ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 338, No. 1, February 1, pp. 97–103, 1997Article No. BB969801

Cyclic AMP Regulation of Mouse Proline-Rich ProteinGene Expression: Isoproterenol Induction of AP-1Transcription Factors in Parotid Glands

Jie Zhou,1 Paul S. Wright,2 Elsie Wong,3 Katayoun Jessen,4

Janice N. Morand, and Don M. Carlson5

Section of Molecular and Cellular Biology, University of California, Davis, California 95616

Received June 14, 1996, and in revised form November 4, 1996

Key Words: proline-rich proteins; isoproterenol; cy-clic AMP; transcription factors.Proline-rich protein mRNAs are increased dramati-

cally in the salivary glands of rats, mice, and hamstersupon treatment with the b-agonist isoproterenol. Se-quence comparisons between mice and hamster pro-

The salivary glands of various animals, including hu-line-rich protein genes identified conserved regionsmans, have the ability to synthesize a group of tissue-upstream from the transcription start site. Reporterspecific proteins, proline-rich proteins (PRPs),6 whichplasmids containing these 5*-flanking sequences fromare unusually high in proline (1, 2). These proteinsa mouse proline-rich protein gene, MP2, were con-normally are either undetectable or are present in verystructed and tested for transcriptional regulation bylow amounts in the rat, mouse, and hamster. NortherncAMP. Transient transfection experiments in mouse

L-M cells showed that the upstream region 0702 to blot analysis and cell free translations show that PRP0322 bp relative to the transcription start site is suffi- mRNAs in these rodents can be induced by the b-ago-cient to confer cAMP induction on a heterologous pro- nist isoproterenol (3–5). Large changes in PRP genemoter. Multiple copies of the AP-1 sequence elements expression occur concurrent with a dramatic increasewithin this region (0625 to 0551) mediate the cAMP in salivary gland hypertrophy and hyperplasia in b-transcriptional response of reporter gene expression agonist-treated animals (2, 6). In addition to salivaryin L-M cells. L-M cell nuclear proteins and purified gland PRPs, a separate class of PRPs called small PRPshuman c-jun protein bind to these upstream elements (sPRPs) has been identified in tracheal epithelial cellsas determined by DNase I footprint analysis. Nuclear of several species, including humans (7). Pig trachealproteins isolated from mouse parotid glands protected sPRP1 is almost identical to the protein encoded bythe consensus AP-1 binding site 5*-TGAGTCA-3 * (0592 the human spr1 gene. sPRP1 levels are regulated byto 0586). The nuclear proteins interacting at this site retinoids and cAMP, whereas human spr1 gene expres-were increased about sixfold in glands isolated from sion is regulated by phorbol esters and uv light (8–isoproterenol-treated mice when compared with

11). The mRNA for sPRP1 is dramatically induced inglands from untreated mice. These results suggest thatsquamous lung tumors of hamsters treated with 4-induction of AP-1 transcription factors in the parotid(methylnitrosoamino)-1-(3-pyridyl)-1-butanone, a to-gland control the upregulation of some mouse salivarybacco-specific nitrosamine (12).proline-rich proteins. q 1997 Academic Press

Salivary PRPs are induced in vitro when primaryhamster parotid cells are incubated with either dibu-1 Present address: Department of Neurology, University of Califor-tyryl cAMP or effectors which elevate intracellularnia, San Francisco, CA 94143.

2 Present address: Hoechst Marion Roussel, Inc., 2110 E. GalbraithRoad, Cincinnati, OH 45215.

6 Abbreviations used: PRP, proline-rich protein; sPRP, small pro-3 Present address: Doheny Vision Research Center, 1355 San PabloSt., Los Angeles, CA 90033. line-rich proteins; RSV, Rous sarcoma virus; CAT, chloramphenicol

acetyltransferase; DTT, dithiothreitol; CRE, cAMP-response ele-4 Present address: Nutrition Department, University of California,Davis, CA 95616. ment; CREB, cAMP-response element binding protein; TRE, tetra-

decanoylphorbol acetate response element; CBP, CREB binding pro-5 To whom correspondence should be addressed. Fax: 916-752-3085. tein.

970003-9861/97 $25.00Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.

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98 ZHOU ET AL.

chloramphenicol for 4 h. The acetylated products were separatedcAMP levels, including isoproterenol and forskolin (13).from the unmodified substrate by thin-layer chromatography (chloro-This transcriptional activation is blocked by cyclohexi-form:methanol, 19:1). After autoradiography, spots corresponding tomide, indicating that protein synthesis is required for the substrate and products were scraped into scintillation vials for

PRP induction. The 5*-flanking sequences of the mouse counting. CAT activities are reported as the percentage of chloram-phenicol converted to acetylated chloramphenicol per 50 ml of cell(MP2) and hamster (H29) PRP genes share several con-extracts. Human growth hormone levels were used to correct forserved elements which likely participate in the induc-transfection efficiencies with each culture.tion of the genes by isoproterenol and cAMP (2). In this

Preparation of nuclear extracts. CD-1 mice were injected dailyreport we show that AP-1 DNA sequence elements in with 0.2 ml 5% isoproterenol per mouse for 4 days. Mice were sacri-the 5*-flanking region of a mouse PRP gene can mediate ficed and parotid glands were removed. Nuclei from mouse parotida cAMP transcriptional response and that nuclear pro- glands were isolated as described by Schibler et al. (18) with some

modifications. Crude nuclear pellets were suspended with a Dounceteins binding to these sequences are increased in thehomogenizer in 20 mM Hepes (pH 7.5), 25% (v/v) glycerol, 0.42 Mparotid glands of isoproterenol-treated mice. These re-NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 0.5 mM dithiothreitol (DTT).sults show that induction of AP-1 transcription factors The suspension was subjected to centrifugation for 30 min at 25,000g.

in the parotid gland likely controls the b-agonist in- The resulting supernatant fraction was dialyzed against 20 mM

duced expression of some mouse salivary PRPs. Hepes (pH 7.5), 20% (v/v) glycerol, 0.1 M KCl, 0.2 mM EDTA, and0.5 mM DTT and subjected to centrifugation at 25,000g for 30 min.The supernatant fluid, designated the nuclear extract, was frozen as

MATERIALS AND METHODS aliquots in liquid nitrogen and stored at 0807C. Nuclear extractsfrom L-M cells were prepared by the method of Dignam et al. (19)Materials. All DNA modifying enzymes, radiochemicals, andand stored as above. Protein concentrations were determined by thechemicals were purchased from GIBCO-BRL (Grand Island, NY),method of Peterson with bovine serum albumin as a standard (20).New England Biolabs (Beverly, MA), Boehringer-Mannheim (India-

napolis, IN), and Amersham Corp. (Arlington Heights, IL). CD-1 Gel mobility shift assay. Probes were prepared by filling in the 3*-mice were from Charles River Laboratories (Wilmington, MA). Oligo- recessed ends of double-stranded oligonucleotides with [a-32P]dATPnucleotides were synthesized at the University of California–Davis using the Klenow fragment of DNA polymerase I. Binding reactionsProtein Structure Laboratory. Human growth hormone assay kit and were performed in 20 ml of 10 mM Tris–HCl (pH 7.5), 40 mM NaCl,pXGH5 were from Nichols Institute Diagnostics (Los Angeles, CA). 1 mM EDTA, 1 mM DTT, 10% glycerol, 2 mg poly(dI 0 dC ), 10 mgDNase I, DPFF grade, was from Cooper Biochemicals. DMEM was nuclear protein, and 5000–10,000 cpm of 32P-labeled DNA (0.1–0.5from GIBCO-BRL and bovine sera were purchased from Hyclone ng). The components were incubated at 237C for 30 min and subjectedLaboratories, Inc. (Logan, UT). Purified c-jun was a gift from Dr. R. to electrophoresis in 5% polyacrylamide–0.51 TBE (89 mM Tris–Tjian, University of California, Berkeley. borate, 2 mM EDTA, pH 8.0) gels. The gels were dried and exposed

to Kodak XAR5 film with intensifying screens for detection of DNA–Plasmid constructions. The upstream sequence (0702 to /78 bp)nucleoprotein complexes. For some experiments an oligonucleotideof the mouse PRP gene, MP2 (14), was subcloned into pUC18 as ancontaining an AP-1 site was used. The sequence of the oligonucleotideEcoRI–SstI fragment. Deletions on both the 5*- and 3 *-ends of theis shown below and the AP-1 site is underlined.0702 to /78 region were made using Bal31 exonuclease. Resulting

fragments were subcloned into pUC18 and sequenced by the methodGATCAAGATATTGACTCATGTATACCTCATATGTGTof Sanger et al. (15). The plasmid pRSVCAT (American Type Culture

Collection No. 37152) was digested with PvuI and BamHI, for isola-TTCTATAACTGAGTACATATGGAGTATACACACTAGtion of a 1.8-kb fragment containing the Rous sarcoma virus (RSV)

promoter and chloramphenicol acetyltransferase (CAT) gene, exclud-ing the strong viral enhancer region. This fragment was inserted in DNase I footprinting assay. DNase I footprinting with crude nu-pUC18 to create the plasmid designated RCAT. Reporter plasmids clear extracts from L-M cells and mouse parotid glands was per-were constructed by joining the PRP MP2 deletions with RCAT. DNA formed as described by Ohlsson and Edlund (21) with some modifica-fragments were modified with linkers and/or Klenow ‘‘filling in’’ to tions. The reactions, in a final volume of 50 ml, contained 25 mMobtain compatible restriction sites for cloning purposes. Hepes (pH 7.5), 50 mM KCl, 0.05 mM EDTA, 0.5 mM DTT, 10%

glycerol, 0.5–5 ng of 32P end-labeled DNA, 1 mg of poly(dI 0 dC ),Transient transfections and CAT assays. The mouse cell lineand 1–25 ml of crude extract. 32P end-labeled DNA was prepared byL-M (ATCC CCL 1.2) was maintained in DMEM plus 5% fetal bovinefilling in with the Klenow fragment of DNA polymerase I. Variousserum and 5% bovine calf serum. Cells were plated at a density ofamounts of extracts were preincubated with poly(dI 0 dC ) for 102.5 1 106 cells/100-mm dish 24 h prior to transfection. Cultures weremin prior to addition of the 32P-labeled DNA fragment. The reactiontransfected with the indicated plasmids (30 mg per dish) using themixture was further incubated on ice for 30 min. DNase I digestioncalcium phosphate method (16). The expression plasmid for humanwas initiated by incubation of reactions at room temperature for 1growth hormone, pXGH5, was transfected with the CAT constructsmin and addition of MgCl2 to a final concentration of 5 mM. A total(4 mg/dish) to assess the relative transfection efficiencies for eachof 30 to 100 ng of freshly diluted DNase I was added to the reactionculture. After the cells were exposed to the DNA–calcium phosphatetube for 1 min at room temperature. DNase I footprinting with puri-precipitate for 4 h, a glycerol shock was performed [25% (v/v) glycerolfied jun protein was performed with modifications from the abovein serum-free medium]. The cells were allowed to recover overnight,protocol as described by Lee et al. (22). The reactions were stopped,then the medium was exchanged and effectors (0.5 mM dibutyrylethanol-precipitated, and analyzed on a 6% sequencing gel. PurinecAMP plus 1 mM theophylline) were added. After 48 h, the mediumand guanosine ladders of DNA probes were prepared and subjectedwas collected and assayed for human growth hormone. The cells wereto electrophoresis along with the footprint reactions (23).harvested and assayed for CAT activity as described previously (17).

Briefly, cytoplasmic extracts were prepared by subjecting the cellsto freeze–thaw in hypotonic buffer. The extracts were heated to 657C RESULTSfor 10 min to inactivate cellular enzymes which compete with CAT

Cyclic AMP regulation of a heterologous promoter byfor substrate. Activities were measured by incubating extracts with4 mM acetyl-CoA plus 0.2 mCi of D-threo-[dichloroacetyl-1-14C]- the 5* flanking region of the mouse PRP gene MP2.

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99AP-1 REGULATION OF PROLINE-RICH PROTEIN GENE EXPRESSION

TABLE IThe 5*-flanking sequences from 0702 to /78 bp, rela-tive to the transcription start site of the mouse PRP Effect of MP2 Upstream Sequences on Expression

of Heterologous Promoter-CAT Constructsagene MP2, were analyzed for cis elements directingcAMP transcriptional responses. The region from0702

CAT activity bto 0198 bp in tandem with the RSV promoter-CAT(RCAT) transfected into L-M cells directed a 3.1-fold

construct Control Dibutyryl cAMP Ratioc

increase in CAT expression in the presence of 0.5 mM

dibutyryl cAMP plus 1 mM theophylline (Fig. 1, Table R-CAT d 21.2 { 2.4 30.2 { 8.1 1.4I, P õ 0.01). Transfection efficiencies were assessed (0702, 0198)e 9.1 { 0.3 28.6 { 3.1 3.1*

(0702, 0252)e 9.9 { 2.8 22.8 { 1.7 2.3*through cotransfection with an expression plasmid for(0702, 0322)e 8.6 { 1.1 20.9 { 4.8 2.4*human growth hormone. The basal construct, RCAT,(0325, 0110)e 18.1 { 2.3 33.0 { 9.2 1.8did not significantly increase CAT expression when(0260, 0110)e 11.8 { 3.2 18.2 { 6.5 1.5tested under these conditions (1.4-fold increase, P õ (0325, 0252)e 0.61 { 0.11 0.90 { 0.39 1.5

0.1). These data show that the 504-bp fragment can (0252, 0325)e 1.26 { 0.55 1.80 { 0.65 1.4(0325, 0252)3*e 7.50 { 1.32 8.48 { 2.75 1.1mediate a cAMP-dependent increase in transcription of

a heterologous promoter-CAT reporter gene construct.a L-M cells were cotransfected with 30 mg of the reporter constructsFurther identification of the sequences involved in this

as indicated plus 4 mg of the human growth hormone expressioncAMP transcriptional response was carried out using plasmid as described under Materials and Methods.constructs containing progressive 5*- and 3 *-end dele- b CAT activity expressed as percentage acetylated chloramphenicol

(means { SD from three cultures) corrected for growth hormonetions. Figure 1 illustrates the sequences cloned into theexpression.RCAT basal plasmid. The response of the individual

c Ratio Å dibutyryl cAMP/control values.plasmids to cAMP in transient transfection assays is d R-CAT Å RSV promoter (minus enhancer) in tandem with theshown in Table I. Deletion from the 3 * orientation to CAT gene.0322 did not abolish cAMP induction of CAT expres- e MP2 upstream sequences linked to RCAT construct as described

under Materials and Methods.sion. Deletion from the 5*-end to 0325 or 0260 bp sig-* P õ 0.01.nificantly weakened the response to cAMP. These data

indicate that the 0702- to 0322-bp region of the mousePRP gene MP2 confers cAMP responsiveness to the

pression. These data suggest that the fragment con-RCAT reporter plasmid.tained a negative regulatory element, or transcrip-Generally, plasmids with the upstream sequences oftional silencer, whose activity was independent of ori-MP2 suppressed CAT expression relative to the RCATentation. When this fragment was cloned into the 3 *-construct. The plasmid (0325, 0252) MP-RCAT wasend of the CAT gene plasmid (0325, 0252) MP-RCATsuppressed to the greatest extent. The reverse orienta-(3 * ), the silencer effect of the sequence element wastion of this fragment relative to the promoter (0252,reduced. The opposite orientation of this fragment at0325) MP-RCAT also exhibited low levels of CAT ex-the 3 *-end of the CAT gene gave similar results (datanot shown).

AP-1 sequence elements within the mouse PRP geneMP2 bind L-M nuclear proteins and can mediate cAMPtranscriptional activation. The 0702- to 0322-bp re-gion of the mouse PRP gene MP2 confers cAMP respon-siveness to the RCAT reporter plasmid. This DNA se-quence does not contain more than 6 bp of the consen-sus CRE (cAMP-response element) (24). A consensusAP-1 element, 5*-TGACTCA-3 *, was identified from0592 to 0586 bp. DNase I footprinting experimentswere performed with crude nuclear extracts fromL-M cells on the 0702- to 0322-bp DNA fragment. Thenuclear proteins protected a region from 0604 to 0575bp within this fragment (Fig. 2). The consensus AP-1 element described above was contained within this

FIG. 1. Reporter gene constructs containing MP2 upstream regula- protected region. Nuclear extracts prepared from L-Mtory sequences. The indicated MP2 sequences were ligated to a Rous cells incubated with dibutyryl cAMP and theophyllinesarcoma virus (RSV) promoter-chloramphenicol acetyltransferase did not contain an increased level of the protective com-(CAT), for reporter gene construction as described under Materials

ponent (data not shown).and Methods. The solid bar represents the RSV promoter (minusenhancer). The hatched bar represents the coding region for CAT. We examined the ability of the AP-1 transcription

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100 ZHOU ET AL.

RCAT and (0702, 0322) MP-RCAT expression levels(Table II). Only the plasmids containing two andthree copies of the DNA fragment showed a cAMPresponse (3- and 2-fold increases, respectively). Thislevel of induction mimicked the fold induction medi-ated by the (0702, 0322) MP-RCAT construct whichcontains all five of the putative AP-1 sites. These re-sults suggested that the multiple AP-1 elementsfound within the MP2 PRP gene function to mediatethe cAMP increase in MP2 transcription.

Isoproterenol induction of nuclear proteins in mouseparotid glands with affinity for the MP2 AP-1 DNAsequence elements. Gel mobility shift experimentswere utilized to determine if nuclear factors in parotidglands were associated with AP-1 DNA sequence ele-ments of MP2. Nuclear extracts from untreated andisoproterenol-treated mice were prepared. Gel mobilityshift assays with an MP2 DNA fragment (0702 to0572

FIG. 2. DNase I footprint analysis of nuclear factors from L-M cells.The noncoding strand of DNA (0702 to 0322 bp) was 32P-labeled atthe 0702 end. Labeled DNA (0.5 ng) was added to each bindingreaction as described under Materials and Methods. Binding reac-tions containing 35 mg L-M nuclear extracts were digested with 60ng (D1) and 100 ng (D2) DNase I. A binding reaction including 10mg bovine serum albumin (F) digested with 30 ng DNase I was runas a control.

factor, c-jun, to protect within this fragment. The puri-fied transcription factor protected the putative AP-1region within the PRP gene (0593 to 0583 bp, Fig.3). Upon increasing the levels of c-jun in the bindingreaction, additional sites of protection within the DNAfragment were observed (Fig. 3). The adjacent regionsfrom 0625 to 0611 bp, 0611 to 0594 bp, 0582 to 0568bp, and 0567 to 0551 bp were partially protected.These four additional sequences were homologous withthe 7-bp AP-1 consensus element with one or two basedifferences (Fig. 4).

The functionality of these putative AP-1 regulatoryelements was tested using the RCAT reporter plas-mid. A synthetic DNA fragment (0591 to 0568 bp)

FIG. 3. DNase I footprint analysis with purified human c-jun pro-containing the third and fourth c-jun protected se-tein. Binding reactions were performed as described for Fig. 2, exceptquences (Fig. 3) and tandem copies of this sequencethat poly(dI 0 dC ) was omitted. DNA was incubated with no protein(up to five) were cloned into RCAT. The AP-1 ele- and digested with 1.0 ng DNase I (F). DNA was incubated with 5,

ments inserted at the 5*-end of RCAT increased the 7, and 9 mg c-jun protein (as indicated on the figure) and was digestedwith 1.75, 2.1, and 2.5 ng DNase I, respectively.level of CAT expression 18- to 34-fold compared with

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101AP-1 REGULATION OF PROLINE-RICH PROTEIN GENE EXPRESSION

of many cAMP-regulated genes, which confers cAMPinducibility to endogenous and heterologous promoters(24, 25). The cAMP-response element binding proteins(CREB) bind to this palindromic sequence which istermed the cAMP-response element (CRE) (26). CREBis activated by serine phosphorylation catalyzed by pro-tein kinase A (27). The phosphorylated CREB then in-teracts with the CREB binding protein (CBP), whichin turn interacts with the basal transcription factorTFIIB to augment transcription (28, 29). Salivarygland-specific CREBs from rats have been identifiedas potentially important components in regulating thecAMP induction of the rat PRP gene RP4 (30). How-ever, the mouse PRP gene MP2 does not contain theconsensus CRE (2, 14).

The consensus sequence of the tetradecanoylphorbolFIG. 4. Comparison of the MP2 upstream sequence with the AP-1acetate response element (TRE) found in the humanconsensus binding sequence. Proposed AP-1 sites are in bold type.collagenase gene (23), as well as several other genes,is a heptamer, 5*-TGACTCA-3 *, which has only onebase different from the consensus CRE. In combinationbp) yielded two major complexes (B1 and B2, Fig. 5A). with certain flanking sequences, the TRE can be stimu-The B1 DNA–protein complex was increased about six- lated by both phorbol ester and cAMP (31). In our stud-fold in isoproterenol-treated mice based on scanning ies, multiple copies of this sequence element were re-desitometry of the autoradiogram (Fig. 5B). DNA foot- quired to reconstitute cAMP induction of RCAT tran-print analysis performed with the 0702- to 0322-bp scription (Table II). AP-1 transcription factors, such asDNA fragment showed that the same AP-1 consensus c-jun and c-fos, interact at the TRE and mediate theelement protected with L-M cell nuclear extracts was activation of transcription (22, 23). We show that puri-protected by parotid nuclear extracts from isoprotere- fied c-jun can protect five AP-1 DNA sequence elementsnol-treated mice (Fig. 6). Nuclear extracts from un- in the upstream regulatory region of MP-2 (Fig. 3). Thetreated mice did not protect this site from DNase I CREB coactivator, CBP, can also interact with the AP-digestion (data not shown).

DISCUSSION TABLE II

Effect of MP2 AP-1 DNA Sequence ElementsThe induction of PRPs in salivary glands of severalon RCAT Expressionamammals by isoproterenol and cAMP has been well

studied both in vivo and in primary cell culture. ResultsCAT activity bpresented here indicate that in mouse parotid glands

there is a trans-acting factor that binds to the AP-1Construct Control Dibutyryl cAMP Ratioc

consensus motif located in the upstream regulatory se-quences of the mouse PRP gene MP2. Transfection ex- R-CATd 10.6 { 0.6 15.5 { 2.3 1.5periments with L-M cells indicate that this sequence (0702, 0302)e 7.9 { 1.2 23.4 { 2.4 3.0

(0591, 0568)RCAT 371 { 82 433 { 127 1.2can mediate cAMP regulation of reporter gene con-(0591, 0568)2RCAT 183 { 21 572 { 95 3.1structs containing a heterologous promoter.(0591, 0568)3RCAT 185 { 31 415 { 33 2.2Transient transfection experiments in L-M cells (0591, 0568)5RCAT 337 { 47 374 { 33 1.1

demonstrated the functionality of MP-2 promoter ele-ments. Although a cAMP-dependent increase in tran- a L-M cells were cotransfected with 30 mg of the reporter constructs

as indicated plus 4 mg of the human growth hormone expressionscription of the heterologous promoter-CAT reporterplasmid as described under Materials and Methods.gene construct was observed, treatment of L-M cells

b CAT activity expressed as percentage acetylated chloramphenicolwith dibutyryl cAMP did not increase the level of nu- (means { SD from three cultures) corrected for growth hormoneclear transcription factors. Clearly, parotid gland cells expression.and the L-M transformed cell line differ in the extent c Ratio Å dibutyryl cAMP/control values.

d R-CAT Å RSV promoter (minus enhancer) in tandem with theof this response. One possibility is that the basal levelCAT gene.of the nuclear factors of interest may be higher in e MP2 upstream sequences and AP-1 oligonucleotide linked toL-M cells than in the cells of the parotid gland. RCAT construct as described under Materials and Methods. The

Several studies have defined an 8-bp palindromic se- subscripts refer to the number of tandem repeats of the 0591, 0568oligonucleotide inserted into RCAT.quence, 5*-TGACGTCA-3 *, in the upstream sequences

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102 ZHOU ET AL.

FIG. 5. Detection of nuclear factors from parotid glands that bind to the sequence 0702 to 0572 bp. (A) Nuclear extracts were preparedfrom parotid glands of control and isoproterenol-treated mice. The MP2 DNA fragment 0702 to 0572 bp was end-labeled with 32P andincubated with 2, 4, 6, 8, and 10 mg parotid gland nuclear proteins from control (lanes 2–6) and from isoproterenol-treated (lanes 7–11)mice. Protein–DNA complexes (B1, B2) were separated from unbound DNA (F). (B) The relative densities of the protein–DNA complex(B1) in A were determined by scanning densitometry.

1 transcription factor, c-jun, to activate transcription amylase genes (32). Another AP-1 transcription factor,c-fos, is induced in the salivary glands of mice treated(29). During rat parotid gland development, increased

c-jun mRNA levels correlate temporally with gland cell with b-agonist (33). Further, Reddy et al. have shownthat the promoter for the small proline-rich proteinproliferation and precede the expression of PRP andSPR1 can be synergistically stimulated by c-jun overex-pression and TPA treatment in transfected tracheo-bronchial epithelial cells (34). Additionally, the expres-sion of c-fos and other protooncogenes is elevated invitro in rat parotid cells by incubation with isoprotere-nol (35). These studies, along with our findings, supportthe idea that isoproterenol-mediated increases in PRPlevels in the mouse parotid gland are regulated in partthrough induction of AP-1 transcription factors, whichin turn induce PRP expression. Future studies will fo-cus on the characterization of the specific AP-1 tran-scription factors which are induced.

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