VHL tumor suppressor regulates Cl-/HCO3- exchange and Na+/H+ exchange activities in renal carcinoma cells

S. ANANTH KARUMANCHI1, LIANWEI JIANG3, BERTRAND KNEBELMANN1, ALAN K. STUART-TILLEY3, SETH L. ALPER1,2,3 and VIKAS P. SUKHATME1,2

1 Renal Division
2 Cancer Center
3 Molecular Medicine Unit, Beth Israel Deaconess Medical Center, Departments of Medicine and Cell Biology, Harvard Medical School, Boston, Massachusetts 02215


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutations in the von Hippel-Lindau (VHL) tumor suppressor gene are thought to play a critical role in the pathogenesis of both sporadic and VHL disease-associated clear-cell renal carcinomas (RCC). Differential display-PCR identified the AE2 anion exchanger as a candidate VHL target gene. AE2 mRNA and polypeptide levels were approximately threefold higher in 786-O VHL cells than in 786-O Neo cells. In contrast, Cl-/HCO3- exchange activity in 786-O VHL cells was 50% lower than in 786-O Neo cells. Since resting intracellular pH (pHi) values were indistinguishable, we postulated that Na+/H+ exchange activity (NHE) might be similarly reduced in 786-O VHL cells. NHE-mediated pHi recovery from acid load was less than 50% that in 786-O Neo cells, whereas hypertonicity-stimulated, amiloride-sensitive NHE was indistinguishable in the two cell lines. The NHE3 mRNA level was higher in 786-O VHL than 786-O Neo cells, but NHE1 mRNA levels did not differ. AE2 and NHE3 are the first transcripts reported to be upregulated by pVHL. Elucidation of mechanisms responsible for downregulation of both ion exchange activities will require further investigation.

von Hippel-Lindau disease; AE2; NHE3; renal cell carcinoma; intracellular pH


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
VON HIPPEL-LINDAU (VHL) disease is an autosomal dominant familial cancer syndrome characterized by bilateral clear-cell renal carcinoma (RCCs), cerebellar hemangioblastoma, pheochromocytoma, and retinal angiomata (33, 41). RCC is the most frequent cause of death in VHL and differs from sporadic RCC in its earlier age of onset and its greater frequency of bilateral and multicentric occurrence. VHL syndrome arises in individuals who inherit a germline mutation in the VHL gene. The RCCs of VHL syndrome are characterized by somatic mutation in the second VHL allele, often in the form of loss-of-heterozygosity. VHL loss-of-heterozygosity has been described not only in solid RCC tumors but also in premalignant epithelial monolayers surrounding renal cysts (44). These data suggest that VHL biology conforms to Knudson’s two-hit hypothesis for tumor suppressor gene function (33). VHL function as a tumor suppressor gene has been confirmed in nude mouse tumor transplantation experiments (26). The VHL gene is also mutated or deleted in up to 75% of sporadic RCC cases (20) but has not been implicated in the less common nonfamilial papillary renal carcinoma. Inactivation of the mouse VHL gene by homologous recombination results in embryonic lethality in homozygotes and suggests a crucial role for this gene in mammalian development (21).

Among the gene products downregulated by pVHL are the hypoxia-inducible genes vascular endothelial-derived growth factor (VEGF) and platelet-derived growth factor B, the constitutive glucose transporter GLUT1, and the hypoxia-inducible transcription factor HIF-1{alpha} (22, 27, 45, 46). VEGF expression is regulated both at the transcriptional level through Sp-1 (46) and at the posttranscriptional level (22, 27). The highly vascular phenotype of RCC and other tumors deficient in pVHL reflects, in part, upregulation of these genes. Additional gene products downregulated by pVHL are transforming growth factor-{alpha} and transforming growth factor-ß1 (4, 36) and transmembrane carbonic anhydrases CA9 and CA12 (30).

The discovery of an in vitro interaction between pVHL and the transcription elongation factors elongin B/C of the elongin (SIII) complex (6, 16, 50) was first interpreted to indicate a central role for pVHL in the regulation of transcriptional elongation. However, no identified pVHL target genes have been found to be regulated at the level of transcriptional elongation.

Klausner and colleagues (51) have identified the F-box protein, Cul2, as a pVHL-interacting protein. The Saccharomyces cerevisiae Cul2 homolog, Skp1-Cdc53, is part of a ubiquitin protein ligase complex that targets cell cycle proteins for ubiquitin-dependent proteolysis (51). pVHL binds Cul2 via elongin C to form the VCB/Cul2 complex, which, together with SCF and APC/C complexes, comprise an E3 ubiquitin ligase superfamily (31, 42, 61). Tumor-derived VHL mutations arise either within the predicted {alpha}-helical elongin C interaction site or within the ß-sheet domain predicted to interact with other target proteins of the ubiquitin ligase complex (54). These findings suggest that VHL mutations may cause pathological accumulation of certain cellular proteins, leading in turn to transformation. Indeed, VHL mutants unable to bind the Cul2-elongin B/C complex are unable to downregulate mRNA levels of the pVHL target gene, GLUT1, (43), previously shown to be upregulated in transformed cells of many lineages (18). VHL-deficient tumor cells subjected to a wide range of experimental stresses to impair protein folding and processing exhibit increased ubiquitination of cellular proteins (23). Thus pVHL appears to participate in the elimination of misprocessed proteins.

We have continued our search for novel pVHL target genes that might contribute to the tumor suppressor function of pVHL by comparing transcript profiles in an pVHL-null RCC cell line with those of the same RCC cell line stably overexpressing heterologous wild-type pVHL, using mRNA differential display (5). We report here that the AE2 anion exchanger and the NHE3 sodium/hydrogen exchanger genes are two novel targets for pVHL. They are the first reported gene products upregulated by heterologous pVHL in VHL-null tumor cells. However, their upregulation is accompanied by contrasting downregulation of anion and cation exchange activities.


    METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

Reagents.
Sodium gluconate, potassium gluconate, calcium gluconate, and N-methyl-D-glucamine (NMDG) were obtained from Fluka Chemical (Milwaukee, WI). Ammonium gluconate was obtained from Pfaltz and Bauer (Waterbury, CT). The acetoxymethyl ester of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM) and nigericin were obtained from Molecular Probes (Eugene, OR). Amiloride and bafilomycin were obtained from Sigma (St. Louis, MO).

Cell culture.
The 786-O human RCC cells, which are null for pVHL (20) and tumorigenic in nude mice, were obtained from the American Type Culture Collection (Manassas, VA) and were stably transfected with an expression vector encoding full-length human VHL with an NH2-terminal FLAG tag (pCMV2-FlagVHL) to generate 786-O VHL cells. 786-O cells were also stably transfected with control vector (pCMV2-FLAG) to generate 786-O Neo cells (36, 46). Pooled clones of the transfected lines were used for all the experiments. 786-O VHL and 786-O Neo were grown in DMEM with 10% FBS supplemented with the antibiotic G418.

mRNA differential display.
Differential display reactions were performed with the GenHunter kit (Nashville, TN) according to the manufacturer’s protocol. In brief, 0.1 µg of poly(A) mRNA was reverse transcribed using the primers H-T11G, H-T11A, and H-T11C. The same primers were used subsequently as 3' primers to amplify cDNA by PCR along with random 13-mer 5' primers using 10 µCi 35S-labeled dATP. The PCR products were then run on 6% sequencing gel. Differentially expressed bands were excised and cDNA eluted. The cDNA was reamplified using the same conditions. Resultant PCR products were then cloned into pCRII (Invitrogen) and sequenced. DNA sequences were analyzed with the National Center for Biotechnology Information BLAST programs.

Northern analysis.
RNA was extracted and Northern analysis performed as described previously (36, 46). cDNA probes used were mouse AE2 and AE1 (64), human bAE3 (63), rat NBC1 (gift of M. Romero), the 1.9-kb BamHI fragment of human NHE1 (gift of D. Biemesderfer), and the 2.5-kb EcoRI/XhoI fragment of rabbit NHE3 cDNA (gift of M. Tse and M. Donowitz).

Immunoblot analysis.
Cells were grown to 70–80% confluence and lysed in lysis buffer as previously described (36, 46). Equal amounts of cell lysates (50 µg) as determined by Bradford assay were loaded. Rabbit polyclonal anti-mouse AE2 raised against amino acids 1224–1237 has been described by Alper et al. (2). Anti-actin antibody was from Santa Cruz (Santa Cruz, CA). Anti-VHL antibody was the gift of W. Kaelin (27).

Immunofluorescence analysis of cells.
The 786-O Neo and 786-O VHL cells, 1 x 104 cells, were plated separately on coverslips placed in six-well plates and grown overnight. These two cell lines exhibit indistinguishable growth rates (26). The cells on coverslips were fixed in 3% paraformaldehyde and subjected to standard indirect immunofluorescence as previously described (1, 57). Rabbit polyclonal anti-mouse AE2 antibody, amino acids 1224–1237, was used at a dilution of 1:1,000 to 1:2,000. Fixed cells were subjected to SDS pretreatment as previously described (2, 11).

Measurement of intracellular pH.
786-O Neo and 786-O VHL were grown to subconfluent density on Cell-Tak-coated 5-cm2 coverslips. Cells were loaded with the pH indicator BCECF by incubation with 3–5 µM of its acetoxymethyl ester (BCECF-AM) derivative in culture medium at 37° for 35 min. Extracellular BCECF-AM was removed by washing twice with normal media. The coverslip was then mounted in a 1-ml perfusion chamber and superfused at 8.5 ml/min. Fluorescence excitation ratio imaging of intracellular pH (pHi) was carried out with an Image-1 digital ratio image system (Universal Imaging, Westchester, PA) as previously described (32).

The BCECF ratio images were recorded at 530-nm emission with dual excitation at 495 and 440 nm over a time period of 20–25 min. In situ calibration of the BCECF fluorescence ratio to pHi in these cells was performed by the nigericin-high K+ method (32). At the end of each experiment, a single point calibration was carried out for each coverslip to convert fluorescence ratio to pHi values from in situ standard calibration curves. Curves of single cell pHi vs. time were generated by the Image-1/FL software and manipulated with Quattro Pro (Borland) and Excel (Microsoft).

Measurement of Cl-/HCO3- and Na+/H+ exchange activities.
In a CO2/HCO3- open system, total intracellular buffer capacity is defined as ßT = ßi + ßCO2, where ßi is the intrinsic buffer capacity for a cell line, and ßCO2 = 2.3 x (HCO3-)i. The pHi dependence of ßi in both 786-O Neo and 786-O VHL cells was determined by the ammonium pulse method as described previously (32) with sequential, stepwise reduction in extracellular NH4Cl concentration from 20 mM through 10, 5.0, 2.5, 1.0. 0.5, and 0 mM. The initial proton flux rate JH+ (mM/s) was calculated as JH+ = ßT x dpHi/dt, where dpHi/dt is the initial rate of change of pHi in response to the applied experimental perturbation.

Cl-/HCO3- exchange activity in single cells was estimated from initial dpHi/dt following bath Cl- removal and subsequent restoration. Cl--containing medium contained (in mM) 126 NaCl, 24 NaHCO3, 5 KCl, 2 MgSO4, 1 CaCl2, and 10 glucose and 5% CO2, pH 7.40. Cl--free medium contained (in mM) 126 sodium gluconate, 24 NaHCO3, 5 potassium gluconate, 2 MgSO4, 3 calcium gluconate, and 10 glucose and 5% CO2, pH 7.40. dpHi/dt was measured by linear regression of the initial portion of each pHi vs. time record. Sodium-free solutions substituted NMDG for sodium.

Na+/H+ exchange was estimated from initial dpHi/dt during pHi recovery from NH4+ prepulse-induced cell acidification in room air. Modified Hanks’ balanced salt solution (HBSS) was replaced by solution in which 20 mM NaCl was substituted with 20 mM NH4Cl, followed by restoration of HBSS. Alternatively, Na+/H+ exchange activity was estimated from initial dpHi/dt during shift from isotonic modified HBSS (310 mosM) to hypertonic medium (460 mosM), with incremental osmoles provided as NaCl as described by Humphreys et al. (25).

Estimation of cell Cl- and Na+ contents.
Cell content of exchangeable Cl- in room air at room temperature was estimated as the 1-h value of 36Cl- uptake from modified HBSS containing 1 mCi/ml Na36Cl. Isotopic Cl- steady-state uptake in human 293 cells requires 20 min (19). Cl- efflux was measured as 36Cl- lost from cells into the isotope-free medium (room air, room temperature).

To measure cell sodium content, monolayers washed three times with ice-cold 300 mM mannitol at 4°C were scraped, pelleted, resuspended in four volumes distilled water, and subjected to three cycles of freeze-thaw and vortexing. The cell lysate was cleared of particulate by microcentrifugation and analyzed by atomic absorption spectroscopy.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

Identification of AE2 as a target for pVHL by mRNA differential display.
Differential display reactions were performed to compare the pattern of gene expression of 786-O RCC cells transfected with pVHL (786-O VHL) compared with the parental RCC cell line (786-O Neo). We identified a 250-bp PCR product that was upregulated in 786-O VHL cells (Fig. 1A). DNA sequence identified it as a 3'-untranslated region fragment of human AE2 cDNA. pVHL immunoblot confirmed that the 786-O and 786-O VHL cells used as mRNA sources respectively lacked and overexpressed pVHL (Fig. 1B).



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Fig. 1. mRNA differential display in 786-O cell lines. A: cDNA (0.1 µg) reverse transcribed from 786-O Neo and 786-O von Hippel-Lindau (VHL) cells was PCR-amplified using primers 5'-AAGC-T11G-3' and 5'-AAGCTTAACGAGG-3' and 35S-dATP. PCR products were size-fractionated by PAGE, and the differentially expressed band (arrow) was reamplified and sequenced. Sequence of the PCR product matched the 3'-untranslated region of human AE2 (GenBank accession no. U76668). B: pVHL expression status of 786-O cell lines used for mRNA differential display. Expression status of flag-tagged pVHL was confirmed by immunoblot of 20 µg whole cell lysates using monoclonal anti-VHL antibody.

 
AE2 mRNA and protein are upregulated by pVHL overexpression.
Northern blots revealed a 3.5-fold upregulation of AE2 mRNA in the 786-O VHL cells compared with 786-O Neo cells (Fig. 2A). Immunoblot analysis confirmed that AE2 polypeptide was similarly upregulated in 786-O VHL cells (Fig. 2B). These data confirm that pVHL overexpression upregulates AE2 mRNA and protein in 786-O VHL cells.



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Fig. 2. AE2 mRNA and protein in 786-O cell lines. A: Northern blot analysis of AE2 mRNA. Agarose gel-fractionated total RNA (20 µg) from 786-O Neo and 786-O VHL cells was blotted and probed with 32P-labeled mouse AE2 cDNA; 786-O VHL cells upregulated AE2 mRNA 3.5-fold compared with 786-O Neo cells. Bottom: same blot as top, but reprobed with ß-actin cDNA. Blot is representative of 3 similar experiments. B: immunoblot analysis of AE2 protein. Whole cell detergent lysates (50 µg protein) from 786-O Neo and 786-O VHL cells were fractionated by gradient SDS-PAGE, transferred to nylon, and probed with anti-AE2 antibody in the presence (lanes 1 and 2) and absence (lanes 3 and 4) of peptide antigen (30 µg/ml). AE2 in 786-O cells comigrated with gastric parietal cell AE2a/b of porcine gastric microsomes (PGM, lane 5). Bottom: same blot as top, but reprobed with anti-actin antibody. Blot is representative of 3 similar experiments.

 
Figure 3, A and B, shows that, as in other cell lines (2, 11), fixed 786-O cells untreated with SDS revealed no AE2 immunostaining at the cell surface, but only in intracellular vesicular compartments. Whereas AE2 in 786-O Neo cells exhibited a Golgi-like pattern, in most 786-O VHL cells AE2 epitope-positive vesicular structures were more widely dispersed. Brief SDS treatment of fixed cells unmasked a surface immunostaining pattern for AE2 (11), while abolishing the Golgi-like intracellular staining pattern (Fig. 3, C and D). Both immunostaining patterns exhibited peptide antigen-specific competition (not shown). Thus, apparent surface AE2 immunostaining in 786-O VHL cells was of similar or greater intensity than in 786-O Neo cells.



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Fig. 3. AE2 immunolocalization in 786-O cell lines. The 786-O Neo and 786-O VHL cells were fixed on coverslips and immunostained with anti-AE2 antibody without (A and B) or with SDS epitope unmasking (C and D). As reported for other tissues and cells, this antibody detected an AE-related epitope with a Golgi-type staining pattern without SDS treatment and detected plasmalemmal AE2 staining after SDS treatment of fixed cells. Both epitopes appeared equally abundant (if not more abundant) in 786-O VHL and Neo cells. Images are representative of 3 similar experiments.

 
pVHL downregulates Cl-/HCO3- exchange activity in 786-O VHL RCC cells.
The difference in levels of AE2 polypeptide in 786-O Neo and 786-O VHL cells prompted measurement of Cl-/HCO3- exchange activity in the two cell lines. Cellular buffer capacities did not differ (Fig. 4, A and B). Resting pHi values in the two cell lines in the presence of CO2/HCO3- similarly did not differ (Table 1). In contrast to the above-noted upregulation of AE2 mRNA and protein levels, Cl-/HCO3- exchange-mediated JH+ was decreased more than half in 786-O VHL cells compared with 786-O Neo cells (Fig. 5, A and B; and Table 1).



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Fig. 4. Intracellular pH (pHi) dependence of intrinsic buffer capacity (ßi, A) and total buffer capacity (ßT, B) in 786-O Neo ({square}) and 786-O VHL cells ({triangleup}).

 

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Table 1. Cl-/HCO3- exchange activity

 


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Fig. 5. Cl-/HCO3- exchange activity in 786-O cell lines. A: mean pHi as a function of time in 15 individual 786-O Neo (squares) and 786-O VHL cells (triangles) on one coverslip during extracellular chloride removal and restoration. See Table 1 for analysis of multiple similar coverslips. B: composite of experiments relating Cl-/HCO3- exchange-mediated proton flux rate (JH+) to pHi in 786-O Neo (squares) and 786-O VHL cells (triangles). Data were obtained during both Cl- removal (open symbols) and Cl- restoration (solid symbols). See Table 1 for summary.

 
This reduced Cl-/HCO3- exchange activity in the face of increased AE2 polypeptide might be explained by decreases in expression of other anion exchangers. However, as shown in Fig. 6, levels of AE3 and AE1 mRNAs did not differ in the two cell lines and were in any case present at much lower levels than AE2 mRNA. Alternatively, cytosolic Cl- concentration might be sufficiently low in magnitude in 786-O VHL cells to explain the observed reduced activity of Cl-/HCO3- exchange. However, the steady-state intracellular Cl- contents (estimated from 1-h 36Cl- uptake values in room air) were, for 786-O Neo and 786-O VHL cells, respectively, 197 ± 9 and 234 ± 19 nmol Cl-/mg protein. If 1 mg protein corresponds to 6.5 µl cell water (19), then these values correspond to intracellular Cl- concentration estimates of 30 and 36 mM (n = 4). The rapid phase of 36Cl- efflux in both cell lines (~75% of the 1-h uptake) was complete within 15 min (not shown).



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Fig. 6. AE3 and AE1 mRNA expression in 786-O cell lines RNA blots prepared as in Fig. 1 were hybridized with 32P-labeled human AE3 cDNA (A) and mouse AE1 cDNA (B). Bottom: same blots reprobed with ß-actin cDNA. One of two similar experiments.

 
pVHL upregulates NHE3 mRNA but not NHE1 mRNA in 786-O VHL cells.
The equivalent resting pHi values in 786-O VHL and 786-O Neo despite the reduced Cl-/HCO3- exchange activity in 786-O VHL cells might be explained by a parallel decrease in base-loading activity. As shown in Fig. 7, NHE3 mRNA level was, indeed, two- to threefold higher in 786-O VHL than in 786-O Neo cells, whereas NHE1 mRNA levels did not differ in the two cell lines. NBC1 Na+-bicarbonate cotransporter mRNA levels did not differ in the two cell lines (data not shown).



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Fig. 7. NHE1 and NHE3 mRNA expression in 786-O cell lines. Northern blot analysis showed 2- to 3-fold upregulation of NHE3 mRNA in 786-O VHL cells compared with 786-O Neo cells (A), but there was no difference in NHE1 mRNA level (B). Bottom: same blot reprobed with ß-actin cDNA. One of two similar experiments.

 
pVHL overexpression reduces acid-stimulated Na+/H+ exchange activity in 786-O VHL cells but does not reduce hypertonic stimulation of activity.
As shown in Fig. 8A and in Tables 2 and 3, the rate of pHi recovery from an acid load induced by a 25 mM NH4+ pulse in room air was 75% lower in 786-O VHL cells than in 786-O Neo cells. In both 786-O VHL and 786-O Neo cells, pHi recovery from acid load required extracellular Na+, was fully inhibited by 100 µM amiloride in the presence of extracellular Na+, and was insensitive to the inhibitor of vacuolar H+-ATPase, bafilomycin (Table 3). These findings confirm that pHi recovery from acid load in both cell lines was mediated by NHE1-like Na+/H+ exchange activity. Interestingly, however, the rate of intracellular alkalinization induced in the two cell lines by hypertonic stimulation (460 mosM) did not differ significantly (Fig. 8B and Table 2). Hypertonic stimulation of intracellular alkalinization in both cell lines was abolished by 100 µM amiloride (n = 3 coverslips for each line, not shown). Intracellular sodium content also did not differ in the two cell lines (not shown), and so altered driving forces did not likely explain the different rates of recovery from acid load.



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Fig. 8. Na+/H+ exchange activity in 786-O cell lines. A: Na+/H+ exchange-mediated recovery from acid load induced by NH4Cl prepulse. Mean pHi changes in 9 individual 786-O Neo cells (squares) and 786-O VHL cells (triangles) on single coverslips during and after NH4+ prepulse. Rate of pHi recovery from acid load was reduced in 786-O VHL cells. See Table 2 for summary of results with multiple similar coverslips. pHi recovery required extracellular Na+, was inhibited by amiloride, and was insensitive to bafilomycin (Table 3). B: Na+/H+ exchange activated by hyperosmolarity. Mean pHi changes in 11 single 786-O Neo cells (squares) and 10 single 786-O VHL cells (triangles) on single coverslips during shift from isotonic to hypertonic extracellular solution. Rates of intracellular alkalinization in the two cell lines did not differ. See Table 2 for summary of results from multiple coverslips.

 

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Table 2. Na+/H+ exchange activity

 

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Table 3. Pharmacology of recovery from acid load

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

AE2 and NHE3 mRNA levels are upregulated by pVHL.
pVHL has been identified as a component of a cellular E3 ubiquitin ligase system (31, 34). This functional link has fueled the hypothesis that pVHL promotes protein ubiquitination and, conversely, that absence of pVHL leads to pathological accumulation of certain cellular proteins. The increased abundance (and/or pathological persistence) of certain proteins throughout the cell cycle may predispose the cell to transformation. This hypothesis agrees with earlier and current findings that most mRNAs regulated by pVHL status are decreased in abundance by pVHL expression. We report here the first mRNAs whose abundance is, in contrast, increased by pVHL: the Cl-/HCO3- exchanger AE2 and the Na+/H+ exchanger NHE3. The abundance of AE2 polypeptide was also increased. Both genes are expressed in proximal tubule epithelial cells (810, 53) from which originate both 786-O RCC cells and (often following loss-of-heterozygosity of the VHL gene) RCCs.

Contrasts between regulation by pVHL of ion exchanger mRNA and protein levels and ion exchange activities.
Curiously, ion exchange activities did not increase in parallel with pVHL overexpression-associated increases in level of AE2 mRNA and protein and in level of NHE3 mRNA. In 786-O VHL cells, Cl-/HCO3- exchange activity was instead reduced 50%. AE2 immunostaining in 786-O cells was not correspondingly decreased, and AE2 polypeptide abundance as determined by immunoblot was increased. Although not yet detected in proximal tubule, AE1 has been detected in LLC-PK1 cells (10, 14). AE3 polypeptide expression has been detected in proximal tubule (3, 53). However, the decreased anion exchange activity of 786-O VHL cells was not easily attributable either to AE1 or AE3, since the levels of their mRNAs were very low and did not differ between the two 786-O cell lines.

AE2 expressed in Xenopus oocytes is inhibited by acidic pHi (24, 55), but resting pHi of 786-O VHL cells did not differ from that of 786-O Neo cells and, so, could not explain the decreased Cl-/HCO3- exchange activity. If indeed AE2-mediated, then the reduced Cl-/HCO3- exchange in 786-O VHL cells could reflect an alkali-shifted pHi dependence of activity. This pH dependence requires both the ~530-amino acid AE2 transmembrane domain and a region in the middle of the 700-amino acid NH2-terminal cytoplasmic domain (56, 64). However, even in Cl--free bath, 786-O cells exhibited pHi values at which recombinant AE2 activity in human 293 (39) or CHOP cells (32) showed little or no pHi dependence. The mechanisms by which both pHi and extracellular pH (pHo) regulate AE2 activity, and how those mechanisms might be modulated in renal carcinoma cells and by pVHL, remain under investigation.

Na+-dependent Cl-/HCO3- exchange, although not evident in rabbit S3 segment (37), appears to be a prominent basolateral Cl- efflux pathway in rabbit proximal convoluted tubule (29). In addition, the Cl-/formate exchanger pendrin (52) may be present in proximal tubule, although its direct contribution to in situ Cl-/HCO3- or Cl-/OH- exchange remains to be shown.

Na+/H+ exchange activity (Na+-dependent, amiloride-sensitive recovery from an acid load) was reduced ~75% in 786-O VHL cells, whereas mRNA levels of NHE3 and NHE1 were increased or unchanged, respectively. This reduced transport activity was not explained by changes in transmembrane H+ or Na+ gradients. Complete inhibition of this Na+/H+ exchange-mediated recovery from acid load by 100 µM amiloride at physiological bath Na+ concentration renders unlikely a major contribution from NHE3 (15) or NHE4 (12) but does not rule out NHE2 (17). However, NHE2 expression has not been detected in proximal tubule (13). Thus the Na+/H+ exchange activity downregulated in 786-O cells by pVHL overexpression likely represents NHE1.

Unlike the downregulation of pHi recovery from acid load in 786-O VHL cells, hyperosmotic stimulation of Na+/H+ exchange was unchanged. Complete inhibition of this hyperosmotic stimulation by 100 µM amiloride renders unlikely the possibility that the lack of change reflects the combination of increased hyperosmotic inhibition of NHE3 plus reduced hyperosmotic stimulation of NHE1. Hyperosmotic activation of NHE1 may reflect in part a shift of pHi set point to more alkaline pHi values, likely via relief of auto-inhibition by a region located between amino acids 635–698. Activation is also mediated by residues proximal to amino acid 566, yet deletion of NHE1 residues proximal to amino acid 635 rendered hypertonicity inhibitory (7). The contrasting downregulation of pHi recovery from acid load in 786-O VHL cells may reflect a pVHL-modulated mechanism for acid shift of the NHE1 pHi activation threshold ("set point") which preserves functional integrity of the above inhibitory domain.

pVHL is a subunit of a ubiquitin ligase (31, 34). Ubiquitination has not been implicated as a control mechanism for AE2, but NHE1-like Na+/H+ exchange activity in mouse mandibular salivary gland undergoes feedback inhibition by intracellular Na+ concentration via a Nedd4-independent ubiquitination process (28). This mechanism would be consistent with the observed pVHL-associated downregulation of acid pHi-activated Na+/H+ exchange activity but does not explain preservation of hypertonic stimulation of Na+/H+ exchange activity.

pVHL has been reported to regulate fibronectin assembly and to modify the structure of extracellular matrix in culture (47). Liganding and cross-linking of integrin receptors activates Na+/H+ exchange in a rhoA-dependent process to enhance cell adhesion and spreading (59). Na+/H+ exchange acts downstream of RhoA to regulate integrin-induced cell adhesion and spreading (58). It is intriguing to speculate that pVHL-altered fibronectin may be a less efficient stimulator of integrin-activated Na+/H+ exchange activity. The influence of integrins on hypertonic activation of Na+/H+ exchange or on Cl-/HCO3- exchange is unknown, despite their apparent requirement at the leading edge of MDCK and melanoma cells for migration (35).

Relationship between pVHL regulation of pH regulatory ion exchangers and of carbonic anhydrase genes.
The particular roles of pHi and pHo regulation in tumor growth, invasion, and metastasis remain unclear (62). Acidic pH at the center of solid tumors has been proposed to limit tumor growth (38) and also to favor tumor invasiveness (30). The contrast between upregulation of AE2 and NHE3 gene products and downregulation of Cl-/HCO3- and Na+/H+ exchange activities in 786-O VHL cells must be considered in light of the regulation by pVHL of two basolateral plasma membrane carbonic anhydrase genes, CA12 and CA9. Both CA12 and CA9 transcripts are expressed at high level in 786-O cells, and pVHL overexpression greatly reduces their mRNA levels (30). CA12 and CA9 are also expressed in RCC cells and tumors (40, 48, 49, 60). However, plasmalemmal carbonic anhydrase activities have not yet been reported in these cell lines or tumors. Inhibition of carbonic anhydrase activity with acetazolamide decreased in vitro invasiveness of some RCC cell lines but not cell proliferation rates (49). Decrease "invasiveness" (measured as transmigration through filters) may have reflected, at least in part, decreased anion exchange activity at the leading edge of the migrating/invading cells (35).

The present data do not clarify whether downregulation of CA9 and CA12 mRNAs or upregulation of AE2 and NHE3 mRNAs (or both) are primary actions of pVHL. The former possibility is consistent with the general pattern of VHL-mediated downregulation of multiple transcripts. It would suggest that parallel downregulation of Cl-/HCO3- and Na+/H+ exchange activities represents appropriate secondary adaptations, perhaps leading to "compensatory" upregulation of AE2 and NHE3 mRNA levels. Alternatively, upregulation of AE2 and NHE3 mRNA levels may represent proximate actions of pVHL, but predicted increases in ion exchange activity may be restricted or reversed by independent primary downregulation of CA9 and CA12. Additional study of bicarbonate handling by RCC and its regulation by pVHL should further understanding of renal cancer.


    ACKNOWLEDGMENTS
 
This work was supported by National Institutes of Health Grants F32-CA-70128 (to S. A. Karumanchi), RO1-CA-77702 (to V. P. Sukhatme), and R01-DK-43495 and P30-DK-34854 (Harvard Digestive Diseases Center; to S. L. Alper) and by a fellowship from the National Kidney Foundation (to B. Knebelmann).


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: S. L. Alper, Molecular Medicine Unit RW 763, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston MA 02215 (E-mail: salper{at}caregroup.harvard.edu).


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
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