| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Laboratory of Developmental Nephrology, Department of Physiology and Biophysics, Faculty of Medicine and 2The Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology; and 3Pediatric Nephrology Unit, Rambam Medical Center, Haifa, Israel
Submitted 16 October 2007 ; accepted in final form 27 February 2008
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIAL AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
acid-base transport; aldosterone; kidney; thyroid; inner ear
In the kidney, pendrin is located in the apical membrane of β-intercalated cells of the cortical collecting duct (CCD). Pendrin has been shown to play a role in acid-base balance by secreting HCO3– into the tubular lumen in exchange for Cl– (14, 36) as well as in blood pressure regulation by reabsorbing Cl– in exchange for secreted HCO3– (32, 50). In the thyroid, pendrin resides on the apical membrane of thyrocytes and, by its activity as a Cl–/I– exchanger, is essential for I– efflux from the cell and accumulation in the follicular lumen (35, 41, 58). In the inner ear, pendrin, possibly by secreting HCO3– into the endolymphatic sac, is thought to play a major role in maintaining endolymph ion balance and pH, and to be essential for normal development of the inner ear (6, 12).
The kidney represents an excellent experimental system for investigating mechanisms of epithelial cell-specific gene expression. The specialized reabsorptive and/or secretory function of each tubule segment depends on its structural arrangement and on the specific pattern of gene expression in each tubular cell type. The promoters of several transporter and channel genes including those encoding aquaporin 2 (29), Na+-phosphate cotransporter (42), Na+-K+-2Cl– cotransporter (20), chloride channels (47), and magnesium channels (8) as well as the promoters of nephrin (56) and cadherin (19) genes have been cloned and shown to direct kidney-specific expression in vitro and/or in transgenic mice. Several transcription factors and elements including myc-associated zinc finger protein and Krüppel-like factor (48), hepatocyte nuclear factor 3 (46), and hepatocyte nuclear factor 1
and 1β (1) were found to be involved in kidney-specific expression of the ClC-K1 chloride channel, thiazide-sensitive Na-Cl cotransporter, and cadherin genes, respectively. The cis-acting peroxisome proliferator response element was found to play a role in renal epithelial cell-specific expression of the Mg2+ channel, paracellin-1, gene (8). However, very little is known about the regulatory elements or nephron-specific transcription factors responsible for cell-specific expression of transporter and channel genes within the kidney.
Three thyroid-specific transcription factors, thyroid transcription factors 1 and 2 (TTF-1, TTF-2) and Pax-8, have been shown to confer exclusive thyroid expression of several genes, including thyroglobulin (Tg), thyroperoxidase, and TSH-receptor genes, and govern the development, differentiation, and the unique three-dimensional structure and function of the thyroid gland (5, 7, 34). Na+/I– symporter TSH-responsive factor 1 was found to be involved in the transcriptional regulation of Na+/I– symporter expression in the thyroid (45). In the inner ear, the promoters of several channel genes including the genes encoding the Cl– channel ClC-Kb (26) and the water channel aquaporin-2 (37), as well as the gene encoding the tight junction protein claudin-14 (2), have been shown to direct inner ear-specific expression in vitro and/or in transgenic mice. As yet, the promoter region of the human pendrin gene has not been characterized and the molecular mechanisms for its epithelial cell-specific activity in the kidney as well as in the thyroid and inner ear have not been investigated.
Several factors are known to control acid-base transport in various nephron segments including the CCD. These factors include, among others, systemic pH, body K+ and Cl– stores, and extracellular fluid volume as well as hormones such as aldosterone, thyroid hormone, and angiotensin II (15, 17, 39). While the modulation of renal acid-base transport has been thoroughly investigated at the tubular and cellular levels, the molecular mechanisms of this modulation are poorly understood. Systemic pH and aldosterone modulate activity of several ion transport processes involved in acid-base transport in the CCD, including pendrin activity (14, 36, 50). It has been shown that acid loading diminishes and alkali loading increases pendrin protein expression and activity in the rodent kidney (14, 31, 53). Administration of the aldosterone analog, deoxycorticosterone, resulted in upregulation of PDS mRNA and protein expression in mouse kidney (50). Nevertheless, it has not been established whether the pH- and aldosterone-induced effect on pendrin activity occurs at the protein or DNA/RNA level, and the direct effect of ambient pH and aldosterone on the PDS promoter has not been explored.
Thyroid function and Na+/I– symporter activity are known to be regulated by several factors including TSH, I–, and Tg (7). Northern blot analysis (35) has demonstrated induction of PDS expression by low concentration of Tg in the rat thyroid cell line FRTL-5. In addition, TTF-1, whose action is a prime factor in controlling thyroid differentiation in vivo, was found to regulate the expression of pendrin in vitro (5). There is evidence that the transcription factor Foxi1 is an upstream regulator of the pendrin gene during inner ear development (18). Moreover, mutations in the Foxi1 binding sites on the hPDS promoter abolish Foxi1-mediated transcriptional activation of hPDS and result in enlarged vestibular aqueduct syndrome (57). However, the cellular and molecular mechanisms controlling pendrin activity in the thyroid and inner ear are essentially unknown.
The purpose of the present study was to define promoter regulatory elements essential for renal, thyroid, and inner ear epithelial cell-specific expression of hPDS. We also explored the effect of modulators of acid-base transport on hPDS promoter activity in kidney, thyroid and inner ear cells. We demonstrate the existence of distinct patterns of pendrin promoter activity in renal, thyroid, and inner ear cells. We show that acidic pH decreases, and alkaline pH increases, hPDS promoter activity in renal and inner ear cells and that aldosterone has an inhibitory effect on the hPDS promoter in renal cells. In addition, we show that this pH- and aldosterone-induced effect occurs via distinct response elements on the hPDS promoter located within 96-bp and 89-bp regions, respectively. Finally, we demonstrate that acidic pH and aldosterone decrease whereas alkaline pH increases endogenous PDS mRNA levels in renal cells. These findings provide the first direct evidence that systemic pH and aldosterone may regulate pendrin activity at the transcriptional level.
|
MATERIAL AND METHODS |
|---|
|
TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Opossum kidney (OK) cells (provided by Dr. J. Green, Technion, Haifa, Israel), human embryonic kidney (HEK293) cells (provided by Dr. K. Skorecki, Technion, Haifa, Israel), mouse distal convoluted tubule (MDCT) cells (provided by Dr. P. Friedman, University of Pittsburgh, Pittsburgh, PA), mouse cortical collecting duct (M1) cells (provided by Dr. Brian Harvey, University College Cork, Ireland), human thyroid follicular carcinoma-derived (LA2) cells (provided by Dr. Z. Kraiem, Technion, Haifa, Israel), human tongue squamous cell carcinoma (SCC-25) cells (provided by Dr. A. Resnick, Technion, Haifa, Israel), and mouse embryonic fibroblast (NIH3T3) cells were grown in monolayer and maintained in DMEM/F-12 supplemented with 10% fetal calf serum, 2 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin (Biological Industries, Beit Ha'Emek, Israel) at 37°C in a humidified atmosphere of 95% air-5% CO2. Mouse embryo ventral otocyst-derived (VOT36) cells (provided by Dr. M. Holley, University of Sheffield, Sheffield, UK) were grown and maintained in MEM with Glutamax (GIBCO-Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum, 50 U/ml IFN-
(PeproTech, Rocky Hill, NJ), 100 IU/ml penicillin, and 100 µg/ml streptomycin at 33°C in a humidified atmosphere of 95% air-5% CO2.
RT-PCR Analysis
Total RNA isolated from the cell lines mentioned above with TRI-reagent (MRB, Cincinnati, OH) was reverse transcribed using Moloney murine leukemia virus (MMLV)-RT and random hexamers (Promega, Madison, WI). The resultant cDNA was amplified by polymerase chain reaction using FastStart Taq DNA polymerase (Roche, Mannheim, Germany) with the intron-spanning primers F1/R1 (Table 1), from exons 3 and 6, respectively, highly homologous between human and mouse PDS. The resultant cDNA was separated on 1% agarose gels. A similar procedure was carried out with actin primers F6/R3 (Table 1) as control.
|
Immunofluorescence Microscopy
HEK293, LA2, VOT36, and NIH3T3 cells grown on Lab-Tek chamber slides (Nunc, Naperville, IL) were fixed with 4% paraformaldehyde for 30 min at room temperature. Cells were washed twice with PBS, and after permeabilization with 0.2% Triton X-100 for 30 min and blocking with 10% normal donkey serum (Santa Cruz Biotechnology, Santa Cruz, CA) for 15 min, the cells were incubated with a previously characterized (21) rabbit polyclonal anti-pendrin antibody (provided by Dr. Dominique Eladari, Université René Descartes, Paris, France) at a final dilution 1/300 for 60 min at room temperature. Cells were then washed three times with PBS, followed by incubation with FITC-conjugated donkey anti-rabbit IgG (Santa Cruz Biotechnology) at a 1/50 final dilution for 60 min in the dark. Immunolabeled cells were mounted with UltraCruz Mounting Medium containing 4,6-diamidino 2-phenylindole dihydrochloride for nuclear staining (Santa Cruz Biotechnology). Immunostained slides were visualized with a Zeiss inverted Axiovert 135 microscope set (Carl Zeiss, Germany) with the appropriate filters.
Immunoprecipitation Followed by Western Blot Analysis of Cell Lines
Pendrin protein was immunoprecipitated from 1 x 107 HEK293, LA2, VOT36, and NIH3T3 cells grown in monolayer in appropriate media using the Protein G-based Immunoprecipitation Kit (Roche) according to the manufacturer's instructions using goat anti-human/mouse pendrin antibody (Santa Cruz Biotechnology). The immunoprecipitated protein extracts were separated on 4–20% gradient SDS-polyacrylamide gels (Pierce, Cheshire, UK) and electrotransferred (40 V for 90 min) to nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany). Membranes were blocked with 5% BSA in TBS, pH 7.4, for 1 h at room temperature, followed by incubation with primary antibody diluted 1:50 in TBS containing 2% BSA and 0.1% Tween 20 (Sigma-Aldrich, St. Louis, MO) overnight at 4°C. After four 10-min washes in TBS containing 0.1% Tween 20, membranes were incubated with 1:50,000 dilution of donkey anti-goat IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology) in TBS containing 2% BSA for 1 h at room temperature. Blots were washed as above and assayed with ECL using SuperSignal West Pico Chemiluminescent Substrate (Pierce) to visualize bound antibodies.
Cloning of hPDS 5'-Flanking DNA
Using the published sequence of hPDS cDNA (accession no. NM-000441.1), a DNA fragment of 4.2 kb in the 5'-flanking sequence region, to but not including the hPDS translation start site (ATG) (Fig. 1A), was synthesized using PCR with human genomic DNA as template and primers F2/R2 (Table 1). The 3', transcription initiation site flanking, 968-bp end of this fragment contained the first exon and intron of the hPDS gene, and it terminated 40 bp upstream of the ATG, which begins 4 bp into exon 2 (Fig. 1B). In a similar manner, a DNA fragment of 3.2 kb in the 5'-flanking sequence region, to but not including the hPDS transcription initiation site (Fig. 1B), was synthesized using PCR with human genomic DNA as template and primers F2/R4 (Table 1). To minimize the possibility of PCR-generated mutations, the High Fidelity Long Range PCR system (Roche) was used. The fragments produced were TA cloned into pCR-XL-TOPO vector (Invitrogen) and sequence-verified. Computer analysis (13, 38) of the 5'-flanking region of hPDS from the gene's translation start site was carried out, and putative transcription factor binding sites were identified.
|
Both 5'-flanking sequences described above (4185 bp and 3217 bp) were subcloned into pGL3-basic vector (Promega) upstream to a luciferase reporter gene (and named pL4 and pL3TI, respectively) (Fig. 1B). In addition, a set of six deletion fragments decreasing in size from the 5' end of the 4145-bp genomic fragment were produced and cloned into pGL3-basic vector (Fig. 1A). Three inserts were prepared using PCR-based strategies with genomic DNA as template and primers F3, F4, F5, and R2 (Table 1). The additional three were prepared by removing specific 5' segments from cloned hPDS sequences in pGL3-basic, using restriction enzymes. All seven constructs were sequenced to verify orientation and integrity of inserts. As illustrated in Fig. 1A, the 5' ends of the deletion fragments were positioned at –2695 (pL2.6), –2060 (pL2.0), –1726 (pL1.7), –1433 (pL1.4), –1044 (pL1.0), and –786 (pL0.75). All fragments extended to the ATG site but did not include it and were cloned into the vector in sense orientation. The 2-kb fragment in pL2 was also cloned in antisense orientation. Next, a set of three reporter constructs decreasing in size between pL1.4 and pL1.0 were prepared using PCR with primers F8, F9, F10, and R2 (Table 1). The 5' end of the promoter fragments cloned were positioned at –1140 (pL1.1), –1253 (pL1.2), and –1342 (pL1.3) (Fig. 1A). Another set of two reporter constructs decreasing in size between pL2.0 and pL1.7 were likewise prepared with primers F11, F12, and R2 (Table 1), generating fragments ending in –1838 (pL1.8) and –1952 (pL1.9).
Generation of Mutated Constructs
Site-specific, 13-bp long mutations in the TTF-1 and TTF-2 binding sites [found by computer analysis (13, 38) of the 4.2-kb 5'-DNA fragment] successively located between –1946 and –1933 on the hPDS promoter were introduced into the pL1.9 plasmid (Fig. 1A). This was performed using PCR with the high-fidelity DNA polymerase Pfu (Promega) with primers F13/R6 (Table 1). The PCR program for this purpose was designed as follows: 95°C for 30 s, 18 cycles of amplification with each step including denaturation at 95°C for 30 s, annealing at 50°C for 1 min, and elongation at 68°C for 15 min; the final elongation was performed at 72°C for 5 min. The primers used contained the desired, centrally located mutations in the TTF binding sites. The parental DNA template was digested with Dpn I overnight, and the mutated plasmid was transformed into competent Escherichia coli strain DH5. Subsequently, small- and large-scale plasmid preparations were performed using Genopure Plasmid Maxi kit (Roche). The presence of the mutation was verified by DNA sequencing.
Transient Transfections and Reporter Gene Assays
In all transfection experiments, luciferase reporter constructs were purified using Genopure Plasmid Maxi kit. Empty pGL3-basic vector containing no insert was used as negative control. The pGL3-control plasmid containing an SV40 enhancer/promoter was used as positive control. Promoter activity was estimated from the ratio of luciferase activity in pGL3-basic construct to promoterless pGL3-basic vector. Plasmid pCH110 (Pharmacia, Uppsala, Sweden) containing a LacZ gene driven by the cytomegalovirus promoter was used to normalize for transfection efficiency.
HEK293, MDCT, M1, LA2, VOT36, and NIH3T3 cells were plated in quadruplicate (5 x 104 cells/well) in 24-well dishes in serum-containing media. Cotransfections were performed using Fugene 6 (1.2 µl/well; Roche) with 0.3 µg reporter plasmid and 0.3 µg pCH110, and cells were incubated at 37°C for 48 h. For enzymatic assays, cells were washed with PBS (150 mM NaCl, 15 mM sodium phosphate, pH 7.4) and lysed by incubation in 200 µl/well M-Per (Pierce) for 5 min at 37°C. Lysed cells were centrifuged, and supernatant was aliquoted (50 µl/well) into 96-well plates. Luciferase Assay Reagent (50 µl; Promega) was automatically added, and the light intensity of the reaction was immediately read in a luminometer (Lucy, Anthos, Austria) for a period of 10 s. Luciferase activity was normalized to β-galactosidase activity, which was measured in identical cell lysates as follows: 160 µl of ONPG substrate (Sigma-Aldrich) were added to 30 µl of cell lysate in each well of a 96-well plate. The reaction mixture was incubated for 30 min at 37°C or until yellow color developed. β-Galactosidase measurements were performed by a spectrophotometer with a 405-nm filter. Measurements of luciferase and β-galactosidase were carried out in duplicate.
In some experiments, 48 h after transfection, growth media were removed and cells were washed with PBS and exposed to fresh media containing 0.2% bovine serum albumin instead of 10% fetal calf serum to prevent artifacts (hormones, growth factors, etc.) arising from the serum. These media had acid pH (7.0–7.1) titered from 1 M HCl solution, normal pH (7.35–7.45) or alkali pH (7.6–7.7) titered from 1 M NaHCO3 solution. In other experiments, the fresh medium (pH 7.35–7.45) contained aldosterone (from a stock solution of 10–5 M in ethanol; Fluka, Sigma-Aldrich) at a final concentration of 10–8 M. Control experiments for aldosterone studies were carried out with 0.1% ethanol. Cells were exposed to experimental media for 24 h and analyzed as explained above.
Quantitative Real-Time PCR Analysis of Cell Lines
Confluent HEK293 cells were plated in six-well dishes (106 cells/well). After 24 h in serum-containing regular media, cells were washed with PBS and placed in media with acidic pH (7.0–7.1), normal pH (7.35–7.45), or alkaline pH (7.6–7.7) containing 0.2% BSA, or in media supplemented with 10–8 M aldosterone. Following 24 h exposure to these media, cells were washed with PBS, and total RNA was prepared using Tri Reagent (Sigma-Aldrich) followed by phenol/chloroform extraction and isopropranol precipitation. Reverse transcription of 2 µg of total RNA was carried out using MMLV (Promega) with random hexamer primers (Promega). Quantitative PCR experiments were performed using TaqMan Universal PCR Master Mix (Roche Molecular Systems) with Assay on Demand primers (Applied Biosystems, Foster City, CA) for gene expression analysis of human PDS (no. Hs 00166504_m1), with human TATA box binding protein (TBP) (no. Hs 99999910_m1) serving as a housekeeping gene for normalization. All measurements were performed in triplicate in an ABI Prism 7000 Cycler (Applied Biosystems).
|
RESULTS |
|---|
|
TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Using the published sequences of PDS cDNA, we localized the human, rat, and mouse pendrin genes on chromosomes 7q31 (accession no. NM-000441.1), 6 (accession no. NM_019214.1), and 12 (accession no. NT_039548.4), respectively. Multiple sequence alignments of pendrin genes, using CLUSTALW engine (http://www.ebi.ac.uk/clustalw), revealed high sequence similarity between the species tested, which was maximal (over 55% resemblance) near the ATG codon.
A DNA fragment corresponding to the hPDS 5'-flanking region, stretching over 4.2 kb from –40 relative to the PDS translation start site, was isolated (Fig. 1A). Computer analysis of this fragment (13, 38) revealed an abundance of SP1 elements throughout this GC-rich region and no TATA box motif. Putative binding sites for several hepatocyte nuclear factors and GATA factors, known to confer renal-specific expression of several genes (1, 46), as well as binding sites for the thyroid transcription factors TTF-1, TTF-2, and Pax8, all conferring thyroid-specific expression of genes (34), were found in the 5'-flanking region of hPDS.
Epithelial Cell-Specific Expression of Pendrin mRNA
RT-PCR analysis detected expression of endogenous PDS mRNA in the renal cell lines HEK293, M1, and MDCT, but not OK, as well as in the thyroid cell line LA2 and the inner ear cell line VOT36 (Fig. 2A). PDS mRNA was not detected in squamous epithelial SCC-25 or in the fibroblast cell line NIH3T3.
|
Immunofluorescence with pendrin-specific primary antibody and FITC-labeled secondary antibody detected pendrin protein expression in HEK293, LA2, and VOT36 but not in NIH3T3 cells (Fig. 2B). Antibody specificity was demonstrated by the absence of staining in all cell lines when the primary antibody was omitted (data not shown).
Immunoprecipitation with pendrin-specific antibody and protein G, followed by Western blot analysis with pendrin-specific antibody and horseradish peroxidase-labeled secondary antibody, detected pendrin protein expression in HEK293, LA2, and VOT36 but not in NIH3T3 cells (Fig. 2C).
On the basis of these findings, the PDS-expressing renal cell lines HEK293, M1, and MDCT, thyroid cell line LA2, and the inner ear cell line VOT36 served as recipient cells in the next set of transfection experiments, and NIH3T3 cells were used as a negative control.
Epithelial Cell-Specific Activity of the hPDS Promoter
To verify that the 5'-flanking region of human PDS contains a functional cell-specific promoter, reporter gene assays were carried out.
We first examined whether the 968-bp end of the 4.2-kb region [containing the first exon and intron of hPDS (Fig. 1B)], located 3' to the transcription initiation site, included any elements with transcriptional activity. For this purpose, the 4.2-kb hPDS 5'-flanking region beginning at the ATG, as well as the 3.2-kb fragment beginning at the transcription initiation site, were cloned in sense orientation upstream to a luciferase reporter gene in pGL3-basic vectors. The resulting plasmids, designated pL4 (4145-bp insert) and pL3TI (3177-bp insert), were transiently transfected into the PDS-expressing cell line HEK293. As shown in Fig. 1B, both constructs induced a similar level of reporter gene activity, a finding that excluded the presence of any regulatory elements of functional significance on the 3' 968-bp end of the 4.2-kb insert. Thus further analysis of the hPDS 5'-flanking region was carried out using the translation initiation site as the 3' end point of the hPDS 5'-flanking region, rather than the transcription initiation site.
In the next set of experiments, the plasmid containing the 4.2-kb hPDS 5'-flanking region, pL4, as well as three 5'-deletion products similarly cloned in sense orientation upstream to a luciferase reporter gene in pGL3-basic vectors, designated pL2.6 (2655-bp insert), pL2 (2020-bp insert), and pL1.4 (1393-bp insert), were transiently transfected into the PDS-expressing cell lines HEK293, M1, and MDCT (renal), LA2 (thyroid), and VOT36 (inner ear). Reporter gene activities were compared with those in transfected fibroblast cell line NIH3T3, which does not express PDS mRNA.
As shown in Fig. 3, HEK293, LA2, and VOT36 cells, which demonstrated the highest levels of endogenous PDS mRNA (Fig. 2A), displayed markedly increased levels of luciferase activity when transfected with pL4, pL2.6, pL2, and pL1.4, compared with activity in the control fibroblast cell line, NIH3T3, which showed no endogenous PDS mRNA expression (Fig. 2A) and negligible induction of luciferase activity (Fig. 3). A mild increase in luciferase activity was observed in M1 and MDCT cell lines (Fig. 3), which also showed lower levels of endogenous PDS mRNA (Fig. 2A). Nevertheless, a different pattern of reporter gene activity was evident between cells displaying maximal activity, namely, a difference between HEK293 cells, on one hand, and LA2 and VOT36 cells, on the other hand. While all of these cell lines transfected with pL4 (4 kb), pL2.6 (2.6 kb), or pL2 (2 kb) displayed a significant and gradual increase in luciferase activity when the insert size was decreased, a further truncation from 2.6 kb to 1.4 kb caused a significant reduction in luciferase activity in LA2 and VOT36 cells but not in HEK293 cells (Fig. 3).
|
The much higher level of induction in HEK293 cells, when compared with induction in M1 and MDCT cells, prompted us to select HEK293 cells as experimental renal cells along with LA2 (thyroid) and VOT36 (inner ear) cells in subsequent experiments.
Deletion Analysis of the hPDS Promoter
To further explore the 5'-flanking region of hPDS and to investigate the different pattern of reporter gene activities in renal, thyroid, and inner ear cells, a second set of deletion constructs was tested (Fig. 1A), two within pL1.4 (designated pL0.75 and pL1.0) and one between pL1.4 and pL2 (designated pL1.7). All seven constructs were transiently transfected into HEK293, LA2, and VOT36 cells. As evident from Fig. 4, the highest level of luciferase activity resulted from transfection of a 2-kb insert-containing plasmid (pL2) in all of the cell lines tested. Nevertheless, decreasing the size of this fragment by 300 bp (pL1.7) caused a 75% and 65% reduction in luciferase activity in LA2 and VOT36 cells, respectively, which remained unchanged or mildly decreased when the fragment was further shortened to 1.4 kb (pL1.4) and 1.0 kb (pL1). On the other hand, decreasing the 2.0-kb fragment size to 1.7 kb and 1.4 kb did not considerably change luciferase activity in HEK293 cells. A marked decrease (60%) in reporter gene activity, however, occurred in this cell line when the fragment size was shortened to 1 kb (pL1). In all three cell lines, another marked decrease (50–80%) was noted when the fragment was further shortened to 0.75 kb (pL0.75).
|
To further explore the 2-kb 5'-flanking region of hPDS, which displayed the highest promoter activity in all three cell lines, HEK293, LA2, and VOT36, an orientation study was performed in these cells. When the 2-kb promoter fragment in pGL3 vector was reversed, luciferase activity was 60–70% lower than activity induced by the same sequence in sense orientation (data not shown). This result demonstrated that the cloned 2-kb fragment (pL2) was orientation-dependent in all three cell lines.
Taken together, these results suggested that positive regulatory elements (PRE), most likely located between positions –1433 and –1044 in renal cells, and between –2060 and –1726 in thyroid and inner ear cells, as well as a negative regulatory element (NRE), likely positioned between –4185 and –2060 in all three cell lines, were involved in PDS promoter activity (Fig. 4).
Deletion Analysis of the PRE-Containing DNA Regions in Cell Lines
To further define the PREs located within the 389-bp (between –1433 and –1044) and the 334-bp (between –2060 and –1726) promoter sequences of functional importance in renal cells and thyroid/inner ear cells, respectively, fine deletion analysis of these segments was carried out. For this purpose, a third set of deletion constructs was tested (Fig. 1A): three between pL1 and pL1.4 (designated pL1.1, pL1.2, and pL1.3) and two between pL1.7 and pL2 (designated pL1.8 and pL1.9).
First, the five constructs including and between pL1 and pL1.4 (pL1, pL1.1, pL1.2, pL1.3, and pL1.4) were transiently transfected into renal HEK293 cells. As seen in Fig. 5A, pL1.4 showed high levels of luciferase activity. A 50% reduction in luciferase activity was evident when this fragment was shortened to 1.3 kb (pL1.3), which remained unchanged with further shortening of the fragments to 1.2 kb (pL1.2), 1.1 kb (pL1.1), and 1 kb (pL1).
|
Taken together, these findings provide evidence for the presence of putative PREs within the 91-bp region (between –1433 and –1342) in renal HEK293 cells, within the 114-bp region (between –1952 and –1838) in thyroid LA2 cells, and, finally, within the 108-bp region (between –2060 and –1952) in inner ear VOT36 cells (Fig. 5).
In silico analysis of these putative PRE-containing regions, using promoter analysis programs (13, 38), revealed a variety of potential transcription factor binding sites. A search for known kidney-specific transcription factor binding sites in the region between –1433 and –1342 yielded putative hepatocyte nuclear factor 3 and Wilm's tumor 1 binding sites. A similar search for thyroid-specific transcription factor binding sites in the region between –1952 and –1838 revealed TTF-1 and TTF-2 binding sites. No known inner ear-specific transcription factor binding sites were found in the region between –2060 and –1952.
Mutation Analysis of the PRE-Containing DNA Region in LA2 Cells
As indicated above, the putative PRE-containing region between –1952 and –1838 identified in the deletion analysis conducted in the thyroid LA2 cells (Fig. 5) was found to harbor TTF-1 and TTF-2 binding sites. These binding sites were located between –1946 and –1938 and between –1942 and –1933, respectively.
It is noteworthy that Dentice and colleagues (5), using small interfering RNA studies, identified on the rat Pds promoter a putative TTF-1 binding site, which was involved in thyroid-specific expression of pendrin in rat FRTL-5 cell line. However, in silico sequence comparison performed by us, using the CLUSTALW engine, detected no significant homology between the rat Pds promoter area found by Dentice et al. (5) to contain a TTF-1 binding site and the corresponding location on the hPDS promoter (data not shown).
To further investigate the PRE-containing DNA region between –1952 and –1838, and to explore the possible role of the TTF binding sites identified within this region in thyroid-specific expression of hPDS, mutation analysis was performed. For this purpose, mutations encompassing a 13-bp region, which includes the TTF-1 and TTF-2 binding sites between –1946 and –1938 and between –1942 and –1933, respectively, were introduced into the 1.9-kb-containing plasmid pL1.9 (Fig. 6A). The resulting plasmid, termed pL1.9mut, as well as pL1.9 were transiently transfected into LA2 cells. As demonstrated in Fig. 6B, luciferase activity in cells transfected with pL1.9mut was diminished by 90% when compared with activity induced by pL1.9. These results suggest that the TTF-1 and/or TTF-2 binding sites between positions –1946 and –1938 and between –1942 and –1933, respectively, may play a key role in thyroid-specific activity of the hPDS promoter.
|
Systemic pH is known to affect H+ and HCO3– transport in the CCD (17, 39), and ambient pH has been shown to modulate pendrin mRNA and protein expression in this nephron segment (14, 31, 53). Furthermore, it has been suggested that pendrin is involved in acid-base balance in the endolymphatic sac (6, 12). However, the exact molecular mechanisms of these actions are unknown. Therefore, we examined the direct effect of ambient pH on the hPDS promoter. For this purpose, HEK293, LA2, and VOT36 cells were transiently transfected with plasmids pL4, pL2, pL1.4, and pL1 and exposed to acidic pH (7.0–7.1), normal pH (7.35–7.45), or alkaline pH (7.6–7.7). As demonstrated in Fig. 7, exposure of transfected HEK293 and VOT36 cells to acidic pH caused a 30–40% decrease in luciferase activity driven by 4-kb, 2-kb, and 1.4-kb hPDS promoter fragments when compared with conditions of normal pH, whereas only a minimal acid-induced inhibition was observed in reporter gene activity driven by the 1-kb promoter fragment. The exact opposite occurred when transfected HEK293 and VOT36 cells were exposed to alkaline pH, namely, a 30–45% increase in luciferase activity driven by fragments 4 kb, 2 kb, and 1.4 kb, along with a minute increase in activity driven by the 1-kb promoter fragment. No such pH-induced effect on PDS promoter activity was evident in similarly transfected LA2 cells.
|
To further define the pH-response element located within the 389-bp promoter region between –1433 bp and –1044 bp, fine deletion analysis of this region was performed. For this purpose, the plasmids pL1, pL1.1, pL1.2, pL1.3, and pL1.4 (Fig. 8A) were transfected into HEK293 and VOT36 cells that were exposed to acidic, normal, and alkaline pH, as above. As demonstrated in Fig. 8B, a similar pattern of reporter gene activity was evident in both cell lines. While transfection of pL1.4 (1.4 kb), pL1.3 (1.3 kb), pL1.2 (1.2 kb), and pL1.1 (1.1 kb) resulted in similar magnitude of acidic pH-induced inhibition and alkaline pH-induced stimulation of luciferase activity in HEK293 and VOT36 cell lines, further truncation of the fragment to 1.0 kb (pL1) caused a marked reduction in acidic pH- and alkaline pH-induced effects in both cell lines.
|
Effect of Aldosterone on hPDS Promoter Activity
Aldosterone is known to affect acid-base transport in the CCD (15) and to modulate pendrin protein and mRNA expression in this nephron segment (50). However, the molecular mechanisms of this aldosterone-induced effect on pendrin activity are unknown.
Hence, we examined the direct effect of aldosterone on the hPDS promoter. To this end, HEK293 as well as LA2 and VOT36 cells were transiently transfected with plasmids pL4, pL2, pL1.4, and pL1 and exposed to 10–8 M aldosterone. As shown in Fig. 7, exposing transfected HEK293 cells to aldosterone caused a marked (30–35%) decrease in pL4-, pL2-, and pL1.4-driven luciferase activity. This decrease greatly diminished in cells transfected with pL1. Aldosterone had no effect on medium pH, which remained identical to the pH in the control medium (7.35–7.45). Aldosterone had no effect on PDS promoter activity in transfected LA2 and VOT36 (Fig. 7).
Taken together, these results suggest that aldosterone may directly affect the hPDS promoter and transcriptionally modulate pendrin activity in the kidney, but not in thyroid and inner ear. The hPDS promoter likely contains an aldosterone-induced element, which appears to be located in the 389-bp region between –1433 and –1044.
To further analyze the aldosterone-induced element residing within this 389-bp promoter region, fine deletion analysis of this region was performed. To this end, plasmids pL1, pL1.1, pL1.2, pL1.2, pL1.3, and pL1.4 (Fig. 8C) were transfected into HEK293 cells, which were exposed to 10–8 M aldosterone. As seen in Fig. 8D, the 35% aldosterone-induced inhibition in luciferase activity driven by the 1.4-kb fragment (pL1.4) remained essentially unchanged when the fragment was shortened to 1.3 kb (pL1.3). Truncation of the fragment to 1.2 kb (pL1.2), however, resulted in a marked reduction in the aldosterone-dependent inhibition of reporter gene activity, which remained at the same level when the fragment was further shortened to 1.1 kb (pL1.1) and 1 kb (pL1).
These data indicate that a putative aldosterone-induced element likely resides within the 89-bp between –1342 and –1253 on the hPDS promoter in the kidney (Fig. 8C). In silico analysis (13, 38) revealed a single MR binding site within this region.
To support our findings on the effect of aldosterone on the pendrin gene promoter, we tested whether the cells responsive to this effect of aldosterone harbor the MR. As shown in Fig. 9, RT-PCR analysis detected MR mRNA in aldosterone-responsive HEK293 cells but not in LA2 and VOT36 cells that showed no response to this hormone. MR is known to mediate aldosterone action by binding the hormone and translocating from the cytoplasm to the nucleus, where the receptor modulates gene expression by functioning as a transcription factor (3, 28). The association of the aldosterone-dependent effect on the pendrin gene promoter in renal HEK293 cells with the evidence for the presence of MR in these cells suggests that aldosterone influences pendrin gene activity in the kidney directly via the genomic pathway.
|
To further explore the influence of ambient pH and aldosterone on transcription of the pendrin gene, we examined the effect of these modulators on endogenous PDS mRNA levels in renal HEK293 cells. For this purpose, cells were grown to confluence and were exposed to acidic pH (7.0–7.1), normal pH (7.35–7.45), alkaline pH (7.6–7.7), or to medium containing aldosterone (10–8 M) for 24 h. Subsequently, cells were lysed and total RNA was extracted and analyzed by real-time PCR for PDS mRNA levels following normalization to the housekeeping gene encoding TBP (TATA-box binding protein). As shown in Fig. 10, acidic pH and aldosterone decreased endogenous PDS mRNA levels by 20–30%, whereas alkaline pH increased these levels by 20%, when compared with conditions of normal pH.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
In addition, we demonstrate that ambient pH affects hPDS promoter activity in renal and inner ear cells and that aldosterone has a kidney-specific modulatory action on this promoter (Figs. 7 and 8). We provide evidence that a 96-bp region between positions –1140 bp and –1044 bp and a 89-bp region between positions –1342 bp and –1253 bp may play key roles in the modulation of hPDS promoter activity by ambient pH and aldosterone, respectively (Fig. 8). These findings were further supported in vitro by demonstrating an acidic pH- and aldosterone-induced decrease and alkaline pH-induced rise in endogenous pendrin mRNA levels in renal cells (Fig. 10).
Epithelial Cell-Specific Expression of hPDS
Identification of regulatory elements and signals governing PDS gene expression in renal, thyroid, and inner ear epithelia may shed light on the molecular mechanisms of bicarbonate secretion, iodide accumulation, and endolymph balance, respectively, and may significantly improve our understanding of epithelial cell differentiation and function in these organs in both health and disease.
Although cis-acting regulatory elements involved in regulating gene transcription may be dispersed throughout the gene locus, often the proximal promoter region contains elements sufficient for high levels of tissue-specific gene transcription. These elements may function as binding sites for tissue-restricted proteins that arbitrate transcriptional activation in expressing cells. In an effort to identify cis-acting elements involved in regulating kidney-, thyroid-, and inner ear-specific expression of hPDS, we focused our study on the 5'-flanking region of this gene.
We isolated a 4.2-kb genomic sequence corresponding to the 5'-flanking region of hPDS and, using reporter gene assays, defined regions necessary for epithelial cell-specific gene transcription. In our study, we examined pendrin mRNA and protein expression as well as PDS promoter activity in renal, thyroid, and inner ear cell lines of epithelial origin as well as in a fibroblast cell line. Our results indicate specific PDS expression and promoter activity in cell lines originating in embryonic kidney (HEK293), distal convoluted tubule (MDCT), and collecting duct (M1), and no expression in a cell line of proximal tubular origin (OK) (Figs. 2 and 3). PDS expression was also detected in thyroid LA2 and inner ear VOT36 epithelial cells, but not in the epithelial cell line SCC-25 or the fibroblast cell line NIH3T3 (Fig. 2). Taken together, these results suggest involvement of the 4.2-kb 5'-flanking region of the PDS gene in driving epithelial cell-specific expression in distal parts of the nephron, as well as in the thyroid and inner ear. Our in vitro findings showing PDS expression in renal cell lines of distal but not proximal tubular origin, indicating nephron segment-specific expression of PDS confined to the distal, but not proximal, tubule, are in accordance with in vivo studies (21, 54) demonstrating no expression of pendrin in the proximal tubule of the mouse kidney. These studies and our findings contradict another study (44) showing pendrin mRNA and protein expression in the proximal tubule of the rat kidney. The discrepancies between these data may be related to species differences (opossum, mouse, and rat), methodological differences, or other factors.
It should be noted that our immunofluorescence experiments detected pendrin protein in the membrane as well as in the cytoplasm of renal HEK293, thyroid LA2, and inner ear VOT36 cells (Fig. 2B). A likely explanation for the cytoplasmic pendrin staining is that this staining may represent a nonfunctional fraction of pendrin residing in the endoplasmic reticulum, a finding commonly observed in thyroid carcinoma-derived cell lines (43).
Deletion analysis of reporter gene constructs containing hPDS promoter DNA transfected into the PDS expressing cell lines HEK293, LA2, and VOT36 showed distinct induction of reporter gene activity, with an expression pattern that differed between the cell lines tested (Fig. 4). The highest levels of promoter activity were evident in all PDS-expressing cells using a 2-kb hPDS 5'-flanking DNA fragment. However, this activity diminished in LA2 and VOT36 with increasing or decreasing DNA length, whereas in HEK293 cells a decrease in promoter activity was evident when DNA length was increased or when the DNA length was shorter than 1.4 kb. Hence, these findings suggest that an NRE between –4185 and –2060 controls PDS promoter activity in renal, thyroid, and inner ear cells. A PRE between –1433 and –1044 controls PDS promoter activity in renal cells, whereas a PRE between –2060 and –1726 controls activity of this promoter in thyroid and inner ear cells.
Further detailed analysis of the two PRE-containing regions controlling hPDS promoter activity in renal and in thyroid/inner ear cells, respectively (Fig. 5), revealed a 91-bp sequence (between –1433 and –1342), a 114-bp sequence (between –1952 and –1838), and a 108-bp region (between –2060 and –1952), which seem to be responsible for modulating kidney-, thyroid-, and inner ear-specific hPDS transcription, respectively.
Mutation analysis targeting the TTF-1 and TTF-2 binding sites, which were found by computer analysis (13, 38) to reside in the 114-bp sequence shown by us to be responsible for thyroid-specific hPDS transcription (Fig. 5), clearly demonstrated that the TTF binding sites at positions –1946 to –1938 and between –1942 to –1933, respectively, are essential for the activity of the hPDS promoter in human thyroid-derived LA2 cells (Fig. 6). Several kidney-specific transcription factor binding sites, including hepatocyte nuclear factor 3 and Wilm's tumor 1 binding sites but no known inner ear-specific transcription factor binding sites, were identified on the 91-bp and 108-bp sequences shown by us to be responsible for kidney- and inner ear-specific hPDS transcription, respectively.
Future in-depth studies are needed to examine the role of the PRE-containing regions identified by us in the epithelial cell-specific activity of the hPDS promoter and to verify the involvement of the transcription factor binding sites outlined above and other, yet to be identified sites in controlling the activity of this promoter.
Regulation of the Pendrin Gene
Acid-base transport in various nephron segments, including the CCD, is modulated by a variety of hormonal and nonhormonal factors (15, 17, 39). Systemic pH regulates activity of several transport pathways participating in acid-base transport in the CCD, including pendrin activity (14, 24, 36). Immunolocalization studies (14, 31, 53) in acid-loaded rats and mice have demonstrated diminished levels of pendrin protein in the apical membrane of intercalated cells accompanied by a shift of the protein to the cytosol, as well as decreased expression of PDS mRNA. In HCO3–-loaded animals, pendrin protein was exclusively expressed in the apical membrane (53). In addition, acid loading resulted in a decline in the relative abundance of pendrin-positive cells, whereas alkali loading increased the number of these cells (53). Pendrin was not detected in the kidneys of PDS-knockout mice, and tubules from alkali-loaded PDS knockout mice failed to secrete HCO3– (36). These studies have suggested that pendrin plays an important role in acid-base regulation and that this anion exchanger is essential for HCO3– secretion following HCO3– loading. However, the exact molecular mechanisms underlying the alterations in pendrin expression and activity in response to pH changes and whether these alterations occur at the level of the pendrin gene have not been investigated.
Pendrin is thought to play a major role in endolymph ion balance in the inner ear (12). Immunohistochemical analysis demonstrating coexpression of pendrin, vacuolar H+-ATPase, and carbonic anhydrase II in epithelial cells of the murine endolymphatic sac (6) suggests that pendrin may exert its action on the endolymph by controlling acid-base balance in this fluid compartment, which is essential for normal hearing. However, nothing is known about the factors modulating pendrin activity in the inner ear and about their mechanisms of action.
In the present study, we have used the powerful model of cell lines transfected with DNA fragments of interest corresponding to the pendrin gene promoter to directly examine the involvement of this promoter in the modulation of pendrin activity by known modulators of pendrin activity, including pH changes and aldosterone. This model is of great advantage since it permits highly controlled investigation and detailed analysis of the control mechanisms involved in cell-specific regulation of the PDS gene. More importantly, in contrast with in vivo studies (14, 31, 50, 53) where the actions of the various modulators of pendrin activity (pH, aldosterone, volume status, etc.) on this anion exchanger are intermingled and hard to differentiate from one another, the in vitro model, used by us, of transfected cell lines enables the dissection and isolation of the specific action on the pendrin gene of each of the factors known to modulate pendrin activity independently of the other factors.
Effect of ambient pH on the hPDS promoter. In our study, we show that ambient pH affects PDS promoter activity in renal and inner ear cells (Figs. 7 and 8). Specifically, acidic pH decreases and alkaline pH increases PDS promoter activity in HEK293 and VOT36 cells. pH changes did not affect PDS promoter activity in the thyroid cell line LA2. These findings are in line with the modulatory effects of acidosis and alkalosis on pendrin protein activity observed in the rodent kidney at the tubular and cellular levels (14, 31, 50, 53) and demonstrate, for the first time, that systemic pH may directly regulate pendrin-mediated, renal tubular HCO3– secretion at the transcriptional level. In addition, these data suggest that systemic pH may modulate endolymph pH by directly regulating transcription of the pendrin gene. The exact molecular mechanisms, whereby this pH-induced effect on the PDS promoter is achieved, remains to be explored. Li and colleagues (25) have shown that the proline-rich tyrosine kinase 2 (Pyk2), a member of the focal adhesion kinase family, is directly activated by acidic pH and that Pyk2 activation is required for acid activation of c-Src kinase and the proximal tubular Na+/H+ exchanger 3 (NHE3). The authors suggest that Pyk2 may serve as a pH sensor that, via phosphorylation of c-Src, provides subsequent activation of the MAPK and JNK signaling pathways, which, in turn, increase transcription of the NHE3 gene (16, 25). There is evidence that the proximal tubular, basolateral-bound Na+-HCO3– cotransporter, NBC, may be regulated by a similar pathway (9).
It is possible that a similar pH-sensing mechanism exists in more distal nephron segments, including the CCD, as well as in other organs in which pH is tightly controlled, such as the endolymphatic sac of the inner ear (6), and that such a mechanism is responsible for the acid and alkali modulation of the pendrin gene promoter demonstrated by us.
In our study, we found that the 96-bp 5'-flanking region of PDS between –1044 and –1140 harbors a putative pH-response element that is active in transfected renal and inner ear cells (Fig. 8). Computer analysis (13, 38) of this DNA region revealed a variety of putative transcription factor binding sites whose binding proteins are known to be regulated by the MAPK signaling pathways. These include, among others, binding sites for Elk-1, c-Jun, ATF-2, CREB, NFAT-4, c-Myc and N-Myc. This finding raises the possibility that, similar to its effect on other H+ and HCO3– transporting pathways, the MAPK pathway may also be involved in pH-induced modulation of the pendrin gene promoter. Alternatively, a direct effect of ambient pH-mediated alterations in intracellular pH on this pH-response element residing on the PDS promoter cannot be excluded. Further studies are needed to elucidate the exact location and the molecular nature of this novel pH-response element.
Our findings on the effects of ambient pH on the hPDS promoter in transfected renal cells were further supported by our experiments demonstrating an acid-induced inhibition and an alkali-induced stimulation of endogenous PDS mRNA levels in renal HEK293 cells (Fig. 10). The combined use of these in vitro models in our study has been of great advantage since it has permitted careful analysis of the molecular pathways responsible for the regulation of pendrin activity by pH and has demonstrated that the pH-induced effect on this anion exchanger involves modulation at the DNA as well as RNA levels.
Effect of aldosterone on the hPDS promoter.
Acid-base transport in the distal segments of the nephron, including the CCD, is known to be regulated by aldosterone (15, 17). Aldosterone stimulates proton secretion in the CCD by activating several transport processes, including epithelial Na+ channels in the apical membrane of principal cells as well as H+-ATPase and H+/K+-ATPase activity in the apical membrane and Cl–/HCO3– exchanger activity in the basolateral membrane of -intercalated cells (15, 17). These actions of aldosterone, which result in net excretion of acid in the distal tubule, are mediated by a variety of genomic mechanisms involving the action of aldosterone-induced regulatory proteins as well as by nongenomic pathways (23, 52, 55). Verlander and colleagues (50) demonstrated upregulation of pendrin mRNA and protein in mouse kidney in response to administration of the aldosterone analog, deoxycorticosterone. To explain this effect of aldosterone, which is not in line with the acid-secreting properties of this hormone, the authors have proposed that in the in vivo model they used, pendrin attenuated the aldosterone-induced metabolic alkalosis by augmenting HCO3– secretion. Nevertheless, the direct effect of aldosterone on the PDS gene has not been investigated.
Using the model of transfected cell lines, we have isolated the effect of aldosterone on the pendrin gene promoter, and we demonstrate that this hormone directly inhibits PDS promoter activity in renal cells (Figs. 7 and 8). Aldosterone had no effect on reporter gene activity in transfected thyroid and inner ear cells. This finding demonstrates, for the first time, that aldosterone may regulate pendrin-mediated HCO3– secretion at the transcriptional level. Moreover, our finding of isolated, direct aldosterone-induced inhibition of the pendrin gene promoter [as opposed to the complex aldosterone/alkalosis-induced stimulation of pendrin activity demonstrated in vivo (50)] is in concert with the H+-secreting and HCO3–-retaining actions of this hormone (15, 17). The PDS promoter-inhibiting action of aldosterone was not secondary to pH changes in the medium (see RESULTS), and it appeared to involve the 89-bp region between –1342 and –1253 of the PDS 5'-flanking region (Fig. 8).
In line with this kidney-specific inhibitory effect of aldosterone on hPDS promoter activity was our finding demonstrating an aldosterone-induced decrease in endogenous pendrin mRNA levels in renal HEK293 cells (Fig. 10), as well as our finding of MR mRNA expression in HEK293 cells but not in thyroid LA2 and inner ear VOT36 cells (Fig. 9).
Taken together, these findings support the notion that the action of aldosterone on the pendrin gene in the kidney may represent a direct genomic effect rather than a nongenomic one. Computer analysis (13, 38) of the functionally significant, aldosterone-sensitive 89-bp region on the hPDS promoter disclosed a single MR binding site within this region. The potential role of this binding site in the aldosterone-dependent effect or the possibility that the aldosterone-induced element demonstrated in our study is a novel transcription factor binding site remains to be explored.
It has been proposed that in addition to the role of pendrin in acid-base balance, mediated by HCO3– secretion into the tubular lumen (36, 53), this anion exchanger may also play an important role in blood pressure regulation by reabsorbing Cl– in exchange for secreted HCO3– (32, 49, 50). Administration of the aldosterone analog, deoxycorticosterone, resulted in hypertension in wild-type PDS(+/+) mice but not in knockout PDS(–/–) mice, suggesting that pendrin may play a role in the pathogenesis of mineralocorticoid-induced hypertension (50). Moreover, it has been known that Cl– depletion increases and Cl– excess decreases pendrin protein abundance in the rodent kidney (32, 49, 51). Hence, our findings on the direct effect of aldosterone on pendrin gene transcription and a possible, to be explored, role of Cl– on transcription of this gene may be of major biological importance and may improve our understanding of blood pressure control at the gene level.
The modulators of acid-base balance used in the present study, namely, ambient pH and aldosterone, had no effect on the hPDS promoter in the thyroid cell line LA2. This finding is not surprising considering the facts that acid-base conditions are not known to exert an effect on thyroid hormone metabolism and that the thyroid gland does not contain aldosterone-responsive epithelial cells. Pendrin functions in the thyroid gland as an exchanger of Cl– and I–. Hence, future studies examining the modulatory effect of these two anions on pendrin activity and exploring whether such an effect occurs at the transcriptional level will be of great importance.
Conclusions
In conclusion, interplay between positive regulatory elements and more distally located negative regulatory elements on the 5'-flanking region of the pendrin gene may determine epithelial cell-specific expression of this gene in the kidney, thyroid, and inner ear. The TTF-1 and TTF-2 binding sites successively located between positions –1946 and –1938 and between –1942 and –1933, respectively, may play a key role in the activity of the hPDS promoter in the thyroid.
Acidic pH and aldosterone decrease, and alkaline pH increases, hPDS promoter activity by acting on distinct response elements on this promoter, which remain to be identified and characterized. Our findings provide the first direct evidence that pendrin-mediated Cl–/HCO3– exchange in the renal tubule, iodide accumulation in the thyroid, and endolymph ion balance in the inner ear may be differentially regulated at the transcriptional level by systemic pH and aldosterone.
Future studies using transgenic animals may verify whether the activity and regulation of the hPDS promoter fragments, examined in the cell culture model, demonstrate the expected organ and epithelial cell activity in the intact organism, where the transgene is exposed to a more physiologically relevant environment (i.e., hormones, proteins, etc.). Once established, it is possible that these transgenic animals will serve as an excellent model to study the transcriptional effects of a variety of physiological as well as pathophysiological factors on the pendrin gene, and thus on acid-base balance, renal salt homeostasis, and blood pressure regulation, as well as on thyroid function and sound perception in health and disease.
|
GRANTS |
|---|
|
TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
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.
|
REFERENCES |
|---|
|
TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
2. Ben-Yosef T, Belyantseva IA, Saunders TL, Hughes ED, Kawamoto K, Van Itallie CM, Beyer LA, Halsey K, Gardner DJ, Wilcox ER, Rasmussen J, Anderson JM, Dolan DF, Forge A, Raphael Y, Camper SA, Friedman TB. Claudin 14 knockout mice, a model for autosomal recessive deafness DFNB29, are deaf due to cochlear hair cell degeneration. Hum Mol Genet 12: 2049–2061, 2003.
3. Brewster UC, Perazella MA. The renin-angiotensin-aldosterone system and the kidney: effects on kidney disease. Am J Med 116: 263–272, 2004.[CrossRef][Web of Science][Medline]
4. Campbell C, Cucci RA, Prasad S, Green GE, Edeal JB, Galer CE, Karniski LP, Sheffield VC, Smith RJ. Pendred syndrome, DFNB4, and PDS/SLC26A4 identification of eight novel mutations and possible genotype-phenotype correlations. Hum Mutat 17: 403–411, 2001.[CrossRef][Web of Science][Medline]
5. Dentice M, Luongo C, Elefante A, Ambrosio R, Salzano S, Zannini M, Nitsch R, Di Lauro R, Rossi G, Fenzi G, Salvatore D. Pendrin is a novel in vivo downstream target gene of the TTF-1/Nkx-2.1 homeodomain transcription factor in differentiated thyroid cells. Mol Cell Biol 25: 10171–10182, 2005.
6. Dou H, Xu J, Wang Z, Smith AN, Soleimani M, Karet FE, Greinwald JH Jr, Choo D. Co-expression of pendrin, vacuolar H+-ATPase alpha4-subunit and carbonic anhydrase II in epithelial cells of the murine endolymphatic sac. J Histochem Cytochem 52: 1377–1384, 2004.
7. Dunn JT, Dunn AD. Update on intrathyroidal iodine metabolism. Thyroid 11: 407–414, 2001.[CrossRef][Web of Science][Medline]
8. Efrati E, Arsentiev-Rozenfeld J, Zelikovic I. The human paracellin-1 gene (hPCLN-1): renal epithelial cell-specific expression and regulation. Am J Physiol Renal Physiol 288: F272–F283, 2005.
9. Espiritu DJ, Bernardo AA, Robey RB, Arruda JA. A central role for Pyk2-Src interaction in coupling diverse stimuli to increased epithelial NBC activity. Am J Physiol Renal Physiol 283: F663–F670, 2002.
10. Everett LA. New insights into the role of pendrin (SLC26A4) in inner ear fluid homeostasis. Novartis Found Symp 273: 213–230, 2006.[Medline]
11. Everett LA, Glaser B, Beck JC, Idol JR, Buchs A, Heyman M, Adawi F, Hazani E, Nassir E, Baxevanis AD, Sheffield VC, Green ED. Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS). Nat Genet 17: 411–422, 1997.[CrossRef][Web of Science][Medline]
12. Everett LA, Morsli H, Wu DK, Green ED. Expression pattern of the mouse ortholog of the Pendred's syndrome gene (Pds) suggests a key role for pendrin in the inner ear. Proc Natl Acad Sci USA 96: 9727–9732, 1999.
13. Farre D, Roset R, Huerta M, Adsuara JE, Rosello L, Alba MM, Messeguer X. Identification of patterns in biological sequences at the ALGGEN server: PROMO and MALGEN. Nucleic Acids Res 31: 3651–3653, 2003.
14. Frische S, Kwon TH, Frokiaer J, Madsen KM, Nielsen S. Regulated expression of pendrin in rat kidney in response to chronic NH4Cl or NaHCO3 loading. Am J Physiol Renal Physiol 284: F584–F593, 2003.
15. Gennari FJ, Maddox DA. Renal regulation of acid-base homeostasis integrated response. In: The Kidney: Physiology and Pathophysiology (3rd ed.), edited by Seldin DW and Giebisch GH. Philadelphia, PA: Lippincott Williams & Wilkins, 2000, p. 2015–2053.
16. Gluck SL. Acid sensing in renal epithelial cells. J Clin Invest 114: 1696–1699, 2004.[CrossRef][Web of Science][Medline]
17. Hamm LL. Renal acidification mechanisms. In: Brenner & Rector's the kidney (7th ed.), edited by Brenner BM and Rector FC. Philadelphia, PA: Saunders, 2004, p. 497–534.
18. Hulander M, Kiernan AE, Blomqvist SR, Carlsson P, Samuelsson EJ, Johansson BR, Steel KP, Enerback S. Lack of pendrin expression leads to deafness and expansion of the endolymphatic compartment in inner ears of Foxi1 null mutant mice. Development 130: 2013–2025, 2003.
19. Igarashi P, Shashikant CS, Thomson RB, Whyte DA, Liu-Chen S, Ruddle FH, Aronson PS. Ksp-cadherin gene promoter. II. Kidney-specific activity in transgenic mice. Am J Physiol Renal Physiol 277: F599–F610, 1999.
20. Igarashi P, Whyte DA, Li K, Nagami GT. Cloning and kidney cell-specific activity of the promoter of the murine renal Na-K-C1 cotransporter gene. J Biol Chem 271: 9666–9674, 1996.
21. Knauf F, Yang CL, Thomson RB, Mentone SA, Giebisch G, Aronson PS. Identification of a chloride-formate exchanger expressed on the brush border membrane of renal proximal tubule cells. Proc Natl Acad Sci USA 98: 9425–9430, 2001.
22. Kopp P. Pendred's syndrome and genetic defects in thyroid hormone synthesis. Rev Endocr Metab Disord 1: 109–121, 2000.[CrossRef][Medline]
23. Koppel H, Christ M, Yard BA, Bar PC, van der Woude FJ, Wehling M. Nongenomic effects of aldosterone on human renal cells. J Clin Endocrinol Metab 88: 1297–1302, 2003.
24. Lacroix L, Mian C, Caillou B, Talbot M, Filetti S, Schlumberger M, Bidart JM. Na+/I– symporter and Pendred syndrome gene and protein expressions in human extra-thyroidal tissues. Eur J Endocrinol 144: 297–302, 2001.[Abstract]
25. Li W, Lee J, Vikis HG, Lee SH, Liu G, Aurandt J, Shen TL, Fearon ER, Guan JL, Han M, Rao Y, Hong K, Guan KL. Activation of FAK and Src are receptor-proximal events required for netrin signaling. Nat Neurosci 7: 1213–1221, 2004.[CrossRef][Web of Science][Medline]
26. Maehara H, Okamura HO, Kobayashi K, Uchida S, Sasaki S, Kitamura K. Expression of CLC-KB gene promoter in the mouse cochlea. Neuroreport 14: 1571–1573, 2003.[CrossRef][Web of Science][Medline]
27. Mount DB, Romero MF. The SLC26 gene family of multifunctional anion exchangers. Pflügers Arch 447: 710–721, 2004.[CrossRef][Web of Science][Medline]
28. Muller OG, Parnova RG, Centeno G, Rossier BC, Firsov D, Horisberger JD. Mineralocorticoid effects in the kidney: correlation between alphaENaC, GILZ, and Sgk-1 mRNA expression and urinary excretion of Na+ and K+. J Am Soc Nephrol 14: 1107–1115, 2003.
29. Nelson RD, Stricklett P, Gustafson C, Stevens A, Ausiello D, Brown D, Kohan DE. Expression of an AQP2 Cre recombinase transgene in kidney and male reproductive system of transgenic mice. Am J Physiol Cell Physiol 275: C216–C226, 1998.
30. Pedemonte N, Caci E, Sondo E, Caputo A, Rhoden K, Pfeffer U, Di Candia M, Bandettini R, Ravazzolo R, Zegarra-Moran O, Galietta LJ. Thiocyanate transport in resting and IL-4-stimulated human bronchial epithelial cells: role of pendrin and anion channels. J Immunol 178: 5144–5153, 2007.
31. Petrovic S, Wang Z, Ma L, Soleimani M. Regulation of the apical Cl–/HCO3– exchanger pendrin in rat cortical collecting duct in metabolic acidosis. Am J Physiol Renal Physiol 284: F103–F112, 2003.
32. Quentin F, Chambrey R, Trinh-Trang-Tan MM, Fysekidis M, Cambillau M, Paillard M, Aronson PS, Eladari D. The Cl–/HCO3– exchanger pendrin in the rat kidney is regulated in response to chronic alterations in chloride balance. Am J Physiol Renal Physiol 287: F1179–F1188, 2004.
33. Reardon W, Trembath RC. Pendred syndrome. J Med Genet 33: 1037–1040, 1996.
34. Riedel C, Dohan O, De la Vieja A, Ginter CS, Carrasco N. Journey of the iodide transporter NIS: from its molecular identification to its clinical role in cancer. Trends Biochem Sci 26: 490–496, 2001.[CrossRef][Web of Science][Medline]
35. Royaux IE, Suzuki K, Mori A, Katoh R, Everett LA, Kohn LD, Green ED. Pendrin, the protein encoded by the Pendred syndrome gene (PDS), is an apical porter of iodide in the thyroid and is regulated by thyroglobulin in FRTL-5 cells. Endocrinology 141: 839–845, 2000.
36. Royaux IE, Wall SM, Karniski LP, Everett LA, Suzuki K, Knepper MA, Green ED. Pendrin, encoded by the Pendred syndrome gene, resides in the apical region of renal intercalated cells and mediates bicarbonate secretion. Proc Natl Acad Sci USA 98: 4221–4226, 2001.
37. Sawada S, Takeda T, Kitano H, Takeuchi Kakigi AS, Azuma H. Aquaporin-2 regulation by vasopressin in the rat inner ear. Neuroreport 13: 1127–1129, 2002.[CrossRef][Web of Science][Medline]
38. Schug J, Overton GC. Modeling transcription factor binding sites with Gibbs Sampling and Minimum Description Length encoding. Proc Int Conf Intell Syst Mol Biol 5: 268–271, 1997.[Medline]
39. Schwartz GJ. Plasticity of intercalated cell polarity: effect of metabolic acidosis. Nephron 87: 304–313, 2001.[CrossRef][Web of Science][Medline]
40. Scott DA, Karniski LP. Human pendrin expressed in Xenopus laevis oocytes mediates chloride/formate exchange. Am J Physiol Cell Physiol 278: C207–C211, 2000.
41. Scott DA, Wang R, Kreman TM, Sheffield VC, Karniski LP. The Pendred syndrome gene encodes a chloride-iodide transport protein. Nat Genet 21: 440–443, 1999.[CrossRef][Web of Science][Medline]
42. Shachaf C, Skorecki KL, Tzukerman M. Role of AP2 consensus sites in regulation of rat Npt2 (sodium-phosphate cotransporter) promoter. Am J Physiol Renal Physiol 278: F406–F416, 2000.
43. Skubis-Zegadlo J, Nikodemska A, Przytula E, Mikula M, Bardadin K, Ostrowski J, Wenzel BE, Czarnocka B. Expression of pendrin in benign and malignant human thyroid tissues. Br J Cancer 93: 144–151, 2005.[CrossRef][Web of Science][Medline]
44. Soleimani M, Greeley T, Petrovic S, Wang Z, Amlal H, Kopp P, Burnham CE. Pendrin: an apical Cl–/OH–/HCO3– exchanger in the kidney cortex. Am J Physiol Renal Physiol 280: F356–F364, 2001.
45. Taki K, Kogai T, Kanamoto Y, Hershman JM, Brent GA. A thyroid-specific far-upstream enhancer in the human sodium/iodide symporter gene requires Pax-8 binding and cyclic adenosine 3',5'-monophosphate response element-like sequence binding proteins for full activity and is differentially regulated in normal and thyroid cancer cells. Mol Endocrinol 16: 2266–2282, 2002.
46. Taniyama Y, Sato K, Sugawara A, Uruno A, Ikeda Y, Kudo M, Ito S, Takeuchi K. Renal tubule-specific transcription and chromosomal localization of rat thiazide-sensitive Na-Cl cotransporter gene. J Biol Chem 276: 26260–26268, 2001.
47. Uchida S, Rai T, Yatsushige H, Matsumura Y, Kawasaki M, Sasaki S, Marumo F. Isolation and characterization of kidney-specific ClC-K1 chloride channel gene promoter. Am J Physiol Renal Physiol 274: F602–F610, 1998.
48. Uchida S, Tanaka Y, Ito H, Saitoh-Ohara F, Inazawa J, Yokoyama KK, Sasaki S, Marumo F. Transcriptional regulation of the CLC-K1 promoter by myc-associated zinc finger protein and kidney-enriched Kruppel-like factor, a novel zinc finger repressor. Mol Cell Biol 20: 7319–7331, 2000.
49. Vallet M, Picard N, Loffing-Cueni D, Fysekidis M, Bloch-Faure M, Deschenes G, Breton S, Meneton P, Loffing J, Aronson PS, Chambrey R, Eladari D. Pendrin regulation in mouse kidney primarily is chloride-dependent. J Am Soc Nephrol 17: 2153–2163, 2006.
50. Verlander JW, Hassell KA, Royaux IE, Glapion DM, Wang ME, Everett LA, Green ED, Wall SM. Deoxycorticosterone upregulates PDS (Slc26a4) in mouse kidney: role of pendrin in mineralocorticoid-induced hypertension. Hypertension 42: 356–362, 2003.
51. Verlander JW, Kim YH, Shin W, Pham TD, Hassell KA, Beierwaltes WH, Green ED, Everett L, Matthews SW, Wall SM. Dietary Cl– restriction upregulates pendrin expression within the apical plasma membrane of type B intercalated cells. Am J Physiol Renal Physiol 291: F833–F839, 2006.
52. Verrey F, Pearce D, Pfeiffer R, Spindler B, Mastroberardino L, Summa V, Zecevic M. Pleiotropic action of aldosterone in epithelia mediated by transcription and post-transcription mechanisms. Kidney Int 57: 1277–1282, 2000.[CrossRef][Web of Science][Medline]
53. Wagner CA, Finberg KE, Stehberger PA, Lifton RP, Giebisch GH, Aronson PS, Geibel JP. Regulation of the expression of the Cl–/anion exchanger pendrin in mouse kidney by acid-base status. Kidney Int 62: 2109–2117, 2002.[CrossRef][Web of Science][Medline]
54. Wall SM, Hassell KA, Royaux IE, Green ED, Chang JY, Shipley GL, Verlander JW. Localization of pendrin in mouse kidney. Am J Physiol Renal Physiol 284: F229–F241, 2003.
55. Winter C, Schulz N, Giebisch G, Geibel JP, Wagner CA. Nongenomic stimulation of vacuolar H+-ATPases in intercalated renal tubule cells by aldosterone. Proc Natl Acad Sci USA 101: 2636–2641, 2004.
56. Wong MA, Cui S, Quaggin SE. Identification and characterization of a glomerular-specific promoter from the human nephrin gene. Am J Physiol Renal Physiol 279: F1027–F1032, 2000.
57. Yang T, Vidarsson H, Rodrigo-Blomqvist S, Rosengren SS, Enerback S, Smith RJ. Transcriptional control of SLC26A4 is involved in Pendred syndrome and nonsyndromic enlargement of vestibular aqueduct (DFNB4). Am J Hum Genet 80: 1055–1063, 2007.[CrossRef][Web of Science][Medline]
58. Yoshida A, Taniguchi S, Hisatome I, Royaux IE, Green ED, Kohn LD, Suzuki K. Pendrin is an iodide-specific apical porter responsible for iodide efflux from thyroid cells. J Clin Endocrinol Metab 87: 3356–3361, 2002.
This article has been cited by other articles:
|
|
 |
|
  P. Hafner, R. Grimaldi, P. Capuano, G. Capasso, and C. A. Wagner Pendrin in the mouse kidney is primarily regulated by Cl- excretion but also by systemic metabolic acidosis Am J Physiol Cell Physiol, December 1, 2008; 295(6): C1658 - C1667. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
0.1 kb led to marked reduction in reporter gene activity, which remained unchanged with further shortening of the fragments. *P < 0.01.
