CREB trans-activates the murine H+-K+-ATPase {alpha}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 final form 21 May 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Despite its key role in potassium homeostasis, transcriptional control of the H+-K+-ATPase {alpha}2-subunit (HK{alpha}2) gene in the collecting duct remains poorly characterized. cAMP increases H+-K+-ATPase activity in the collecting duct, but its role in activating HK{alpha}2 transcription has not been explored. Previously, we demonstrated that the proximal 177 bp of the HK{alpha}2 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 HK{alpha}2 transcriptional control in murine inner medullary collecting duct (mIMCD)-3 cells. Forskolin and vasopressin induced HK{alpha}2 mRNA levels, and CREB overexpression stimulated the activity of HK{alpha}2 promoter-luciferase constructs. Serial deletion analysis revealed that CREB inducibility was retained in a construct containing the proximal 100 bp of the HK{alpha}2 promoter. In contrast, expression of a dominant negative inhibitor (A-CREB) resulted in 60% lower HK{alpha}2 promoter-luciferase activity, suggesting that constitutive CREB participates in basal HK{alpha}2 transcriptional activity. A constitutively active CREB mutant (CREB-VP16) strongly induced HK{alpha}2 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-specific 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 HK{alpha}2 promoter. In contrast, mutation of the neighboring –104/–94 {kappa}{beta} element did not alter CREB-VP16 trans-activation of the HK{alpha}2 promoter. Thus CREB-1, binding to one or more CRE-like elements in the –86/–60 region, trans-activates the HK{alpha}2 gene and may represent an important link between rapid and delayed effects of cAMP on HK{alpha}2 activity.

transcription; promoter; cAMP; potassium


POTASSIUM AND ACID-BASE HOMEOSTASIS 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 {alpha}2-subunit gene (HK{alpha}2, 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 mutations in this gene experience profound hypokalemia during dietary K+ (18) and Na+ restriction (31). HK{alpha}2 may also play a role in HCO3 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 HK{alpha}2 gene remains poorly characterized.

The HK{alpha}2 gene contains 23 exons and spans 23.5 kb of genomic DNA on chromosome 14C3 (38). It shares an exon-intron organization comparable to that of human ATP1AL1. Our laboratory (38) previously demonstrated that the proximal 177 bp of the 5'-flanking 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-{kappa}{beta}, binding to a {kappa}{beta} DNA-binding element at –104 to –94, recruits histone deacetylase (HDAC)-6 to the DNA protein complex to suppress HK{alpha}2 transcription. Analysis of putative binding elements for transcription factors within the proximal promoter also revealed potential binding sites for cAMP-responsive 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 specifically upregulated in cortical collecting ducts (CCD) and outer medullary collecting ducts (OMCD) with chronic hypokalemia (15) and thus may represent the functional correlate of HK{alpha}2.

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 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 identified 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 trans-activation 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 identification of a defined region of the HK{alpha}2 promoter required for vasopressin- and forskolin-stimulated, CREB-1-mediated transcription of the HK{alpha}2 gene in mIMCD-3 renal medullary collecting duct cells. We demonstrate that CREB-1, acting through CRE-like elements in the HK{alpha}2 –86/–60 region and without participation of the neighboring {kappa}{beta} element, significantly activates transcriptional activity of the HK{alpha}2 gene.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 37°C 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-{kappa}{beta} p50, and anti-CREB-1 antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A CREB consensus oligonucleotide 5'-AGAGATTGCCTGACGTCAGAGAGCTAG-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 first-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 HK{alpha}2 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 10–8 M vasopressin for various times as indicated or with 15 µM 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 first-strand synthesis system for RT-PCR (GIBCO-Invitrogen). The cDNA was then quantified using quantitative RT-PCR with the DyNAmo SYBR green qPCR kit (Finnzymes). The HK{alpha}2 primers to amplify nucleotides +28 to +476 were forward, 5'-GGTGCCTTGTCTCTGTAAC-3', and reverse, 5'-GACCCTGGATGATGTTTG-3'. Normalization was performed using {beta}-actin mRNA level as a housekeeping control. The {beta}-actin primers were forward, 5'-GTGGGCCGCTCTAGGCACCAA-3', and reverse, 5'-CTCTTTGATGTCACGCACGATTTC-3', amplifying nucleotides +25 to +564. The specificity of these primer sets for their targets was confirmed 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 HK{alpha}2 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'-flanking region of the murine HK{alpha}2 gene nucleotides were previously described (38) and named pGL3-0.1mHK{alpha}2, pGL3-0.18mHK{alpha}2, pGL3-0.48mHK{alpha}2, pGL3-1.3mHK{alpha}2, pGL3-2.8mHK{alpha}2, pGL3-4.3mHK{alpha}2, and pGL3-5.7mHK{alpha}2. A PCR fragment spanning nucleotides +253 to –109 of the murine HK{alpha}2 proximal promoter was cloned into pGL3-Basic at the MluI and BglII sites to generate the construct pGL3-0.1mHK{alpha}2. pGL3-0.1mHK{alpha}2{Delta}–86/–60, which harbors mutated mHK{alpha}2 CRE-like sites at –86TGAGCTGC–79, –79CGTCG–75, and –65CGTGG–61 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 pGL3-0.1mHK{alpha}2 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 DNA-binding 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). pGL3-0.1mHK{alpha}2{Delta}{kappa}{beta}, which harbors a mutated NF-{kappa}{beta} site at the –104 to –94 position (5'-GGGGCGTCCCC-3' converted to 5'-TAGCCGTCCCC-3', mutated bases underlined) that ablates NF-{kappa}{beta} DNA-binding activity and enhancer activity was described previously (27).



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Fig. 1. Map of the proximal 5'-flanking region of the murine H+-K+-ATPase {alpha}2-subunit gene (HK{alpha}2) 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.

 
In vitro DNase I footprinting. DNase I footprinting analysis was performed with the Core footprinting system (Promega) according to the manufacturer's instructions and previously published work done at our laboratory (37). A PCR fragment corresponding to –148 to –2 of the native HK{alpha}2 5'-flanking region was generated using pGL3-0.18mHK{alpha}2 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 final volume of 50 µl with or without 1.5 µg of recombinant protein of the CREB DNA-binding domain (amino acids 254–327) or NF-{kappa}{beta} 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 Promega's technical manual.

EMSA and supershift assays. Double-stranded oligonucleotides corresponding to the CREB consensus oligonucleotide (5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3'; Santa Cruz Biotechnologies), –28/–74 (designated 0.1mHK{alpha}2), and the HK{alpha}2 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 pGL3-0.1mHK{alpha}2{Delta}–86/–60, were end labeled with [{gamma}-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 DH5{alpha}F after transfection with GST-CREB plasmid, according to previous work done at our laboratory (37). In the EMSA, 1.5 µg of recombinant CREB-1 DNA-binding domain (amino acids 254–327) or 12 µg 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 µM forskolin. In the supershift assay, 3 µg of GST-CREB or GST were preincubated with or without 4 µg 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 µg poly(dI-dC)] for 1 h on ice by adding 1.75 pmol of duplex DNA probe (~2 x 105 cpm) in 20 µl of reaction binding buffer (Promega). In competition experiments to determine specificity, 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 film with an enhancing screen at –70°C 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 efficiency and other assay-to-assay variability. Trans-repression/trans-activation experiments used 0.8 µg of pGL3-0.1mHK{alpha}2, pGL3-0.18mHK{alpha}2, pGL3-0.48mHK{alpha}2, pGL3-1.3mHK{alpha}2, pGL3-2.8mHK{alpha}2, pGL3-4.3mHK{alpha}2, and pGL3-5.9mHK{alpha}2, as well as 0.1 µg 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 mHK{alpha}2 region was tested by transient cotransfection of pGL3-0.1mHK{alpha}2{Delta}–86/–60 or pGL3-0.1mHK{alpha}2{Delta}–86/–60 together with CREB-VP16. The potential involvement of the –104/–94 NF-{kappa}{beta} site in CREB-mediated trans-activation of the HK{alpha}2 promoter was examined by transient cotransfection of pGL3-0.1mHK{alpha}2{Delta}{kappa}{beta} together with CREB-VP16. Forty-eight hours later, firefly and Renilla luciferase activities in 5- to 10-µl lysate samples were measured in a Turner Systems 20/20 luminometer using the Dual-Luciferase reporter assay system according to the manufacturer's protocol. Firefly 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 HK{alpha}2 promoter activity.

Data analysis. Potential regulatory motifs in the HK{alpha}2 gene were identified 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 significance using ANOVA. P < 0.05 was considered significant.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Vasopressin and forskolin stimulate HK{alpha}2 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 HK{alpha}2 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 (10–8 M) promoted a time-dependent increase in normalized HK{alpha}2 mRNA levels, with the peak levels apparent at 4 h and falling to near-baseline levels at 24 h (Fig. 2A). Similarly, forskolin (15 µM) treatment of mIMCD-3 cells for 8 h resulted in twofold greater HK{alpha}2 mRNA levels normalized to {beta}-actin mRNA levels, which were invariant between the groups (Fig. 2B).



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Fig. 2. Vasopressin and forskolin induce HK{alpha}2 mRNA expression in murine inner medullary collecting duct (mIMCD)-3 cells. A: mIMCD-3 cells were treated with vehicle or vasopressin (AVP; 10–8 M) for the indicated times, after which total RNA was harvested and cDNA was prepared. Quantitative real-time PCR was then performed to assess HK{alpha}2 and {beta}-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 µM) for 8 h, after which total RNA was harvested and cDNA was prepared. Quantitative real-time PCR was then performed to assess HK{alpha}2 and {beta}-actin mRNA levels in each sample (n = 3). *P < 0.05 vs. vehicle-treated control.

 
CREB trans-activates the HK{alpha}2 promoter. To determine whether CREB participates in the forskolin-stimulated induction of HK{alpha}2 gene expression, the effect of CREB overexpression on HK{alpha}2 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 pGL3-5.7mHK{alpha}2 promoter activity compared with vector-transfected controls (Fig. 3A). Serial deletion analysis of the 5'-flanking region of the HK{alpha}2 gene revealed that CREB inducibility was retained in a HK{alpha}2 promoter construct containing the proximal 100 bp of the promoter (pGL3-0.1mHK{alpha}2) (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 pGL3-0.1mHK{alpha}2, suggesting that constitutive CREB also plays a role in basal HK{alpha}2 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 pGL3-0.1mHK{alpha}2 luciferase activity about 16-fold, whereas expression of CREBdLZ-VP16, which lacks the CREB DNA-binding domain, abolished the constitutive activation of pGL3-0.1mHK{alpha}2, indicating that CREB binding to target cis elements in the proximal 100 bp of the HK{alpha}2 promoter mediates HK{alpha}2 gene trans-activation (Fig. 3B).



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Fig. 3. cAMP/Ca2+-responsive element (CRE)-binding protein (CREB)-1 trans-activates the HK{alpha}2 promoter. A: mIMCD-3 cells were transfected separately with a series of deletion mutants of the HK{alpha}2 5'-flanking region fused to the firefly 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 HK{alpha}2 5'-flanking region deletions are indicated numerically. Forty-eight hours after transfection, cell lysates were prepared and firefly and Renilla luciferase activities in lysates of the cells were assayed. Firefly luciferase activity was normalized to Renilla luciferase activity and reported as HK{alpha}2 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: pGL3-0.1mHK{alpha}2 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 firefly and Renilla luciferase activities in lysates of the cells were assayed. Firefly luciferase activity was normalized to Renilla luciferase activity. Values are means ± SE of 4 separate experiments. *P < 0.05 vs. CMV500-transfected controls.

 
The –86/–60 region of the HK{alpha}2 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 HK{alpha}2 promoter that had been implicated in the promoter-reporter gene studies. DNase I footprinting assay with recombinant GST-CREB-1 (Fig. 4), NF-{kappa}{beta} p50, or no protein added was performed with a radiolabeled PCR amplicon corresponding to nucleotides –148/–2 of the HK{alpha}2 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-{kappa}{beta} 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-{kappa}{beta} p50 did not bind to –94 to –104 as reported previously using a different binding buffer (37).



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Fig. 4. DNase I footprinting analysis of HK{alpha}2 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-{kappa}{beta} p50 as a negative control. A representative gel is shown (n = 4). A sequencing ladder was also generated for definition 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 HK{alpha}2 gene.

 
Binding of recombinant CREB-1 to the –86/–60 element was further investigated by performing EMSA using both a consensus CRE oligomer and the 0.1mHK{alpha}2 oligomer, which contains the –86/–60 sequence, as a template. As shown in Fig. 5A, GST-CREB-1 bound in a sequence-specific manner to both DNA templates in an identical pattern. GST alone produced no DNA-protein complexes. Incubation with anti-CREB1 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 significantly enhanced protein binding to the –86/–60 probe (Fig. 5B). This DNA-binding activity was sequence specific 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 elements in this region (86TGAGCTGC–79, –79CGTCG–75, and –65CGTGG–61) (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 anti-CREB-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.



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Fig. 5. CREB-1 binds the –86/–60 region of the HK{alpha}2 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 HK{alpha}2 gene (0.1mHK{alpha}2). Polyclonal antibody specific 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 (10–8 M), or forskolin (15 µM) and the 32P-labeled –86/–60 binding element oligomer (mHK{alpha}2–86/–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 specificity, reactions were also conducted in the presence of a 50-fold molar excess of unlabeled CREB-1 consensus oligomer, –86/–60 binding element oligomer (mHK{alpha}2–86/–60), –86/–60 oligomer harboring mutations in the CRE-like elements (mHK{alpha}2–86/–60 as in Fig. 1), or nonspecific 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 (mHK{alpha}2–86/–60), or a –86/–60 oligomer harboring mutations in the CRE-like elements (mHK{alpha}2{Delta}–86/–60). The autoradiograms are representative of 3 independent experiments performed in separate preparations of nuclear extracts.

 
The HK{alpha}2 –86/–60 region, but not the –104/–94 {kappa}{beta} element, participates in CREB-mediated trans-activation of the HK{alpha}2 promoter. To address the functional importance of the putative CREB binding site at –86/–60 in the CREB-mediated activation of the HK{alpha}2 promoter, we constructed pGL3-0.1mHK{alpha}2{Delta}–86/–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 CREB-VP16-stimulated promoter activity of pGL3-0.1mHK{alpha}2{Delta}–86/–60 was half that of wild-type pGL3-0.1mHK{alpha}2 (Fig. 6). Taken together, these data support the conclusion that CREB-1 binding to –86/–60 results in trans-activation of the HK{alpha}2 gene.



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Fig. 6. Mutations in the CRE-like sequences of the –86/–60 region limit the CREB-mediated induction of HK{alpha}2 proximal promoter activity. The pGL3-0.1mHK{alpha}2 reporter construct or pGL3-0.1mHK{alpha}2{Delta}–86/–60, which harbors mutations in the CRE-like elements of the HK{alpha}2 –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 firefly and Renilla luciferase activities in lysates of the cells were assayed. Firefly luciferase activity was normalized to Renilla luciferase activity. Values are means ± SE of 4 separate experiments. *P < 0.05 vs. pGL3-0.1mHK{alpha}2.

 
To confirm that the neighboring –104/–94 NF-{kappa}{beta} element does not participate in the CREB-mediated trans-activation of the HK{alpha}2 promoter, the CREB-VP16-stimulated promoter activity of pGL3-0.1mHK{alpha}2{Delta}{kappa}{beta}, which contains a mutated and functionally inactive –104/–94 NF-{kappa}{beta} element, was tested. As shown in Fig. 7, the CREB-VP16-stimulated promoter activity of pGL3-0.1mHK{alpha}2{Delta}{kappa}{beta} was comparable to that of the wild-type HK{alpha}2 promoter.



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Fig. 7. Mutations in the –104/–94 {kappa}{beta} element do not limit the CREB-mediated induction of HK{alpha}2 proximal promoter activity. The pGL3-0.1mHK{alpha}2 reporter construct or pGL3-0.1mHK{alpha}2{Delta}{kappa}{beta}, which harbors inactivating mutations in the –104/–94 {kappa}{beta} element of the HK{alpha}2 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 firefly and Renilla luciferase activities in lysates of the cells were assayed. Firefly luciferase activity was normalized to Renilla luciferase activity. Values are means ± SE of 4 separate experiments. *P < 0.05 vs. pGL3-0.1mHK{alpha}2.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Analysis of the regulation of active K+ reabsorption in the renal collecting duct and distal colon requires knowledge of transcription control mechanisms governing HK{alpha}2 gene expression. In this study, we have built on earlier work performed at our laboratory to define such control elements by demonstrating that CREB-1 trans-activates the HK{alpha}2 gene by binding to CRE-like elements in the –86/–60 region of the HK{alpha}2 proximal promoter. These results provide the first evidence for a specific 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 specifically 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 HK{alpha}2 promoter (Fig. 3A). The binding of proteins to the –86/–60 region was specific 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 anti-CREB-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 HK{alpha}2 gene has thus far revealed two functional elements: a {kappa}{beta} element at –104/–94 that binds NF-{kappa}{beta}, as well as the CRE-like elements at –86/–60 that bind CREB-1 reported here. The –104/–94 {kappa}{beta} element does not appear to participate in the CREB-mediated trans-activation of the HK{alpha}2 promoter, because an HK{alpha}2 promoter harboring a mutated –104/–94 {kappa}{beta} element exhibited trans-activation by CREB-VP16 comparable to that of the wild-type HK{alpha}2 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 HK{alpha}2 gene regulation remains unknown. The transcriptional coactivator CBP and closely related p300 are known to function as bridging factors between sequence-specific 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 HK{alpha}2 promoter activity because interaction and recruitment of HDAC-6 to NF-{kappa}{beta} bound to the HK{alpha}2 promoter inhibits HK{alpha}2 promoter activity (37). Thus it 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 HK{alpha}2 gene.

In addition to inducible effects, CREB-1 also trans-activates, to a much lesser degree, basal transcription of the HK{alpha}2 gene through the –86/–60 region. Overexpression of the dominant negative mutant A-CREB inhibited basal promoter activity of pGL3-0.1mHK{alpha}2 (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 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 specific 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 phosphorylation-independent manner (8). TORC recruitment appears to enhance the interaction of CREB with the polymerase complex without significantly altering CREB DNA-binding activity. Further studies are needed to define the specific mechanisms that underlie basal CREB-mediated HK{alpha}2 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 first 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: –86TGAGCTGC–79 is a 6/8 nucleotide match of the full palindrome, and –79CGTCG–75 and –65CGTGG–61 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 specific roles of HK{alpha}2 in the collecting duct in vivo are unclear. Because HK{alpha}2-null mice exhibit profound fecal rather 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 HK{alpha}2 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 HK{alpha}2-null than in the HK{alpha}2 wild-type mice during chronic K+ restriction, although this difference was not statistically significant. 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 specific physiological and pathophysiological roles for vasopressin-stimulated and CREB-mediated trans-activation of the HK{alpha}2 promoter in IMCD cells remain to be defined. It is known that chronic K+ deprivation, a strong stimulus for renal medullary HK{alpha}2 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 HK{alpha}2 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 HK{alpha}2-mediated 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 HK{alpha}2 expression and H+-K+-ATPase-mediated H+ secretion that would facilitate correction of the metabolic acidosis.

We would like to have tested whether enhanced expression of HK{alpha}2 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 indirectly inhibits all K+-ATPases (Na+-K+- as well as H+-K+-ATPase) (6). Because the method of distinguishing H+-K+-ATPase {alpha}1- and {alpha}2-subunits is based primarily on the Sch-28080 sensitivity of the former and the Sch-28080 insensitivity of the latter, we cannot specifically 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 HK{alpha}2 by vasopressin and cAMP/protein kinase A. The combination of rapid nongenomic effects (15) and delayed and sustained genomic effects provides the HK{alpha}2 gene with substantial versatility in meeting challenges to K+ and acid-base homeostasis.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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).


    ACKNOWLEDGMENTS
 
We thank Lei Zou and Hui-Wen Lo for technical help in gel shift and footprinting experiments.


    FOOTNOTES
 

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{at}uth.tmc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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