Effect of STAT5b on Rat Liver Alcohol Dehydrogenase

Archives of Biochemistry and Biophysics Vol. 391, No. 1, July 1, pp. 41– 48, 2001 doi:10.1006/abbi.2001.2393, available online at http://www.idealibrary.com on

Effect of STAT5b on Rat Liver Alcohol Dehydrogenase 1 James J. Potter and Esteban Mezey 2 Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2195

Received November 21, 2000, and in revised form April 10, 2001; published online June 6, 2001

Growth hormone (GH) enhances rat liver alcohol dehydrogenase (ADH) due to an increase in enzyme synthesis, which is mediated at the level of transcription. Previous studies have shown that the effect of GH in enhancing activation of the ADH promoter is mediated by C/EBP␤ binding to region ⴚ22 to ⴚ11 relative to the start of transcription. In this study, STAT5b and C/EBP␤ were found to bind to adjacent nucleotide sequences on a region between ⴚ226 and ⴚ194. Expression vectors for both STAT5b and C/EBP␤ independently activated the promoter. Furthermore, the expression vector for the GH receptor also activated the ADH promoter, and this effect was abrogated by mutations of the adjacent STAT5b and C/EBP␤ binding sites. These observations indicate that the enhancing effect of GH is mediated by both STAT5b and C/EBP␤. © 2001 Academic Press

Key Words: alcohol dehydrogenase; STAT5b; C/EBP␤; growth hormone.

Liver alcohol dehydrogenase (ADH, 3 alcohol:NAD oxidoreductase, EC 1.1.1.1.) is principally responsible for ethanol oxidation. The enzyme activity is regulated by hormones and increases in the activity of the enzyme result in increased formation of metabolites of ethanol such as acetaldehyde, which are important in the pathogenesis of alcoholic liver disease (1). We previously demonstrated that GH enhances rat class I 1 This work was supported by Grant AA00626 from the United States Public Health Service. 2 To whom correspondence and reprint requests should be addressed. Fax: (410) 955-9677. E-mail: [email protected]. 3 Abbreviations used: ADH, alcohol dehydrogenase; C/EBP, CCAAT/enhancer binding protein; STAT, signal transducers and activators of transcription; GH, growth hormone; RGHR, rabbit growth hormone receptor NE, nuclear extract; DMEM, Dulbecco’s modified Eagle medium; FBS, fetal bovine serum; DMSO, dimethylsulfoxide; EMSA, electrophoretic mobility shift assay; CAT, chloramphenicol acetyl transferase; UV, ultraviolet; RLU, relative light units.

0003-9861/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

ADH due to an increase in enzyme synthesis mediated at the level of transcription (2). Furthermore, the effect of GH in enhancing the ADH promoter was found to be mediated by the CCAAT/enhancer binding protein ␤ (C/EBP␤) (3). Both C/EBP␣ and C/EBP␤ bind to the promoter at ⫺22 to ⫺11 relative to the start of transcription and independently activate the promoter (4, 5) in transient transfection experiments. GH increased C/EBP␤ mRNA, but not C/EBP␣ mR⌵〈, and the activating effect of GH was abrogated by a mutation of the promoter at the C/EBP binding site. Since the above studies were completed, new knowledge has been gained into molecular mechanisms by which GH activates gene transcription. GH binds to a specific cell surface receptor, which dimerizes, following which the complex binds to and activates cytoplasmic Janus tyrosine kinase (JAK2), and which in turn phosphorylates the receptor as well as JAK 2 itself. JAK 2, as well as other serine/threonine kinases phosphorylate a family of signal transducers and activators of transcription (STAT), which are translocated into the nucleus and activate transcription by binding to specific regulatory sequences (6). A STAT regulatory sequence that consists of a motif TT(N) 5AA (first identified as a ␥-interferon-activated sequence (GAS)) is present in the regulatory sequence of many GH regulated genes (7, 8). In a recent study two members of the STAT family, STAT 5a and STAT5b were found to mediate the GH-dependent enhancement of the cytochrome P450 3A10/lithocholic acid 6␤ hydroxylase promoter (9). On analysis of the nucleotide sequence of the proximal class I rat ADH promoter we found the presence of the above STAT regulatory motif (5⬘-TTGGGAAAA-3⬘) at ⫺211 to ⫺203. The purpose of this study was to determine whether STAT5a or STAT5b are also mediators of the effect of GH in enhancing ADH. MATERIALS AND METHODS Animals and chemicals. Adult male Sprague–Dawley rats were obtained from Charles River Laboratories (Wilmington, MA). All 41

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animals received humane care in compliance with the guidelines from the Animal Care and Use Committee of the Johns Hopkins University. Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), dithiothreitol (DTT), agarose, cesium chloride, the restriction enzymes XbaI and HindIII were purchased from Life Technologies, Inc. (Gaithersburg, MD). Plastic 10-cm 2 culture dishes and 75 and 150-cm 2 tissue culture flasks were purchased from Becton Dickinson and Co. (Franklin Lakes, NJ). ). [␥- 32P] ATP, [␣- 32P] dATP, [␣- 32P] dCTP, and [ 35S]methionine were purchased from DuPont-New England Nuclear (Boston, MA). Human GH was obtained from the National Hormone and Pituitary Program of NIDDK (Torrance, CA). An oligonucleotide containing the consensus binding site for CCAAT enhancer binding protein (C/EBP) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Plasmids. The rat class I 240ADH-CAT construct containing the proximal 240 bp of the rat class I ADH promoter sequence fused to the coding sequence of the reporter gene chloramphenicol acyltransferase (CAT), obtained from Dr. David W. Crabb of Indiana University School of Medicine (Indianapolis, IN), has been described previously (3). The STAT expression vectors pCMV-STAT5a and pCMVSTAT5b, the rabbit GH receptor vector pCMV-RGHR and the empty vector pCMV5 were a gift from Dr. Gregorio Gil of The Department of Biochemistry and Molecular Biophysics, Medical College of Virginia (Richmond, VA). The expression vectors pMSV-C/EBP␤ and the empty vector pCMX-L1 were obtained from Dr. M. Daniel Lane of the Department of Biological Chemistry at out institution. Generation of luciferase constructs of the ADH promoter. The 240 proximal rat ADH promoter was cut out from the 240ADH-CAT construct using HindIII and XbaI endonucleases (Life Technologies, Inc., Gaithersburg, MD). The pGL3-Enhancer vector (Promega Corp., Madison, WI) bearing the luciferase reporter gene was digested with NheI. Digestion mixtures were electrophoresed on a 1% agarose gel, bands of the appropriate size were excised and purified of agarose using the QIAEX Gel Extraction protocol (Qiagen, Studio City, CA). The concentration of each DNA fragment was determined spectrophotometrically. Ligation of the inserts into the pGL3 enhancer vector was performed by blunt end ligation of the 5⬘ HindIII site and direct ligation of the respective compatible protruding ends resulting from the XbaI and NheI digestions. Site-directed mutagenesis. Oligonucleotides containing a minimum of 2 bp and a maximum of 4-bp substitutions were designed for site directed mutagenesis. The method employed for the mutagenesis of the ADH promoter was based on the strategy of overlap extension using PCR described by Ho et al. (10). Successful mutations were confirmed by the DNA sequencing facility of our Department of Biological Chemistry. The partial nucleotide sequences of the wild type and mutated ADH promoters are shown in Table I. Nuclear protein extraction. Nuclear extracts from livers of adult male Sprague–Dawley rats were prepared as described previously (4). The nuclear protein extracts were aliquoted and stored under nitrogen at ⫺100°C. Protein content of the nuclear extract was determined by the method of Lowry et al. (11). DNase I protection. The DNase I protection (footprint) procedure of the ADH promoter was performed as described previously (4). The coding strand of the 240 bp proximal ADH promoter labeled at the 3⬘-end was incubated with increasing concentrations of rat liver nuclear extracts in a buffer containing 20 mM Hepes, 5 mM DDT, 1 mM MgCl 2, and 60 mM KCL. The DNA was resolved on a 6% polyacrylamide sequencing gel containing 8 mM urea. The nucleotide sequence of the protected bands was determined by the chain termination sequencing method (12). Electrophoretic mobility shift assays (EMSA). The sequence of the wild-type oligonucleotide used initially for EMSA was 5⬘-CAATTGGCTGTACATTTGGGAAAATAAAACTTT-3⬘ (⫺226 to ⫺194). Mutated oligonucleotides contained the bp nucleotide substitutions shown in Table I and Fig 4. Complimentary strands of each oligonu-

cleotide were annealed and the double-stranded oligonucleotides were labeled with [␣- 32P]dATP and [␣- 32P]dCTP, using Klenow enzyme according to the method of Feinberg and Vogelstein (13). DNAprotein binding reactions were performed following the previously described EMSA procedure (3). Nuclear extracts (8 ␮g protein) from normal adult rat liver were incubated with the labeled oligonucleotide probes (2.5–25 fmol) at room temperature for 30 min in 25 ␮l of reaction buffer containing 25 mM Hepes (pH 7.8), 50 mM KCl , 0.1 mM ZnCl 2, 1 mM DTT, 2 ␮g poly(dIdC), and 10% glycerol. Competition assays were performed by incubating 10-fold molar excess of unlabelled oligonucleotides for 30 min with nuclear proteins prior to the addition of labeled oligonucleotide probes. For “supershift” EMSA experiments, rabbit polyclonal antibodies to STAT5a, STAT5b, and C/EBP␤ (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were used. These antibodies were added separately to the reaction at the completion of DNA–protein binding and incubated for additional 30 min at room temperature. The resultant samples were resolved on a 5% nondenaturing polyacrylamide gel and visualized on X-ray film. Transient transfection and luciferase assay. Transient transfection experiments were carried out in cultured HepG2 cells using the calcium phosphate precipitation method (14). HepG2 cells were seeded on 75-cm 2 polystyrene flasks and allowed to grow over night into 60 –70% confluency in DMEM containing 10% FBS. The medium was renewed 1 h prior to transfection. To each flask 10 ␮g of pGL3ADH or mutant pGL3-ADH, 5 ␮g of ␤-galactosidase vectors, and 5 ␮g of salmon sperm DNA were added in the form of CaHPO 3-DNA precipitates. For each experiment, pGL3-basic and pGL3-control luciferase vectors were used as a negative and a positive control. After overnight incubation, the cells were shocked with 10% DMSO in DMEM for 3 min and then washed and refed with fresh DMEM containing 10% FBS. The concentration of GH in 10% FBS is approximately 10 –20 ng/ml (15, 16). At 40 h after transfection, the cells were harvested and subjected to one freeze–thaw cycle in 200 ␮l of the reporter lysis buffer (Promega Corp., Madison, WI). Luciferase activity and ␤-galactosidase activity were determined by respective chemiluminescent assays (17, 18). Ultraviolet cross-linking of nuclear proteins to oligonucleotides. The binding of nuclear proteins to the oligonucleotide probe was performed as for EMSA. Reactions using 8 ␮g of nuclear protein and 50 fmol of radioactively labeled oligonucleotides were used. Following the binding reaction, samples were UV-irradiated on ice for 15 min at 120,000 ␮J. Following denaturation, samples were resolved on a 10% SDS–polyacrylamide gel. Immunoblot analysis. UV cross-linking of nuclear protein to the wild-type ADH and mutated oligonucleotides was carried out as described above and the complexes were resolved on a 8% SDS– polyacrylamide gel. Rainbow prestained protein molecular weight markers (Amersham Life Science Inc., Arlington Heights, IL) were used for molecular weight estimation. The resolved proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Hybond-P, Amersham Life Science Inc., Arlington Heights, IL) in a Trans-Blot Cell at 30 V/0.24 A overnight, according to the manufacturer’s protocol (Bio-Rad Laboratories, Richmond,CA). The membranes were washed in Tris-buffered saline pH 7.6 containing 0.1% Tween 20 (TBS-T) and subsequently blocked with 5% w/v dried nonfat milk and 0.5% FBS in TBS-T for 1 h at room temperature. The membranes were then incubated with rabbit polyclonal antibody to STAT 5b (1:200 dilution) or to C/EBP␤ (C-19, 1:500 dilution) (Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 1 h. After repeated washing for 1 h, the membranes were incubated with horseradish peroxidase conjugated goat anti-rabbit IgG (1: 50,000 dilution, Amersham Life Science Inc. Arlington Heights, IL) or for 1 h. The membranes were again washed for 1 h and then immersed in lumigen PS-3 acridan substrate solution (ECL ⫹ Plus, Amersham Life Science Inc. Arlington Heights, IL) for 5 min. The antigens were visualized by exposing the membrane to X-ray film.

EFFECT OF STAT5b ON ALCOHOL DEHYDROGENASE

43

Coimmunoprecipitation experiments. HepG2 cells transfected with 20 ␮g of pCMV-STAT5b alone, 20 ␮g of pMT2-C/EBP␤ alone or with the combination of both vectors using the calcium precipitation method as described above. After a 20-h incubation the cells were shocked with 10% DMSO in DMEM and 2 h later the media replaced by methionine-free DMEM containing 500 ␮Ci [ 35S]methionine (1175 Ci/mmol) at 37°C for 3 h. The cells were washed three times with 10 ml ice-cold PBS, lysed, collected by scrapping in 1 ml of PBS containing 0.1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, and 1 ␮g/ml each of leupeptin, pepstatin, and aprotinin, and centrifuged. The labeled cell extracts were incubated with either preimmune serum, antibody to C/EBP␤ or antibody to STAT5b at 4°C for 2 h. The antigen–antibody complexes were precipitated from the incubation mixtures by the addition of protein A bearing Staphylocccus aureus (Pansorbin, Calbiochem, San Diego, CA) at 4°C for 1 h. The precipitates were centrifuged, washed three times with PBS, and resuspended in 0.125 M Tris–HCl buffer, pH 6.2, containing 6 M urea, 3% sodium dodecyl sulfate, and 5% mercaptoethanol, heated for 5 min at 95°C, and then subjected to sodium dodecyl sulfate–polyacrylamide electrophoresis. Data analysis. All data points are expressed as means ⫾ SE. The differences between means of paired groups and between means of more than two paired groups were examined by the Student’s t test and by two-way analysis of variance plus appropriate multiple comparisons, respectively.

RESULTS

DNase I protection analysis demonstrates nuclear protein binding to the ADH promoter between positions ⫺221 and ⫺197 (Fig. 1), which includes the STAT binding motif 5⬘-TTGGGAAAA-3⬘ located at ⫺211 to ⫺203. Three other areas of nuclear protein binding to the ADH promoter at positions ⫺10 to ⫺22, ⫺36 to ⫺44, and ⫺52 to ⫺80 were demonstrated by DNase protection analysis by us previously (4, 5). Nuclear protein binding to the oligonucleotide specifying the wild-type and mutant ⫺226 to ⫺194 region of the ADH promoter is shown in Fig. 2. Four protein– DNA complexes were found with the wild-type and mutant oligonucleotides. The intensity of the binding of all four proteins was less with the mutant than with the wild-type oligonucleotide. Antibody to STAT5b resulted in the disappearance of the uppermost protein– DNA complex formed with the wild-type oligonucleotide, although no supershifted complex was detectable. Antibodies to STAT5a and to C/EBP␤ did not affect the protein–DNA complexes. Antibodies to either STAT5a, STAT5b, or C/EBP␤ decreased the intensity of the two middle protein–DNA complexes formed with the mutant oligonucleotide without the appearance of supershifted complexes. To identify proteins that bind to the ⫺226 to ⫺194 region of the promoter, nuclear proteins were UVcross-linked with the labeled wild-type (ADHwt) or mutated oligonucleotide (ADHmut) in the presence or absence of competing unlabelled oligonucleotides and resolved on a SDS–polyacrylamide gel. UV cross-linking demonstrates two major proteins of approximate molecular weight of 80 and 42 kDa (Fig. 3) binding to

FIG. 1. DNase I footprint analysis of the rat class I ADH promoter with nuclear extracts obtained from rat liver. The coding strand of the ADH promoter was labelled at the 3⬘-end for analysis. Increasing amounts of nuclear extract (from 0 to 100 ng) were used. The region of DNA protected is marked with a rectangular box.

both ADHwt and ADHmut oligonucleotides. The upper 80-kDa protein is competed away by a proximal oligonucleotide which includes 2 bp of the proximal end of the STAT binding site (Table I), while the lower 42kDa protein is competed away by a distal oligonucleotide adjacent to the distal end of the STAT binding site. A C/EBP protein binding consensus sequence (Table I) also competes away the lower 42-kDa protein, but not

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FIG. 2. EMSA with supershift showing the binding of rat liver nuclear protein to the wild-type (ADHwt) and mutated (ADHmut) oligonucleotides, specifying the STAT binding site (region ⫺226 to ⫺194) in the ADH. The EMSAs were performed with 8 ␮g of nuclear extract and the labeled oligonucleotides. For supershifts, the same reaction mixture was incubated with preimmune serum (PI) or antibodies to STAT5a (1:200 dilution), STAT5b (1:200 dilution), or C/EBP␤ (1:500 dilution). F indicates the position of the free probe.

the upper 80-kDa protein. Both bands are competed away by the ADHwt and ADHmut oligonucleotides. A mutational analysis of the STAT binding site was performed by EMSA of protein–DNA complexes formed with labeled oligonucleotides which were sequentially mutated by two base pairs between ⫺226 and ⫺194 (Fig. 4). This analysis reveals that the distal mutation of the oligonucleotide (M4) eliminates the upper of two middle complexes of protein binding and decreases the lowermost complex of binding, while a more proximal mutation of the oligonucleotide (M9) located in the STAT binding site, eliminates the uppermost complex. Immunoblot analysis of the UV-cross linked proteins to the wild-type STAT (ADHwt), mutated STAT (ADHmut), and the mutatedADHmut4 and ADHmut9 oligonucleotides is shown in Fig. 5. Three immunoblot experiments were done. The mean percentage densitometry of the autoradiographs of the immunoblots is shown in Table II. The greatest reduction in binding of STAT5 and C/EBP␤ as compared with binding to the wild-type oligonucleotide is demonstrated for ADHmut9 and ADHmut4, respectively. The STAT5b expression vector (pCMV-STAT5b) results in a twofold activation of the alcohol dehydrogenase promoter (pGL3-ADH) in transfected HepG2 cells, while the STAT5a expression vector (pCMVSTAT5a) suppresses the activity of pGL3-ADH (Tables

FIG. 3. Ultraviolet cross-linking of proteins from rat liver nuclear extracts to the labeled (ADHwt) and mutated (ADHmut) oligonucleotides specifying the STAT binding site (region ⫺226 to ⫺194) in the ADH promoter in the absence or presence of their respective unlabeled oligonucleotides, shorter distal (D) or proximal (P) wild-type oligonucleotides (A and B) or the C/EBP protein consensus binding sequence (B) (Table I). The resolving gels are 10% denaturing (SDS) polyacrylamide gels. The calculated approximate molecular weight of the cross-linked proteins are indicated.

III and IV). The expression vector of the rabbit GH receptor (pCMV-RGHR) results in a five- to ninefold activation of pGL3-ADH. The combination of pCMVRGHR with either pCMV-STAT5a or pCMV-STAT5b results in a lesser activation of pGL3-ADH than found with pCMV-RGHR alone. The continuous presence of GH (1 ␮g/ml) resulted in a fourfold activation of pGL3ADH, from 13,502 ⫾ 3184 to 41,867 ⫾ 2236 RLU per mg of protein (P ⬍ 0.05), confirming prior studies with transfected 240ADH-CAT (4). The addition of GH, however, did not have any additional or different effect on pGL3-ADH with cotransfection of pCMV-STAT5a, pCMV-STAT5b, pCMV-RGHR, or the combination of

TABLE I

Partial Nucleotide Sequences of the Coding Strand of the Wild-Type and Mutated ADH Promoters ⫺226

⫺211

⫺203

⫺194

ADHwt 5⬘-CAATTGGCTGTACATTTGGGAAAATAAAACTTT-3⬘ ADHmut 5⬘-CAATTGGCTGTACATCCGGGGCAATAAAACTTT-3⬘ ADHmut4 5⬘-CAATTGGTAGTACATTTGGGAAAATAAAACTTT-3⬘ ADHmut9 5⬘-CAATTGGCCGTACATTTTTGAAAATAAAACTTT-3⬘ Proximal oligo 5⬘-AATAAAACTTT-3⬘ Distal oligo 5⬘-TTGGCCGTA-3⬘ C/EBP a 5⬘-TGCAGATTGCGCAATCTGCA-3⬘ Note. The STAT regulatory motif is underlined. The mutated oligonucleotides are shown in bold letters. a C/EBP protein consensus binding sequence.

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EFFECT OF STAT5b ON ALCOHOL DEHYDROGENASE

FIG. 4. Mutational analysis of the STAT binding sequence of the ADH promoter. EMSA was performed with 1 pg of the labeled wild-type oligonucleotide and 8 ␮g of rat liver nuclear protein extract. Unlabeled competitor oligonucleotides were added in 10-fold molar excess over the probe. The bold case letters indicate the mutated sequences in each mutant oligonucleotide. F is free probe. WT 5⬘-CAATTGGCTGTACATTTGGGAAAATAAAACTTT-3⬘ M1 5⬘-CTTTTGGCTGTACATTTGGGAAAATAAAACTTT-3⬘ M2 5⬘-CAAAAGGCTGTACATTTGGGAAAATAAAACTTT-3⬘ M3 5⬘-CAATTTTCTGTACATTTGGGAAAATAAAACTTT-3⬘ M4 5⬘-CAATTGGTAGTACATTTGGGAAAATAAAACTTT-3⬘ M5 5⬘-CAATTGGCTTAACATTTGGGAAAATAAAACTTT-3⬘ M6 5⬘-CAATTGGCTGTTTATTTGGGAAAATAAAACTTT-3⬘ M7 5⬘-CAATTGGCTGTACTATTGGGAAAATAAAACTTT-3⬘ M8 5⬘-CAATTGGCTGTACATAAGGGAAAATAAAACTTT-3⬘ M9 5⬘-CAATTGGCTGTACATTTTTGAAAATAAAACTTT-3⬘ M10 5⬘-CAATTGGCTGTACATTTGGTTAAATAAAACTTT-3⬘ M11 5⬘-CAATTGGCTGTACATTTGGGAATTTAAAACTTT-3⬘ M12 5⬘-CAATTGGCTGTACATTTGGGAAAAATAAACTTT-3⬘ M13 5⬘-CAATTGGCTGTACATTTGGGAAAATATTACTTT-3⬘ M14 5⬘-CAATTGGCTGTACATTTGGGAAAATAAATTTTT-3⬘ M15 5⬘-CAATTGGCTGTACATTTGGGAAAATAAAACAAT-3⬘

pCMV-RGHR with the STAT expression vectors than demonstrated above in the presence of GH found in 10% FBS (data not shown). The individual effects of the STAT vectors and of pCMV-RGHR were not apparent with transfection of the ADH promoter mutated at the STAT site (ADHmut), although minimal activation was found with the combination of the STAT vectors with pCMV-RGHR (Table III). In further experiments the demonstrated effects of the STAT expression vectors and of pCMV-RGHR on the wild-type pGL3-ADH are shown to be abrogated with transfection of pGL3ADHmut9, which has different bp substitution mutation in the STAT binding site (Table IV). By contrast, studies with the transfected ADHmut4 promoter, that has a mutation in a proximal C/EBP binding site, shows enhanced activation of the promoter by both pCMV-STAT5a and pCMV-STAT5b in the presence or absence of cotransfected pCMV-RGHR, while pCMVRGHR alone fails to activate pGL3-ADHmut4. The C/EBP␤ expression vector pMT2-C/EBP␤ resulted in activation of pGL3-ADH (Table V), confirm-

FIG. 5. Immunoblot showing the presence of STAT5a and C/EBP␤ proteins in nuclear extracts from rat liver UV-cross linked to the unlabelled wild type oligonucleotide (ADHwt) or the oligonucleotides mutated at the STAT binding site (ADHmut) or mutated at either the distal (ADHmut4) or proximal sites (ADHmut9). The molecular weights of the identified proteins is indicated.

ing prior studies with transfected 240ADH-CAT (4). pMT2-C/EBP␤ activated both pGL-ADHmut9 and pGL-ADHmut4, to a greater extent than the activation of pGL3-ADH (Table V). Cotransfection with pCMVSTAT5b resulted in marked inhibition of the activating effect of pMT2-C/EBP␤ on pGL3-ADH, and partial inhibition on the activating effect of pMT2-C/EBP␤ on pGL3-ADHmut4 and pGL3-ADHmut9. The antagonism between pMT2-C/EBP␤ and pCMVSTAT5b in activating pGL3-ADH was explored by investigation of protein–protein interactions. Labeled C/EBP␤ and STAT5b were immunoprecipitated from transfected HepG2 cells which were labeled with [ 35S]methionine following the transfections. As shown in Fig. 6, antibody to C/EBP␤ precipitated C/EBP␤ in cells transfected with pMT2-C/EBP␤ (lane 2), while antibody to STAT5b precipitated STAT5b in cells transfected with pCMV-STAT5b (lane 4). Antibody to C/EBP␤ did not precipitate STAT5b in cells transfected with pCMV-STAT5b alone, while antibody STAT5b did

TABLE II

Relative Densitometry Readings of the Autoradiographs of the Immunoblots of UV-Cross Linked Proteins to the WildType (ADH wt) and Mutated (ADH mut) Alcohol Dehydrogenase (ADH) Oligonucleotides Densitometry expressed as a percentage of the value with the wild-type ADH oligonucleotide Oligonucleotide ADH ADH ADH ADH

wt mut mut 4 mut 9

STAT 5b

C/EBP␤

100 80 ⫾ 9 53 ⫾ 5 39 ⫾ 4

100 69 ⫾ 6 42 ⫾ 5 62 ⫾ 7

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POTTER AND MEZEY TABLE III

Effect of STAT5a, STAT5b, and GHR on the Activation of the Wild-Type and Mutated Alcohol Dehydrogenase (ADH) Promoter Luciferase activity expressed as a percentage of the wild or mutated ADH promoters a Expression vector

pGL3-ADH

pGL3-ADH mut

Control pCMV-STAT 5a pCMV-STAT 5b pCMV-RGHR pCMV-STAT 5a ⫹ pCMV-RGHR pCMV-STAT5b ⫹ pCMV-RGHR

100 ⫾ 12 41 ⫾ 11 b 185 ⫾ 19 b 506 ⫾ 69 c 295 ⫾ 26 c 266 ⫾ 24 c

100 ⫾ 13 96 ⫾ 12 43 ⫾ 12 68 ⫾ 11 166 ⫾ 19 127 ⫾ 15

Note. Data are expressed as means of 3– 4 determinations. The controls were transfected with the empty pCMV5 vector. a The mean pGL3-ADH and pGL3-ADHmut luciferase activities were 15,830 and 58,615 and RLU/mg protein, respectively. b P ⬍ 0.05 as compared with respective control. c P ⬍ 0.01 as compared with respective control.

not precipitate C/EBP␤ in cells transfected with pMT2C/EBP␤ alone (not shown). However, when the cells were cotransfected with both vectors, antibody to either C/EBP␤ alone (lane 6) or to STAT5b alone (lane 7) precipitated both C/EBP␤ and STAT5b. These results indicate the existence of a physical protein–protein interaction between C/EBP␤ and STAT5b. DISCUSSION

This study shows that STAT5b binds and activates the rat class I ADH promoter. STAT5b and C/EBP␤ bind to adjacent nucleotide sequences on a region between ⫺226 and ⫺194 relative to the start-site of tran-

scription. The binding of STAT5b was demonstrated by supershift EMSA, while the binding of C/EBP␤, which was not apparent by supershift EMSA, was demonstrated by immunoblot using specific C/EBP␤ antibody of nuclear protein UV-cross linked to the oligonucleotide sequence specifying the above region. The activation of the ADH promoter by STAT5b was abrogated by 2 different mutations of the promoter in the STAT binding motif 5⬘-TTGGGAAAA-3⬘. The initial mutation (ADHmut) of the STAT binding motif to 5⬘-TCCGGGCAA-3⬘ resulted in decreased binding on EMSA of all 4 nuclear proteins observed binding to the wild-type oligonucleotide encompassing this site, while the later mutation (ADHmut9) (5⬘-TTTTGAAAA-3⬘) specifically eliminated binding of STAT5b, identified by supershift with STAT5b antibody. The more general decrease of protein binding caused by the first mutation is associated with an inhibitory effect of STAT5b on the pGL3-ADHmut promoter, while the specific mutation of STAT5b binding eliminates the effect of STA5b in activating pGL3-ADHmu9. C/EBP␤ binding was found to occur distal to the STAT binding motif. This was demonstrated by competitive elimination of the UV cross-linking of C/EBP␤ with an unlabeled distal oligonucleotide or an oligonucleotide containing the C/EBP protein binding consensus sequence. Previously only one site of C/EBP binding located in region ⫺22 to ⫺11 in the promoter had been demonstrated (4). Both C/EBP␣ and C/EBP␤ bound to this region and activated the promoter (4, 5). Mutation of the C/EBP binding site (ADHmut4) eliminates one of four nuclear protein complexes formed with the wild-type oligonucleotide (ADHwt), and, as noted above, this complex was identified as being C/EBP␤ by immunoblot of the UV-cross linked complexes. C/EBP␤ activated the wild-type promoter

TABLE IV

Effect of STAT5a, STAT5b, and GHR on the Activation of the Wild-Type and Mutated Alcohol Dehydrogenase (ADH) Promoter Luciferase activity expressed as a percentage of the wild or mutated ADH promoters a Expression vector

pGL3-ADH

pGL3-ADH mut 4

pGL3-ADH mut 9

Control pCMV-STAT 5a pCMV-STAT 5b pCMV-RGHR pCMV-STAT 5a ⫹ pCMV-RGHR pCMV-STAT 5b ⫹ pCMV-RGHR

100 ⫾ 11 55 ⫾ 11 b 237 ⫾ 19 c 971 ⫾ 88 c 509 ⫾ 45 c 733 ⫾ 62 c

100 ⫾ 12 540 ⫾ 48 c 1442 ⫾ 94 c 128 ⫾ 15 1230 ⫾ 110 c 1865 ⫾ 168 c

100 ⫾ 8 121 ⫾ 15 145 ⫾ 18 127 ⫾ 3 113 ⫾ 13 63 ⫾ 9 b

Note. Data are expressed as means of 3– 4 determinations. The controls were transfected with the empty pCMV5 vector. a The mean pGL3-ADH, pGL3-ADHmut4, and pGL3-ADHmut9 luciferase activities were 11,162; 1,320; and 107,632 RLU/mg protein, respectively. b P ⬍ 0.05 as compared with respective control. c P ⬍ 0.01 as compared with respective control.

47

EFFECT OF STAT5b ON ALCOHOL DEHYDROGENASE TABLE V

Effect of the C/EBP␤ Expression Vector on the Activation of the Wild-Type and Mutated Alcohol Dehydrogenase (ADH) Promoters Luciferase activity expressed as percentage of the wild-type and mutated ADH promoters a Expression vector

pGL3-ADH

pGL3-ADH mut 4

pGL3-ADH mut 9

Control pMT2-C/EBP␤ pMT2-C/EPP␤ ⫹ pCMV-STAT 5b

100 ⫾ 22 192 ⫾ 15 b 5 ⫾ 0.7

100 ⫾ 15 605 ⫾ 21 c 278 ⫾ 19 c

100 ⫾ 23 1117 ⫾ 5 c 428 ⫾ 26 c

Note. The controls were transfected with the empty pCMV-L1 vector. a Data are expressed as means ⫾ 4 – 8 determinations. b P ⬍ 0.05 as compared with respective control. c P ⬍ 0.001 as compared with respective control.

pGL3-ADH as demonstrated previously (4). This activation was markedly enhanced with the promoters mutated at the STAT (pGL3-mut9) or at the newly described C/EBP binding site (pGL3-ADHmut4), suggesting that the principal site of binding and activation of the ADH promoter is at the proximal ⫺22 to ⫺11 binding site. The combination of C/EBP␤ and STAT5b expression vectors in cotransfection experiments is inhibitory on pGL3-ADH activation, and this interaction appears to occur at their adjacent binding sites since the inhibition is less with the promoters mutated either at the STAT (pGL3-ADHmut9) or the C/EBP (pGL3-ADHmut4) binding sites. A direct protein–protein interaction between Stat5b and C/EBP␤, shown by their coimmunopreciptitation in this study, probably resulting in their decreased binding to the ADH promoter is the most likely cause for the decreased activation of pGL3ADH observed with the cotransfection of the STAT5b and C/EBP␤ expression vectors. Direct interactions between STAT proteins and other proteins have been described previously. For instance, STAT5b was found to have a direct protein–protein interaction with the glucocorticoid receptor resulting in down regulation of the glucocorticoid response, but up-regulation of a STAT5b response, in the mouse mammary-tumor virus long terminal repeat (MMTV-LTR) promoter (19). The continuous exposure of hepatocytes in culture to GH enhances rat class I ADH due to an increase in enzyme synthesis which is mediated at the level of transcription (2). This effect of GH was initially found to be mediated by C/EBP␤ binding to the originally identified C/EBP binding site at ⫺22 to ⫺11 in the promoter (3). In this study we show that an expression vector for the GH receptor activates the wild-type promoter pGL3-ADH and that this effect is abrogated by transfection of the ADH promoter with mutations of the STAT binding site as well as by mutation of the C/EBP binding site indicating that the enhancing ef-

fect of the GH receptor is mediated by both the STAT and C/EBP binding sites. The effect of GH in increasing ADH may be a mechanism for known higher ADH activity and rates of ethanol elimination in female than in male rats (20, 21). The continuous presence of GH in culture media, which results in increased ADH in cultured hepatocytes from male rats (2), is akin to feminization, since male rats have intermittent blood peaks of GH every

FIG. 6. Coimmunoprecipitation of STAT5b and C/EBP␤. HepG2 cells were transfected with 20 ␮g of pCMV-STAT alone, 20 ␮g of pMT2-C/EBP␤ alone, or with the combination of both vectors. After transfection the cells were labeled with [ 35S]methionine. The cells were harvested, lysed, and incubated with rabbit preimmune serum (PI), antibody (Ab) to C/EBP␤, or antibody to STAT5b. The immune complexes were precipitated with protein A bearing Staphylocccus aureus and then resolved by gel electrophoresis. The arrow indentifies STAT5b, while the arrowhead identifies C/EBP␤.

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3– 4 h during the day, while female rats have a more steady continuous presence of GH in the blood (22). Short exposure of cultured liver CWSV-1 cells to GH results in a transient high peak level of STAT5b EMSA signal intensity (phase 1 signal), while continuous exposure to GH results in lower steady levels of STAT5b (Phase 2 signal) (23). Both phase 1 and phase 2 signals, which are characteristic for male and female GH secretion respectively, can activate gene transcription (23, 24). Increased ethanol elimination in women as compared to men (25) with more rapid formation of acetaldehyde may be an important factor in the greater susceptibility of women to alcoholic liver disease. In summary, this study shows that STAT5b and C/EBP␤ bind to adjacent nucleotide sequences on a region between ⫺226 and ⫺194 relative to the startsite of transcription in the rat class I ADH promoter. C/EBP␤ was known previously to bind only to region ⫺22 to ⫺11 in the promoter. Both C/EBP␤ and STAT5b activate the promoter and mediate the effect of GH in enhancing ADH. REFERENCES 1. Mezey, E. (1993) Alcohol Alcohol. Suppl. 2, 57– 62. 2. Potter, J. J., Yang, V. W., and Mezey, E. (1989) Arch. Biochem. Biophys. 274, 548 –555. 3. Potter, J. J., Yang, V. W., and Mezey, E. (1993) Biochem. Biophys. Res. Commun. 191, 1040 –1045. 4. Potter, J. J., Mezey, E., Christy, R. J., Crabb D. W., Stein, P. M., and Yang, V. W. (1991). Arch. Biochem. Biophys. 285, 246 –251. 5. Potter, J. J., Cheneval, D., Dang, C. V., Resar, L. M. S., Mezey, E., and Yang, V. W. (1991). J. Biol. Chem. 266, 15457–15463. 6. Carter-SU, C., Schwartz, J., and Smit, L. S. (1996). Annu. Rev. Physiol. 58, 187–207.

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