Activator Protein-1 Contributes to High NaCl-induced Increase in Tonicity-responsive Enhancer/Osmotic Response Element-binding Protein Transactivating Activity
2007; Elsevier BV; Volume: 283; Issue: 5 Linguagem: Inglês
10.1074/jbc.m703490200
ISSN1083-351X
AutoresCarlos E. Irarrázabal, Chester K. Williams, Megan Annette Ely, Michael J. Birrer, Arlyn García-Pérez, Maurice B. Burg, Joan D. Ferraris,
Tópico(s)Genomics, phytochemicals, and oxidative stress
ResumoTonicity-responsive enhancer/osmotic response element-binding protein (TonEBP/OREBP) is a Rel protein that activates transcription of osmoprotective genes at high extracellular NaCl. Other Rel proteins NFAT1–4 and NF-κB complex with activator protein-1 (AP-1) to transactivate target genes through interaction at composite NFAT/NF-κB·AP-1 sites. TonEBP/OREBP target genes commonly have one or more conserved AP-1 binding sites near TonEBP/OREBP cognate elements (OREs). Also, TonEBP/OREBP and the AP-1 proteins c-Fos and c-Jun are all activated by high NaCl. We now find, using an ORE·AP-1 reporter from the target aldose reductase gene or the same reporter with a mutated AP-1 site, that upon stimulation by high extracellular NaCl, 1) the presence of a wild type, but not a mutated, AP-1 site contributes to TonEBP/OREBP-dependent transcription and 2) AP-1 dominant negative constructs inhibit TonEBP/OREBP-dependent transcription provided the AP-1 site is not mutated. Using supershifts and an ORE·AP-1 probe, we find c-Fos and c-Jun present in combination with TonEBP/OREBP. Also, c-Fos and c-Jun coimmunoprecipitate with TonEBP/OREBP, indicating physical association. Small interfering RNA knockdown of either c-Fos or c-Jun inhibits high NaCl-induced increase of mRNA abundance of the TonEBP/OREBP target genes AR and BGT1. Furthermore, a dominant negative AP-1 also reduces high NaCl-induced increase of TonEBP/OREBP transactivating activity. Inhibition of p38, which is known to stimulate TonEBP/OREBP transcriptional activity, reduces high NaCl-dependent transcription of an ORE·AP-1 reporter only if the AP-1 site is intact. Thus, AP-1 is part of the TonEBP/OREBP enhanceosome, and its role in high NaCl-induced activation of TonEBP/OREBP may require p38 activity. Tonicity-responsive enhancer/osmotic response element-binding protein (TonEBP/OREBP) is a Rel protein that activates transcription of osmoprotective genes at high extracellular NaCl. Other Rel proteins NFAT1–4 and NF-κB complex with activator protein-1 (AP-1) to transactivate target genes through interaction at composite NFAT/NF-κB·AP-1 sites. TonEBP/OREBP target genes commonly have one or more conserved AP-1 binding sites near TonEBP/OREBP cognate elements (OREs). Also, TonEBP/OREBP and the AP-1 proteins c-Fos and c-Jun are all activated by high NaCl. We now find, using an ORE·AP-1 reporter from the target aldose reductase gene or the same reporter with a mutated AP-1 site, that upon stimulation by high extracellular NaCl, 1) the presence of a wild type, but not a mutated, AP-1 site contributes to TonEBP/OREBP-dependent transcription and 2) AP-1 dominant negative constructs inhibit TonEBP/OREBP-dependent transcription provided the AP-1 site is not mutated. Using supershifts and an ORE·AP-1 probe, we find c-Fos and c-Jun present in combination with TonEBP/OREBP. Also, c-Fos and c-Jun coimmunoprecipitate with TonEBP/OREBP, indicating physical association. Small interfering RNA knockdown of either c-Fos or c-Jun inhibits high NaCl-induced increase of mRNA abundance of the TonEBP/OREBP target genes AR and BGT1. Furthermore, a dominant negative AP-1 also reduces high NaCl-induced increase of TonEBP/OREBP transactivating activity. Inhibition of p38, which is known to stimulate TonEBP/OREBP transcriptional activity, reduces high NaCl-dependent transcription of an ORE·AP-1 reporter only if the AP-1 site is intact. Thus, AP-1 is part of the TonEBP/OREBP enhanceosome, and its role in high NaCl-induced activation of TonEBP/OREBP may require p38 activity. Among Rel-proteins, TonEBP/OREBP activates target genes that are osmoprotective at elevated extracellular NaCl. Transcriptional targets of TonEBP/OREBP include aldose reductase (AR), 2The abbreviations used are: AP-1activator protein-1ARaldose reductaseARRE-2antigen receptor-response element-2BGT1betaine/γ-aminobutyric acid transporterDBDDNA binding domainOREosmotic response elementOREBPORE-binding proteinSMITsodium-myo-inositol co-transporterTauTtaurine transporterTonEBPtonicity-responsive enhancer (TonE)-binding proteinAQP2aquaporin 2ATFactivating transcription factorHSP70heat shock protein 70siRNAsmall interfering RNANFATnuclear factor of activated T cell. the betaine/γ-aminobutyric acid transporter (BGT1), the sodium-myo-inositol co-transporter (SMIT), the taurine transporter (TauT), heat shock protein 70 (HSP70), and aquaporin 2 (AQP2). AR converts glucose to the osmoprotectant sorbitol, whereas BGT1, SMIT, and TauT transport the organic osmolytes betaine, inositol, and taurine, respectively, into cells (1Burg M.B. Kwon E.D. Kultz D. Annu. Rev. Physiol. 1997; 59 (437–455): 437-455Crossref PubMed Scopus (331) Google Scholar). HSP70, a protein chaperone, also serves a protective function at elevated NaCl (2Rauchman M.I. Pullman J. Gullans S.R. Am. J. Physiol. 1997; 273: F9-P17PubMed Google Scholar). AQP2 increases water permeability of the renal collecting duct during adaptation to dehydration (3Nielsen S. Frokiaer J. Marples D. Kwon T.H. Agre P. Knepper M.A. Physiol. Rev. 2002; 82: 205-244Crossref PubMed Scopus (1041) Google Scholar). TonEBP/OREBP target genes often have multiple TonEBP/OREBP cognate DNA elements known as osmotic response elements (OREs) or tonicity enhancer-responsive elements (TonEs) (Tables 1 and 2). Additionally, all of the above noted genes except TauT have one or more activator protein-1 (AP-1) sites within 35 bp of an ORE (Tables 1 and 2). In the SMIT gene, an AP-1 site overlaps an ORE in the antisense direction (Table 1). In the AR gene, which has been mapped in multiple species, the AP-1 site is highly conserved across all species (Table 2).TABLE 1ORE and associated AP-1 sites and their relative positions in BGT1, SMIT, and HSP70 genesORE sequencePositionAP-1 sequencePosition BGT1-canineTonE2AGGAAAATCCC-144, -134TonE1TGGAAAAGTCC-62, -52TGATTCA-45, -39 SMIT-humanTonEATGGAAAACTAC∼-55 kbTGAGTAG10 bp 5′TonEB2TGGAAAATTCC∼-55 kbTGAGTCA34 bp 3′TonEC1TGGAAAATTAC∼-15 kbTonEC2TGGAAAGTTAC∼-15 kbTGAGTAAoverlap 4-bp antisenseTonEPTGGAAAGTTCC-331, -321 HSP70-mouseTonEATGGAAAGTTTT-1070, -1060TGAGGCA-1034, -1028TonEBTGGAAAATTTT-2326, -2316TGACTCA-2281, -2275TonECTGGAAATCTCC-3698, -3688TonEDTGGAAAAACAC-3715, -3705 AQP2-mouseTonETGGAAATTTGT-489, -479TGATTAA-528, -522 Open table in a new tab TABLE 2Relative positions of OREs and associated AP-1 sites in AR genes across multiple speciesORE sequencePositionAP-1 sequencePosition AR-humanORE-ATGGAAAAATAT-1230, -1220ORE-BTGGAAAATTTA-1198, -1188ORE-CTGGAAAATCAC-1157, -1147TGAGTCA-1117, -1111 AR-rabbitORE-ACGGAAAAATAT-1181, -1171ORE-BTGGAAAAATTT-1148, -1138ORECGGAAAATCAC-1105, -1095TGAGTCA-1072, -1066 AR-mouseORE-ATGGAAAATATC-1128, -1118ORE-BGGGAAAATTTA-1108, -1098ORE-CTGGAAAATCAC-1053, -1043TGACTCA-1012, -1006 AR-ratORE-ATGGAAAATATC-1146, -1136ORE-BGGGAAAATTTA-1106, -1116ORE-CTGGAAAATCAC-1071, -1061TGACTCA-1031, -1025 Open table in a new tab activator protein-1 aldose reductase antigen receptor-response element-2 betaine/γ-aminobutyric acid transporter DNA binding domain osmotic response element ORE-binding protein sodium-myo-inositol co-transporter taurine transporter tonicity-responsive enhancer (TonE)-binding protein aquaporin 2 activating transcription factor heat shock protein 70 small interfering RNA nuclear factor of activated T cell. TonEBP/OREBP, also known as NFAT5, has characteristics intermediate between NFAT1–4 and NF-κB (4Lopez-Rodriguez C. Aramburu J. Jin L. Rakeman A.S. Michino M. Rao A. Immunity. 2001; 15: 47-58Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 5Stroud J.C. Lopez-Rodriguez C. Rao A. Chen L. Nat. Struct. Biol. 2002; 9: 90-94Crossref PubMed Scopus (101) Google Scholar). Like other Rel proteins, the DNA binding domain (DBD) of TonEBP/OREBP is in an N-terminal rel-homology region. The TonEBP/OREBP DBD is highly similar to the DBD in NFAT1–4 (up to 43% sequence identity), which was the basis for the cloning of this protein by one group of investigators (6Lopez-Rodriguez C. Aramburu J. Rakeman A.S. Rao A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7214-7219Crossref PubMed Scopus (319) Google Scholar). Although the TonEBP/OREBP rel-homology region is similar to those of NFAT1–4, it binds DNA as a dimer, similar to NF-κB, whereas NFAT1–4 bind as monomers (5Stroud J.C. Lopez-Rodriguez C. Rao A. Chen L. Nat. Struct. Biol. 2002; 9: 90-94Crossref PubMed Scopus (101) Google Scholar). Both NFAT1–4 and NF-κB interact with AP-1 transcription factors in the transactivation of downstream genes. The AP-1 factors c-Jun and c-Fos interact with NFAT1–4 proteins at multiple rel-homology region residues resulting in strong stabilization of the ternary complex on DNA (7Macian F. Lopez-Rodriguez C. Rao A. Oncogene. 2001; 20: 2476-2489Crossref PubMed Scopus (623) Google Scholar). In the transactivation of many genes, the activity of the NFAT1–4 transcription factors depends on physical cooperativity with active AP-1 factors at composite NFAT:AP-1 sites found in promoter and regulatory regions. NF-κB also interacts with AP-1 in activation of target genes. This interaction may involve functional synergy (8Thomas R.S. Tymms M.J. McKinlay L.H. Shannon M.F. Seth A. Kola I. Oncogene. 1997; 14: 2845-2855Crossref PubMed Scopus (137) Google Scholar) as well as physical cooperativity with AP-1 proteins (9Xiao W. Hodge D.R. Wang L. Yang X. Zhang X. Farrar W.L. Cancer Biol. Ther. 2004; 3: 1007-1017Crossref PubMed Scopus (86) Google Scholar). ARRE-2 is an NFAT binding site in the interleukin-2 promoter (6Lopez-Rodriguez C. Aramburu J. Rakeman A.S. Rao A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7214-7219Crossref PubMed Scopus (319) Google Scholar, 7Macian F. Lopez-Rodriguez C. Rao A. Oncogene. 2001; 20: 2476-2489Crossref PubMed Scopus (623) Google Scholar). TonEBP/OREBP binds to it in vitro, as does NFAT1 (6Lopez-Rodriguez C. Aramburu J. Rakeman A.S. Rao A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7214-7219Crossref PubMed Scopus (319) Google Scholar). However, unlike NFAT1, TonEBP/OREBP does not activate a luciferase reporter gene driven by three copies of ARRE-2 (6Lopez-Rodriguez C. Aramburu J. Rakeman A.S. Rao A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7214-7219Crossref PubMed Scopus (319) Google Scholar). Although c-Fos and c-Jun cooperate with NFAT1 in binding to the ARRE-2 site in vitro, they do not cooperate with TonEBP/OREBP (amino acids 252–548) in its binding, which led to the conclusion that TonEBP/OREBP does not interact with AP-1 (6Lopez-Rodriguez C. Aramburu J. Rakeman A.S. Rao A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7214-7219Crossref PubMed Scopus (319) Google Scholar). In another study the high NaCl-induced increment of activity of a luciferase reporter containing a reconstructed ORE region that lacks the AP-1 site is not significantly different from one that contains it since the decrease in activation occurred at both normo- and hypertonicity. This led to the conclusion that the AP-1 site does not play an important role in the osmoregulation of AR gene transcription (10Ko B.C.B. Ruepp B. Bohren K.M. Gabbay K.H. Chung S.S. J. Biol. Chem. 1997; 272: 16431-16437Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Nevertheless, not only TonEBP/OREBP, but also the AP-1 proteins c-Fos and c-Jun are activated by high NaCl (11Cohen D.M. Wasserman J.C. Gullans S.R. Am. J. Physiol. 1991; 261: C594-C601Crossref PubMed Google Scholar, 12Ying Z. Reisman D. Buggy J. Brain Res. Mol. Brain Res. 1996; 39: 109-116Crossref PubMed Scopus (30) Google Scholar, 13Wiese S. Schliess F. Haussinger D. Biol. Chem. 1998; 379: 667-671Crossref PubMed Scopus (27) Google Scholar). Additionally, as noted, although a lack of the AP-1 site does not significantly affect the relative increase in ORE reporter activity induced by high NaCl, the lack does reduce the absolute activity by 37% when the level of extracellular salt is high and by 23% when it is not (10Ko B.C.B. Ruepp B. Bohren K.M. Gabbay K.H. Chung S.S. J. Biol. Chem. 1997; 272: 16431-16437Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Noting that neither of these negative studies (6Lopez-Rodriguez C. Aramburu J. Rakeman A.S. Rao A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7214-7219Crossref PubMed Scopus (319) Google Scholar, 10Ko B.C.B. Ruepp B. Bohren K.M. Gabbay K.H. Chung S.S. J. Biol. Chem. 1997; 272: 16431-16437Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar) utilized a composite ORE·AP-1 DNA sequence in its native context, which may have affected the results, we determined to reinvestigate the question using native constructs that contain the cognate DNA element of TonEBP/OREBP. Using the ORE·AP-1 DNA sequence from the AR gene, we now find that mutating the AP-1 DNA site reduces TonEBP/OREBP-dependent reporter activity at elevated extracellular NaCl. Also, when NaCl is high, dominant negative AP-1 constructs reduce transcriptional and transactivating activity of TonEBP/OREBP, and siRNA knockdown of c-Fos or c-Jun reduces the mRNA abundance of its target genes, AR and BGT1. Furthermore, inhibition of p38, which is known to stimulate TonEBP/OREBP transcriptional activity (14Ko B.C. Lam A.K. Kapus A. Fan L. Chung S.K. Chung S.S. J. Biol. Chem. 2002; 277: 46085-46092Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), reduces high NaCl-dependent transcription of an ORE·AP-1 reporter only if the AP-1 site is intact. We conclude that TonEBP/OREBP requires AP-1 for its full high NaCl-dependent increase in transactivation, and its role in high NaCl-induced activation of TonEBP/OREBP may require p38 activity. Cell Culture and Treatment—PAP-HT25 cells (passages 60–78) were cultured in 300 mosmol/kg medium as previously described (15Uchida S. Green N. Coon H. Triche T. Mims S. Burg M. Am. J. Physiol. 1987; 253: C230-C242Crossref PubMed Google Scholar). HEK293 cells (passages 38–48) were cultured in 300 mosmol/kg medium according to ATCC instructions. At experiment-specific time points, medium was replaced with medium that was 300, 200 (NaCl added to NaCl-free medium, Biofluids, Rockville, MD), or 500 mosmol/kg (NaCl added). For inhibitor experiments cells were pretreated with Me2SO or the p38 inhibitor, SB203508 (10 μm) in 300 mosmol/kg medium. After 1 h, fresh 300 or 500 mosmol/kg (NaCl added) medium containing either Me2SO or SB203508 was substituted. Me2SO was 0.01% in all inhibitor experiments. Plasmids and siRNAs—The ORE reporter construct contains bp –3497 to +27 of the rabbit aldose reductase gene upstream of the Photinus pyralis luciferase gene (described previously as ARLuc9) (16Ferraris J.D. Williams C.K. Martin B.M. Burg M.B. Garcia-Perez A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10742-10746Crossref PubMed Scopus (60) Google Scholar). The sequence –3497 to +27 includes the aldose reductase promoter, three OREs (–1105, –1095; –1181, –1171; –1148, –1138), and an AP-1 site (–1072, –1066) in native gene context (GenBank™ U12317). The promoter reporter construct is the same but includes only bp –209 to +27. The mutant AP-1 construct reporter is the same as the ORE reporter with bp –1070 to –1068 changed from AGT to CTG. The mutation was made using site-directed mutagenesis (QuikChange, Stratagene, La Jolla, CA), and the construct was sequence-verified. The ORE-X luciferase reporter construct contains two copies of human ORE-X (17Ferraris J.D. Williams C.K. Ohtaka A. Garcia-Perez A. Am. J. Physiol. 1999; 276: C667-C673Crossref PubMed Google Scholar) within a minimal interleukin-2 promoter (18Trama J. Lu Q. Hawley R.G. Ho S.N. J. Immunol. 2000; 165: 4884-4894Crossref PubMed Scopus (135) Google Scholar) (hTonE-GL3, a gift from S. N. Ho, University of California, San Diego, CA), as previously described (19Irarrazabal C.E. Liu J.C. Burg M.B. Ferraris J.D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8809-8814Crossref PubMed Scopus (99) Google Scholar). Human TonEBP/OREBP cDNA clone KIAA0827 was a gift from Dr. Takahiro Nagase (Kazusa DNA Research Institute, Chiba, Japan). Sequence coding for amino acids 1–547 or 1–1531 of KIAA0827 was cloned into expression vector pcDNA6V5-His (Invitrogen) to generate 1–547 or 1–1531V5-His as previously described (19Irarrazabal C.E. Liu J.C. Burg M.B. Ferraris J.D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8809-8814Crossref PubMed Scopus (99) Google Scholar, 20Zhang Z. Ferraris J. Irarrazabal C.E. Dmitireva N.I. Park J.H. Burg M.B. Am. J. Physiol. Renal Physiol. 2005; 289: 506-511Crossref Scopus (34) Google Scholar). The binary GAL4 reporter system has been described (21Ferraris J.D. Williams C.K. Persaud P. Zhang Z. Chen Y. Burg M.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 739-744Crossref PubMed Scopus (121) Google Scholar). In brief, plasmid pFR-Luc (Stratagene) contains the yeast GAL4 binding site (upstream activating sequence) upstream of a minimal promoter and the P. pyralis luciferase gene. Expression plasmid pFA-CMV (Stratagene) contains sequence coding for the yeast GAL4 DNA binding domain. A fusion protein was generated by in-frame insertion of the sequence coding for amino acids 548–1531 of clone KIAA0827 into pFA-CMV to generate GAL4dbd-548–1531. TonEBP/OREBP amino acids 548–1531 contain a NaCl-dependent transactivation domain (21Ferraris J.D. Williams C.K. Persaud P. Zhang Z. Chen Y. Burg M.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 739-744Crossref PubMed Scopus (121) Google Scholar). GAL4dbd contains no transactivation domain but expresses the GAL4dbd (pFC2-dbd, Stratagene). A-Fos dominant negative construct (22Olive M. Krylov D. Echlin D.R. Gardner K. Taparowsky E. Vinson C. J. Biol. Chem. 1997; 272: 18586-18594Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar) generously was provided by Dr. Charles Vinson (NCI, National Institutes of Health, Bethesda, MD). Tam-67, lacking the major transactivation domain of c-Jun, was previously described (23Chowdhury M. Kundu M. Khalili K. Oncogene. 1993; 8: 887-892PubMed Google Scholar). We designed the siRNA against c-Jun (24Liu G. Ding W. Liu X. Mulder K.M. Mol. Carcinog. 2006; 45: 582-593Crossref PubMed Scopus (25) Google Scholar) as a synthetic double-stranded RNA Dicer substrate to enhance the RNA interference potency and efficacy (25Kim D.H. Behlke M.A. Rose S.D. Chang M.S. Choi S. Rossi J.J. Nat. Biotechnol. 2005; 23: 222-226Crossref PubMed Scopus (742) Google Scholar). Duplex sequences were: sense, 5′-Phos-AGUCAUGAACCACGUUAACUUCAdAdG-3′ and antisense 5′-CUUGAAGUUAACGUGGUUCAUGACUGG-3′ (Integrated DNA Technologies, Coralville, IA). The siRNA against c-Fos was a pool of 4 target-specific 20–25-nucleotide siRNAs (sc-29221 Santa Cruz Biotechnology, Santa Cruz, CA). The control (nontargeting) siRNA duplex sequences were: sense, 5′-Phos-UGAACCUGACCCAGGGGAGGGAGdTdT-3′ and antisense sequence 5′-AACUCCCUCCCCUGGGUCAGGUUCAUU-3′ (26Irarrazabal C.E. Burg M.B. Ward S.G. Ferraris J.D. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8882-8887Crossref PubMed Scopus (61) Google Scholar) (Integrated DNA Technologies). Transfection and Luciferase Assays—PAP HT25 cells were grown in 300 mosmol/kg medium in 6-well plates and transfected with 0.5 μg of wild type ORE or mutant AP-1 reporter using CellPhect (GE Healthcare) according to supplier instructions. HEK293 cells were grown in 300 mosmol/kg medium. Five million cells were transfected by electroporation (Gene Pulser, Bio-Rad) according to manufacturer's instructions. For ORE-X reporter assays, cells were transfected with 5 μg of ORE-X reporter. For ORE reporter assays, cells were co-transfected with 5 μg of ORE or mutant AP-1 reporter and 4 μg of TAM-67, 10 μg of A-Fos, or empty vector. For the GAL4 binary assay, cells were co-transfected with 5 μg of the GAL4 upstream activating sequence reporter, 0.5 μg of GAL4dbd or GAL4dbd-548–1531, and 10 μg of A-Fos or empty vector. Twenty-four hours after transfection at 300 mosmol/kg, fresh 200, 300, or 500 mosmol/kg medium was substituted. Luciferase activity was measured 16 h later with the Bright-Glo Luciferase assay system (Promega, Madison, WI). Total protein was measured (BCA protein assay kit; Pierce). Luciferase activity was expressed in relative light units per μg of total cell protein. Electrophoretic Mobility Shift Assay—HEK293 cells stably expressing recombinant TonEBP/OREBP 1–1531V5-His were grown at 300 mosmol/kg. Fresh medium at 300 or 500 mosmol/kg (NaCl added) was substituted. Two hours later, nuclear pellets were prepared using NE-PER nuclear and cytoplasmic extraction reagents (Pierce) according to supplier instructions. Nuclear pellets were resuspended in lysis buffer (50 mm Tris HCl, pH 8.0, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100 with protease (Complete Mini, Roche Applied Science) and phosphatase (Phosphatase Inhibitor Cocktails 1 and 2, Sigma) inhibitors (27Lee S.D. Woo S.K. Kwon H.M. Biochem. Biophys. Res. Commun. 2002; 294: 968-975Crossref PubMed Scopus (35) Google Scholar) and centrifuged 10 min at 15,000 × g, and the supernatant was retained. Double-stranded ORE probe (bp –1238 to –1,104 of the human aldose reductase gene), containing three OREs and an AP-1 site in native gene context, was generated by annealing complementary 5′-biotinylated oligonucleotides (Integrated DNA Technologies). The AP-1 mutant probe is the same as the wild type ORE probe with the 5′ to 3′ bp substitutions in the AP-1 site –1115 to –1113 from AGT to CTG. We combined 0.25–1 μg of nuclear extract with 0.5–1 μg of poly(dA-dT) in binding buffer (LightShift chemiluminescent electrophoretic mobility-shift assay kit, Pierce). Anti-TonEBP/OREBP (1 μg, NFAT5, Affinity Bioreagents, Neshanic Station, NJ), anti-c-Jun (4 μg), or anti-c-Fos (2 μg) (Santa Cruz Biotechnology) was added to some reactions and incubated at 4 °C for 1 h. This was followed by the addition of 100 fmol of ORE or AP-1 mutant probe with or without 1000 fmol of nonbiotinylated ORE probe and incubation for 20 min. Total binding reaction volume was 20 μl. Reaction products were separated by gel electrophoresis in 0.4% SeaKem Gold-agarose in 0.5× Tris borate/EDTA buffer at 4 °C, transferred to nylon membranes, and UV cross-inked (Stratalinker, Stratagene). Biotinylated probes were detected using the LightShift chemiluminescent electrophoretic mobility-shift assay kit (Pierce) according to manufacturer instructions. Immunoprecipitation—HEK293 cells stably expressing recombinant TonEBP/OREBP 1–1531-V5-His, 1–547-V5-His, GAL4dbd-548–1531, or GAL4dbd were grown in 300 mosmol/kg medium. Fresh medium at 200, 300, or 500 mosmol/kg was substituted. Two hours later cells were trypsinized and pelleted by centrifugation. Subsequent steps were at 4 °C. For whole cell extracts, the pellet from one 10-cm dish was extracted for 5 min with 1 ml of lysis buffer containing 50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% Triton X-100, protease (Complete Mini, Roche Applied Science), and phosphatase inhibitor cocktails (Phosphatase Inhibitor Cocktails 1 and 2; Sigma) and centrifuged (15,000 × g, 10 min). Nuclear and cytoplasmic extracts were prepared using NE-PER reagents (Pierce) according to supplier instructions. Nuclear and cytoplasmic extracts were diluted with lysis buffer as above. For immunoprecipitation of 1–1531-V5-His or 1–547-V5-His and any associated proteins, samples were precleared with 1 mg of Dynabeads (Invitrogen) and 2 μg of rabbit IgG biotin-conjugated (Santa Cruz Biotechnology) for 1 h and centrifuged. Precleared supernatants were incubated overnight with 4 μg of rabbit anti-V5 biotin-conjugated (Immunology Consultants Laboratory, Inc.) and 1 mg of Dynabeads (Invitrogen). For immunoprecipitation of GAL4dbd or GAL4dbd-548–1531 and any associated proteins, the samples were precleared with 1 mg of Dynabeads (Invitrogen) and 2 μg of mouse IgG biotin-conjugated (Santa Cruz Biotechnology) for 1 h and centrifuged. Precleared supernatants were incubated overnight with 5 μg of mouse anti-Gal4dbd biotin-conjugated (Immunology Consultants Laboratory) and 1 mg of Dynabeads (Invitrogen). As negative controls, IgG-biotin conjugate (Santa Cruz Biotechnology) was substituted for anti-V5 or anti-Gal4dbd. Ethidium bromide (100 μg/ml), which disrupts protein-DNA association (28Lai J.S. Herr W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6958-6962Crossref PubMed Scopus (398) Google Scholar), was included in some immunoprecipitations to test whether DNA is involved in the coimmunoprecipitation of associated proteins. For immunoprecipitation of c-Fos or c-Jun and any associated proteins, samples were precleared with 20 μl of protein A/G-Plus-agarose beads (Santa Cruz Biotechnology) and 0.5μg of rabbit IgG (Santa Cruz Biotechnology) for 1 h and centrifuged. Precleared supernatants were mixed overnight with 2 μg of rabbit anti-c-Jun (Santa Cruz Biotechnology) or 2 μg of rabbit anti-c-Fos (Santa Cruz Biotechnology) and 20 μl of protein A/G-Plus-agarose beads (Santa Cruz Biotechnology). As negative controls, IgG (Santa Cruz Biotechnology) was substituted for anti-c-Fos or c-Jun. Beads were resuspended in Laemmli sample buffer and incubated for 5 min at 95 °C, and after centrifugation, supernatant proteins were separated on a Tris-HCl, 4–15% polyacrylamide gel (Bio-Rad). Proteins were transferred to a nitrocellulose membrane, which was then cut. One part of the membrane was incubated at 4 °C overnight with mouse anti-V5 (Invitrogen), anti-Gal4dbd (Santa Cruz Biotechnology), or anti-TonEBP/OREBP (NFAT5, Affinity Bioreagents); the others were incubated with rabbit anti-c-Fos or c-Jun (Santa Cruz Biotechnology). Blots were visualized using a LI-COR Odyssey Infrared Imager. siRNA Knockdown of c-Jun and c-Fos and Quantitative Real Time PCR—HEK293 cells were grown in 300 mosmol/kg medium and transfected with 20 nm control, c-Jun, or c-Fos siRNA using Lipofectamine 2000 according to supplier instructions. After 48 h, fresh medium at 300 or 500 mosmol/kg (NaCl added) was substituted. Sixteen hours later, total RNA was isolated (RNeasy, Qiagen, Valencia, CA), and cDNA was prepared using a TaqMan reverse transcription kit (Applied Biosystems, Foster City, CA) according to supplier instructions. PCR was performed on 8 and 80 ng of cDNA samples/20-μl reaction in triplicate for each experiment (Taqman PCR master mix, Applied Biosystems). Amplicons were detected with an ABI Prism 7900HT sequence detection system (Applied Biosystems). Primers directed against the human sequence of aldose reductase (AR) were 5′-ATCGCAGCCAAGCACAATAA-3′ and 5′-AGCAATGCGTTCTGGTGTCA-3′. The 6-carboxyfluorescein-labeled probe was 5′-CAGCCCAGGTCCTGATCCGGTTC-3′ (29Cai Q. Ferraris J.D. Burg M.B. Am. J. Physiol. Renal Physiol. 2004; 286: F58-F67Crossref PubMed Scopus (35) Google Scholar). The primers for human BGT1 were 5′-CCCGAGGAGGGAGAGAAGTT-3′ and 5′-TCCATCTTGTTGGTCCATTGG-3′. The 6-carboxyfluorescein-labeled probe was 5′-AAAGACGAGGACCAGGTGAAGGATCGG-3′ (29Cai Q. Ferraris J.D. Burg M.B. Am. J. Physiol. Renal Physiol. 2004; 286: F58-F67Crossref PubMed Scopus (35) Google Scholar). Primers directed against the human sequence of the cyclophilin gene were 5′-TGTGCCAGGGTGGTGACTT-3′ and 5-TCAAATTTCTCTCCGTAGATGGACTT-3′. The 6-carboxyfluorescein-labeled probe was 5′-CCACCAGTGCCATTATGGCGTGT-3′. The detection system records the number of PCR cycles (Ct) required to produce an amount of product equal to a threshold value, which is a constant. From the Ct values we calculated the mRNA abundance in each experimental condition relative to that of control cells at 300 mosmol/kg, as described (21Ferraris J.D. Williams C.K. Persaud P. Zhang Z. Chen Y. Burg M.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 739-744Crossref PubMed Scopus (121) Google Scholar). Statistical Analysis—Data were compared by repeated measures of analysis of variance followed by Bonferroni multiple comparison test for separation of significant means. Normalized data were log-transformed before analysis of variance. Differences were considered significant for p ≤ 0.05. The 5′-flanking region of the rabbit AR gene contains three OREs (16Ferraris J.D. Williams C.K. Martin B.M. Burg M.B. Garcia-Perez A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10742-10746Crossref PubMed Scopus (60) Google Scholar, 30Ferraris J.D. Williams C.K. Jung K.Y. Bedford J.J. Burg M.B. Garcia-Perez A. J. Biol. Chem. 1996; 271: 18318-18321Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) and an AP-1 site (31Ferraris J. Garcia-Perez A. Am. Zool. 2001; 41: 731-742Google Scholar) (Table 2). We used an ORE luciferase reporter and the same reporter mutated at the AP-1 site to determine the relative contribution of this binding site to TonEBP/OREBP-mediated transcriptional activity in PAP-HT25 and HEK293 cells at 300 and at 500 mosmol/kg (NaCl added) (Fig. 1). When osmolality is raised from 300 to 500 mosmol/kg by adding NaCl, transcriptional activity of the ORE reporter increases significantly. Mutation of AP-1 reduces transcriptional activity by 46% in PAP-HT25 cells and 44% in HEK293 cells. Transcriptional activity at 300 mosmol/kg is unaffected by mutation at the AP-1 site. We conclude that at 500 mosmol/kg, AP-1 is a potentiating element. To determine whether the AP-1 transcription factors c-Fos and c-Jun associate with TonEBP/OREBP in the protein complex that binds to the wild type DNA probe containing three OREs and an AP-1 site, we performed electrophoretic mobility shift assay binding reactions using HEK293 nuclear protein extracts with and without added antibody to c-Fos or c-Jun. Electrophoretic mobility shift assays using nuclear extracts from cells at 300 mosmol/kg required 4× more extract to generate a mobility shift than nuclear extracts from cells at 500 mosmol/kg because high NaCl causes TonEBP/OREBP to translocate to the nucleus, which results in more TonEBP/OREBP in the nucleus at 500 mosmol/kg (Fig. 2, A and B). The additi
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