CREB trans-activates the murine H+-K+-ATPase 2 -subunit gene

Xiangyang Xu, Wenzheng Zhang and Bruce C. Kone

Am J Physiol Cell Physiol 287:C903-C911, 2004. First published 26 May 2004; doi: 10.1152/ajpcell.00065.2004 You might find this additional info useful... This article cites 38 articles, 27 of which you can access for free at: http://ajpcell.physiology.org/content/287/4/C903.full#ref-list-1 This article has been cited by 4 other HighWire-hosted articles: http://ajpcell.physiology.org/content/287/4/C903#cited-by Updated information and services including high resolution figures, can be found at: http://ajpcell.physiology.org/content/287/4/C903.full Additional material and information about American Journal of Physiology - Cell Physiology can be found at: http://www.the-aps.org/publications/ajpcell This information is current as of December 5, 2012.
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American Journal of Physiology - Cell Physiology is dedicated to innovative approaches to the study of cell and molecular physiology. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright 2004 the American Physiological Society. ISSN: 0363-6143, ESSN: 1522-1563. Visit our website at http://www.the-aps.org/.

Am J Physiol Cell Physiol 287: C903C911, 2004. First published May 26, 2004; 10.1152/ajpcell.00065.2004.

CREB trans-activates the murine H-K-ATPase 2-subunit gene

Xiangyang Xu, Wenzheng Zhang, and Bruce C. Kone
Department of Internal Medicine and Department of Integrative Biology and Pharmacology, The University of Texas Medical School at Houston, and Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, The University of Texas Health Science Center at Houston, Houston, Texas 77030
Submitted 5 February 2004; accepted in nal form 21 May 2004

Xu, Xiangyang, Wenzheng Zhang, and Bruce C. Kone. CREB trans-activates the murine H-K-ATPase-2 subunit gene. Am J Physiol Cell Physiol 287: C903C911, 2004. First published May 26, 2004; 10.1152/ajpcell.00065.2004.Despite its key role in potassium homeostasis, transcriptional control of the H-K-ATPase 2-subunit (HK2) gene in the collecting duct remains poorly characterized. cAMP increases H-K-ATPase activity in the collecting duct, but its role in activating HK2 transcription has not been explored. Previously, we demonstrated that the proximal 177 bp of the HK2 promoter confers basal collecting duct-selective expression. This region contains several potential cAMP/Ca2-responsive elements (CRE). Accordingly, we examined the participation of CRE-binding protein (CREB) in HK2 transcriptional control in murine inner medullary collecting duct (mIMCD)-3 cells. Forskolin and vasopressin induced HK2 mRNA levels, and CREB overexpression stimulated the activity of HK2 promoter-luciferase constructs. Serial deletion analysis revealed that CREB inducibility was retained in a construct containing the proximal 100 bp of the HK2 promoter. In contrast, expression of a dominant negative inhibitor (A-CREB) resulted in 60% lower HK2 promoter-luciferase activity, suggesting that constitutive CREB participates in basal HK2 transcriptional activity. A constitutively active CREB mutant (CREB-VP16) strongly induced HK2 promoter-luciferase activity, whereas overexpression of CREBdLZ-VP16, which lacks the CREB DNA-binding domain, abolished this activation. In vitro DNase I footprinting and gel shift/supershift analysis of the proximal promoter with recombinant glutathione S-transferase (GST)-CREB-1 and mIMCD-3 cell nuclear extracts revealed sequence-specic DNA-CREB-1 complexes at 86/ 60. Mutation at three CRE-like sequences within this region abolished CREB-1 DNA-binding activity and abrogated CREB-VP16 trans-activation of the HK2 promoter. In contrast, mutation of the neighboring 104/94 element did not alter CREB-VP16 transactivation of the HK2 promoter. Thus CREB-1, binding to one or more CRE-like elements in the 86/60 region, trans-activates the HK2 gene and may represent an important link between rapid and delayed effects of cAMP on HK2 activity. transcription; promoter; cAMP; potassium

must be strictly maintained and restored for the proper functioning of all eukaryotic cells. Epithelial cells of the kidney and colon play a critical role in adjusting K, Na, and acid elimination to accommodate changes in dietary intake. Physiological and molecular biological studies in animals have pointed to the H-K-ATPase 2-subunit gene (HK2, also termed colonic H-K-ATPase), which is principally expressed in the distal colon and renal collecting duct, as a key participant in the control of body K homeostasis (14, 29, 30, 35). For example, mice with null
Address for reprint requests and other correspondence: B. C. Kone, Dept. of Internal Medicine and Dept. of Integrative Biology, Pharmacology and Physiology, Univ. of Texas Medical School at Houston, 6431 Fannin Ave., MSB 1.150, Houston, TX 77030 (E-mail: Bruce.C.Kone@uth.tmc.edu). http://www.ajpcell.org

POTASSIUM AND ACID-BASE HOMEOSTASIS

mutations in this gene experience profound hypokalemia during dietary K (18) and Na restriction (31). HK2 may also play a role in HCO 3 absorption by the kidney (20, 21) and the distal colon (18), as well as increased ammonium secretion in the inner medullary collecting duct (IMCD) during chronic hypokalemia (34) and the chronic adaptation to changes in Na (27) and aldosterone (11, 12, 23) balance. Although it plays a central role in these important adaptive responses, transcriptional control of the HK2 gene remains poorly characterized. The HK2 gene contains 23 exons and spans 23.5 kb of genomic DNA on chromosome 14C3 (38). It shares an exonintron organization comparable to that of human ATP1AL1. Our laboratory (38) previously demonstrated that the proximal 177 bp of the 5-anking region of this gene confer basal transcriptional activity in mIMCD-3 cells and appear to be essential for collecting duct-selective expression. This proximal promoter region contains several consensus sequences for transcription factors that we have been systematically characterizing. Our laboratory (37) also has shown that NF-, binding to a DNA-binding element at 104 to 94, recruits histone deacetylase (HDAC)-6 to the DNA protein complex to suppress HK2 transcription. Analysis of putative binding elements for transcription factors within the proximal promoter also revealed potential binding sites for cAMPresponsive element (CRE) binding (CREB) protein. In the kidney, three different K-ATPase activities, distinguished by their kinetic and pharmacological properties and adaptation to chronic K depletion, have been described. One activity (type I) is K dependent but not Na dependent, ouabain resistant, Sch-28080 sensitive, and expressed in collecting ducts. A second activity (type II) is K dependent but not Na dependent, Sch-28080 and ouabain sensitive, and expressed basally in proximal tubules and the thick ascending limbs. This activity is virtually abolished during chronic K depletion. A third activity (type III) is activated by either Na or K and exhibits higher sensitivities than type II activity to ouabain and Sch-28080 and a lower sensitivity than type I activity to Sch-28080. This activity is not expressed basally but is specically upregulated in cortical collecting ducts (CCD) and outer medullary collecting ducts (OMCD) with chronic hypokalemia (15) and thus may represent the functional correlate of HK2. Hormone signaling through cAMP/protein kinase A has been shown to rapidly stimulate type III K-ATPase in principal cells of both CCD and OMCD and in OMCD intercalated cells in K-depleted rats (15). To date, chronic effects of
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cAMP/protein kinase A on K-ATPase activity have not been demonstrated. In addition to such nongenomic effects, cAMP mediates the hormonal stimulation of a number of eukaryotic genes through the binding of the CREB/activating transcription factor (ATF) family of proteins, members of the larger basic leucine zipper (bZIP) family of transcription factors, to a conserved CRE to recruit RNA polymerase to a promoter (17). CRE have been identied in numerous gene promoters and generally consist of minor variations in the 8-bp palindrome 5-TGANNTCA-3 (7, 19, 28). The minimum sequence required for a functional CRE is the downstream half-site, 5-NGTCA-3, although binding to this site is generally weaker and sequence exceptions have occurred (9). The transactivation of genes through a CRE is proposed to occur by binding of phosphorylated ATF/CREB transcription factors to the CRE with recruitment of CREB binding protein (CBP) (5). The CREB family includes CREB-1, cAMP-responsive element modulatory (CREM) protein, and ATF-1. Whereas CREB and ATF-1 are ubiquitously expressed, CREM is expressed most highly in neuroendocrine tissues. Of the members of the CREB/ATF family, both CREB-1 and ATF-1 have been shown to be responsive to both the cAMP/protein kinase A and Ca2/calmodulin-dependent protein kinase pathways (17). We report here the identication of a dened region of the HK2 promoter required for vasopressin- and forskolin-stimulated, CREB-1-mediated transcription of the HK2 gene in mIMCD-3 renal medullary collecting duct cells. We demonstrate that CREB-1, acting through CRE-like elements in the HK2 86/60 region and without participation of the neighboring element, signicantly activates transcriptional activity of the HK2 gene.
MATERIALS AND METHODS

Cell culture and reagents. mIMCD-3 cells, an immortalized cell line derived from the IMCD (24), were cultured in DMEM supplemented with 10% FBS at 37C in a 5% CO2 environment. The Dual-Luciferase reporter assay system and the luciferase vectors pGL3-Basic and pRL-SV40, human recombinant CREB-1 dimerization domain (amino acids 254 327), human recombinant NF- p50, and anti-CREB-1 antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A CREB consensus oligonucleotide 5AGAGATTGCCTGACGTCAGAGAGCTAG-3 (26) and the DNase I footprinting kit were purchased from Promega (Madison, WI). The QuickChange site-directed mutagenesis kit was obtained from Stratagene (La Jolla, CA). The Superscript rst-strand synthesis system was purchased from GIBCO-Invitrogen (Carlsbad, CA). The DyNAmo SYBR green qPCR kit was obtained from Finnzymes (Espoo, Finland). The bicinchoninic acid protein estimation kit was purchased from Pierce Chemical. Enhanced chemiluminescence reagents were

obtained from Amersham Pharmacia Biotech (Piscataway, NJ). All oligonucleotides were synthesized by Genosys (The Woodlands, TX). Quantitative real-time RT-PCR analysis of HK2 mRNA expression. The assays were performed using the MJ Research DNA Engine Opticon 2 System (South San Francisco, CA). mIMCD-3 cells were incubated with vehicle or 108 M vasopressin for various times as indicated or with 15 M forskolin or vehicle for 8 h, and total RNA was then extracted using RNA-Bee (Tel-Test, Friendswood, TX). The RNA was reverse transcribed to cDNA using the Superscript rststrand synthesis system for RT-PCR (GIBCO-Invitrogen). The cDNA was then quantied using quantitative RT-PCR with the DyNAmo SYBR green qPCR kit (Finnzymes). The HK2 primers to amplify nucleotides 28 to 476 were forward, 5-GGTGCCTTGTCTCTGTAAC-3, and reverse, 5-GACCCTGGATGATGTTTG-3. Normalization was performed using -actin mRNA level as a housekeeping control. The -actin primers were forward, 5-GTGGGCCGCTCTAGGCACCAA-3, and reverse, 5-CTCTTTGATGTCACGCACGATTTC-3, amplifying nucleotides 25 to 564. The specicity of these primer sets for their targets was conrmed by agarose gel electrophoresis. Each quantitative PCR assay was repeated three times, and the results were averaged. Plasmids and constructs. A series of deletion constructs of the murine HK2 proximal promoter beginning at nucleotide 253, which is between the transcription start site and the translation initiation codon ATG, and various lengths of the contiguous proximal 177, 477, 1,329, 2,833, 4,306, and 5,667 bp of the 5-anking region of the murine HK2 gene nucleotides were previously described (38) and named pGL3-0.1mHK2, pGL3-0.18mHK2, pGL3-0.48mHK2, pGL3-1.3mHK2, pGL3-2.8mHK2, pGL3-4.3mHK2, and pGL35.7mHK2. A PCR fragment spanning nucleotides 253 to 109 of the murine HK2 proximal promoter was cloned into pGL3-Basic at the MluI and BglII sites to generate the construct pGL3-0.1mHK2. pGL3-0.1mHK286/60, which harbors mutated mHK2 CRE-like sites at 86TGAGCTGC79, 79CGTCG75, and 65CGTGG61 in the 86/60 region (wild type 5-TGAGCTGCGTCGCCCCAGGTACGTGGG-3 converted to 5-TGAGAAGCGTTTCCCCATATACATGGG-3 with mutated bases underlined; Fig. 1), was generated by PCR using pGL3-0.1mHK2 as a template. Plasmids CMV500, CREB, the dominant negative CREB mutant A-CREB (1), the constitutively active CREB mutant CREB-VP16 (25, 36), and pCREBdLZ-VP16, which is identical to CREB-VP16 except that the DNAbinding and dimerization domains of CREB are deleted (25), were a gift from Dr. D. D. Ginty (Department of Neuroscience, Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, MD). Plasmid glutathione S-transferase (GST)CREB-1 (10) was a gift from Dr. T. F. Osborne (Department of Biology and Biochemistry, University of California, Irvine, CA). pGL3-0.1mHK2, which harbors a mutated NF- site at the 104 to 94 position (5-GGGGCGTCCCC-3 converted to 5-TAGCCGTCCCC-3, mutated bases underlined) that ablates NF- DNA-binding activity and enhancer activity was described previously (27).

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Fig. 1. Map of the proximal 5-anking region of the murine H-K-ATPase 2-subunit gene (HK2) promoter. Consensus sites for the binding of selected transcription factors are indicated. Sequence of the 86/60 region and the mutated sequence used for EMSA and trans-activation assays also is indicated.

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In vitro DNase I footprinting. DNase I footprinting analysis was performed with the Core footprinting system (Promega) according to the manufacturers instructions and previously published work done at our laboratory (37). A PCR fragment corresponding to 148 to 2 of the native HK2 5-anking region was generated using pGL30.18mHK2 as a template. This fragment was used as a radiolabeled DNA template for footprinting and as an unlabeled fragment for DNA sequencing performed with the fmol DNA cycle sequencing system (Promega). For footprinting, the labeled template DNA was incubated in a nal volume of 50 l with or without 1.5 g of recombinant protein of the CREB DNA-binding domain (amino acids 254 327) or NF- p50 (as a negative control) in binding buffer (Promega). DNase I digestion, extraction with phenol/chloroform/isoamyl alcohol, electrophoresis, and band visualization were performed according to Promegas technical manual. EMSA and supershift assays. Double-stranded oligonucleotides corresponding to the CREB consensus oligonucleotide (5-AGAGATTGCCTGACGTCAGAGAGCTAG-3; Santa Cruz Biotechnologies), 28/74 (designated 0.1mHK2), and the HK2 putative CREB binding region 86/60, derived from footprinting analysis, as well as the mutated 86/60 region (5-TGAGAAGCGTTTCCCCATATACATGGG-3) used in the design of pGL3-0.1mHK286/60, were end labeled with [-32P]ATP using T4 polynucleotide kinase. Nuclear extracts were prepared from mIMCD-3 cells as detailed in earlier work done at our laboratory (37). GST-CREB fusion protein was harvested from Escherichia coli strain DH5F after transfection with GST-CREB plasmid, according to previous work done at our laboratory (37). In the EMSA, 1.5 g of recombinant CREB-1 DNA-binding domain (amino acids 254 327) or 12 g of mIMCD-3 nuclear extract was used. For the experiment in which the effect of forskolin was examined, nuclear extracts were prepared from mIMCD-3 cells treated for 2 h with either vehicle or 15 M forskolin. In the supershift assay, 3 g of GST-CREB or GST were preincubated with or without 4 g of anti-CREB-1 antibody or IgG. Binding reactions were performed in binding buffer [10 mM Tris, pH 7.5, 50 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 4% glycerol, and 1 g poly(dI-dC)] for 1 h on ice by adding 1.75 pmol of duplex DNA probe (2 105 cpm) in 20 l of reaction binding buffer (Promega). In competition experiments to determine specicity, binding reactions were conducted in the presence or absence of a 50-fold molar excess of nonradiolabeled competitor oligonucleotides or an unrelated oligonucleotide (Sp1). In all cases, aliquots of the reactions were resolved on 4% native polyacrylamide gels in 0.5 M Tris-borate-EDTA buffer. The gels were dried and exposed to X-ray lm with an enhancing screen at 70C to detect the DNA-protein and DNA-protein-antibody complexes. Each observation represents a binding reaction performed in a new nuclear extract preparation. Experiments were replicated a minimum of three times. Transient transfection and reporter gene assays. mIMCD-3 cells grown in 24-well plates were transiently transfected using Lipofectamine 2000 reagent (Life Technologies) as detailed in previously published work performed in our laboratory (37). For comparative purposes, the cells were cotransfected with the Renilla luciferase expression plasmid pRL-SV40 (20 ng/well) to control for transfection efciency and other assay-to-assay variability. Trans-repression/ trans-activation experiments used 0.8 g of pGL3-0.1mHK2, pGL30.18mHK2, pGL3-0.48mHK2, pGL3-1.3mHK2, pGL3-2.8mHK2, pGL3-4.3mHK2, and pGL3-5.9mHK2, as well as 0.1 g of plasmids for CREB, CREB-VP16, CREBdLZ-VP16, A-CREB (1), or insertless expression vector CMV500. The functionality of the putative CRE in the 86/60 mHK2 region was tested by transient cotransfection of pGL3-0.1mHK286/60 or pGL3-0.1mHK286/60 together with CREB-VP16. The potential involvement of the 104/94 NF- site in CREB-mediated trans-activation of the HK2 promoter was examined by transient cotransfection of pGL3-0.1mHK2 together with CREBVP16. Forty-eight hours later, rey and Renilla luciferase activities in 5to 10-l lysate samples were measured in a Turner Systems 20/20
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luminometer using the Dual-Luciferase reporter assay system according to the manufacturers protocol. Firey luciferase activity was normalized for Renilla luciferase activity in the lysates. The data were then normalized to the data of the parent vector pGL3 alone and recorded as HK2 promoter activity. Data analysis. Potential regulatory motifs in the HK2 gene were identied with TESS: Transcription Element Search System software (http://www.cbil.upenn.edu/tess/tess33; Computational Biology and Informatics Laboratory, School of Medicine, University of Pennsylvania) using the TRANSFAC version 4.0 database. Quantitative data are expressed as means SE and were analyzed for statistical signicance using ANOVA. P 0.05 was considered signicant.
RESULTS

Vasopressin and forskolin stimulate HK2 gene expression in mIMCD-3 cells. cAMP and the adenylate cyclase agonist forskolin have been shown to rapidly activate H-K-ATPase activity in the rat OMCD via posttranslational mechanisms (15, 16), but the ability of PKA-dependent activation of HK2 gene expression has not been studied. Vasopressin is a physiological agonist of cAMP/PKA and is known to act in this manner in the renal collecting duct. Forskolin is known to activate PKA and thereby CREB in many cell types. Quantitative real-time RT-PCR analysis revealed that compared with vehicle-treated controls, vasopressin (108 M) promoted a time-dependent increase in normalized HK2 mRNA levels, with the peak levels apparent at 4 h and falling to near-baseline levels at 24 h (Fig. 2A). Similarly, forskolin (15 M) treatment of mIMCD-3 cells for 8 h resulted in twofold greater HK2 mRNA levels normalized to -actin mRNA levels, which were invariant between the groups (Fig. 2B). CREB trans-activates the HK2 promoter. To determine whether CREB participates in the forskolin-stimulated induction of HK2 gene expression, the effect of CREB overexpression on HK2 promoter-luciferase activity was measured in trans-activation/trans-repression assays in mIMCD-3 cells. Overexpression of CREB resulted in 2.5-fold higher rates of pGL3-5.7mHK2 promoter activity compared with vectortransfected controls (Fig. 3A). Serial deletion analysis of the 5-anking region of the HK2 gene revealed that CREB inducibility was retained in a HK2 promoter construct containing the proximal 100 bp of the promoter (pGL30.1mHK2) (Fig. 3A). In contrast, expression of A-CREB, a potent and selective dominant negative inhibitor of CREB DNA-binding activity, resulted in 60% lower basal activity of pGL3-0.1mHK2, suggesting that constitutive CREB also plays a role in basal HK2 transcriptional activity (Fig. 3B). It is important to note that the DNA-binding activity of CREB and its closely related family members is potently and selectively inhibited by A-CREB, while the DNA binding activity of other bZIP transcription factors, including ATF-2, are not altered even at very high expression levels of CREB (1). The constitutively active CREB mutant CREB-VP16, which interacts with CRE through the basic leucine zipper domain of CREB and activates transcription through the activation domain of the herpes simplex virus VP16 protein (32) induced pGL3-0.1mHK2 luciferase activity about 16-fold, whereas expression of CREBdLZ-VP16, which lacks the CREB DNAbinding domain, abolished the constitutive activation of pGL30.1mHK2, indicating that CREB binding to target cis elewww.ajpcell.org

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consensus CRE oligomer and the 0.1mHK2 oligomer, which contains the 86/60 sequence, as a template. As shown in Fig. 5A, GST-CREB-1 bound in a sequence-specic manner to both DNA templates in an identical pattern. GST alone produced no DNA-protein complexes. Incubation with antiCREB1 antibody caused a supershift of the DNA-protein complex and near-complete diminution of the intensity of the original DNA-protein complexes compared with incubation with IgG. Similar results were obtained when EMSA was performed with nuclear extract proteins from mIMCD-3 cells and the radiolabeled 86/60 probe (Fig. 5, BD). A small amount of protein binding to the 86/60 probe was observed under basal conditions, but vasopressin or forskolin treatment of mIMCD-3 cells resulted in signicantly enhanced protein binding to the 86/60 probe (Fig. 5B). This DNA-binding activity was sequence specic because binding was competed by a molar excess of unlabeled 86/60 oligonucleotide but not by the unrelated oligomer for Sp1 or a mutated 86/60 oligomer designed to disrupt three potential CRE-like ele-

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Fig. 2. Vasopressin and forskolin induce HK2 mRNA expression in murine inner medullary collecting duct (mIMCD)-3 cells. A: mIMCD-3 cells were treated with vehicle or vasopressin (AVP; 108 M) for the indicated times, after which total RNA was harvested and cDNA was prepared. Quantitative real-time PCR was then performed to assess HK2 and -actin mRNA levels in each sample (n 4). *P 0.05 vs. vehicle-treated control (Ctrl). B: mIMCD-3 cells were treated with vehicle or forskolin (15 M) for 8 h, after which total RNA was harvested and cDNA was prepared. Quantitative realtime PCR was then performed to assess HK2 and -actin mRNA levels in each sample (n 3). *P 0.05 vs. vehicle-treated control.

ments in the proximal 100 bp of the HK2 promoter mediates HK2 gene trans-activation (Fig. 3B). The 86/60 region of the HK2 promoter binds CREB-1. In vitro DNase I footprinting and gel shift/supershift analyses were performed to identify potential regulatory proteins bound within the proximal 100 bp of the HK2 promoter that had been implicated in the promoter-reporter gene studies. DNase I footprinting assay with recombinant GST-CREB-1 (Fig. 4), NF- p50, or no protein added was performed with a radiolabeled PCR amplicon corresponding to nucleotides 148/2 of the HK2 promoter as template. Protected sites in the region 86/60 were detected in the binding reactions that included GST-CREB-1 compared with the reactions with no protein added or with NF- p50 added (Fig. 4). The sequence of this protected region read as 5-TGAGCTGCGTCGCCCCAGGTACGTGGG-3. It should be noted that under this assay condition, recombinant NF- p50 did not bind to 94 to 104 as reported previously using a different binding buffer (37). Binding of recombinant CREB-1 to the 86/60 element was further investigated by performing EMSA using both a
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Fig. 3. cAMP/Ca2-responsive element (CRE)-binding protein (CREB)-1 trans-activates the HK2 promoter. A: mIMCD-3 cells were transfected separately with a series of deletion mutants of the HK2 5-anking region fused to the rey luciferase gene or the empty vector pGL3-Basic (as a control) together with the expression vector for CREB-1 and the Renilla luciferase expression plasmid pRL-SV40. The positions of the 5-end of the HK2 5-anking region deletions are indicated numerically. Forty-eight hours after transfection, cell lysates were prepared and rey and Renilla luciferase activities in lysates of the cells were assayed. Firey luciferase activity was normalized to Renilla luciferase activity and reported as HK2 promoter activity. Values are means SE of 5 separate experiments and represent the relative increase over controls transfected with pGL3-Basic. *P 0.05. B: pGL3-0.1mHK2 reporter construct and pRL-SV40 were cotransfected with the expression vector for CREB-1, a constitutively active CREB mutant CREB-VP16, CREBdLZ-VP16, the CREB dominant-negative mutant A-CREB, or an insertless mammalian expression vector containing the cytomegalovirus promoter CMV500. Forty-eight hours after transfection, cell lysates were prepared and rey and Renilla luciferase activities in lysates of the cells were assayed. Firey luciferase activity was normalized to Renilla luciferase activity. Values are means SE of 4 separate experiments. *P 0.05 vs. CMV500-transfected controls. www.ajpcell.org

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activity of pGL3-0.1mHK2 was comparable to that of the wild-type HK2 promoter.

DISCUSSION

Fig. 4. DNase I footprinting analysis of HK2 proximal promoter. A DNA probe (476 to 82) labeled with 32P at the 5-end on the noncoding strand was incubated with and without recombinant CREB-1 DNA-binding domain protein (amino acids 254 327) or NF- p50 as a negative control. A representative gel is shown (n 4). A sequencing ladder was also generated for denition of footprinted or hypersensitive sites (not shown). Filled bar represents region with footprinted sites produced by recombinant CREB-1 DNA-binding domain protein. 86 and 60 are nucleotide positions relative to the transcription start site of the HK2 gene.

ments in this region (86TGAGCTGC79, 79CGTCG75, and 65 CGTGG61) (Fig. 5C). The mutated 86/60 oligomer was shown to disrupt binding of recombinant CREB-1 and nuclear proteins from mIMCD-3 cells to the wild-type sequence (Fig. 5D). Because a CRE is a common recognition site of the CREB/ATF family, supershift assays were performed to determine whether CREB-1 is a component of the protein complex that binds the element in mIMCD-3 cells. Supershift analysis showed that nuclear extracts incubated with antiCREB-1 antibody exhibited much less CREB DNA-binding activity compared with controls incubated with IgG (Fig. 5C) but not supershift. This result most likely indicates that the antibody disrupted the CREB-1-DNA interaction, resulting in a reduction in the amount of the characteristic gel shift but no supershift. The HK2 86/60 region, but not the 104/94 element, participates in CREB-mediated trans-activation of the HK2 promoter. To address the functional importance of the putative CREB binding site at 86/60 in the CREB-mediated activation of the HK2 promoter, we constructed pGL30.1mHK286/60, which harbors the same mutations in the 86/60 region that prevented CREB-1 DNA-binding activity in the EMSA (Fig. 5D). In trans-activation experiments, the CREBVP16-stimulated promoter activity of pGL3-0.1mHK286/60 was half that of wild-type pGL3-0.1mHK2 (Fig. 6). Taken together, these data support the conclusion that CREB-1 binding to 86/60 results in trans-activation of the HK2 gene. To conrm that the neighboring 104/94 NF- element does not participate in the CREB-mediated trans-activation of the HK2 promoter, the CREB-VP16-stimulated promoter activity of pGL3-0.1mHK2, which contains a mutated and functionally inactive 104/94 NF- element, was tested. As shown in Fig. 7, the CREB-VP16-stimulated promoter
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Analysis of the regulation of active K reabsorption in the renal collecting duct and distal colon requires knowledge of transcription control mechanisms governing HK2 gene expression. In this study, we have built on earlier work performed at our laboratory to dene such control elements by demonstrating that CREB-1 trans-activates the HK2 gene by binding to CRE-like elements in the 86/60 region of the HK2 proximal promoter. These results provide the rst evidence for a specic trans-activator pathway for this gene and identify alternative sequences for binding of CREB-1 that may be more broadly applicable to other gene promoters. The effect of CREB-1 on the promoter was direct and mediated principally by DNA-binding because the 86/60 region was specically footprinted in vitro by GST-CREB-1 (Fig. 4), and mutation of this DNA sequence disrupted the CREB DNA-binding activity (Fig. 5D) as well as abrogated CREB-mediated trans-activation of the HK2 promoter (Fig. 3A). The binding of proteins to the 86/60 region was specic because the formation of the major complexes was inhibited by the addition of excess unlabeled sequence but not by an excess of unlabeled sequence in which the CRE-like elements were mutated (Fig. 5C). However, because antiCREB-1 antibody did not completely disrupt the 86/60 DNA-protein complex in supershift assays (Fig. 5, A and C), other transcription factors or coregulatory proteins may contribute, quantitatively to a much lesser degree, to the complex. Further studies are needed to identify such proteins. Promoter regions of eukaryotic genes are generally composed of multiple binding sites for transcriptional activators and repressors that act in combination to regulate the expression of a linked gene. Our analysis of the proximal promoter of the HK2 gene has thus far revealed two functional elements: a element at 104/94 that binds NF-, as well as the CRE-like elements at 86/60 that bind CREB-1 reported here. The 104/94 element does not appear to participate in the CREB-mediated trans-activation of the HK2 promoter, because an HK2 promoter harboring a mutated 104/94 element exhibited trans-activation by CREB-VP16 comparable to that of the wild-type HK2 promoter. Activator protein AP-2, which has a consensus binding site in this general region, also is not involved, because our laboratory previously showed by in vitro DNase I footprinting that this transcription factor does not footprint this region (37). Whether other CREB/ATF family members participate in HK2 gene regulation remains unknown. The transcriptional coactivator CBP and closely related p300 are known to function as bridging factors between sequence-specic transcriptional activators and basal transcriptional machinery and to assemble them into multiprotein complexes. CBP/p300 also possesses intrinsic histone acetyltransferase (HAT) activity, which increases the accessibility of the basal transcriptional machinery to the promoter and activates transcription (33). Interestingly, our laboratory has already established that histone acetylation is important for activation of HK2 promoter activity because interaction and recruitment of HDAC-6 to NF- bound to the HK2 promoter inhibits HK2 promoter activity (37). Thus it
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Fig. 5. CREB-1 binds the 86/60 region of the HK2 proximal promoter. A: a glutathione S-transferase (GST)-CREB-1 fusion protein or GST alone was subjected to EMSA with 32P-labeled oligomers containing a CREB consensus sequence or nucleotides 28 to 74 of the HK2 gene (0.1mHK2). Polyclonal antibody specic for CREB-1 or IgG (as a negative control) was used in supershift experiments. Autoradiogram is representative of 3 independent experiments performed on separate preparations of nuclear extracts. B: EMSA was conducted with nuclear extracts from mIMCD-3 cells that had been treated with vehicle, vasopressin (108 M), or forskolin (15 M) and the 32P-labeled 86/60 binding element oligomer (mHK286/60). The autoradiogram is representative of 3 independent experiments performed in separate preparations of nuclear extracts. C: nuclear extracts from forskolin-treated mIMCD-3 cells were examined using EMSA with the 32P-labeled 86/60 binding element oligomer. To demonstrate binding specicity, reactions were also conducted in the presence of a 50-fold molar excess of unlabeled CREB-1 consensus oligomer, 86/60 binding element oligomer (mHK286/60), 86/60 oligomer harboring mutations in the CRE-like elements (mHK286/60 as in Fig. 1), or nonspecic Sp1 oligomer. Supershift experiments were performed with anti-CREB-1 antibody or IgG. The autoradiogram is representative of 3 independent experiments performed on separate preparations of nuclear extracts. D: nuclear extracts from mIMCD-3 cells were incubated in an EMSA with radiolabeled CREB consensus oligomer, the 86/60 binding element oligomer (mHK286/60), or a 86/60 oligomer harboring mutations in the CRE-like elements (mHK286/60). The autoradiograms are representative of 3 independent experiments performed in separate preparations of nuclear extracts.

will be interesting to determine whether CBP couples with CREB-1 bound in the 86/60 region and, through HAT activity, contributes to the trans-activation potential of CREB-1 on the HK2 gene. In addition to inducible effects, CREB-1 also trans-activates, to a much lesser degree, basal transcription of the HK2
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gene through the 86/60 region. Overexpression of the dominant negative mutant A-CREB inhibited basal promoter activity of pGL3-0.1mHK2 (Fig. 3B), and low levels of CREB-1 DNA-binding activity were apparent under basal conditions (Fig. 5B). It is known that CREB stimulates basal transcription of many CRE-containing genes in addition to its
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Fig. 6. Mutations in the CRE-like sequences of the 86/60 region limit the CREB-mediated induction of HK2 proximal promoter activity. The pGL30.1mHK2 reporter construct or pGL3-0.1mHK286/60, which harbors mutations in the CRE-like elements of the HK2 86/60 binding element (see Fig. 1), and the Renilla luciferase expression plasmid pRL-SV40 in the presence of the expression vector for CREB-1. Forty-eight hours after transfection, cell lysates were prepared and rey and Renilla luciferase activities in lysates of the cells were assayed. Firey luciferase activity was normalized to Renilla luciferase activity. Values are means SE of 4 separate experiments. *P 0.05 vs. pGL3-0.1mHK2.

induction of target gene transcription upon phosphorylation by protein kinases. The basal activity of CREB resides in the constitutive activation domain (CAD) at the COOH terminus, whereas phosphorylation and inducibility map to a centrally located kinase-inducible domain (KID). The CAD interacts with a specic TATA binding protein-associated factor, which recruits a polymerase complex and activates transcription (13). In addition, in some cell types, interaction of CREB via its bZIP DNA binding/dimerization domain with the TORC (transducers of regulated CREB activity) family of coactivators promotes CRE-dependent transcription in a phosphorylationindependent manner (8). TORC recruitment appears to enhance the interaction of CREB with the polymerase complex without signicantly altering CREB DNA-binding activity. Further studies are needed to dene the specic mechanisms that underlie basal CREB-mediated HK2 transcriptional activation in mIMCD-3 cells. CRE are typically located in the proximal 100 bp of the TATA box in most genes, and the CRE-like elements described here also share proximity to the transcriptional initiation region. About half of the known genes with a functional CRE contain a full palindrome, with the other half containing a single CGTCA motif. However, several variations have been noted. For example, the TAT promoter contains a CRE that differs from the consensus sequence at the rst three nucleotides (CTGCGTCA), and the c-fos promoter contains a CRE that differs from the consensus CRE by nucleotides in the seventh and eighth positions (TGACGTAG) (22). Sequence comparisons revealed three near-matches to the consensus CRE full and half-sites: 86 TGAGCTGC79 is a 6/8 nucleotide match of the full palindrome, and 79CGTCG75 and 65CGTGG61 are both 4/5 nucleotide matches of the consensus CRE half-site. Mutation of all three sites dramatically limited CREB-1 binding to the 86/60 oligomer in the EMSA and the activity of the pGL3-0.1mHK-luc promoter construct, indicating that they are indeed functional CRE. The specic roles of HK2 in the collecting duct in vivo are unclear. Because HK2-null mice exhibit profound fecal rather
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than urinary K losses during K restriction (18), a major role in K reabsorption has not been convincingly established. However, as Meneton et al. (18) pointed out, the data do not rule out the possibility that HK2 is involved in renal K conservation by further limiting urinary K losses. In their report, the mean values for daily K excretion were consistently 20% greater in the HK2-null than in the HK2 wild-type mice during chronic K restriction, although this difference was not statistically signicant. However, because plasma K levels were 25% lower in the knockout mice, one would expect greater K conservation, not a trend toward less K conservation. Similarly, the specic physiological and pathophysiological roles for vasopressin-stimulated and CREB-mediated trans-activation of the HK2 promoter in IMCD cells remain to be dened. It is known that chronic K deprivation, a strong stimulus for renal medullary HK2 expression, causes impairment of urine-concentrating ability (2). Chronic K depletion has been shown to result in impaired sensitivity of cAMP production to vasopressin in the renal medulla as well as polyuria (4). This decrease in cAMP would also limit the stimulus for HK2 gene transcription, as shown here. Thus the blunting of responsiveness to vasopressin in the medullary collecting duct would cause renal water losses and potentially aggravate the K depletion by limiting HK2mediated renal K conservation. Further physiological studies are required. In terms of acid-base balance, metabolic acidosis has been shown to be associated with an increase in AVP synthesis and secretion, with a resultant increase in aquaporin-2 expression and water reabsorption in the collecting duct (3). Such an increase in vasopressin levels would be expected to enhance HK2 expression and H-K-ATPasemediated H secretion that would facilitate correction of the metabolic acidosis. We would like to have tested whether enhanced expression of HK2 by vasopressin and cAMP is associated with increased H/K exchange activity in mIMCD-3 cells. However, we previously showed that Sch-28080 depletes intracellular ATP levels in mIMCD-3 cells and thereby

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Fig. 7. Mutations in the 104/94 element do not limit the CREBmediated induction of HK2 proximal promoter activity. The pGL30.1mHK2 reporter construct or pGL3-0.1mHK2, which harbors inactivating mutations in the 104/94 element of the HK2 promoter, and the Renilla luciferase expression plasmid pRL-SV40 in the presence of the expression vector for the constitutively active CREB mutant CREB-VP16. Forty-eight hours after transfection, cell lysates were prepared and rey and Renilla luciferase activities in lysates of the cells were assayed. Firey luciferase activity was normalized to Renilla luciferase activity. Values are means SE of 4 separate experiments. *P 0.05 vs. pGL3-0.1mHK2. www.ajpcell.org

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CREB TRANS-ACTIVATES MURINE H-K-ATPASE 2-SUBUNIT GENE 14. Kone BC. Renal H,K-ATPase: structure, function and regulation. Miner Electrolyte Metab 22: 349 365, 1996. 15. Laroche-Joubert N, Marsy S, and Doucet A. Cellular origin and hormonal regulation of K-ATPase activities sensitive to Sch-28080 in rat collecting duct. Am J Physiol Renal Physiol 279: F1053F1059, 2000. 16. Laroche-Joubert N, Marsy S, Michelet S, Imbert-Teboul M, and Doucet A. Protein kinase A-independent activation of ERK and H,KATPase by cAMP in native kidney cells: role of Epac I. J Biol Chem 277: 18598 18604, 2002. 17. Mayr B and Montminy M. Transcriptional regulation by the phosphorylation-dependent factor CREB. Nat Rev Mol Cell Biol 2: 599 609, 2001. 18. Meneton P, Schultheis PJ, Greeb J, Nieman ML, Liu LH, Clarke LL, Duffy JJ, Doetschman T, Lorenz JN, and Shull GE. Increased sensitivity to K deprivation in colonic H,K-ATPase-decient mice. J Clin Invest 101: 536 542, 1998. 19. Montminy MR, Sevarino KA, Wagner JA, Mandel G, and Goodman RH. Identication of a cyclic-AMP-responsive element within the rat somatostatin gene. Proc Natl Acad Sci USA 83: 6682 6686, 1986. 20. Nakamura S, Amlal H, Galla JH, and Soleimani M. Colonic H-KATPase is induced and mediates increased HCO 3 reabsorption in inner medullary collecting duct in potassium depletion. Kidney Int 54: 1233 1239, 1998. 21. Nakamura S, Wang Z, Galla JH, and Soleimani M. K depletion increases HCO 3 reabsorption in OMCD by activation of colonic H -K ATPase. Am J Physiol Renal Physiol 274: F687F692, 1998. 22. Pearman AT, Chou WY, Bergman KD, Pulumati MR, and Partridge NC. Parathyroid hormone induces c-fos promoter activity in osteoblastic cells through phosphorylated cAMP response element (CRE)-binding protein binding to the major CRE. J Biol Chem 271: 2571525721, 1996. 23. Perrone RD and McBride DE. Aldosterone and PCO2 enhance rubidium absorption in rat distal colon. Am J Physiol Gastrointest Liver Physiol 254: G898 G906, 1988. 24. Rauchman MI, Nigam SK, Delpire E, and Gullans SR. An osmotically tolerant inner medullary collecting duct cell line from an SV40 transgenic mouse. Am J Physiol Renal Fluid Electrolyte Physiol 265: F416 F424, 1993. 25. Riccio A, Ahn S, Davenport CM, Blendy JA, and Ginty DD. Mediation by a CREB family transcription factor of NGF-dependent survival of sympathetic neurons. Science 286: 2358 2361, 1999. 26. Roesler WJ, Vandenbark GR, and Hanson RW. Cyclic AMP and the induction of eukaryotic gene transcription. J Biol Chem 263: 90639066, 1988. 27. Sangan P, Rajendran VM, Mann AS, Kashgarian M, and Binder HJ. Regulation of colonic H-K-ATPase in large intestine and kidney by dietary Na depletion and dietary K depletion. Am J Physiol Cell Physiol 272: C685C696, 1997. 28. Short JM, Wynshaw-Boris A, Short HP, and Hanson RW. Characterization of the phosphoenolpyruvate carboxykinase (GTP) promoter-regulatory region: II. Identication of cAMP and glucocorticoid regulatory domains. J Biol Chem 261: 97219726, 1986. 29. Shull GE, Miller ML, and Schultheis PJ. Lessons from genetically engineered animal models. VIII. Absorption and secretion of ions in the gastrointestinal tract. Am J Physiol Gastrointest Liver Physiol 278: G185 G190, 2000. 30. Silver RB and Soleimani M. H-K-ATPases: regulation and role in pathophysiological states. Am J Physiol Renal Physiol 276: F799 F811, 1999. 31. Spicer Z, Clarke LL, Gawenis LR, and Shull GE. Colonic H-KATPase in K conservation and electrogenic Na absorption during Na restriction. Am J Physiol Gastrointest Liver Physiol 281: G1369 G1377, 2001. 32. Tao X, Finkbeiner S, Arnold DB, Shaywitz AJ, and Greenberg ME. Ca2 inux regulates BDNF transcription by a CREB family transcription factor-dependent mechanism. Neuron 20: 709 726, 1998. 33. Vanden Berghe W, De Bosscher K, Boone E, Plaisance S, and Haegeman G. The nuclear factor- engages CBP/p300 and histone acetyltransferase activity for transcriptional activation of the interleukin-6 gene promoter. J Biol Chem 274: 3209132098, 1999. 34. Wall SM, Truong AV, and DuBose TD Jr. H-K-ATPase mediates net acid secretion in rat terminal inner medullary collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 271: F1037F1044, 1996. www.ajpcell.org

indirectly inhibits all K-ATPases (Na-K- as well as H-K-ATPase) (6). Because the method of distinguishing H-K-ATPase 1- and 2-subunits is based primarily on the Sch-28080 sensitivity of the former and the Sch-28080 insensitivity of the latter, we cannot specically distinguish the two and cannot reliably distinguish the effects of the H-K-ATPase from those of the Na-K-ATPases in the presence of Sch-28080 in intact mIMCD-3 cells. The data reported here highlight the complex transcriptional and posttranslational control of HK2 by vasopressin and cAMP/protein kinase A. The combination of rapid nongenomic effects (15) and delayed and sustained genomic effects provides the HK2 gene with substantial versatility in meeting challenges to K and acid-base homeostasis.
ACKNOWLEDGMENTS We thank Lei Zou and Hui-Wen Lo for technical help in gel shift and footprinting experiments. GRANTS This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-47981 and by endowment funds from The James T. and Nancy B. Willerson Chair (both to B. C. Kone). REFERENCES 1. Ahn S, Olive M, Aggarwal S, Krylov D, Ginty DD, and Vinson C. A dominant-negative inhibitor of CREB reveals that it is a general mediator of stimulus-dependent transcription of c-fos. Mol Cell Biol 18: 967977, 1998. 2. Amlal H, Krane CM, Chen Q, and Soleimani M. Early polyuria and urinary concentrating defect in potassium deprivation. Am J Physiol Renal Physiol 279: F655F663, 2000. 3. Amlal H, Sheriff S, and Soleimani M. Upregulation of collecting duct aquaporin-2 by metabolic acidosis: role of vasopressin. Am J Physiol Cell Physiol 286: C1019 C1030, 2004. 4. Beck N and Webster SK. Impaired urinary concentrating ability and cyclic AMP in K-depleted rat kidney. Am J Physiol 231: 1204 1208, 1976. 5. Chrivia JC, Kwok RP, Lamb N, Hagiwara M, Montminy MR, and Goodman RH. Phosphorylated CREB binds specically to the nuclear protein CBP. Nature 365: 855 859, 1993. 6. Codina J, Cardwell J, Gitomer JJ, Cui Y, Kone BC, and Dubose TD Jr. Sch-28080 depletes intracellular ATP selectively in mIMCD-3 cells. Am J Physiol Cell Physiol 279: C1319 C1326, 2000. 7. Comb M, Birnberg NC, Seasholtz A, Herbert E, and Goodman HM. A cyclic AMP- and phorbol ester-inducible DNA element. Nature 323: 353356, 1986. 8. Conkright MD, Canettieri G, Screaton R, Guzman E, Miraglia L, Hogenesch JB, and Montminy M. TORCs: transducers of regulated CREB activity. Mol Cell 12: 413 423, 2003. 9. Craig JC, Schumacher MA, Mansoor SE, Farrens DL, Brennan RG, and Goodman RH. Consensus and variant cAMP-regulated enhancers have distinct CREB-binding properties. J Biol Chem 276: 11719 11728, 2001. 10. Dooley KA, Bennett MK, and Osborne TF. A critical role for cAMP response element-binding protein (CREB) as a co-activator in sterolregulated transcription of 3-hydroxy-3-methylglutaryl coenzyme A synthase promoter. J Biol Chem 274: 52855291, 1999. 11. Foster ES, Jones WJ, Hayslett JP, and Binder HJ. Role of aldosterone and dietary potassium in potassium adaptation in the distal colon of the rat. Gastroenterology 88: 41 46, 1985. 12. Jaisser F, Escoubet B, Coutry N, Eugene E, Bonvalet JP, and Farman N. Differential regulation of putative K-ATPase by low-K diet and corticosteroids in rat distal colon and kidney. Am J Physiol Cell Physiol 270: C679 C687, 1996. 13. Kim JK, Summer SN, and Berl T. The cyclic AMP system in the inner medullary collecting duct of the potassium-depleted rat. Kidney Int 26: 384 391, 1984. AJP-Cell Physiol VOL

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