Proc. Nati. Acad. Sci. USAVol. 85, pp. 767-771, February 1988Biochemistry

Regulation of gene expression of class I alcohol dehydrogenaseby glucocorticoids

(glucocorticoid action)

Yu DONG*, LORENZ POELLINGER*, SAM OKRET*, JAN-OLOV HOOGt, HEDVIG VON BAHR-LINDSTROMt,HANS JORNVALLt, AND JAN-AKE GuSTAFSSON**Department of Medical Nutrition, Karolinska Institutet, Huddinge University Hospital F-69, S-141 86 Huddinge, Sweden; and tDepartment of Chemistry I,Karolinska Institutet, S-104 01 Stockholm, Sweden

Communicated by Sune Bergstrom, October 26, 1987

ABSTRACT The effect of glucocorticoids on gene expres-sion of rat class I alcohol dehydrogenase (ADH; alcohol:NAD+oxidoreductase, EC 1.1.1.1) was investigated. A cDNA clonefor the (3-subunit of human ADH (ADH2) was used to analyzeclass I ADH mRNA levels in rat hepatoma cells, which areknown to contain a functional glucocorticoid receptor. RNAgel blot analysis of total cellular RNA isolated from these cellsshowed hybridization of the human ADH2 cDNA probe to asingle -1500-base RNA species. Treatment of the cells withdexamethasone (0.1 nM to 1 ,uM) caused a dose-dependentincrease in total cellular class I ADH mRNA levels by a factorof 2-4. Maximal levels were reached within 18-24 hr oftreatment. This effect was reversible following withdrawal ofdexamethasone. The glucocorticoid induction of class I ADHmRNA does not seem to require ongoing protein synthesissince treatment of the cells with cycloheximide did not affectthe increase in class I ADH mRNA levels by dexamethasone.The humanADH2 gene contains both upstream and within thecoding region sequence motifs that display homology withresponse elements of genes positively regulated by glucocorti-coids. These data suggest a receptor-mediated transcriptionalenhancement of the ADH2 gene as the mechanism of regula-tion. However, analysis of RNA decay in cells treated withactinomycin D indicates that the dexamethasone-induced in-crease in class I ADH mRNA might, at least in part, be due toenhanced ADH mRNA stability.

Mammalian alcohol dehydrogenase (ADH; alcohol:NAD+oxidoreductase, EC 1.1.1.1) is a complex system of dimericzinc metalloenzymes capable of oxidizing a wide variety ofaliphatic and aromatic alcohols. Three classes (I-III) of thehuman enzyme have been distinguished (1) and structurallycharacterized (2-4), revealing both interclass enzyme differ-ences and intraclass isozyme differences (5). Different iso-zymes of class I constitute the "typical" enzymes ofADH inmammalian liver (for review, see ref. 2). The subunits of thehuman class I enzymes, a, 3, and y, are clearly relatedpolypeptide chains (2, 6) encoded by separate genes (7, 8).There exists a paucity of data on endocrine regulation of

ADH gene expression. However, it has been shown thatandrogens stimulate class I ADH gene expression in mousekidney both at the protein level (9) and at the level of cellularmRNA prevalence (10). Steroid hormones generally act byaltering the rates of transcription of specific target genes.The model for steroid hormone action entails hormonebinding to a soluble receptor protein, whereafter the hor-mone-receptor complex is thought to associate with specificDNA sequences adjacent to or within the transcribed seg-ments of regulated genes (for reviews, see refs. 11 and 12). In

the case of glucocorticoids, a number of genes have beenshown to contain such regions of DNA, termed glucocorti-coid response elements. They exhibit specific binding of theglucocorticoid-receptor complex in vitro and are necessaryand sufficient for hormone-regulated expression (12, 13).

In this study, we describe the induction of class I ADHgene expression in rat hepatoma cells by glucocorticoids.We also show that the human ,B-subunit ADH (ADH2) andrat class I ADH cDNA contain internal sequence motifsdisplaying similarity to previously defined (12) consensussequences of glucocorticoid response elements in positivelyregulated genes. Similar sequences have also been describedin the 5' flanking region of the human ADH2 gene (8).

MATERIAL AND METHODSCell Culture and Preparation of RNA. Rat Rueber hepa-

toma cells (H4IIE, hepatoma tumor cell line HTC) weregrown in monolayer culture in RPMI 1640 medium (FlowLaboratories) supplemented with heat-inactivated and char-coal-treated fetal calf serum (GIBCO) at a final concentra-tion of 8% (vol/vol), 2 mM L-glutamine (Flow Laboratories),and bensylpenicillin (400 units/ml; Astra, Sodertalje, Swe-den), streptomycin (0.2 mg/ml; Novo, Copenhagen). Re-moval of steroids from fetal calf serum was achieved bystirring the serum with 20%o (wt/vol) dextran-coated char-coal (Norit A; Sigma) for 2 hr at 37°C. Dexamethasone(Sigma) was added to the medium from a 10 mM stocksolution prepared in ethanol. The same volume of ethanolwas added to control cells. When indicated, cells weretreated with cycloheximide (1.5 pkg/ml) (Sigma) or actino-mycin D (5 ,ug/ml) (Sigma). Cell viability was determined byexclusion of 0.04% trypan blue. Greater than 95% viabilitywas observed for all conditions of treatment during theexperimental time periods. Total cellular RNA was isolatedby homogenization of cells in 4 M guanidinium thiocyanateand centrifugation in cesium chloride (14). RNA concentra-tions were determined from the optical density at 260 nm.Recombinant Plasmids. Plasmid pADH2 carries a 1.4-

kilobase (kb) cDNA insert encoding human ADH2, clonedinto the vector pT4 (15). The insert covers the full-length1122-base-pair (bp) ADH2 reading frame, a 5' 72-bp noncod-ing segment, and a 3' noncoding tail. A cDNA probe for13-actin was from Cleveland et al. (16).

Hybridization Analysis of RNA. In RNA gel blot experi-ments, equal amounts (20 ug) of heat- and formamide-denatured total cellular RNA were separated in 0.9%(wt/vol) agarose gels containing 2.2 M formaldehyde. TheRNA was transferred by capillary flow to nitrocellulosefilters (Schleicher & Schuell) and hybridized as described(17). Hybridization was performed for 16 hr at 50°C in buffer

Abbreviations: ADH, alcohol dehydrogenase; ADH2, P-subunit ofADH.

767

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Proc. Natl. Acad. Sci. USA 85 (1988)

containing radioactive probe (-2 x 106 dpm/ml). Probeswere labeled with [a-32P]dATP (3000 Ci/mmol; 1 Ci = 37GBq; Amersham) by nick-translation (17) to a specific activ-ity of =4 x 108 dpm/pAg. The filters were washed at 550C asdescribed (18). For slot blot experiments, RNA was immo-bilized on nitrocellulose filters with the use of a blottingapparatus (Schleicher & Schuell), after which the filterswere hybridized with 32P-labeled probes and washed asdescribed above. The signal intensity of the autoradiogramswas measured densitometrically with a Shimadzu dual-wavelength TLC scanner CS-930 (Kyoto, Japan).

RESULTSBasal and Enhanced Total Cellular Class I ADH mRNA

Levels. Class I ADH gene expression in rat hepatoma cellswas investigated by RNA gel blot analysis. Total cellularRNA was isolated from nontreated cells and subjected toanalysis with the plasmid pADH2 as a probe. This probecarries the full-length human ADH2 cDNA clone and hybrid-izes to a single RNA band at an electrophoretic positioncorresponding to a size of =1.5 kb (Fig. 1, lane -). This isthe same size as that reported for class I ADH mRNA in ratand mouse liver and kidney (10, 19). When rat hepatomacells were treated with 0.5 uM dexamethasone for 24 hr, anincrease in cellular class I ADH mRNA levels was detect-able (Fig. 1, lane +). Densitometric scanning of severalautoradiograms following both RNA gel blot and RNA slotblot analyses indicated that cellular ADH mRNA levels wereincreased by a factor of 2-4 by treatment with this concen-tration of dexamethasone. The size of the class I ADHtranscript is the same in dexamethasone-treated cells as incontrol cells (Fig. 1).To investigate the dose dependence of the effect of dexa-

methasone on class I ADH mRNA levels, rat hepatoma cellswere treated with a wide range of hormone for 24 hr.Subsequently, cellular class I ADH mRNA levels weremeasured by slot blot RNA hybridization. The results indi-cate no effect of dexamethasone at concentrations below 10nM but a maximal increase in ADH mRNA levels at =100nM dexamethasone (data not shown).

- + DEX

28S-

The time course of the effect of dexamethasone on cellularADH prevalence levels was studied. Within 12 hr of treat-ment with 0.5 ,M dexamethasone, there was an increase intotal cellular class I ADH mRNA, which reached maximallevels within 24 hr (Fig. 2) and remained stable up to 60 hr oftreatment. Withdrawal of hormone from cells treated with0.5 AM dexamethasone for 24 hr caused the induced cellularclass I ADH mRNA levels to return to control values withina further 24-48 hr (Fig. 3).

Effect of Cycloheximide on Glucocorticoid Induction ofClass I ADH mRNA. The dexamethasone-induced increasein cellular ADH mRNA levels also occurred in cells in whichprotein synthesis had been inhibited by treatment withcycloheximide for 24 hr (Fig. 4). At the concentration ofcycloheximide used (1.5 ,g/ml), protein synthesis was in-hibited by _95%, as measured by the incorporation of[35S]methionine into cytosolic protein precipitable by tri-chloroacetic acid. In both the absence and presence ofcycloheximide, treatment with 0.5 ,M dexamethasonecaused an =3-fold increase in cellular class I ADH mRNAlevels. During all treatments, cellular 13-actin mRNA levelsremained relatively constant.

Effect of Dexamethasone on Class I ADH mRNA Stability.The effect of dexamethasone on the decay of class I ADHmRNA in the presence of actinomycin D was investigated.RNA synthesis was inhibited by an average of 90% at theconcentration of actinomycin D used (5 ,&g/ml). Analysis ofADH mRNA in untreated cells revealed a decay from whicha half-life of the ADH mRNA of 5-6 hr can be deduced (Fig.5). In contrast, the class I ADH mRNA was stabilized in thepresence of hormone and exhibited no significant degrada-tion during a 7-hr period of treatment with actinomycin D, atthe end of which >90% of the class I ADH mRNA levelsremained detectable (Fig. 5). In control experiments, the,3-actin mRNA showed a decay rate with a half-life of 9-10hr, which was not altered by treatment with dexamethasone.

A

B

18S-S -ADH

400

- C 300:0

0 0< o 200-

0o

%ae 100

actin0

FIG. 1. RNA gel blot analysis of class I ADH mRNA in rathepatoma cells before and after treatment with dexamethasone(DEX). Total cellular RNA was isolated from control cells (lane -)or from cells treated with 0.5 ,uM dexamethasone for 24 hr (lane +),separated on O.9o agarose-formaldehyde gels, and transferred tonitrocellulose. Plasmid pADH2 or ,8-actin cDNA were labeled with32P by nick-translation and hybridized to the various RNAs asindicated. Each lane contains 20 ,ug of total RNA. The apparent sizeof the hybridization signal was judged from the electrophoreticmobilities of 18S and 28S rRNAs, the positions of which areindicated.

0 fi 1 2 2 4 6 () lit

* * **-ADH

606 12 18 24TIME (hr)

FIG. 2. Time course of alterations in cellular class I ADHmRNA levels in response to dexamethasone treatment. Rat hepa-toma cells were treated with 0.5 ,uM dexamethasone for the timesindicated. Total RNA was purified, applied to nitrocellulose filtersin dots, and hybridized with 32P-labeled cDNA probes for ADH2and 3-actin as described. Each dot contains 2 ,ug of total RNA. (A)Autoradiogram. (B) Relative hybridization signal intensities for classI ADH mRNA as calculated by densitometric scanning of theautoradiograms and related to the expression of ,B-actin.

I I I I %% I

M a kN

I I 1. I

768 Biochemistry: Dong et al.

Proc. Natl. Acad. Sci. USA 85 (1988) 769

1 2 3 4 A B-actin

* -ADH

" -B-actinl

B300

-

0

Q ' 2001 c

m 0

<

O 0 100

0Lib1 2 3 4

FIG. 3. Reversibility of the effect of dexamethasone treatmenton cellular class I ADH mRNA prevalence. Rat hepatoma cells weregrown in the presence of 0.5 uM dexamethasone for 24 hr. There-after, dexamethasone was withdrawn by change of medium, and thecells were grown for a further 48 hr. RNA measurements were madeby RNA gel blot analyses as described in the legend to Fig. 1. Totalcellular RNA was isolated as follows: at 0 hr of treatment (control;lane 1, bar 1); at 24 hr of treatment (lane 2, bar 2); 24 hr afterwithdrawal (lane 3, bar 3), and 48 hr after withdrawal (lane 4, bar 4).(A) Autoradiogram. (B) Mean values from the results of twoexperiments analyzed by densitometry and related to the expressionof 3-actin.

Identification of a Consensus Sequence for GlucocorticoidResponse Elements Within Class I ADH Genes. Computeranalysis of the cDNA sequence for human ADH2 (15) andrat class I ADH (19) revealed a region with 70-76% similarityto a 17-mer consensus sequence formulated for glucocorti-coid response elements of target genes such as mousemammary tumor virus and human metallothionein IIA (20).This sequence motif is located within the open reading frameof both the human ADH2 and rat class I cDNA from bases173-189 (Leu-57-Pro-62) and from bases 207-223 (Leu-57-Pro-62), respectively (Fig. 6). In addition, a similarsequence is located from bases 334-350 (Cys-111-Leu-116)in the human ADH2 cDNA but is poorly conserved in thecorresponding rat cDNA (76% and 59o similarity with theconsensus sequence, respectively).

DISCUSSIONIn the present study, rat hepatoma cells were used todemonstrate that dexamethasone can increase cellular classI ADH mRNA levels in a dose-dependent manner. Gluco-corticoids, and other steroid hormones, appear to regulategene expression via hormone-specific receptor proteins thatinteract with the steriod ligand and associate with nuclearbinding sites (11, 12). Many of the effects of glucocorticoidsinvolve altered transcription of a subset of genes in targetcells. In general, this effect is exerted as a direct or primaryresponse not influenced by inhibitors of protein synthesisand characterized by a rapid increase in the transcriptionrate of the inducible gene. This has been shown to be thecase in one of the best-studied examples of a primaryresponse, the induction of mouse mammary tumor virus

B300

0

: 200c< 0

I100

-

0 12E+

+

+ DEX+ CYC

FIG. 4. Effect of cycloheximide on induction of cellular class IADH mRNA levels by dexamethasone. Rat hepatoma cells wereexposed to 0.5 AM dexamethasone (DEX) for 24 hr in the presenceor absence of cycloheximide (CYC) (1.5 ug/ml). CYC was added 1hr before addition of DEX. Total cellular RNA was purified, and 2,ug was applied to nitrocellulose filters in slots. The filters werehybridized and analyzed by autoradiography as described. (A)Autoradiogram. (B) Average from the results of two experimentsanalyzed by densitometry and related to the expression of ,B-actin.

transcription by glucocorticoids (for reviews, see refs. 12and 13). A number of other glucocorticoid-responsive tran-scription units have been identified and characterized-e.g.,the genes of human growth hormone (22-24), human metal-lothionein (20), and rat tyrosine aminotransferase (25). Thecurrent model of action of steroid hormones entails associ-ation ofthe hormone-receptor complex to promoter/enhanc-er elements of specific target genes, resulting in activation oftranscription (12).The glucocorticoid-stimulated production of class I ADH

mRNA in rat hepatoma cells appears to represent a primaryevent following treatment with hormone. Induction of ADHmRNA was not inhibited by the presence of cycloheximide,an inhibitor of protein synthesis, indicating no requirementof ongoing protein synthesis for the cellular response to

100

o zz

Z7 6< 60

40zE 20E

0

2 4 6 8 10TIME (hr)

FIG. 5. Effect of dexamethasone treatment on class I ADHmRNA stability. Rat hepatoma cells were grown in the absence (A)or presence (A) of 0.5 ,uM dexamethasone for 24 hr. Thereafter,cells were treated with actinomycin D (5 ,ug/ml) for the indicatedtimes. Cellular class I ADH mRNA levels were determined asdescribed in the legend to Fig. 4.

A28S-

1 8S-

ADH

Mw-

_w +

._ + +

DEX CYC

Biochemistry: Dong et A

Proc. Natl. Acad. Sci. USA 85 (1988)

T G G T - A C A C - T G T T C T

-186 T G G T T A C A A A C T G T T C T

-226 T G T T A C A A T TT|T C AmC

-187 T G A A A C A A A A T C T T NT

173 T G G T G A C[CD C CT T C C T

334 T G T T A A A A T G9T C T

207 T GUT C AC CI JTiT C C T

-1 70

-21 0

-1 71

189

350

223

FIG. 6. Sequence similarity within the human ADH2 (J3ADH)and rat class I ADH cDNA glucocorticoid response elements.Putative- human ADH2 (15) and rat class I (rADH; ref. 19) internalglucocorticoid response element sequences are aligned with twosimilar sequences from the 5' flanking region of the human ADH2gene (8) and the consensus sequence (20). Also shown is a sequencemotif of a glucocorticoid response element in mouse mammarytumor virus (MMTV; glucocorticoid receptor-binding region 1.3;ref. 21). Mismatches are boxed.

hormone treatment. The hormone induction of ADH mRNAlevels could be due to an increase in the rate of its synthesisand/or a decrease in the rate of its degradation. The timecourse for the induction response (maximal mRNA levelswithin 18-24 hr of treatment) indicates a longer time framefor the response than expected for a predominantly tran-scriptional effect of the hormone. For instance, induction oftranscription of mouse mammary tumor virus is a rapidevent, where the maximal rate of transcription is reachedwithin 15 min after hormone treatment (26, 27). Therefore, itis possible that dexamethasone may affect the apparentstability of the 1.5-kb ADH mRNA species. In the case ofregulation of vitellogenin gene expression by estrogen, it hasbeen shown that the hormone stabilizes the vitellogeninmRNAs against cytoplasmic degradation (28). In line withthis model, the ADH mRNA was considerably more stablein the presence of hormone than in the absence of hormone,as assessed by determinations of mRNA decay in cellstreated with actinomycin D.

Glucocorticoids are important regulators of gene expres-sion in target cells. It is interesting that they also affect theexpression of a major hepatic gene product such as ADH. Inaddition, it has recently been reported that glucocorticoidsmodulate the expression of the rat hepatic albumin gene, themRNA of which constitutes -10% of the total poly(A)+RNA fraction in adult liver (29). This may imply a function ofglucocorticoids in maintaining/modulating the expression ofgenes that characterize the typical differentiated function ofhepatocytes and that have not previously been thought of as

target sites for endocrine stimuli because of the large poolsize of their mRNAs. For instance, it has been reported thatandrogens induce cellular class I ADH mRNA levels inmouse kidney but not in mouse liver, where class I ADH was

suggested to be constitutively expressed (10).In vitro, the purified glucocorticoid receptor interacts

selectively with target genes at discrete contact sites notonly upstream of the start site of transcription but also atseveral widely separated loci within transcribed and trans-lated sequences. In several cases, these contact sites havebeen shown to confer hormone responsiveness in cis (forreview, see ref. 12). Several sequences upstream of andwithin the coding region of human ADH2 gene have a closematch with such response and receptor-binding elements ofglucocorticoid-sensitive genes. Two sequence motifs up-stream of the ADH2 gene have been described (8), and twoadditional motifs reside within transcribed and, translatedsequences between bases 173-189 and bases 334-350, re-

spectively, one of which is poorly conserved in the rat (Fig.6). Internal binding sites for the glucocorticoid receptor have

been demonstrated in mouse mammary tumor virus and thehuman growth hormone gene, which are positively regulatedby glucocorticoids (21, 23, 24), as well as in the rat gluco-corticoid receptor cDNA, the corresponding mRNA ofwhich is negatively regulated by glucocorticoids (18). Theinternal sequence motifs within the coding region of theADH2 gene correspond to amino acids 57-62 and 111-116,respectively. These sequences reside in a variable region ofthe class I polypeptide, which displays interspecies differ-ences as compared both to other class I isozymes and toclass II and III forms ofADH (2). Whether some or all of theupstream and internal ADH sequence motifs are relevant fora possible transcriptional activation of the class I ADH geneby glucocorticoids is presently not known. Alternatively, itis conceivable that internal sequence elements might beimportant for the stability of the mRNA-e.g., by pro-tein-RNA interaction via the receptor protein, in line withthe effect of dexamethasone on class I ADH mRNA stabil-ity.

This work was supported by grants from the Swedish MedicalResearch Council (13X-2819, 03X-3592, 03X-3532, and 03X-7148).L.P. is a recipient of a research fellowship from the SwedishMedical Research Council, and S.O. is a research fellow of theKarolinska Institute.

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