NF-kappa B inhibits transcription of the H+-K+-ATPase alpha 2-subunit gene: role of histone deacetylases

Wenzheng Zhang and Bruce C. Kone

Departments of Internal Medicine and of Integrative Biology, Pharmacology, and Physiology, The University of Texas Medical School at Houston, Houston, Texas 77030


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The H+-K+-ATPase alpha 2 (HKalpha 2) gene plays a central role in potassium homeostasis, yet little is known about its transcriptional control. We recently demonstrated that the proximal promoter confers basal transcriptional activity in mouse inner medullary collecting duct 3 cells. We sought to determine whether the kappa B DNA binding element at -104 to -94 influences basal HKalpha 2 gene transcription in these cells. Recombinant NF-kappa B p50 footprinted the region -116/-94 in vitro. Gel shift and supershift analysis revealed NF-kappa B p50- and p65-containing DNA-protein complexes in nuclear extracts of mouse inner medullary collecting duct 3 cells. A promoter-luciferase construct with a mutated -104/-94 NF-kappa B element exhibited higher activity than the wild-type promoter in transfection assays. Overexpression of NF-kappa B p50, p65, or their combination trans-repressed the HKalpha 2 promoter. The histone deacetylase (HDAC) inhibitor trichostatin A partially reversed NF-kappa B-mediated trans-repression of the HKalpha 2 promoter. HDAC6 overexpression inhibited HKalpha 2 promoter activity, and HDAC6 coimmunoprecipitated with NF-kappa B p50 and p65. These results suggest that HDAC6, recruited to the DNA protein complex, acts with NF-kappa B to suppress HKalpha 2 transcription and identify NF-kappa B p50 and p65 as novel binding partners for HDAC6.

kidney; colon; trichostatin A; promoter


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE H+-K+-ATPASE alpha 2-SUBUNIT (HKalpha 2) is a member of the X+-K+-ATPase multigene family, which includes the gastric H+-K+-ATPase and the Na+-K+-ATPase isoforms. The X+-K+-ATPases share homologous structures, common catalytic mechanisms, and a requirement for heterodimeric (alpha /beta ) assembly (22). HKalpha 2 is principally expressed in distal colon and the renal collecting duct, in which it plays a critical role in potassium and acid-base homeostasis. Mice with targeted ablation of the HKalpha 2 gene develop fecal K+ wasting and profound hypokalemia during potassium (27) and sodium restriction (34). HKalpha 2 also appears to contribute to bicarbonate absorption by the kidney (29) and distal colon (27) and increased ammonium secretion in the inner medullary collecting duct (IMCD) during chronic hypokalemia (37). Recent studies also suggest that HKalpha 2, possibly functioning as an Na+-K+ exchanger, participates in the chronic adaptation to altered sodium and aldosterone balance (32).

The HKalpha 2 gene is differentially expressed in kidney and distal colon under basal conditions and in response to chronic potassium or sodium deprivation. Under basal conditions, the gene is robustly expressed in distal colon, but only weakly expressed in the renal medulla. After chronic K+ deprivation, however, HKalpha 2 gene expression is upregulated in the rat and mouse kidney outer medulla but not in distal colon (1, 32). In contrast, chronic sodium deprivation upregulates HKalpha 2 expression in distal colon but not in the renal collecting duct (32).

We recently isolated and completely sequenced the cDNA and structural gene encoding the mouse HKalpha 2 gene and localized it to mouse chromosome 14C3 (41). Using deletion analysis of promoter-reporter gene constructs, we demonstrated functional activity of the HKalpha 2 promoter in cultured renal collecting duct cells and found that the proximal 177 bp of the promoter appear to be essential for collecting duct-selective expression. This proximal promoter region contains several consensus sequences for transcription factors. Among these is an NF-kappa B site -104 GGGGCGTCCCC -94. The mammalian NF-kappa B/Rel family comprises five known members: p50, p52, p65, c-Rel, and RelB. NF-kappa B subunits form homo- or heterodimers through the Rel homology domain, forming transcription factor complexes that exert a broad range of DNA-binding and -activation potentials (4). The protein is bound in the cytoplasm with members of the inhibitor of kappa B (Ikappa B) family, which prevents phosphorylation of the active unit and its translocation to the nucleus. A variety of stimuli and signaling events leads to activation of NF-kappa B, and there is evidence that NF-kappa B can transport between cytoplasm and nucleus even in unstimulated cells. In several tissues, including kidney, NF-kappa B DNA-binding activity is evident under basal conditions. It has been suggested that NF-kappa B in these settings suppresses or activates basal gene expression in these cells (10, 18). In this context, it has been recently shown that NF-kappa B can suppress target gene transcription by means of interactions of p65 with histone deacetylase (HDAC)1 and HDAC2 corepressor proteins (3). Indeed, interaction with other transcription factors and accessory proteins appears to lend NF-kappa B versatility and specificity in mediating transcriptional responses.

Histone acetylation is a dynamic process regulated by the activities of two histone-modifying enzymes, histone acetyltransferase(s) and HDACs. Present models indicate that HDACs are recruited to target sequences through protein-protein interactions (23). HDAC activity results in histone hypoacetylation, chromatin condensation, and, generally, transcriptional repression (23). HDACs are typically found as components of large corepressor complexes. They affect transcriptional activity not only by modifying chromatin structure but also by deacetylating transcription factors and altering their transcriptional competency. For example, HDAC1 and HDAC2 have been shown to repress transcription through direct interaction with transcription factors. HDAC1 directly interacts with MyoD to silence MyoD-dependent transcription of p21 (25). HDAC2 interacts with the YY1 transcription factors, converting them from activators to repressors (39). Deacetylation of RelA by HDAC3 serves as a molecular switch within the nucleus that controls the NF-kappa B transcriptional response (11).

In this report, we examined the role of basal NF-kappa B expression on HKalpha 2 promoter activity in renal medullary collecting duct cells. We demonstrate that NF-kappa B acting through the -104/-94 element significantly suppresses transcriptional activity of the HKalpha 2 gene and that this inhibition involves recruitment of HDAC6 to the NF-kappa B activation complex through protein-protein interactions. These results provide the first evidence for regulatory components of HKalpha 2 gene transcription and identify novel interactions of NF-kappa B p65 and p50 with specific HDAC proteins.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture and reagents. Mouse IMCD-3 (mIMCD-3) cells, an immortalized cell line derived from mIMCD (31), were cultured in DMEM supplemented with 10% FBS at 37°C in a 5% CO2 environment. Polyclonal antibodies recognizing NF-kappa B p65, p52, p50, c-Rel, and STAT3 were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-FLAG M2 antibody was from Sigma. Oligonucleotides were custom synthesized by Genosys (The Woodlands, TX). Lipofectamine 2000 reagent was from Invitrogen (Carlsbad, CA). The Dual-Luciferase Reporter Assay System and the luciferase vectors pGL3-Basic and pRL-SV40, recombinant human NF-kappa B p50 (rhNF-kappa B), and activator protein-2 (AP-2) nuclear extract were from Promega (Madison, WI). The BCA protein estimation kit was from Pierce Chemical. Enhanced chemiluminescence reagents were from Amersham Pharmacia Biotech (Piscataway, NJ).

Plasmids and constructs. pGL3-0.48MHKalpha 2, which contains a fragment spanning from -477 to +253 of the murine HKalpha 2 gene upstream of the luciferase gene, has been previously characterized (41). pGL3-0.48MHKalpha 2mut, which harbors a mutated NF-kappa B site at the -104 to -94 position (TAGCCGTCCCC; mutated bases underlined), was created by PCR overlap extension. pCMV500, a mammalian expression vector containing the cytomegalovirus promoter, NF-kappa B expression plasmids pRSV-p65 and dominant-negative pCMV-Ikappa Balpha Delta 1-36 (8), and the NF-kappa B reporter construct p36B(-)(NF-kappa B)3-luc, which contains three tandem copies of the kappa B binding element (GGGGACTCTCCC) upstream of the prolactin promoter sequence and fused to the coding sequence for the luciferase gene (12), were gifts from Dr. Bharrat Agarwal (The University of Texas M. D. Anderson Cancer Center). pRSV-p50, which encodes NF-kappa B p50, was kindly provided by Dr. Warren S.-L. Liao (The University of Texas M. D. Anderson Cancer Center). The FLAG-tagged HDAC2 expression plasmid pME18S-FLAG-HDAC2 (38) was from Dr. Edward Seto (University of South Florida). FLAG-tagged mammalian expression plasmids pBJ-HDAC1, pBJ-HDAC4, pBJ-HDAC5, and pBJ-HDAC6 (13) were from Dr. S. L. Schreiber (Harvard University).

In vitro DNase I footprinting. DNase I footprinting analyses were performed with the Core Footprinting System (Promega), according to the manufacturer's instructions. A PCR fragment corresponding to -476 to +82 of the native murine HKalpha 2 5'-flanking region was obtained by using two primers: WZ162 (5'-ATCGAGACGCGTATAGATTCCCCGCCCCACCCTCATTTACAC-3', sense, MluI site attached for cloning) and 32P-labeled or unlabeled oligo 3 (5'-GTCCGGGTCCCTGAGTGGTGA-3', antisense). This fragment was used as the DNA template for footprinting, and the unlabeled fragment also was sequenced with oligo 3 as a marker (T7 Sequenase, version 2.0, DNA sequence kit, Amersham Pharmacia Biotech). The transcription factors examined were NF-kappa B p50 and, as a control, AP-2. For the NF-kappa B experiment, the same final binding buffer was used as described (21). Briefly, the labeled template DNA was incubated in a final volume of 50 µl with or without 2 µl of recombinant human NF-kappa B in the final buffer containing 10 mM HEPES (pH 7.9), 0.2 mM EDTA, 50 mM KCl, 2.5 mM dithiothreitol, 10% glycerol, and 0.05% Nonidet P-40 for 10 min on ice. After addition of 50 µl Ca2+/Mg2+ solution (5 mM CaCl2, 10 mM MgCl2), 3 µl of DNase I was used for digestion for 1 min at room temperature. The reaction was terminated by adding 90 µl of stop solution (200 mM NaCl, 30 mM EDTA, 1% SDS, 100 µg/ml yeast RNA). Samples were extracted with phenol/chloroform/isoamyl alcohol, precipitated, resuspended in sequencing loading buffer, and separated on 8% sequencing gels. Bands were visualized by autoradiography.

EMSAs and supershift assays. Nuclear extracts were prepared from mIMCD-3 cells as detailed in earlier work from our laboratory (15). Double-stranded oligonucleotides were generated corresponding to nucleotides -109 to -90 containing the kappa B element (sense strand: wild-type, 5'-CCCCAGGGGCGTCCCCAGTG -3', kappa B binding element underlined) and a mutated kappa B element (5'-CCCCA<UNL>TA</UNL>G<UNL>C</UNL>CGTCCCCAGTG-3', mutations double underlined) of the native murine HKalpha 2 promoter and were end-labeled with [gamma -32P]ATP (3,000 Ci/mmol) using T4 polynucleotide kinase. Nuclear extract proteins (12 µg) were preincubated with or without 4 µg antibodies specific for NF-kappa B p65, p50, p52, c-Rel, or HDAC6 in the final 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)] at 4°C for 1 h or overnight. Binding reactions were performed for 30 min at room temperature by adding 1.75 pmol of duplex DNA probe (~2 × 105 cpm) in the presence or absence of a 10-fold molar excess of nonradiolabeled competitor oligonucleotides. The final reaction volume was adjusted to 20 µl. Aliquots of the reactions were resolved on 4% native polyacrylamide gels in 0.5× 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 on a new nuclear extract preparation. Experiments were replicated a minimum of three times, as indicated in the figure legends.

Transient transfection and reporter gene assays. mIMCD-3 cells grown in 24-well plates were transiently transfected using the LipofectAMINE 2000 Reagent (Life Technologies) as detailed in our laboratory's previous work (41). For comparative purposes, the cells were cotransfected with the Renilla luciferase expression plasmid pRL-SV40 (10 ng/well) to control for transfection efficiency and other assay-to-assay variability. Trans-repression/trans-activation experiments used 0.8 µg of pGL3-0.48MHKalpha 2, pGL3-0.48MHKalpha 2mut, or p36B(-)(NF-kappa B)3-luc and 0.2 µg of pRSV-p65, pRSV-p50, dominant-negative pCMV-Ikappa Balpha Delta 1-36 or insertless expression vector, or the FLAG-tagged expression vectors pBJ-HDAC1, pME18S-FLAG-HDAC2, pBJ-HDAC4, pBJ-HDAC5, or pBJ-HDAC6. In the cotransfection experiments with pRSV-p65 and pRSV-p50 together, 0.1 µg of each plasmid was used. Twenty-four 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 (Promega) following the manufacturer's protocol. Firefly luciferase activity was normalized for Renilla luciferase activity in the lysates. The results were recorded as "normalized MHKalpha 2 promoter activity." In some experiments, the cells were treated after transfection with vehicle or trichostatin A (TSA; 100 nM) for 16 h, after which cell lysates were prepared and luciferase assays performed. As TSA treatment also dramatically affects expression of Renilla luciferase expression plasmid pRL-SV40, firefly luciferase activities in these experiments were normalized to the total lysate protein used in the assay. Each experimental observation represents the mean of results from at least three independent transfections. HDAC isoform overexpression was confirmed by analyzing immunoblots of cell lysates prepared from the transfected cells with antibodies against the specific HDAC isoform and with the anti-FLAG M2 antibody. Roughly comparable levels of overexpression were observed for HDAC1, HDAC2, HDAC4, HDAC5, and HDAC6 (not shown).

Coimmunoprecipitation. mIMCD-3 were harvested, lysed in JLB buffer (50 mM Tris · HCl, pH 8, 150 mM NaCl, 10% glycerol, 0.5% Triton X-100) containing 1 mM PMSF, and 3% protease inhibitor cocktail, and precleared by incubating with 20 µl/ml protein A/G-agarose beads (Santa Cruz) for 1 h at 4°C. After briefly spinning down, the supernatant was added with anti-FLAG M2, anti-NF-kappa B p65, or anti-NF-kappa B p50 antibodies overnight at 4°C, followed by the addition of 20 µl of protein A/G-agarose beads. Immunoprecipitates were washed four times in JLB buffer, resuspended in SDS sample buffer, boiled for 5 min, and analyzed on 4-15% linear gradient Tris · HCl gels (Bio-Rad). Proteins were electrophoretically transferred to polyvinylidene difluoride membranes and incubated with anti-FLAG M2 overnight, followed by extensive washes and incubation with secondary antibody conjugated to horseradish peroxidase. Antigen-antibody complexes were detected with enhanced chemiluminescence reagent (ECL, Amersham).

Data analysis. Potential regulatory motifs in the HKalpha 2 gene were identified with TESS software (http://www.cbil.upenn.edu/tess). Quantitative data were analyzed for significance using t-test, two-sample assuming equal variances, with significance assigned at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NF-kappa B p50 footprints the HKalpha 2 gene in vitro at -116 to -94. The proximal promoter of the HKalpha 2 gene was initially analyzed by TESS software. A putative NF-kappa B and an overlapping AP-2 binding site were identified at -104 to -93, with the NF-kappa B site ranging from -104 to -94 and the AP-2 site from -99 to -93. To determine the significance of these sites, DNase I footprinting assay with recombinant NF-kappa B p50 (Fig. 1) and AP-2 was performed. Protected or hypersensitive sites in the region -116/-94 were detected in the binding reactions that included NF-kappa B p50, when compared with the reaction in which p50 was omitted. The antisense of this protected region reads as 5'-GGGGACGCCCCTGGGGTAGGAAA-3', with the NF-kappa B site underlined. In addition, no footprint was observed with recombinant AP-2 under the condition tested (data not shown).


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Fig. 1.   DNase I footprinting analysis of H+-K+-ATPase alpha 2 (HKalpha 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 NF-kappa B p50 protein. A sequencing ladder was also generated for definition of footprinted or hypersensitive sites. Filled bar, region with hypersensitive sites produced by NF-kappa B p50 protein; -104, -94, nucleotide positions relative to the transcription start site of the HKalpha 2 gene.

-109/-90 Sequence binds NF-kappa B p65 and p50. Binding of NF-kappa B to the -104/-94 element was further investigated by gel shift assays with mIMCD-3 cell nuclear extracts and the radiolabeled -109/-90 sequence containing the kappa B element as probes. Sequence-specific DNA-protein complexes were detected (Fig. 2A). Supershift assays demonstrated that antibodies to NF-kappa B p65 and p50 supershifted the DNA-protein complex (Fig. 2B). In contrast, the mobility of the DNA-protein complex in the presence of antibodies to p52, c-Rel, or STAT3 (as a negative control) was not altered. Furthermore, mutation of these sites dramatically decreased, although did not completely prevent, NF-kappa B binding to this site (data not shown). These data are consistent with the conclusion that NF-kappa B p50/p65 heterodimers are basally expressed in the nuclei of mIMCD-3 cells.


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Fig. 2.   NF-kappa B p50 and p65 bind the -104/-94 kappa B element of HKalpha 2 proximal promoter. A: nuclear proteins extracted from mouse inner medullary collecting duct 3 (mIMCD-3) cells were subjected to EMSA with a 32P-labeled oligomer containing the -104/-94 kappa B binding element of the HKalpha 2 gene (S*). To demonstrate binding specificity, reactions were also conducted in the presence of a 10-fold molar excess of unlabeled -104/-94 kappa B binding element oligomer (S) or nonspecific (NS) oligomers (the corresponding, mutated version of -104/-94 kappa B binding element oligomer). The autoradiogram is representative of 3 independent experiments performed on separate preparations of nuclear extracts. B: polyclonal IgGs specific for NF-kappa B p50, p52, p65, c-Rel, and STAT3 (as a negative control) were used in supershift experiments with nuclear extracts from mIMCD-3 cells and the 32P-labeled -104/-94 kappa B binding element oligomer. The autoradiograms are representative of 3 independent experiments performed on separate preparations of nuclear extracts. SS, S, supershifted and shifted complexes, respectively; no Ab, no antibody.

NF-kappa B p65 and p50 trans-repress the HKalpha 2 promoter. To determine the functional effect of NF-kappa B on HKalpha 2 promoter activity, trans-activation/trans-repression assays were performed in mIMCD-3 cells. Overexpression of NF-kappa B p65 or p50 resulted in ~22 and ~30%, respectively, lower rates of HKalpha 2 promoter activity compared with vector-transfected controls (Fig. 3A). Overexpression of NF-kappa B p65 and p50 together resulted in a dramatic, synergistic inhibition (~87%) of HKalpha 2 promoter activity (Fig. 3A). In concert with these findings, overexpression of an Ikappa Balpha mutant designed to limit NF-kappa B translocation to the nucleus resulted in a 40% increase in HKalpha 2 promoter activity (Fig. 3A). Importantly, overexpression of NF-kappa B p65 or p50 in these same cells strongly activated activity of the NF-kappa B reporter plasmid p36B(-)(NF-kappa B)3-luc (Fig. 3B), indicating that the classic gene activation by NF-kappa B signaling under these conditions was intact and that the observed inhibitory effects on HKalpha 2 promoter activity likely reflected the influence of the promoter context and/or coregulatory molecules.


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Fig. 3.   NF-kappa B p50 and p65 trans-repress the HKalpha 2 promoter. A: mIMCD-3 cells were transfected with the pGL3-0.48MHKalpha 2 reporter construct (Wild-Type) or pGL3-0.48MHKalpha 2mut (Mutant), which harbors a mutation of the -104/-94 kappa B binding element, and the Renilla luciferase expression plasmid pRL-SV40 in the presence of the expression vector for NF-kappa B p50 (pRSV-p50), p65 (pRSV-p65), p50+p65 (pRSV-p50+pRSV-p65), an Ikappa Balpha dominant-negative mutant (pCMV-Ikappa Balpha Delta 1-36), or an insertless mammalian expression vector containing the cytomegalovirus promoter (pCMV-500). Twenty-four 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. B: identical methods as in A, except that an NF-kappa B consensus element reporter construct p36B(-)(NF-kappa B)3-luc and its parent plasmid p36-luc were used instead of pGL3-0.48MHKalpha 2 plasmid and pCMV500, respectively. Values are means + SE of 4 separate experiments. *P < 0.05.

To address the functional importance of the NF-kappa B binding site at -104/-94 in the NF-kappa B-mediated repression of the HKalpha 2 promoter, we constructed pGL3-0.48MHKalpha 2mut, which harbors the same mutation in the NF-kappa B binding site as that generated for the mutant probes used in the gel shift assay. When compared with expression of pGL3-0.48MHKalpha 2, expression of pGL3-0.48MHKalpha 2mut was significantly augmented in all cases examined (Fig. 3A). In the control cells transfected with pCMV500 vector, the mutation augmented expression by ~110%, suggesting that the binding of NF-kappa B to this site is important for the endogenous NF-kappa B-mediated repression of the HKalpha 2 promoter. Similarly, the repression resulting from overexpression of p65, p50, or both was at least partially relieved by the mutation, resulting in higher level expression of the HKalpha 2 promoter-reporter (Fig. 3A). HKalpha 2 promoter-reporter activity was greatest when nuclear expression of NF-kappa B was limited by overexpressing the Ikappa Balpha mutant and the HKalpha 2 promoter harboring the mutated kappa B element (Fig. 3A). Taken together, these data support the conclusion that NF-kappa B binding to -104/-94 results in downregulation of activity of the HKalpha 2 promoter. It is interesting to note that the HKalpha 2 promoter harboring the mutated kappa B element, while exhibiting higher activities compared with the wild-type promoter when p65, p50, p65/p50, or the Ikappa Balpha mutant was overexpressed, was still partially inhibited by p65 and p65/p50, but not p50, and augmented by the Ikappa Balpha mutant. This result probably reflects, in part, the fact that the mutation did not completely abolish NF-kappa B binding on gel shift assays and the likelihood that NF-kappa B may also mediate repression through other mechanisms independent of its binding to -104/-94.

TSA augments and HDAC6 overexpression inhibits HKalpha 2 promoter activity. NF-kappa B is known to interact with HDAC1, HDAC2, and HDAC3 (3, 11, 19) in other cell types and promoter contexts (3), and HDACs are known to act as corepressors in some instances. To determine whether the state of histone acetylation influences the ability of NF-kappa B to trans-repress the HKalpha 2 promoter in mIMCD-3 cells, HKalpha 2 promoter-luciferase activity was measured in the presence and absence of the potent and specific HDAC inhibitor TSA. TSA treatment promoted a 2.5-fold increase in basal HKalpha 2 promoter activity (Fig. 4A). As shown in Table 1, TSA treatment resulted in significantly greater activity of the NF-kappa B consensus element reporter in the vector-transfected controls c-transfected with either the wild-type HKalpha 2 promoter (pGL3-0.48HKalpha 2) or the HKalpha 2 promoter harboring the mutation in the NF-kappa B site (pGL3-0.48HKalpha 2mut). However, p65, p50, and p65+p50 had less ability to inhibit pGL3-0.48HKalpha 2mut promoter activity compared with the wild-type pGL3-0.48HKalpha 2 construct in the presence of TSA, indicating that TSA partially reversed the NF-kappa B-dependent trans-repression of the HKalpha 2 promoter (Table 1). These findings are consistent with the involvement of an HDAC in the process. In accordance with these findings, overexpression of HDAC6 inhibited HKalpha 2 promoter activity (Fig. 5A). Interestingly, the inhibitory effect appeared to be specific for HDAC6, because overexpression of HDAC1, HDAC3, HDAC4, and HDAC5 had no appreciable effect on HKalpha 2 promoter activity (Fig. 5A), despite roughly comparable levels of expression (not shown).


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Fig. 4.   Trichostatin A (TSA) partially relieves NF-kappa B-mediated trans-repression of the HKalpha 2 promoter. A: mIMCD-3 cells were transfected with the pGL3-0.48MHKalpha 2 reporter construct. Twenty-four hours later, the cells were treated with vehicle or 100 nM TSA for 16 h, after which cell lysates were prepared and luciferase activities measured. Values are means ± SE; n = 4. *P < 0.05.


                              
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Table 1.   Effects of trichostatin A on activity of the wild-type H+-K+-ATPase alpha 2-promoter and promoter harboring a mutation in the -104/-90 NF -kappa B element



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Fig. 5.   Histone deacetylase (HDAC) 6 overexpression inhibits activity of the HKalpha 2 promoter. mIMCD-3 cells were transfected with the pGL3-0.48MHKalpha 2 reporter construct and a Renilla expression plasmid together with expression plasmids for HDAC1, HDAC2, HDAC4, HDAC5, or HDAC6 or an insertless vector containing the CMV promoter (pCMV-500). Cell lysates were prepared and luciferase activities measured. Values are means ± SE; n = 4. *P < 0.05.

TSA treatment also accentuated basal activity of the NF-kappa B reporter plasmid p36B(-)(NF-kappa B)3-luc (Table 2). However, in contrast to its effects on NF-kappa B-dependent trans-repression of the HKalpha 2 promoter, TSA did not affect the ability of overexpressed p65, p50, or p65+p50 to trans-activate the NF-kappa B reporter plasmid p36B(-)(NF-kappa B)3-luc (Table 2). This result suggests that promoter context is important for the functional effects of HDAC activity on transcription.

                              
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Table 2.   Effects of TSA on ability of NF-kappa B proteins to trans-activate NF-kappa B consensus element reporter construct p36B(-)NF-kappa B)3-luc

HDAC6 and NF-kappa B interact in mIMCD-3 cells in vivo. EMSA and supershift studies using the -104/-94 kappa B element as probe, nuclear extracts from mIMCD-3 cells, and anti-HDAC6 antibodies or IgG (as a negative control) demonstrated that anti-HDAC6 partially supershifted the kappa B-specific DNA-protein complex (Fig. 6). In agreement with these results and those of the HKalpha 2 promoter assays, coimmunoprecipitation experiments demonstrated interaction of HDAC6 and NF-kappa B proteins (Fig. 7). HDAC6 was overexpressed as a FLAG-tagged construct in mIMCD-3 cells, and the cells were then subjected to coimmunoprecipitation with antibodies to NF-kappa B p50, NF-kappa B p65, the FLAG epitope (as a positive control), or IgG (as a negative control), followed by blotting with the anti-FLAG M2 antibody. As seen in Fig. 7, both NF-kappa B p50 and p65 coprecipitated with HDAC6.


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Fig. 6.   HDAC6 contributes to the -104/-94 kappa B element-protein complex of the HKalpha 2 proximal promoter. Supershift experiments with nuclear extracts from mIMCD-3 cells and the 32P-labeled -104/-94 kappa B binding element oligomer were performed in the presence and absence of polyclonal antibodies against HDAC6 or with nonimmune IgG. The autoradiograms are representative of 3 independent experiments performed on separate preparations of nuclear extracts.



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Fig. 7.   HDAC6 interacts with NF-kappa B p65. HDAC6 was overexpressed as a FLAG-tagged protein in mIMCD-3 cells, cell extracts were prepared and immunoprecipitated (IP) with polyclonal antibodies directed against the FLAG epitope, NF-kappa B p50, NF-kappa B p65, or IgG, separated by SDS-PAGE, and immunoblotted with anti-FLAG M2 antibody. Data are representative of 3 independent experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we demonstrated that NF-kappa B p65 and p50 DNA binding activities are basally expressed in cultured renal collecting duct cells (Fig. 2) and that these transcription factors serve to trans-repress the HKalpha 2 gene by binding to a kappa B element in the proximal promoter region of this gene (Fig. 3A). The fact that blockade of HDAC activity with TSA relieves NF-kappa B-mediated trans-repression of the HKalpha 2 gene (Fig. 4) and that HDAC6 overexpression inhibits HKalpha 2 promoter activity (Fig. 5) indicates that the state of histone or NF-kappa B acetylation strongly influences HKalpha 2 promoter activity. We further demonstrate that NF-kappa B p50 and p65 interact with HDAC6 in supershift experiments with the -104/-94 kappa B element (Fig. 6) and in coimmunoprecipitation assays (Fig. 7). These results provide the first data concerning transcriptional regulation of the HKalpha 2 gene, give a novel example of the ability of NF-kappa B to suppress basal gene expression, and identify for the first time HDAC6 as a binding partner with NF-kappa B p50 and p65. However, it remains to be defined whether p50 and p65 are subject to acetylation/deacetylation and whether HDAC6 is directly involved in this process.

The fact that the -104/-94 region was footprinted in vitro by NF-kappa B p50 (Fig. 1) and that mutation of the kappa B element partially relieved repression of HKalpha 2 promoter activity (Fig. 3A) suggests that much of the effect of NF-kappa B on the promoter was direct and mediated by DNA binding. However, because the effect was only partial, and because overexpression of NF-kappa B p65 or p65/p50, but not p50 alone, still inhibited the mutant HKalpha 2 promoter (Fig. 3A), other undefined mechanisms are apparently involved. The kappa B element rests 53-bp upstream of the TATA box and is flanked by an AP-2 site. However, AP-2 did not footprint the region in vitro (Fig. 1). Because the combination of p50 and p65 overexpression synergistically suppressed HKalpha 2 promoter activity (Fig. 3A) and because these NF-kappa B proteins contributed to the kappa B-specific DNA-protein complexes in supershift assays (Fig. 2B), we conclude that p50/p65 heterodimers are likely the dominant NF-kappa B species binding the HKalpha 2 promoter and the most effective in trans-repressing the HKalpha 2 gene.

NF-kappa B is subject to complex control. The principal mode of regulation has long been considered to be the retention of NF-kappa B in the cytoplasm by association with Ikappa B proteins and its release for translocation to the nucleus on Ikappa B phosphorylation and degradation (4). This regulatory pathway appears to be operative at least to a degree in our study, because overexpression of an Ikappa Balpha dominant-negative mutant augmented basal HKalpha 2 promoter activity (Fig. 3A). In addition, NF-kappa B has been shown to shuttle between cytoplasm and nucleus under basal conditions. Chen et al. (11) showed that deacetylation of RelA by interaction with HDAC3 promotes effective binding to Ikappa B and results in Ikappa Balpha -dependent nuclear export of the complex. Ashburner et al. (3) established that NF-kappa B p65 in the nucleus of unstimulated cells interacts with HDAC1 and HDAC2 to suppress basal gene expression. The glucocorticoid receptor has also been shown to recruit HDAC2 to the p65-cAMP response element binding protein (CBP) histone acetyltransferase complex to inhibit IL-1beta -induced gene expression (19). NF-kappa B has been found in the nucleus of unstimulated cells, in which it is theorized to repress and/or activate basal gene expression (10). The differential association of NF-kappa B with coregulatory proteins may regulate this function. CBP and p300 coactivators interact with p65 to enhance its ability to activate transcription (4). The histone acetyltransferase function of the p300/CBP-associated factor coactivator (9) and the steroid receptor coactivator-1 (28) were shown to interact with p50 to potentiate NF-kappa B-mediated trans-activation.

Histone acetylation and deacetylation play essential roles in modifying chromatin structure and regulating expression of eukaryotic genes. HDACs are part of transcriptional corepressor complexes. HDAC interacts with NcoR (nuclear corepressor) and SMRT (silencing mediator of receptor transcription) to mediate nuclear receptor repression, as well as with the Mad-Max complex to confer transcriptional repression (2, 16, 17). In our study, TSA treatment increased HKalpha 2 promoter activity, consistent with the model that TSA blocks activity of HDAC, resulting in hyperacetylation of histones and, consequently, a higher level of gene expression (40). Consistent with this, overexpression of HDAC6 inhibited HKalpha 2 promoter activity (Fig. 6). We do not know whether TSA promotes dissociation of HDAC from p65 or p50, facilitating the binding of CBP or other coactivators.

Acetylation regulates transcription factors other than NF-kappa B, including p53 (6), GATA-1 (7), and MyoD (25), and it alters transcription factor function in several ways, including altering protein-protein interactions (5), affecting conformation (33), and altering half-life (26). Direct interaction with HDACs has only been demonstrated for a few transcription factors. Retinoblastoma protein (24) and DNA topoisomerase II (36) interact directly with HDAC1, SP1 (20) and YY1 (35) interact with HDAC2, GATA-2 couples with HDAC3 and HDAC5 (30), and myocyte enhancer factor 2 was shown to interact directly with HDAC4 and HDAC5 (14). Our finding of an interaction between HDAC6 and NF-kappa B p50 and p65 adds to this list. Why specific HDAC isoforms are active in some cell types but not others likely relates to the specific promoter context and other coregulatory molecules. Nonetheless, this protein-protein interaction could lend cell specificity and versatility to the regulatory response. In addition, the ability of NF-kappa B to function as both activator and repressor in mIMCD-3 cells indicates that this transcription factor may exist in different complexes with different acetylation/deacetylation patterns, resulting in differential DNA binding specificities and regulation of expression of its target genes.


    ACKNOWLEDGEMENTS

The authors thank Dr. Bharrat Agarwal, The University of Texas M.D. Anderson Cancer Center, for the gift of the NF-kappa B and mutant Ikappa Balpha expression plasmids, Dr. Warren S.-L. Liao, The University of Texas M.D. Anderson Cancer Center, for the gift of the NF-kappa B expression plasmid, Dr. Edward Seto, University of South Florida, for the FLAG-tagged HDAC2 expression plasmid pME18S-FLAG-HDAC2, and Dr. S. L. Schreiber, Harvard University, for the FLAG-tagged expression plasmids pBJ-HDAC1, pBJ-HDAC4, pBJ-HDAC5, and pBJ-HDAC6. The authors also thank Dr. Jun Chen and Sandra Higham for technical assistance.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-47981.

Address for reprint requests and other correspondence: B. C. Kone, Depts. of Internal Medicine and of Integrative Biology, Pharmacology, and Physiology, Univ. of Texas Medical School at Houston, 6431 Fannin, MSB 4.138, 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.

July 9, 2002;10.1152/ajprenal.00156.2002

Received 22 April 2002; accepted in final form 19 June 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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