport in normal isotonic medium. Importantly, there was a three- to fourfold increase in transport after 24 h hypertonic stress (Fig. 3, transfected). Thus the hypertonic upregulation of BGT1 transport in MDCK cells was reproduced in C3H/10T
fibroblasts expressing EGFP-tagged BGT1 transporters.
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The transport functions of the fusion proteins were characterized further by determining their sensitivity to a number of inhibitors. Cells were first exposed to 24 h hypertonic stress to induce full expression of transport activity. As previously reported (52), the Na+/Cl--dependent uptake of GABA by normal MDCK cells was markedly inhibited by omission of either Na+ or Cl-, or by the addition of -alanine, nipecotic acid, phloretin, and quinidine to the uptake medium. Nipecotic acid,
-alanine, and quinidine are potent inhibitors of the related Na+/Cl--dependent neurotransmitter (GAT) transporters (47). GABA uptake was also inhibited by the substrate betaine but not by phlorizin (Fig. 4A). This pattern of inhibitor sensitivity was closely reproduced in fibroblasts expressing the EGFPBGT fusion protein (Fig. 4B), but, in contrast, only quinidine was an effective inhibitor of uptake by the BGT-EGFP fusion protein (Fig. 4C).
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Additional experiments focused on the EGFP-BGT fusion protein because it retained normal transport functions. The kinetics of GABA uptake after hypertonic stress for 24 h were analyzed by double-reciprocal plots, as in previous studies (10, 23), by using GABA concentrations in the range 0.01-0.10 mM (Fig. 4). The apparent Km value for EGFP-BGT transiently expressed in fibroblasts was identical to the apparent Km for endogenous BGT1 in normal MDCK cells (Fig. 5). These values were derived from the Na+-dependent component of GABA uptake, and their similarity provides further evidence that the transport function of BGT1 was not altered when EGFP was attached to the NH2 terminus.
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MDCK cells transiently expressing EGFP-BGT fusion protein were used to directly compare the subcellular distribution of this protein with that of endogenous BGT1, after hypertonic stress. Endogenous BGT1 was detected by immunostaining by using a secondary antibody tagged with Cy5. The two fluorescent signals showed almost complete overlap and were colocalized in the plasma membranes (Figs. 6, A and B). This overlap is also an important control that demonstrates that EGFP fluorescence represents the intact EGFPBGT protein because it will be detected by both EGFP fluorescence and immunostaining. The antibody also stained nontransfected cells, but, as expected, the signal was much more intense in the cells that had been transfected with EGFP-BGT (Fig. 6C). This panel provides a direct comparison of endogenous BGT1 and EGFP-BGT in the same group of cells. Note that endogenous BGT1 in the nontransfected cells is also located predominantly in the plasma membranes (Fig. 6C). These observations strongly suggest that the EGFP-BGT protein is trafficked normally when MDCK cells adapt to hypertonic stress.
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Western blotting of cell lysates with anti-GFP antibody was used to determine whether hypertonic upregulation of transport was accompanied by a change in the abundance of expressed EGFP-BGT protein. Antibody specificity was validated by the absence of any binding to lysates from nontransfected fibroblasts. As expected, the antibody bound to a 26-kDa band, corresponding to free EGFP, in lysates from fibroblasts transfected with pEGFPC3 vector (Fig. 7). The expressed EGFP-BGT was detected as a broad band at 95-100 kDa, a size consistent with 70 kDa for BGT1 (HM Kwon, personal communication) plus 26 kDa for EGFP, and there was no increase in abundance after 24 h of hypertonic stress (Fig. 7). Reprobing with anti-actin antibodies revealed no consistent difference in sample loading. These findings were confirmed in four separate experiments. In each experiment, the abundance of EGFP-BGT in Western blots of lysates from hypertonic cells was compared with isotonic controls by densitometry. The hypertonic:isotonic ratio was 1.02 ± 0.24 (means ± SE, n = 4). The same ratio for actin was 0.98 ± 0.18.
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Presumably, the previous data reflect redistribution of EGFP-BGT from cytosol to plasma membrane. This might indicate that overexpression of EGFP-BGT in transfected cells provides the cell with abundant protein so that de novo synthesis is not essential during the subsequent onset of hypertonic stress. In theory, under these circumstances, the EGFP-BGT protein would be available for early insertion in the plasma membrane. The time course of membrane insertion during hypertonic stress was studied online in live MDCK cells at 24 h posttransfection (Fig. 8). At time 0, the fluorescence was excluded from the nucleus but restricted to an area in the cytosol close to the nucleus, possibly the endoplasmic reticulum (ER)-Golgi region. The fluorescence is most likely due to EGFP-BGT rather than free EGFP, which floods all cell compartments (Fig. 1). Close inspection reveals that EGFPBGT fluorescence appeared in the plasma membrane of the upper cell after 2.5 h (arrowhead) of hypertonic stress, possibly earlier (1.5-2.0 h). By 3 h, the plasma membrane location of EGFP-BGT was quite distinct (arrowhead), and by 4 h the cytosol adjacent to the plasma membrane appeared empty of fluorescence (upper cell). The response in the lower cell was the same but slower. By 14 h, both cells showed the same plasma membrane fluorescence with none in adjacent cytosol. A second study confirmed the time course shown here in Fig. 8.
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Independent verification of these data was obtained by surface biotinylation of transfected MDCK cells after exposure to hypertonic growth medium for 1-6 h (Fig. 9). The cells were used at 24 h after transfection with EGFP-BGT and, as expected, EGFP-BGT protein was abundant in the Triton lysate at all time points. Due to relatively low transfection efficiency (20%), only a small amount of surface EGFP-BGT was available for biotin labeling. Nevertheless, a weak signal for EGFPBGT was detectable in the streptavidin bead fractions obtained at 3-6 h, but none was detected at 1-2 h. Cadherin was readily biotinylated at all time points. These findings are based on a population of cells and confirm the time course observed in individual cells by microscopy (Fig. 8). Taken together, the data show that an increase in surface expression of EGFP-BGT protein in MDCK cells can be detected within 2-3 h of hypertonic stress.
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When the time course study (Fig. 8) was repeated in the presence of cycloheximide at 10 µM (32), the membrane insertion of EGFP-BGT protein was blocked completely (not shown). Cycloheximide was added when the cells were switched to the hypertonic medium and there was no pretreatment. However, over the 14-h treatment period, there was significant cell detachment. To reduce adverse effects on cell viability, the experiment was repeated with a lower cycloheximide concentration (3 µM), and the cells were incubated for 14 h in normal isotonic or hypertonic growth medium (±cycloheximide) in a CO2 incubator. The results confirmed that cycloheximide blocked translocation of EFGP-BGT to the plasma membrane in response to hypertonicity. No cell rounding or detachment occurred, and representative confocal images are shown in Fig. 10.
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DISCUSSION |
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Regulated insertion of proteins into the plasma membrane has been implicated in the control of numerous membrane transporters. Most recently, tagging membrane transporters with fluorescent proteins has been used successfully to monitor such events for the Na+/phosphate cotransporter (NaPi2) (17), the Na+/H+ exchanger (NHE3) (19, 20), the cystic fibrosis transmembrane conductance regulator (21, 28), subunits of the epithelial sodium channel (6), and the glucose transporter (GLUT4) (41). In the cited studies, the fluorescent tag did not interfere with the normal behavior and transport function of the proteins.
One aim of the present work was to document the transport properties of BGT1 after fusion with EGFP. Such work could not be done with precision in MDCK cells due to endogenous BGT1, so this work introduced the use of fibroblasts with low endogenous BGT1 activity (Fig. 3). The fusion protein formed by attachment of EGFP to the NH2 terminus of BGT1 (EGFP-BGT) functioned normally when expressed in C3H/10T fibroblasts. The Na+- and Cl--dependent transport of GABA, the hypertonic upregulation of transport, the sensitivity to several inhibitors of normal BGT1, and the apparent Km for GABA were indistinguishable from normal BGT1. The EGFP-BGT protein also trafficked normally in response to hypertonic stress, based on its localization vs. normal BGT1 in the plasma membrane of MDCK cells. The low but measurable level of BGT1 transport activity in nontransfected cells in isotonic solution (Fig. 2) was entirely consistent with the findings of previous studies (3, 7, 34) and suggests that a small amount of BGT1 protein must reside in the plasma membrane even in the absence of osmotic stress. It was therefore not surprising to observe a small amount of EGFP-BGT fluorescence associated with the plasma membrane under isotonic conditions (Fig. 1).
Interestingly, the COOH-terminal fusion protein (BGT-EGFP) had showed similar trafficking in response to hypertonic stress despite having clearly compromised sensitivity to inhibitors. Nipecotic acid, a cyclic analog of GABA, and -alanine are competitive inhibitors of GABA transporters (14) and likely bind to the same site as the GABA (or betaine) substrate (46). These results suggest that the conformational stability of the transport site, or the fidelity of the transport function, is not required for the signals that regulate insertion of protein into the plasma membrane. Given the altered behavior of BGT-EGFP, only the EGFPBGT protein was studied in more detail.
Results in transfected fibroblasts unequivocally show that hypertonic stress can activate BGT1 transport function in the absence of changes in BGT1 protein abundance. Direct visualization of the EGFP-BGT protein (Figs. 1 and 6) suggests that in transfected fibroblasts, the upregulation of BGT1 transport during hypertonic stress is due primarily to redistribution of intracellular BGT1 protein from cytoplasm to plasma membrane rather than synthesis of additional BGT1 protein. This provides strong support for the possibility that a redistribution mechanism may also operate in normal kidney cells during the initial phase of hypertonic adaptation. Translocation of endogenous BGT1 protein in MDCK cells has been suggested by previous studies (3). Direct studies of the time course of redistribution in transfected MDCK cells (Figs. 8 and 9) showed that EGFP-BGT appeared in the plasma membrane within 2-3 h after the onset of hypertonic stress. Overexpression, due to transfection, provides a plentiful supply of EGFP-BGT protein under isotonic conditions without the need for synthesis de novo. Under these conditions, the EGFP-BGT was detected at the cell surface relatively quickly when the cells were transferred to hypertonic medium, consistent with a redistribution mechanism.
Hypertonic upregulation of BGT1 transport is dependent on de novo RNA and protein synthesis, as shown by blockade of the upregulation by actinomycin D and cycloheximide in kidney (3), bone (11), and endothelial (40) cells. A proper understanding of how this dependence on RNA and protein synthesis can be reconciled with the lack of an increase in EGFP-BGT protein abundance, reported here, will require additional investigation. One possibility is that insertion of the BGT1 protein into the plasma membrane requires synthesis of accessory proteins, such as certain SNARE proteins involved in membrane docking and fusion. Alternatively, BGT1 protein may simply be unstable under hypertonic conditions. In this case, an increased production of BGT1 protein would be required for maintaining the adaptation established initially by redistribution. Either explanation is consistent with blockade of the overall adaptive mechanism by nonselective inhibitors of RNA and protein synthesis. However, blockade of the redistribution by cycloheximide, even when the supply of EGFP-BGT protein is already plentiful (Fig. 10), strongly supports the notion that accessory proteins are required for the membrane insertion step. Until adequate amounts of these proteins are made available through de novo synthesis, the intracellular EGFP-BGT cannot gain access to the plasma membrane.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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