Glucocorticoid-Dependent Action of Neural Crest FactorAP-2: Stimulation of Phenylethanolamine N-Methyltransferase
Gene Expression
Steven N. Ebert, Mary Beth Ficklin, Song Her, Brenda J. Siddall, Rose Ann Bell,Karunesh Ganguly, Kyoji Morita, and Dona L. Wong
Nancy Pritzker Laboratory, Department oJ Psychiatry and Behavioral Sciences, Stanford University School of Medicine,Stanford, California, U.S.A.
Abstract: AP-2 is a vertebrate transcription factor ex- DNA element consisting of the consensus sequencepressed in neural crest cells and their derivative tissues, 5’-GCCNNNGGC-3’ (Williams and Tjian, 1991a,b).including the adrenal medulla, where epinephrine is pro- The 5’ promoter-regulatory regions of many genesduced. AP-2 is shown to stimulate expression of the gene contain similar sequences and hence potential AP-2encoding the epinephrine biosynthetic enzyme phenyl- binding elements. In particular, AP-2 has been shownethanolamine N-methyltransferase (PNMT). However, to bind to and stimulate expression of several genesstimulation of the PNMT gene by AP-2 requires glucocor-ticoids and appears to be mediated through the interac- expressed in neural crest-derived tissues, including thetion of AP-2 with activated type II glucocorticoid recep- human insulin-like growth factor binding protein-Stors. Mutation of AP-2 and/or glucocorticoid receptor gene (Duan and Clemmons, 1995), the human proen-binding elemertts within the PNMT promoter disrupts the kephalin gene (Hyman et al., 1989), the rat neuronalability of AP-2 and glucocorticoids to induce PNMT pro- nicotinic acetylcholine receptor cr3 subunit gene (Yangmoter activity. These findings suggest, in the case of et al., 1995), and the human AP-2 gene itself (BauerPNMT, that AP-2 stimulates gene expression through a et al., 1994).novel glucocorticoid-dependent mechanism. Key Words: One tissue arising from the neural crest is the adrenalAP-2 —Glucocorticoids— Phenylethanolamine N-meth- medulla, the major site of peripheral catecholamineyltransferase gene—Adrenergic expression.J. Neurochem. 70, 2286—2295 (1998). production. Recently, AP-2 has been shown to stimu-
late expression of the gene encoding dopamine ~3-hy-droxylase (DBH), the catecholamine biosynthetic en-zyme responsible for the production of norepinephrinefrom dopamine (Greco et al., 1995). Through consen-
AP-2 is a developmentally regulated vertebrate tran- sus sequence matching, we have found that AP-2scription factor that is primarily expressed in neural DNA binding elements are also present in the genecrest cells and neural crest-derived tissues (Williams et encoding the final enzyme in the catecholamine bio-al., 1988; Mitchell et al., 1991; Winning et al., 1991). synthetic pathway, phenylethanolamine N-methyl-Recent studies suggest that this factor may be critical transferase (PNMT; EC 2.1.128), whose activity isduring development because transgenic mice lacking responsible for the conversion of norepinephrine toAP-2 die at or before birth due to “severe congenital epinephrine. Like DBH, PNMT is abundantly ex-defects” (Schorle et al., 1996; Zhanget al., 1996). pressed in adrenal chromaffin cells. However, AP-2The most notable of these defects include skeletal ab- regulation of PNMT gene expression has not been pre-normalities, extensive structural deficits in craniofacial viously demonstrated.development, and failure of the cranium, thorax, abdo- The PNMT gene is known to be responsive to gluco-men, and neural tube to close. AP-2 is thereforethought to play a critical role in regulating the expres-
Received November 21, 1997; revised manuscript received Janu-sion of genes required for the development of these ary 19, 1998; accepted January 20, 1998.structures. Address correspondence and reprint requests to Dr. D. L. Wong
AP-2 stimulates gene transcription by binding to at Nancy Pritzker Laboratory, Department of Psychiatry and Behav-specific DNA sequences in the 5’ promoter-regulatory toral Sciences, Stanford University School of Medicine. MSLSregion of target genes (Williams and Tjian, 199la,b). Building, Room P-106, Stanford, CA 94305-5485, U.S.A.Abbreviations used: DBH, dopamine /3-hydroxylase; GC, gluco-Binding is thought to occur through interaction of AP- corticoid; GR, glucocorticoid receptor; GRE, glucocorticoid2 homodimers, via a helix—span—helix motif, with a sponse element; PNMT, phenylethanolamine N-methyltransferase.
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AP-2 AND GLUCOCORTICOID REGULATION OF PNMT GENE 2287
corticoids (GCs) (Wong et al., 1992a,b; Kim et al., EXPERIMENTAL PROCEDURES1993; Ebert et al., 1994), and a functional GC responseelement (GRE) has been identified between —515 and DNase I footprinting—529 bp 5’ to the site of transcription initiation (Ross The plasmid pRP863LUC, which contains the PNMT pro-
moter sequences extending from —863 to +20 bp fused up-et al., 1990). According to classical mechanisms, the stream of the firefly luciferase gene, was digested with Hin-GRE is the target site for transcriptional activation dill, NheI, and XhoI to generate two restriction fragments,by GC-activated GC receptors (GRs) (Yamamoto, NheI—XhoI (—393 to —863) and HindIII—NheI (+24 to1985), and studies using transgenic mice lacking the —389), and the fragments were radiolabeled with [y32PJ-GR gene have recently shown that this receptor is nec- ATP (Du Pont NEN, Wilmington, DE, U.S.A.) using T4essary for the normal development of adrenergic chro- polynucleotide kinase (Life Technologies, Gaithersburg,maffin cells (Cole et al., 1995). Neither adrenergic MD, U.S.A.) as previously described (Ebert and Wong,chromaffin cells nor epinephrine was present in the 1995). To define the boundaries of AP-2 binding on bothadrenal glands of these GR-deficient mice. strands of PNMT promoter DNA,each fragment was radiola-beled uniquely at one end or the other. Thus, four sets of
It has become apparent, however, that although es- probes were generated (Fig. 1). Binding reactions were per-sential, GCs and GRs alone are insufficient for the formed on ice in the absence or presence of purified AP-2initiation of PNMT expression and differentiation to (4 fpu per reaction; I fpu = 10—25 ng of AP-2; Promega,the adrenergic phenotype. For example, intrauterine Madison, WI, U.S.A.). Each reaction contained 5 X io~injection of GCs in vivo (Teitelman et al., 1979; Bohn dpm of freshly isolated probe, 20 mM Tris-HC1 (pH 7.5),et al., 1981) or exposure of cultured adrenal explants or 40 mMKC1, 12% glycerol, 0.5 mg/ml bovine serum albuminpheochromoblasts to GCs does not induce precocious (fraction V; Boehringer Mannheim), 5 mM MgCI
2, and 1expression of PNMT in vitro (Teitelman et al., 1982; mM dithiothreitol, in a total volume of 50 ,ul. Following 2h on ice, samples were cleaved with RQ1 DNase 1(0.03 unitAnderson and Michelson, 1989; Michelson and Ander- per sample; Promega) and processed as described previouslyson, 1992), even though functional GRs appear to be (Ebert and Wong, 1995). To map the nucleotides, chemicalpresent in adrenergic precursor cells at least 24—48 h sequencing ladders (Maxam and Gilbert, 1977), generatedbefore the normal onset of PNMT expression (Seidi from these same probes, were electrophoresed simultane-and Unsicker, 1989; Michelson and Anderson, 1992; ously on the DNA sequencing gels (7% acrylamide). ResultsAnderson, 1993). Similarly, PNMT mRNA expression were visualized by autoradiography of the dried gel.is only slightly stimulated by GC exposure in PC12cells (Kim et al., 1993), a pheochromocytoma cell Plasmids
The plasmids pSPRSV-AP-2 and pSPRSV-NN (Williamsline. Furthermore, in RS 1 cells, a clonal derivative of and Tjian, 199 la,b) were kindly provided by Dr. T. Williamsthe PC12 cell line selected to express functional GR (Yale University, New Haven, CT, U.S.A.). The plasmidactivity, basal PNMT expression remains undetectable, pSPRSV-AP-2 is a construct containing the full-length mu-and it is not substantially induced by treatment with rine AP-2 eDNA, whereas the plasmid pSPRSV-NN is aGCs (Ebert et al., 1994). Therefore, other transcription control vector that does not contain any AP-2 eDNA. Thefactors may be required to enable GC-mediated stimu- plasmid pRP863LUC contains the region of the rat PNMTlation of PNMT gene expression. promoter extending from —863 to +20 bp fused to the firefly
luciferase gene (Ebert et al., 1994). Site-directed mutagene-As an important developmental transcriptional acti- sis of the PNMT promoter was performed as previously
vator, AP-2 is one candidate factor. The present study described using the mutagenic oligonucleotides shown be-investigates the potential involvement of AP-2 in low (Ebert et al., 1994). All plasmids were purified throughPNMT gene expression using transient cotransfection two successive CsC1 density gradients or using Qiagen (Va-assays with PNMT promoter—reporter gene constructs lencia, CA, U.S.A.) columns, and their identities were yen-and an AP-2 expression vector. Our findings suggest fled by restriction mapping and DNA sequencing analyses.that AP-2 cannot independently stimulate PNMT pro-moter activity, despite its ability to bind to multiple OligonucleotidesAP-2 sites in the 5’ promoter-regulatory region of the The oligonucleotides used for site-directed mutagenesisof the PNMT promoter were as follows, with mutant residuesPNMT gene. However, in the presence of GCs, AP-2 indicated by lowercase letters and the core consensus Se-significantly and markedly activates the PNMT pro- quence underlined: mut57 I = 5 ‘-TGCCATCAAATGaaC-moter. Mutagenesis studies demonstrate that AP-2 and GGGGCAGAGACTGCTC-3’; mut653 = 5 ‘-AGCAGC-the activated GR must bind to their cognate consensus GCATAGCCCCAttGCCCACAGGGAT-3’; mutGRE 1 =
elements in the 5’ PNMT promoter-regulatory region 5’ - CACAAGACAGAGGCCAGAgggGAGaaaCCTTTC-to achieve maximal PNMT promoter activation. More- TGAAGGAGGATA-3’; mutGRE2 = 5 ‘-GTACCAGGGCover, endogenous PNMT gene expression appears to CACCAGACAGAGCTGAAGGAGGATAGAGAbe similarly induced by AP-2 and GCs. Finally, we CGGGG-3’ (GRE deleted); mutGRE3 = 5’-GGCCAGctC-
AcAcTGatCTTTCTGAAGGAGG-3” and mutGRE4 = 5’-show that AP-2 is expressed in adrenal chromaffin cells GGCCAGCACAcACTaTTaTTTCTGAAGGAGG-3’.in vivo. These findings implicate AP-2 as a potential For gel mobility-shift assays, the following complemen-regulator of PNMT gene expression and suggest a tary 31-mer oligonucleotides (sense strand shown) were syn-novel, hormone-dependent mechanism for AP-2 ac- thesized: wtGRE = 5 ‘-CAGAGGCCAGAACAGAGTGTC-tion. CTTTCTGAAG-3’; mutGRE = 5 ‘-CAGAGGCCAGAggg-
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2288 S. N. EBERT ET AL.
from DNA International (Lake Oswego, OR, U.S.A.) or Op-eron Technologies (Alameda, CA, U.S.A.).
Gel mobility-shift assaysGel mobility-shift assays were performed essentially as
described previously (Ebert et al., 1994). To analyze GR—GRE interactions, T7X556, a purified protein fragment con-sisting of the DNA binding domain of the rat type II GR(Freedman et a!., 1988), was added to binding reactions(=60 ng per reaction) containing the radiolabeled duplexwtGRE oligomer probe (=0.25 ng per reaction; specific ac-tivity, 1 X io~dpm/~tg)and poly(dA~dT).poly(dA~dT)(0.4 ~zgpçr reaction) in binding buffer identical to that usedfor the footpninting reactions. Binding reactions for AP-2were conducted similarly. Purified AP-2 protein (=5 ng perreaction; Promega) was incubated with the radiolabeled du-plex wtAP-2 (—653) probe (=0.25 ng per reaction; specificactivity, 1 x l0~dpmI~.eg)in binding buffer containing poly-(dA.dT)~poly(dA~dT)(4.0 jsg per reaction). Unlabeledcompetitor duplex oligomers were included in some of thereactions, as indicated in the text and figure legends. Analy-sis of the resulting protein—DNA complexes and calculationof IC50 values were performed as described previously (Ebertand Wong, 1995).
Cell culture and transient transfection assaysRS1 cells were maintained as previously described (Ebert
et al., 1994). Serum was depleted of steroids by pretreatmentwith activated charcoal, and RS 1 cells were passaged a mini-mum of three times in medium containing charcoal-treatedserum before experiments were performed. Transient trans-fection assays were also conducted as previously described(Ebert et al., 1994). To control for differences in transfectionefficiency, luciferase activity was expressed relative to totalprotein and ~l-galactosidaseactivity produced from a controlcotransfected plasmid (pCMV-/3GAL) (Ebert et al., 1994).Drug treatment was initiated 16—20 h after transfection, andthe cells were harvested 6 h later. Corticosterone and dexa-methasone were purchased from Sigma Chemical Co. (St.
FIG. 1. AP-2 binding sites in the PNMT promoter. A: Schematic Louis, MO, U.S.A.), and the type II GR antagonist, RUrepresentation of the rat PNMT promoter. The site of transcrip- 38486, was obtained from Roussel-UCLAF (Romainville,tion initiation (arrow) is designated at +1. Relevant restriction France). Stock solutions (10 mM) of all steroids were pre-enzyme sites and consensus binding sites for Egr-1 and Spi pared in dimethyl sulfoxide and stored at —80°C. Before(—45 and —165 bp), the GRE (—515 bp), and the putative AP- use, drugs were diluted in serum-free Dulbecco’s modified2 binding sites (—103, —571, and —653 bp) are shown. B: DNase ,
I footprinting analysis of AP-2 binding to the rat PNMT promoter. Eagle s medium to bOx the final desired concentration. ForThe probes designated *Hind3(+24) and *Nhel(_389) repre- the antagonist experiment, RU 38486 was added to the cellsent Hindlll—Nhel restriction fragments, whereas the probes culture medium I h before dexamethasone treatment. Alldesignated *Nhel (—393) and *Xhol (—868) represent Nhel—Xhol transfection assays represent the combined results from arestriction fragments derived from pRP863LUC. All probes were minimum of two experiments where n = 3—6 samples peruniquely radiolabeled at one end (asterisks). Each fragment was experiment. Statistical significance was determined by one-incubated in the absence (odd lanes) or presence (even lanes) way ANOVA.of purified AP-2 and subjected to cleavage by DNase I, and theresulting fragments were resolved on a DNA sequencing gel. RNase protection assayOnly those portions of the autoradiogram containing regions of Poly( A) + RNA was isolated from RS 1 cells using theprotection are shown. The protected nucleotides were mapped Micro-FastTrack kit from Invitrogen (San Diego, CA,by comparison with adjacent sequencing ladders and are mdi- U.S.A.). To detect PNMT mRNA, 2.5-~.tg samples ofcated by the vertical bars to the right of each footprint. poly (A) + RNA from RS 1 cells were hybridized with an
antisense PNMT riboprobe and analyzed as previously de-GAG a a a C CT T T CT GA AG -3’; wtlO3 = 5’- scribed (Ebert et a!., 1994). Total RNA from an adult ratGAGATGTGGCGGCCTCGGCGCCTCATCCCTC-3’; adrenal gland (2.5 ~g per hybridization) and known concen-wt57 1 = 5 ‘-TGCCATCAAATGCTCGGGGCAGAGACT- trations of PNMT sense RNA standards were used as con-GCTC-3’; and wt653 = 5 ‘-AGCAGCGCATAGCCCCAG- trols. All hybridizations contained a total of 50 ~zgof RNA,GGCCCACAGGGAT-3’. with yeast tRNA (Life Technologies) making up the differ-
The 31-hp oligonucleotides with mutated —571 and —653 ence. As an additional control, a rat /3-actin antisense ribo-bp AP-2 sites are the those shown above, mut57l and probe was used to evaluate ~3-actinmRNA concentrationsmut653, respectively. All oligonucleotides were obtained in the same RNA samples (Wong et al., 1993).
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AP-2 AND GLUCOCORTICOID REGULATION OF PNMT GENE 2289
Immunohistochemistry lanes 6 and 8), and —87 to —120 bp (Fig. 1B, lanesPregnant Sprague—Dawley rats, obtained from Simonsen 2 and 4), spanning the AP-2 binding sites identified
Labs (Gilroy, CA, U.S.A.), were maintained on a 12:12 by consensus sequence identity above.day—light cycle (lights on at 6:00 am.) and provided food Other minor regions of protection were observed,and water ad libitum. On postnatal day 10, rat pups were but they were not of sufficient length to constitute akilled by decapitation. Their adrenal glands were immedi- legitimate AP-2 binding site. Moreover, the three ma-ately removed, rinsed in phosphate-buffered saline, and im-mersion-fixed in phosphate-buffered saline containing 4% jor sites were the only ones that were consistently resis-paraformaldehyde for 2 h at 0°C.The adrenal glands were tant to DNase I cleavage following binding of AP-2then equilibrated at 4°Cin phosphate-buffered saline con- to the overlapping regions of both strands of PNMTtaming 30% sucrose, frozen, and stored at —80°C. Sections promoter DNA (HindIII—NheI, +24 to —389 bp;(10 tim) were mounted on Superfrost/Plus slides (Fisher NheI—XhoI, —393 to —863). Numerous DNase I hy-Scientific, Pittsburgh, PA, U.S.A.), and immunohistochemi- persensitive sites were also apparent at positions imme-cal staining was performed as described previously (Ebert diately flanking the major regions protected by AP-2et a!., 1994). The animal protocols used for these studies binding. In addition, footprinting analysis using probeshave been approved by the Institutional Animal Care and overlapping the NheI site at the junction between theUse Committee at Stanford University School of Medicine two sets of probes shown in Fig. 1 did not reveal anyand meet the guidelines designated by the National Institutes
AP-2 binding sites in the junctional region (data notof Health.
The AP-2 antibody and peptides were obtained from Santa shown). Thus, the patterns of DNase I protection andCruz Biotechnology (Santa Cruz, CA, U.S.A.). The peptide hypersensitivity depicted in Fig. I B demonstrate thatused to generate the AP-2 antibody corresponds to amino AP-2 binds to the PNMT promoter at the three posi-acids 420—437 from the human AP-2 protein. The control tions depicted in Fig. 1A.peptide used in these experiments represents the carboxy-terminal 19 amino acids from the mouse Egr- I protein. Pro- AP-2 stimulation of PNMT promoter activityduction and characterization of the rabbit polyclonal anti- The ability of AP-2 to bind to multiple discrete re-PNMT antiserum have been described elsewhere (Wong et gions of the rat PNMT promoter suggested that AP-2a!., 1987).
Primary antibody incubations were performed with either might be involved in regulating PNMT gene expres-sion. To test this hypothesis, the firefly luciferase gene
the AP-2 antibody (1:5,000 dilution) or the PNMT antibody(1:4,000 dilution) in buffer containing 0.3% Triton x-lOO, under the control of the rat PNMT promoter5% nonfat powdered milk, and 20 mM phosphate-buffered (pRP863LUC, —863 to +20 bp) was used as a reportersaline. For blocking experiments, peptides (0.4 ~.tg/ml)were of PNMT promoter activity in transient transfectionincubated with the primary antibody solution for 16 h at assays. The PNMT—buciferase plasmid construct was4°Cbefore use. Primary and secondary antibody incubations cotransfected into RS I cells with either an AP-2 ex-were performed as previously described (Ebert et al., 1994). pression vector (pSPRSV-AP-2) or a control vectorPositive PNMT immunoreactivity was visualized by 3,3 ‘ backing any AP-2 eDNA (pSPRSV-NN). AP-2 proteindiaminobenzidine staining using the Vectastain Elite ABC was abundantly expressed in RS 1 cells transfected withkit (Vector Laboratories, Burlingame, CA, U.S.A.). Nickel the AP-2 expression vector as shown by immunostain-chloride (4 mg/ml) was added to the 3,3 ‘-diaminobenzidinesolution to enhance AP-2 immunoreactivity. ing (see Fig. 6), although it is not normally present
in RS 1 cells as determined by western analysis, immu-nohistochemistry, and gel mobility-shift assays with
RESULTS RS 1 cell nuclear extracts (data not shown). However,AP-2 binding to the PNMT promoter no stimulation of buciferase was observed in the pres-
Examination of the rat PNMT 5’ promoter-regula- ence of AP-2 alone. Instead, AP-2 consistently reducedtory region (Fig. IA) revealed the presence of DNA basal luciferase activity up to 50% compared with thesequences that resemble consensus DNA bitiding ele- activity produced in control cotransfections (Fig. 2A).ments for the AP-2 transcription factor (5 ‘-GCCNNN- However, in the presence of dexamethasone, a potentGGC-3’). There are two sites where five of six nucleo- synthetic GC, AP-2 did stimulate luciferase activity.tides match the AP-2 consensus sequence, located at Significant stimulation of buciferase was apparent at—653 and —571 bp, respectively, and a site with a four 0.1 1i.tM dexamethasone (p < 0.0001), with maximalof six nucleotide match at —103 bp. To determine if induction (=4.5-fold) occurring at 1.0 ,uM dexa-these and/or other regions of the PNMT promoter methasone (Fig. 2A). In this study, dexamethasonecould serve as AP-2 binding sites, DNase I footprinting did not independently alter luciferase activity, whereasanalysis was performed using purified AP-2 protein in other studies (Ross et al., 1990; Ebert et a!., 1994)and radiolabeled DNA probes from the PNMT pro- up to a twofold activation of the reporter gene hasmoter (Fig. 1B). The footprinting results showed that been observed. Thus, although AP-2 alone does notthere were three major binding sites for AP-2 within stimulate PNMT promoter activity, it markedly in-the region of the PNMT gene extending from +20 to duces reporter gene expression from the PNMT pro-—863 bp (relative to the transcriptional start site at +1 moter in the presence of dexamethasone.bp). These sites are located between —646 to —671 We further demonstrated that GC-dependent AP-2(Fig. lB, lanes 6 and 8), —571 to —592 (Fig. lB. induction of the PNMT promoter could be blocked by
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2290 S. N. EBERT ET AL.
FIG. 2. Dose-dependent actions of dexamethasone and RU 38486 on luciferase expression from the PNMT promoter. The PNMTpromoter-driven luciferase plasmid construct pRP863LUC was cotransfected with either an AP-2 expression vector or a control vector(NN) into RS1 cells. Approximately 20 h posttransfection, the cells were treated with increasing concentrations of (A) dexamethasoneor (B) RU 38486. In A, the samples were harvested after 6 h and analyzed for reporter gene activity. In B, cells were pretreated withRU 38486, followed by treatment with 0.1 p~Mdexamethasone; the cells were then harvested for reporter gene level measurementsafter an additional 6 h of incubation. Relative luciferase activity is expressed as luciferase activity (square root of dpm produced) per/3-galactosidase unit (A420) per microgram of total protein (Ebert et al., 1994). ***Significantly different from AP-2 control, p < 0.0001.
the type Il-specific GR antagonist RU 38486 in a dose- the core consensus sequence) of the moderate-affinitydependent manner (Fig. 2B). The transfected cells —571 bp site were replaced with thymine and adeninewere pretreated with increasing concentrations of RU residues (lowercase letters), respectively, both sites38486 (0—10 ,aM) followed by incubation with dexa- became low-affinity AP-2 binding elements (IC50methasone (0.1 1.tM). At concentrations of 1 tiM, >2,000 nM). Because the AP-2 site at —103 bp al-RU 38486 completely prevented buciferase induction ready shows low affinity for AP-2 binding, no pointby AP-2 and dexamethasone (Fig. 2B), whereas alone, mutations were introduced into this element. Finally,it did not significantly affect luciferase activity pro- when targeted mutations were introduced into theduced in control cotransfections (NN). Thus, PNMT GRE, GR binding affinity declined >45-fold (relativepromoter stimulation by AP-2 and dexamethasone ap- IC50 increased from 43 to >2,000 nM).pears to require the activation of a type II GR. The oligonucleotides containing these mutated DNA
consensus binding sequences were then incorporatedFunctionality of AP-2 and GR binding elements individually or in combination into PNMT—luciferasein the PNMT promoter
To determine the contributions of the AP-2 bindingsites and the GRE to PNMT promoter activation byAP-2 and GCs, site-directed mutagenesis of the DNA TABLE 1. Relative binding affinities of wild-type (wt)binding elements was performed, and !uciferase re- and mutant (mat) AP-2 and GR DNA binding elements in
the PNMT promoterporter gene expression was examined in mutant _______________________________________________
PNMT—buciferase reporter gene constructs. Methy!a- Competitor Core consensus 1C5()tion interference assays, which identify bases im- oligomer sequence (nM)portant for protein—DNA interaction, were used to se-lect nucleotides for point mutation (Williams et al., AP-2 “consensus” G C C N N N G G C ND1988; S.N.E. and D.L.W., data not shown). Wild-type wt653 G CCC CA G G G 11mut653 GCCCCAtLG >2,000and mutant oligonucleotides were synthesized, and rel- wt571 G C T C G G G G C 160ative binding affinities for the wild-type and mutant mutS7l G a a C G G G G C >2,000DNA binding sequences were determined by gel mo- wtlO3 G C C T C G G C G >2,000bility-shift competition assays. As shown in Table 1, GRE “consensus” AGA ACA NNN TGT TCT ND
wtGRE AGA ACA GAG TGT CCT 43the AP-2 element at —653 bp is a relatively high- mutGRE AGA ggg GAG aaa CCT >2,000affinity binding site forAP-2 (IC50 = 11 nM), whereasthe AP-2 element at —571 bp is a moderate-affinity Purified recombinantAP-2 and GR (T7X556) proteins were incu-site (IC50 = 160 nM), and the AP-2 element at —103 bated with oligomer probes containing the wt PNMT AP-2 and GRbp is a low-affinity site (IC50 2,000 nM). When the DNA binding elements, respectively. Gel mobility-shift assays were
used to separate and quantify the amount of AP-2 or GR proteintwo guanine residues at —650/1 bp (3’ end of the core bound in the presence of increasing concentrations of wt or mutconsensus sequence) of the high-affinity —653 bp site competitor duplex oligomers (0—2,000 nM). IC50 values were calcu-and the cytosine residues at —573/4 bp (5’ end of lated as described previously (Ebert and Wong, 1995). ND, not done.
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AP-2 AND GLUCOCORTICOID REGULATION OF PNMT GENE 229]
FIG. 3. Site-specific mutagenesis of AP-2 and GR DNAbinding elements. Wild-type and mutant PNMT promoter constructs used fortransient transfection analysis are shown with “X” designating a mutated element. Oligonucleotides used to generate the mutationsare specified in Experimental Procedures. The specific base changes and their effects on AP-2 binding affinity are listed in Table 1.For dexamethasone treatment, the drug (1
1iM) was applied to the cultures 20 h posttransfection, and the cells were harvested forreporter gene measurements 6 h later. Luciferase activity is expressed as fold induction in response to dexamethasone and AP-2relative to the NN control. Fold induction was determined by first calculating the ratio of luciferase activity in the presence of AP-2versus NN when glucocorticoids were absent or present. These ratios were then used to calculate the ratio of luciferase expressionin the presence of both AP-2 and GC5 versus luciferase expression with AP-2 alone. aSignificantly different from control (pRP863LUC),p < 0.01; bsignificantly different from control (pRP863LUC), p < 0.0001; °significantlydifferent from pRPmut653LUC, p < 0.0001;4significantly different from pRPmut571LUC, p < 0.0001; esignificantly different from pRPmutGRE1LUC, p < 0.01.
reporter gene constructs, and the ability of the mutant methasone (I jiM) on luciferase expression from theseconstructs to respond to AP-2 and dexamethasone was constructs were examined in transient transfectionexamined by transient cotransfection assay (Fig. 3). assays (Fig. 4). All contructs, even that with the GREAgain, treatment with the combination of AP-2 and eliminated, showed luciferase induction equivalent todexamethasone induced wild-type PNMT—luciferase =50% of that observed with the wild-type constructactivity (pRP863LUC) =4.5-fold. It is surprising that or approximately twofold basal levels in the absencemutation of the distal AP-2 site (pRPmut653) resulted of AP-2.in a small but significant induction of luciferaseby AP- Thus, it appears that the moderate-affinity —571 bp2 and dexamethasone (4.9-fold), whereas disruption of AP-2 site is essential for most of AP-2 and GC/GReither the intermediate-affinity AP-2 site (pRPmut57 1) activation of thePNMT promoter, although stimulationor the GRE (pRPmutGREI) reduced luciferase activa- is still observed with the other two AP-2 sites.tion >2.0-fold. When both the —653 and —571 bp AP-2 sites were altered, induction of luciferase was only Biological significance of AP-21.5-fold compared with the wild-type PNMT pro- To examine the biological significance of AP-2/GCmoter—luciferase reporter gene construct, and with the induction of PNMT promoter activity, endogenousadditional mutation of the GRE (pRPmut653/57l / PNMT mRNA concentrations were analyzed in RS1GRE 1LUC), no significant activation of luciferase was cells transfected with either the functional AP-2 ex-observed with dexamethasone alone or in the presence pression vector or the corresponding control vectorof AP-2 (Fig. 3). (NN), followed by incubation in the presence or ab-
Despite the fact that the mutation of the GRE re- sence of corticosterone, the endogenous GC in the rat.duces the IC
511 of the GR for the GRE to >2,000 nM, PNMT mRNA ~1pg/jig of poly(A)~RNA] was onlytransient transfection assay with the corresponding detected in the presence of both AP-2 and corticoste-plasmid construct (pRPmutGREb) still showed a re- rone (Fig. 5, bane 4). In the absence of AP-2 andsidual 50% activation of luciferase activity (Table I corticosterone or in the presence of either factor alone,and Fig. 3). Three additional mutant GRE/PNMT pro- PNMT mRNA appeared to be either absent or presentmoter—luciferase pbasmid constructs were therefore at levels below the lower limits of sensitivity of thegenerated, including two alternative point mutants RNase protection assay 1<0.2 pg/jig of poly(A)~(pRPmutGRE3 and pRPmutGRE4) (Bruzdzinski et RNA; Fig. 5, lanes 1—3; control RNA samples areal., 1993) and one mutant with the entire GRE deleted shown in lanes 5—101. Similar results have been ob-(pRPmutGRE2), and the effects of AP-2 and dexa- served using dexamethasone(b jiM) instead of cortico-
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2292 S. N. EBERT ET AL.
FIG. 4. Alternative site-specific muta-genesis of GR DNA binding element.The specific base changes introducedinto the GRE are shown, and the re-sponse of each mutant GRE plasmidconstruct to dexamethasone and AP-2is designated adjacent to the sche-matic. Dexamethasone (1 pM) was ap-plied directly to the cultures at 20 hposttransfection, and the cells wereharvested for reporter gene level mea-surements 6 h later. Luciferase activityis expressed as fold induction in re-sponse to dexamethasone and AP-2.eSignificantly different from wild-typecontrol, p < 0.0001; bsignificafltly dif-ferent from pRPmutGRE1LUC, p <0.001; °significantly different from allother constructs, p < 0.05.
sterone (data not shown). These results therefore dem- PNMT antiserum (Fig. 6b) showed a PNMT stainingonstrate that the endogenous PNMT gene in RS 1 cells pattern remarkably similar to that seen for AP-2 (com-can be induced by AP-2 and GCs just as they activate pare Fig. 6a and b), except that PNMT staining wasthe PNMT promoter to stimulate luciferase expression cytoplasmic. Preincubation of the primary antibodyfrom the PNMT—luciferase reporter gene construct. with the AP-2 peptide completely blocked AP-2-im-
The above finding is consistent with the apparent munoreactive staining (Fig. 6c), whereas inclusion ofrole of AP-2 in the differentiation of tissues of neural an equivalent amount of a nonspecific peptide had nocrest origin, because the adrenal medulla is neural effect (Fig. 6d). These results indicated that the ob-crest-derived. We therefore examined AP-2 protein ex- served staining was specific for AP-2. Thus, AP-2 ispression in vivo in adrenal chromaffin cells of rat pups expressed in the rat adrenal medulla in vivo at a timeon postnatal day 10, the developmental time point point when PNMT and adrenergic differentiation iswhen the adrenal PNMT level reaches its peak (Wong occurring in the adrenal chromaffin cells.et al., l992b). Serial adrenal sections were immunohis-tochemicalby stained with either an anti-AP-2 peptide DISCUSSIONantibody or an anti-PNMT antiserum (Fig. 6). DiscreteAP-2-specific staining was concentrated in the nuclei In this report, we have shown that AP-2 stimulatesof cells throughout the medulla but was absent in the PNMT promoter activity only in the presence of GCs.cells of the cortex (Fig. 6a). Higher magnification us- In the absence of GCs, AP-2 reduced PNMT promotering phase-contrast microscopy confirmed the nuclear activity up to 50%, suggesting that AP-2 may alsolocalization of AP-2 (data not shown). have a negative influence on PNMT gene expression.
Staining of an adjacent adrenal section with anti- Although examples of negative regulation by AP-2have been reported (Buettner et al., 1993; Gaubatz etal., 1995), negative regulation of PNMT gene expres-sion is difficult to measure in RSI cells because unin-duced endogenous PNMT mRNA is undetectable inthese cells. In fact, there are no cell lines currentlyknown to express appreciable amounts of PNMT. Con-sequently, for the purpose of the present study, wehave focused solely on the activation of PNMT geneexpression.
AP-2 binding sites and PNMT promoterAll three AP-2 binding elements in the rat PNMT
FIG. 5. Endogenous PNMT mRNA induction. Endogenous promoter, identified from consensus sequence similar-PNMT mRNA was quantified by the RNase protection assay ity, were shown to bind AP-2 protein. However tran-described previously (Ebert et al., 1994). RS1 cells were trans- . . .
fected with either a control vector (NN; lanes 1 and 2) or the sient transfection assays with site-directed mutantAP-2 expression vector (lanes 3 and 4). Corticosterone (CaRT; PNMT promoter—luciferase reporter gene constructs10 pM) was applied to half of the samples (lanes 2 and 4) —20 demonstrated that the —571 bp AP-2 binding elementh,after transfection, and the cells were harvested for RNAanaly- was the most important site for PNMT promoter acti-sis 24 h later. Lane 5 contains total RNAfrom an adult rat adrenal . .
gland. As an additional control, rat /l-actin mRNA was also ana- vation by AP-2 and dexamethasone. Disruption of thelyzed in the same RNAsamples. Lanes 6—10 contain 0, 0.5, 2.0, —571 bp binding site permitted only a twofold induc-5.0, and 20.0 pg of PNMT sense RNA, respectively. tion of buciferase by AP-2 and dexamethasone com-
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FIG. 6. AP-2 expression in the rat adrenal medulla in vivo. Serial sections (10 pm) from a rat adrenal gland were immunohistochemicallystained for either (a) AP-2 or (b) PNMT. Positive staining is indicated by 3,3’-diaminobenzidine precipitation, which appears dark. Todemonstrate the specificity of the observed AP-2 staining, additional sections were incubated with the AP-2 antibody (AB) andprocessed in a manner similar to that used in (a), except that either (c) the specific peptide (PEP) recognized by the AP-2 AB or (d)an equivalent concentration of a control PEP was included during the incubation with the primary antibody. The adrenal cortex (CTX)and medulla (MED) are indicated in each section.
pared with the four- to fivefold induction seen with affinity for the —571 bp AP-2 binding element thanthe wild-type reporter gene construct. Mutation of the for the —653 bp site, the binding affinities and func-—653 bp AP-2 binding site caused a slight, but signifi- tionality of the AP-2 sites would then in fact be consis-cant, activation of luciferase (4.9- vs. 4.4-fold induc- tent.tion by comparison with wild-type control). The fact As described earlier, the proximal AP-2 binding ele-that mutation of this site did not lead to a decline in ment at —103 bp shows very low affinity for AP-2luciferase activity indicates that this site alone does (Table 1) and is a weak consensus match (four of sixnot appear important for GC-mediated AP-2 activation bp), thereby making it an impractical target for site-of the PNMT promoter. Because the —653 bp AP-2 directed mutagenesis. Although the participation ofsite had the highest binding affinity for AP-2 (IC50 = this site in GC-mediated AP-2 activation of the PNMT
1 nM, Table 1), the results suggest that proximity of promoter cannot be completely excluded, the fact thatthe AP-2 element to the GRE may be a more critical site-directed mutagenesis studies showed that lucifer-parameter than binding affinity. When AP-2 bound to ase was not activated by AP-2 and dexamethasonethe —571 bp site interacts with the GRE, perhaps fur- when both distal AP-2 sites and the GRE were mutatedther interaction between the GR and AP-2 bound to suggests that any contribution from this site is likelythe —653 bp site is not possible. Recently, another minimal. However, we cannot exclude the possibilityAP-2 transcription factor, AP-2/3 (Bauer et al., 1994), that the AP-2 sites and and the activated GR functionwhich apparently arises from a second AP-2 gene, was interdependently.identified in the mouse. AP-2/3 and the originally iden-tified AP-2a have distinct temporal and spatial patterns Type II GR activity and the PNMT promoterof expression during devebopment, with AP-2/3 being GREsolely expressed in the adrenal medulla. An AP-2j3 GCs mediate transcriptional control through theirhomologue may be uniquely expressed in the rat adre- activation of intracellular, cytosolic receptors (Yama-nal medulla as well, and if this homologue has a higher moto, 1985; Funder, 1992), and in the case of the
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2294 S. N. EBERT ET AL.
PNMT promoter, this stimulation is likely effected ulate the PNMT gene, and to determine the significancethrough a type II or classical GR, as the type Il-specific of these findings in vivo.GR antagonist, RU 38486, blocked the AP-2/dexa-methasone-mediated increase in luciferase production. Acknowledgment: The work was supported by grantBecause mutation of the solely identified GRE (—515 DK5 1025 from the National Institute of Diabetes and Diges-bp) in the PNMT promoter significantly reduced the tive and Kidney Diseases, the Marianne Gerschel Charitabledegree of luciferase stimulation produced in response Account, the Spunk Fund, the Edward and Marjorie Grayto AP-2 and dexamethasone, the target site for the Endowment Fund, the Sobel Account, and the endowmentof the Nancy Pritzker Laboratory. S.N.E. was supported byactivated GR appears to be this consensus GRE. I-loW NSRA MHIO3SO from the National Institute of Mentalever, GC activation (twofold) still occurred when the Health.GRE was altered, despite the reduction in relative bind-ing affinity to >2,000 nM, rendering a very-low-affin-
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