heparin decreases activator protein-1 binding to dna in ... file15 heparin decreases activator...

15

Heparin Decreases Activator Protein-1 Bindingto DNA in Part by Posttranslational

Modification of Jun B

Yin Ping Tina Au, Grazyna Dobrowolska, David R. Morris, Alexander W. Clowes

Abstract Heparin is a potent inhibitor of the proliferationand migration of vascular smooth muscle cells. This agentselectively inhibits the transcription of tissue-type plasmino-gen activator and interstitial collagenase, probably by decreas-ing the binding of activator protein-1 (AP-1) to phorbolester-responsive elements in the promoters of these genes.Decreased AP-1 binding is not due to a direct inhibition byheparin, since heparinase digestion of nuclear extracts pre-pared from heparin-treated smooth muscle cells does notrestore AP-1 binding activity. Treatment of cells with heparinsuppresses the expression of Jun B, one of the components of

AP-1. The major effect of heparin is at the level of posttrans-lational modification of Jun B. Results from pulse-chaselabeling experiments show that the newly synthesized Jun B israpidly converted to a higher-molecular-weight form and thatconversion is suppressed by heparin. Evidence is presentedsuggesting that the heparin-inhibited event is phosphorylationof Jun B. (Circ Res. 1994;75:15-22.)

Key Words * Jun B * posttranslational phosphorylation* tissue-type plasminogen activator * interstitialcollagenase * activator protein-1

P roliferation and migration of vascular smoothmuscle cells (SMCs) contribute to the resteno-sis of arteries subjected to angioplasty, and

effective treatment of this disorder should regulatethese responses to injury. Heparin may be an appro-priate drug for this purpose because it inhibits SMCgrowth and migration both in vitro' 2 and in vivo.34 Itis of interest to understand how heparin producesthese inhibitory effects, since heparin-like glycosami-noglycans secreted by endothelial cells may be endog-enous inhibitors of SMCs in vivo.56 The mechanism ofheparin action is still not known, even though there isa vast literature describing its effects. Heparin hasbeen shown to affect a number of cellular functions: itdecreases the expression of oncogenes,78 it decreasesthe binding of different growth factors,9-11 it enhancesthe activity of a growth inhibitor,12 and it alters thedeposition of matrix proteins.13 It is not clear whetherthese effects represent different pathways or whetherthey are related to a common pathway that is modu-lated by heparin. It is also not known whether heparinenters the cells and acts directly or whether it actsindirectly via secondary mediators to produce theseeffects.To understand the mechanism of heparin action, we

have identified a pathway that is modulated by heparinand hope to determine the molecular basis of the actionof heparin by unraveling this pathway. We have previ-ously shown that heparin selectively inhibits the expres-sion of two proteases, tissue-type plasminogen activator

Received September 10, 1993; accepted March 31, 1994.From the Departments of Surgery (Y.P.T.A., A.W.C.), Phar-

macology (G.D.), and Biochemistry (D.R.M.), University ofWashington, Seattle.

Correspondence to Yin Ping Tina Au, PhD, RF-25, Departmentof Surgery, University of Washington, Seattle, WA 98195.C 1994 American Heart Association, Inc.

(TPA) and interstitial collagenase, in an in vitro mito-genesis model using baboon SMCs.14,5 Heparin alsodecreases the expression of TPA in rat SMCs induced tomigrate by arterial injury.16 Further investigation hassuggested that heparin inhibits the transcription of TPAand collagenase by decreasing the binding of a trans-acting factor, activator protein-1 (AP-1), to the phorbolester-responsive element (TRE)." A consensus TREsequence is present in the promoter of collagenasegene,17 and a similar, but not identical, sequence isfound in TPA.18 The binding of AP-1 to the TRE issufficient to activate the transcription of collagenase.17Hence, the inhibitory effect of heparin on TPA andcollagenase may be related to its inhibitory effect onAP-1 binding.

It has been suggested that heparin decreases AP-1binding by direct inhibition in HeLa cells.19 We haveexamined this possibility in baboon SMCs and havefound that digestion of the nuclear extracts with hepa-rinase does not restore the decreased AP-1 binding.This result suggests that the decrease in AP-1 bind-ing is not by direct inhibition. We also examine thepossibility that heparin decreases AP-1 binding by in-hibiting the expression of one or more proteins of theAP-1 complexes.

Materials and MethodsMaterialsHeparin (isolated from porcine intestine mucosa), phorbol

12-myristate 13-acetate (PMA), heparinase (isolated fromFlavobacterium heparinum), and cell culture media were ob-tained from Sigma Chemical Co. Recombinant heparinase wasa generous gift from Dr Robert Rosenberg, MIT. Arcticshrimp alkaline phosphatase (SAP) was obtained from USB.Antibodies and blocking peptides to Jun B and c-Jun werepurchased from Santa Cruz Biotechnology, Inc, and OncogeneScience. Reagents used for RNA isolation and Northern

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16 Circulation Research Vol 75, No 1 July 1994

blotting were of molecular biology grade. Radioisotopes wereobtained from Amersham Corp.

Cell CultureAortic SMCs isolated from normal baboons were used be-

tween passages 10 and 18.14 After the cells have been growth-arrested for 3 days in serum-free medium (Dulbecco's modifiedEagle's medium/F-12 Ham [1:1] containing 6 ,.g insulin permilliliter, S ,g transferrin per milliliter, 1 mg ovalbumin permilliliter, 200 U penicillin per milliliter, and 200 gg strepto-mycin per milliliter), the experiments were started by theaddition of fresh serum-free medium containing PMA (5ng/mL)+heparin (100l g/mL).

Gel Retardation AssaysNuclear protein extracts prepared from SMCs were used for

gel retardation as described previously.15

Supershift Gel Retardation AssaysNuclear extracts were preincubated with 2 ,ug of the Jun B

antibody (described in Western blotting section) for 10 minutesat room temperature before the addition of the reaction buffercontaining the 32P-labeled probe. The probe for the gel retar-dation assay was a synthetic oligonucleotide containing thebinding sequence for AP-1 from the human collagenase pro-moter region (5'-AAGCATGAGTCAGACACCTCTGGC-3').

Northern AnalysisTotal RNA was isolated from SMCs20 and used for North-

ern blotting as described in our previous report.14 cDNAprobes forjun B (1.2-kb mouse cDNA),2' c-jun (1.8-kb mousec-jun),21 and glyceraldehyde-3-phosphate dehydrogenase(1.2-kb human cDNA)22 were labeled by nick translation.Signals were detected by autoradiography and quantified byphosphoimaging.

Western BlottingNuclear proteins were used for the Western blotting.23 The

antibodies to Jun B and c-Jun are polyclonal antibodies raisedagainst peptides corresponding to residue 45-61 of murine JunB and residue 73-87 of c-Jun. These antibodies are specific toJun B and c-Jun and do not cross-react with each other or withJun D. The secondary antibody was alkaline phosphatase-conjugated rabbit antibody. The signal was detected by incu-bating the membrane in nitroblue tetrazolium and 5 -bromo-4-chloro-3-indolyl phosphate.

Continuous and Pulse-Chase LabelingFor continuous-labeling experiments, growth-arrested

SMCs were incubated in serum-free medium lacking methio-nine for 1 hour before addition of PMA+heparin. Cells weretreated with PMA±heparin for 1 hour and then labeled with100 1,Ci/mL of [35S]methionine for 3 hours. For pulse-chaselabeling experiments, SMCs that have been incubated inmethionine-free medium were treated with PMA±heparin for2 hours and then pulsed with 200 ,uCi/mL of [35S]methioninefor 30 minutes. Cells were chased for 0, 10, 20, 40, 80, 160, and210 minutes in medium without [35S]methionine and contain-ing 18 mg/L unlabeled methionine. For immunoprecipitationunder nondenaturing conditions, cells were lysed in RIP buffer(9.1 mmol/L Na2PO4, 1.7 mmol/L NaHPO4, 150 mmol/LNaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1%sodium dodecyl sulfate (SDS), and 0.02% sodium azide, pH7.25) with 1 mg/mL bovine serum albumin, 0.2 U/mL aproti-nin, and 1 mmol/L phenylmethylsulfonyl fluoride. For immu-noprecipitation under denaturing conditions, the cells werelysed in 50 mmol/L Tris, pH 7.5, 0.5% SDS, and 70 mmol/Ll3-mercaptoethanol and then boiled for 10 minutes. The boiledmixtured was diluted with 4 vol RIP without SDS.24 Cell debriswere removed by centrifuging at 14 00(g for 30 minutes at 4°C.

Aliquots of the supernatant were used to determine theincorporation of [355]methionine into total cellular proteins bytrichloroacetic acid precipitation. An equal amount of trichlo-roacetic acid-precipitable radioactivity from each sample wasused for the immunoprecipitation. The supernatants wereprecleared with protein A agarose. The precleared superna-tant was incubated with the Jun B antibody (1 1£g/mL) for 1hour at 4°C, followed by the addition of protein A agarose andcontinued incubation overnight at 4°C. The immunoprecipi-tates were washed four times with RIP buffer and analyzed bySDS-polyacrylamide gel electrophoresis (PAGE). Labeledproteins were detected by fluorography, and the signals werequantified by phosphoimaging.

Dephosphorylation by Alkaline PhosphataseAfter washing four times with RIP buffer, the 35S-Jun B

immunoprecipitates were washed three times with 100 mmol/LTris, pH 8.8, 10 mmol/L MgCl2, and 0.2 U aprotinin permilliliter before the addition of 5 U SAP and incubated at 37°Covernight.

Rephosphorylation of SAP-Treated35S-Jun B Immunoprecipitates

Nuclear or cytoplasmic extracts prepared from PMA-stim-ulated SMCs were used as a source of kinases to phosphory-late SAP-treated samples. SAP was inactivated by incubationat 65°C for 15 minutes. After inactivation of SAP, sampleswere rephosphorylated by either nuclear or cytoplasmic ex-tracts in 20 mmol/L HEPES, pH 7.5, 15 mmol/L MgC12,phosphatase inhibitors (0.15 mmol/L sodium molybdate, 0.15mmol/L f-glycerophosphate, 15 mmol/L sodium fluoride, and0.3 nmol/L sodium orthovanadate), and 1.5 mmol/L ATP at30°C for 30 minutes. After phosphorylation, the reactionmixture was washed once with RIP buffer before analysis bySDS-PAGE.

In Vitro Phosphorylation Using [1/2PJATPNuclear extract isolated from cells treated with PMA for 4

hours was phosphorylated in the presence of [_32PJATP at30°C for 30 minutes. After phosphorylation, the nuclearextracts were lysed under denaturing conditions before immu-noprecipitation with the Jun B antibody.

Statistical AnalysisA paired t test was used to analyze the differences between

PMA and PMA+heparin treatments. Values are presented asmean± SEM.

ResultsEffect of Heparinase on AP-1 BindingWe have previously shown that PMA increases AP-1

binding in SMCs and that heparin decreases the in-duced AP-1 activity. To determine whether the de-creased AP-1 binding was due to a direct inhibition byheparin in the extracts, nuclear extracts from SMCswere predigested with heparinase before the bindingassays. Digestion with heparinase of nuclear extractsprepared from heparin-treated cells did not restore thedecreased AP-1 binding (Fig 1, lane 4 versus 5). Hence,the decreased AP-1 binding was not due to a directinhibition by heparin. We have examined the possibilitythat heparinase inhibitory activity might be present inthe nuclear extracts by deliberately adding exogenousheparin to the extracts. The addition of 17 ,ug/mLexogenous heparin abolished the AP-1 binding (lane 6versus 4), and digestion with heparinase restored it(lane 7 versus 6). A similar experiment was also per-formed in nuclear extracts prepared from PMA-treated



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Au et al Regulation of Jun B by Heparin 17

P f H

0 1 2 4

a 9 10

FIG 1. Effect of heparinase on activator protein-1 (AP-1) bind-ing. Nuclear extracts prepared from smooth muscle cells treatedwith phorbol 12-myristate 13-acetate (PMA) and heparin for 4hours were used for the gel retardation assays. Digestion withrecombinant heparinase was performed by addition of theenzyme (2 U) to the nuclear extracts in the presence of 1 mmol/Lphenylmethyisulfonyl fluoride and 5 mmol/L CaCI2 and incu-bated at 37°C for 1 hour before the gel-retardation assays.Nuclear extracts were prepared from cells subjected to thefollowing conditions: medium alone (lane 1), PMA without hep-arinase digestion (lane 2) and with digestion (lane 3), andPMA+heparin without heparinase digestion (lane 4) and withdigestion (lane 5). Whether there was heparinase-inhibitoryactivity in the nuclear extracts prepared from PMA+heparin-treated cells was examined by direct addition of exogenousheparin (17 gg/mL) to the extracts in the absence (lane 6) orpresence (lane 7) of heparinase. A similar experiment was alsoperformed to determine the activity of heparinase (lanes 8through 10). Nuclear extracts were prepared from PMA (lane 8)and by direct addition of exogenous heparin (17 gg/mL) to theextracts in the absence (lane 9) or presence (lane 10) ofheparinase (n=4 experiments).

cells to determine the activity of heparinase (lanes 8through 10). Direct addition of exogenous heparinabolished AP-1 binding (lane 9 versus 8), and digestionwith heparinase restored the binding (lane 10 versus 9).These results were obtained by using either recombi-nant (shown) or commerically available (not shown)heparinase.

Effect of Heparin on jun and fos mRNAsIn a time-course study, jun B and c-jun mRNAs

increased transiently after PMA stimulation; the levelpeaked at 2 hours and was still slightly elevated at 4hours (Fig 2). The responses ofjun B and c-jun mRNAsto PMA were different in magnitude. PMA increased

2 4h

jun B

c jun

APD

FIG 2. Northern analysis of jun B and c-jun in the presence ofphorbol 12-myristate 13-acetate (P) and heparin (H). Total RNAwas isolated from smooth muscle cells treated with either P orP+H for 0, 1, 2, and 4 hours. The blot was reprobed withglyceraldehyde-3-phosphate dehydrogenase (GAPD) to normal-ize the amount of RNA loaded into each lane.

jun B mRNA by 6±1.2-fold and c-jun mRNA by 2+0.3-fold (n-5). Heparin slightly decreased the PMA-in-duced jun B and c-jun mRNAs by 33±+10% and37±13%, respectively (n=5). jun D mRNA was consti-tutively expressed and was not modulated by PMA or

heparin (data not shown). We previously have shownthat PMA increases c-fos mRNA and that heparin hasno significant effect on the induction.'5 fos B mRNA was

not detected in either unstimulated cells or PMA-stimulated cells (data not shown).

Heparin Decreases Jun B ExpressionHeparin decreased expression of Jun B as determined

by Western blot analysis. It was found mainly in thenuclear fraction; only a trace amount was found in thecytoplasmic fraction (data not shown). Antibody specificto Jun B detected two specific polypeptides, a majorband at 43 kD and a minor band at 39 kD, plus a

nonspecific band at 49 kD (Fig 3). The two specificbands could be blocked by a Jun B peptide (amino acid45-61), against which the antibody was made (Fig 3).This region (amino acid 45-61) is specific to Jun B andhas little homology with the c-Jun or Jun D sequence.25In addition, these two bands were not observed withantibodies specific to c-Jun or Jun D (data not shown).The level of Jun B slightly increased at 2 hours, reacheda maximum at 4 hours, and remained high at 6 hoursafter stimulation with PMA. Heparin decreased PMA-induced Jun B at all time points (Fig 3). The inhibitoryeffect on Jun B was specific to heparin, since other

2 h 4 h 6 h

.n p (4t)

SF P H SF P H SF P H CS + peptide

_~~~~m_ ~- __ c 43 id*

FIG 3. Western analysis of Jun B expression in thepresence of phorbol 12-myristate 13-acetate (P) andheparin (H). Nuclear proteins isolated from smooth mus-cle cells treated with serum-free medium (SF) alone, P,and P+H for 2, 4, and 6 hours were used for Westernblotting. The level of Jun B was also determined in cellstreated with P+chondroitin-4-sulfate (CS). Two Jun B

kDa0.un B polypeptides were detected at 43 kD and 39 kD. TheDa Jun B specificity of these two bands determined by incu-

bating a Western blot containing nuclear proteins pre-pared from cells treated with P for 4 hours with the Jun Bpeptide (amino acid 45-61).

HEPARINASE

EXOGENOUS

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HEPARIN + +

P11A -+ +

1 2 3 4 5 6 7

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HEPARIN +

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4.0 -

3.0 -

2.0-

1-0

PMA

HEPARIN

43 kDa Jun B

39 kDa Jun B

T*

o

43 kDaJun B

St < c-Jun FiG 4. Bar graphs showing the effect of phor-bol 12-myristate 13-acetate (PMA) and hepa-rin on Jun B and c-Jun synthesis. 35S-Jun Band c-Jun were recovered by immunoprecip-itation from cells treated with serum-free me-dium (light hatched bars), PMA (solid bars), orPMA+heparin (dense hatched bars). Insetsdisplay autoradiograms of immunoprecipi-tated Jun B and c-Jun after sodium dodecylsulfate-polyacrylamide gel electrophoresis.Bar graphs depict the mean,SEM fold in-creases over serum-free control for Jun B (leftpanel, *P<.005, **P<.01, n=9) and c-Jun(right panel, n=8). The molecular weight ofc-Jun is 40 kD; a nonspecific band is ob-served at 43 kD.

39 kDac Jun

glycosaminoglycans such as chondroitin-4-sulfate (Fig3) or chondroitin-6-sulfate (data not shown) did notreduce Jun B expression.The increase in Jun B induced by PMA was due to

new protein synthesis, since the increase could beblocked by cycloheximide (data not shown). This was

further confirmed by metabolically labeling SMCs with[35S]methionine for 3 hours, followed by immunoprecip-itation with the Jun B antibody (Fig 4, left panel). Thetotal amount of [VSlmethionine incorporated into cel-lular proteins determined by precipitation with trichlo-roacetic acid was not different in cells treated witheither PMA or PMA+heparin. Similar to the resultsobtained with Western blotting, immunoprecipitation ofJun B detected both 43-kD (major) and 39-kD (minor)bands (Fig 4, left panel). These two bands were specificto Jun B, since they could be blocked by the Jun Bpeptide (data not shown). Furthermore, both bandswere detected by immunoprecipitation under nondena-turing or denaturing conditions, which suggested thatthese two bands were not a result of coimmunoprecip-itation. PMA increased the labeling of the 43-kD and39-kD Jun B bands by 3.5±0.6- and 3.0±0.4-fold,respectively, above the serum-free control (n=9). Hep-armn decreased the PMA-induced 43-kD Jun B by61±9% (P<.005, n=9) and the 39-kD Jun B by38±22% (P<.01, n=9) (Fig 4, left panel). Heparin hadno effect on the efficiency of immunoprecipitation ofJun B, since addition of heparin (20 ,ug/mL) directly tothe assay did not affect the amount of Jun B recovered(data not shown).The effect of heparin on c-Jun expression was deter-

mined by immunoprecipitation (Fig 4, right panel). Theantibody to c-Jun detected a 40-kD c-Jun band and a

43-kD nonspecific band. The 40-kD band is specific toc-Jun, since it could be completely eliminated by theblocking peptide (residue 73-87) (data not shown). Thelevel of c-Jun was not significantly increased by PMA(22±17%, n=8). Heparin slightly decreased the level ofc-Jun (Fig 4, right panel).

Jun B in AP-1 ComplexesWhether heparin decreases Jun B in the AP-1 com-

plexes was determined by supershift gel-retardationassay. The presence of Jun B in the complexes was

detected by the formation of a higher-molecular-weightcomplex with the antibody, as indicated by a supershiftband (Fig 5, lane 5). This supershift band was specific toJun B, as it could be eliminated by the Jun B blockingpeptide (Fig 5, lane 9 versus 8). Also, the Jun Bsupershift was not a result of artifactual shift due to theaddition of excess antibody, since this supershift bandwas not observed in the presence of an irrevelantantibody such as NF-kB (data not shown). PMA in-creased the level of Jun B in the AP-1 complexes, and

PMA

HEPARIN

JUN B Ab

JUN B Peptide

+ + + + + + +

+ + +

1 2 3 4 5 60,

JUN B>

AP-1 >

__mWW _ _

7 8 9

JUN B>

AP-1 >

_4

FIG 5. Effect of phorbol 12-myristate 13-acetate (PMA) andheparin on Jun B in the activator protein-1 (AP-1) complexes.AP-1 binding was performed in the presence (lanes 4 through 6)or absence (lanes 1 through 3) of the Jun B antibody (Jun B Ab).The presence of Jun B in the AP-1 complexes was detected bythe formation of a higher-molecular-weight band (indicated by>) with the antibody in supershift gel-retardation assay. Thissupershift band was specific to Jun B, since it could be elimi-nated by the blocking peptide (lane 9 versus 8).

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a

p P+H

cc5 -coboo - 1-t co- -c>; t cc--

t SR 95t * ~ *- ~43kD)a

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,P + H

0 40 80 120 160 200CHASE TIME (MIN)

FIG 6. Blot (a) and graph (b) showing the effect of phorbol12-myristate 13-acetate (P) and heparin (H) on posttranslationalmodification of Jun B. a, 35S-Jun B was recovered by immuno-precipitation from cells labeled for 30 minutes with [35S]methio-nine and then chased with unlabeled medium for 0, 10, 20, 40,80, and 160 minutes. Cells were treated with either P or P+H. b,The conversion of the 39-kD Jun B to the 43-kD Jun B wasillustrated by the 43-kD-to-39-kD ratio. The ratio in cells treatedwith P (n) and P+H (A) is plotted for each time point (averagevalue from three experiments).

heparin suppressed the induction by -70% (Fig 5, lane5 versus 6). These results indicate that the decreasedJun B expression by heparin (Fig 4, left panel) corre-

sponded to a reduced level of Jun B in the AP-1complexes.

Effect of Heparin on PosttranslationalModification of Jun B

The small reduction of jun B mRNA by heparin(33+10%) could not account for the decrease in theprotein (61+±9%), suggesting that the major effect ofheparin on Jun B was at the posttranscriptional level.To explore this possibility, we next examined the eftectof heparin on the synthesis, posttranslational modifica-tion, and degradation of Jun B with pulse-chase labelingexperiments. Cells were treated with PMA and heparinfor 2 hours, labeled for 30 minutes with [37S]methionine,and then chased with unlabeled methionine medium forvarious periods. 5S-labeled Jun B was recovered byimmunoprecipitation and analyzed by SDS-PAGE.

Results from pulse-chase experiments showed thatthe 39-kD Jun B was the precursor to the 43-kD Jun B.The 39-kD Jun B was the predominant species after abrief labeling (Fig 6a, 0 minutes), and it rapidly con-

verted to the 43-kD form during the chase (Fig 6a). Thisconversion was illustrated by the 43-kD-to-39-kD ratioat various periods. In PMA-stimulated cells, the ratioincreased from 0.8 to 3.1 after 80 minutes of chase (Fig6b). The 39-kD Jun B was the predominant speciesafter brief labeling (Fig 6a, 0 minutes), whereas the43-kD Jun B was the predominant species after contin-uous labeling (Fig 3). This difference was due to the

rapid conversion of the newly synthesized 39-kD Jun Bto the 43-kD form.Heparin slowed the conversion of the 39-kD Jun B to

the 43-kD Jun B (Fig 6a). Results from three experi-ments showed that heparin consistently decreased theapparent conversion of the 39-kD Jun B to the 43-kDJun B, as demonstrated by a lower ratio at a range oftime points (Fig 6b). The decreased conversion in thepresence of heparin also was reflected in the longerhalf-life of the 39-kD Jun B, which was estimated to be71 versus 56 minutes in PMA-stimulated cells. In thisparticular experiment, heparin appeared to decreasethe amount of Jun B synthesized (Fig 6a, 0 minutes).However, average results from three experimentsshowed that heparin had no effect on the newly synthe-sized 39-kD Jun B (1+17%, P=NS), and it only had asmall effect on decreasing the 43-kD Jun B (25+14%,P=NS) at 0 minutes.

Posttranslational Phosphorylation of Jun BWhether the posttranslational modification of the

39-kD Jun B to the 43-kD Jun B was due to phosphor-ylation was addressed by performing dephosphorylationand rephosphorylation studies. Dephosphorylation usingalkaline phosphatase should lead to the disappearance ofthe 43-kD Jun B and the reappearance of the 39-kD JunB, whereas rephosphorylation of the alkaline phospha-tase-treated Jun B should result in the reappearance ofthe 43-kD Jun B. 35S-Jun B immunoprecipitates wereused in both studies in order to follow the conversionsbetween the 39-kD Jun B and the 43-kD Jun B.

35S-Jun B was isolated by immunoprecipitation fromPMA-stimulated SMCs, which were metabolically la-beled with [355]methionine. Dephosphorylation withSAP of the 5S-Jun B caused the disappearance of the43-kD Jun B and the reappearance of the 39-kD Jun B(Fig 7a, lane 1 versus 2). This change was best illus-trated by comparing the 43-kD-to-39-kD ratio beforeand after SAP treatments. The ratio was 2.2 beforetreatment with SAP, and it decreased to 0.6 after treat-ment. The decrease in the ratio was not observed withheat-inactivated SAP or in the presence of phosphataseinhibitors (p-nitrophenyl phosphate and /3-glycerophos-phate) (data not shown), indicating that the changeswere not due to proteolysis. Similar results were alsoobserved with calf intestine alkaline phosphatase (datanot shown). Thus, the conversion of the 39-kD Jun B tothe 43-kD Jun B was due to phosphorylation.

Nuclear or cytoplasmic extracts isolated from PMA-stimulated SMCs were used as a source of kinases torephosphorylate SAP-treated 35S-Jun B immunoprecip-itates. SAP-treated 5S-Jun B was incubated at 65°C for15 minutes to inactivate SAP and then phosphorylatedwith either nuclear or cytoplasmic extracts in the pres-ence of unlabeled ATP. The control was an SAP-treated sample that had not been incubated at 65°C toinactivate SAP before being phosphorylated with nu-clear extracts. Rephosphorylation with either nuclear orcytoplasmic extracts caused an increase in the 43-kDJun B and a decrease in the 39-kD Jun B, as shown byan increase in the 43-kD-to-39-kD ratio from 0.6 to 1.2and 1.1 (Fig 7a, lane 2 versus 3 and 4). This change wasnot observed in the control sample with active SAPduring rephosphorylation, as shown by the ratio of 0.7(Fig 7a, lane 5).



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1.2 1.1 0.7

SF P P+H

A^ 43 kDa39 kDa

43kDa

1 2

FIG 7. Posttranslational phosphorylation of Jun B. a, Dephos-phorylation of 35S-Jun B immunoprecipitates by shrimp alkalinephosphatase (SAP) is shown under the following conditions:control (lane 1), dephosphorylation with SAP (lane 2), rephos-phorylation of SAP-treated 35S-Jun B immunoprecipitates witheither nuclear or cytoplasmic extract as a source of kinases afterincubation at 65°C for 15 minutes to inactivate SAP beforerephosphorylation by the extracts (lanes 3 and 4); and controlSAP-treated sample without heat inactivation of SAP beforebeing rephosphorylated (lane 5) (n=3 experiments). b, In vitrophosphorylation of nuclear extracts isolated from phorbol 12-myristate 13-acetate (P)-stimulated smooth muscle cells with[932P]ATP is shown. 32P-Jun B was recovered by immunoprecip-itation and migrated at the 43-kD band (lane 1). This band wasspecific to Jun B, since it could be blocked by Jun B peptide(lane 2) (n=4 experiments). c, The effect of heparin (H) onphosphorylation of Jun B is shown. In vitro phosphorylationusing [932]ATP was performed in nuclear extracts isolated fromsmooth muscle cells treated with serum-free medium alone (SF),P, or P+H for 4 hours. 32P-Jun B was recovered by immunopre-cipitation and analyzed by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (n=3 experiments).

The sum of the 43-kD and 39-kD Jun B signals was

compared between samples treated with and withoutSAP. The amount recovered from the SAP-treatedsample was 87±8% (P= NS, n= 3) of the nontreatedsample, indicating that SAP treatment did not cause

proteolysis. However, incubation of the SAP-treated35S-Jun B immunoprecipitates with either nuclear or

cytoplasmic extracts resulted in a decrease of the sum ofthe 43-kD and 39-kD Jun B signals; the amount recov-

ered was 60% of the untreated sample. Althoughsome proteolytic activity was present in the extracts, theincrease in the ratio observed in the SAP-treated sam-

ple phosphorylated with the extracts (Fig 7a, lane 3 and4) was due to an actual increase in the 43-kD Jun B anda decrease in the 39-kD Jun B. This indicated that theincrease in the ratio was not a result of selectiveproteolytic degradation of the 39-kD Jun B.

In vitro phosphorylation of nuclear extracts isolatedfrom PMA-stimulated SMCs was performed using[y32PJATP. 32P-Jun B in the nuclear extract was recov-

ered by immunoprecipitation using the denaturing con-

dition and then analyzed by SDS-PAGE. Resultsshowed that 32P-Jun B migrated only as the 43-kD bandand that no band of 39 kD was detected (Fig 7b). These

observations suggested that the 43-kD band is thephosphorylated form of Jun B at steady state.The effect of heparin on phosphorylation of Jun B

was determined by in vitro phosphorylation of nuclearextracts isolated from PMA+ heparin-treated SMCs.Results showed that the amount of 32P-Jun B (43 kD)recovered from PMA+heparin-treated cells was lessthan that from PMA-treated cells (Fig 7c). These resultssupported the notion that heparin decreases the phos-phorylation of Jun B.

DiscussionHeparin inhibits the expression of TPA and collage-

nase in part by lowering the cellular activity of transcrip-tion factor AP-1.15 In the present study, we examinedthe effect of heparin on components of AP-1 complexes.Jun B and c-Fos, but not c-Jun, Jun D, or Fos B, wereinduced by PMA. Heparin suppressed the expression ofJun B as demonstrated by Western blot analysis andimmunoprecipitation. Heparin reduced the amount ofJun B in the AP-1 complexes, as demonstrated bysupershift gel-retardation assay. We also have investi-gated the effect of heparin on c-Fos by Western blottingand immunoprecipitation. Because the c-Fos antibodies(obtained from two different sources) did not work ineither assay, we could not quantify the effect of heparinon c-Fos. However, results obtained from the supershiftgel retardation assay showed that the c-Fos supershiftband was not reduced by heparin (unpublished data).The effect of heparin on Jun B expression was both at

the transcriptional and posttranscriptional levels. It hada small, but significant, influence in decreasing jun BmRNA induced by PMA, which probably explains thedecrease in synthesis of Jun B protein when it wasmeasured with short labeling times. The major effect ofheparin was at the level of posttranslational modifica-tion of Jun B. Western blot analysis and immunoprecip-itates obtained from continuous-labeling experimentsdetected two species of Jun B, a major band at 43 kDand a minor band at 39 kD. Results from pulse-chaselabeling experiments are consistent with the 39-kD JunB being the precursor and the 43-kD Jun B being theposttranslational modified product in PMA-stimulatedcells. The conversion of 39-kD to 43-kD Jun B wassuppressed by heparin and seemed to be due to phos-phorylation, on the basis of results obtained from bothdephosphorylation and rephosphorylation studies. Con-sistent with these conclusions, it has been shown in 3T3cells that newly synthesized Jun B is rapidly phosphor-ylated, resulting in an increase in the apparent molec-ular weight.24 These observations suggest that an impor-tant influence of heparin is on the posttranslationalphosphorylation of Jun B.

Unlike c-Jun, phosphorylation of Jun B has not beenwell characterized. However, sequence comparisonsshow interesting differences in potential phosphoryla-tion sites in Jun B. At least five phosphorylation siteshave been identified in c-Jun26-301; two of them (Ser 63and Ser 73) are located in the N-terminus, and the otherthree (Thr 231, Ser 243, and Ser 249) are adjacent to theDNA-binding domain in the C-terminal portion of themolecule. It is not clear which enzymes phosphorylatec-Jun in vivo, although in vitro studies have shown thatc-Jun can be phosphorylated by various kinases. Forinstance, mitogen-activated protein kinase (MAPK),29,31

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p34CdC2"32 and a 67-kD protein33 have been demonstratedto phosphorylate Ser 63, Ser 73, and Ser 243, respec-tively, whereas casein kinase-II (CK-II)30 and glycogensynthase kinase-3 (GSK-3)26,34,35 phosphorylate Thr 231and Ser 249, respectively. Phosphorylation of the N-ter-minus increases the transactivation activity of c-Jun,whereas phosphorylation of the C-terminus decreasesthe DNA binding activity of c-Jun.26 283035 Whetherphosphorylation modulates Jun B in the same manneras c-Jun is not clear. Others have shown that MAPKphosphorylates the N-terminus of c-Jun but not Jun B36and that GSK-3 phosphorylates the C-terminus of bothc-Jun and Jun B.35 By sequence inspection, Jun Bappears to have multiple potential CK-JI and MAPKphosphorylation sites. Results obtained from labelingserum-stimulated 3T3 cells with 32p show that Jun B isphosphorylated at a much higher level than is c-Jun.24The possibility that Jun B is phosphorylated at multiplesites and that heparin affects phosphorylation of specificsites might explain the muted effect of heparin on theoverall phosphorylation of Jun B. It will be important toidentify the heparin-modulated phosphorylation site(s)in Jun B. We speculate that the magnitude of the effectof heparin might be larger when we examine the phos-phorylation of these sites.An intriguing possibility is that heparin affects Jun B

phosphorylation by inhibiting one of the kinases. Arecent report has shown that heparin reduced MAPKactivity by inhibiting the activation of the enzyme in ratSMCs.37 Heparin also might inhibit CK-II, since it is apotent competitive inhibitor of CK-II with Ki of 1.4nmol/L.38 CK-II is a constitutively expressed enzymeand is found mainly in the nucleus.39 It is conceivablethat heparin might enter the nucleus and inhibit CK-II.Busch et al19 concluded from heparitinase experimentsusing HeLa cells that heparin was present in the nuclearextract and was interfering with AP-1 binding. Weperformed similar experiments with baboon SMCs andfound that heparinase digestion of the nuclear extractsfrom heparin-treated cells did not restore the decreasedAP-1 binding. On the other hand, trace amounts ofheparin might be present in the nucleus at levelssufficient to inhibit CK-II but too low to inhibit AP-1binding to DNA. On the basis of the K, value, wecalculate that the amount of heparin required to inhibitCK-II by 50% is :'20 ng/mL, which is at least 150-foldless than the amount (3 ,g/mL) needed to inhibit AP-1binding.15 In fact, heparin has been shown to bind toSMCs and be internalized.40 Furthermore, smallamounts of heparan sulfates have been recovered fromnuclear preparations.41 Recent studies suggest that themechanism for nuclear translocation of heparin maydepend on the binding to other proteins. A likelycandidate is fibroblast growth factor (FGF), which has anuclear translocation sequence and a high affinity forheparin.42 The nuclear translocation of basic FGF hasbeen shown to occur in bovine endothelial cells.43 It ispossible that after internalization, heparin binds tocytoplasmic basic FGF and is transported into thenucleus.CK-II phosphorylates a number of nuclear and cyto-

plasmic proteins, many of which are involved in geneexpression and protein synthesis.44 For example, phos-phorylation of serum-responsive factor by CK-II in-

responsive element, which then leads to an increase intranscription of c-fos.45 According to the hypothesisproposed here, heparin, which has been shown todecrease c-fos gene expression in rat SMCs,8 may act byinhibiting CK-II phosphorylation of serum-responsivefactor. Likewise, the small effect of heparin on decreas-ingjun B mRNA found in the present study may be dueto an effect on CK-II-phosphorylated transcriptionfactors. Thus, it would be of interest to find out if theinfluence of heparin is on selected CK-II phosphory-lated proteins or if it affects all of them.What is the role of Jun B in activating the expression

of AP-1-responsive genes? The current biochemicalevidence suggests that because of its decreased DNA-binding activity, Jun B is not as potent as c-Jun inactivating gene transcription.46 In cell types such asHeLa cells, in which c-Jun is the major protein of theJun family, Jun B most likely does not play a significantrole in activating AP-1-responsive genes. However, thesituation may be different in other cell types in whichthe content of Jun B is higher, since it will be able toform abundant heterodimers with Fos proteins. TheDNA-binding affinity of Jun B: c-Fos or Jun B: Fos B iscomparable to that of c-Jun complexes.47 The impor-tance of Jun B in collagenase and TPA gene expressionin baboon SMCs is currently under investigation.

In conclusion, we have demonstrated that heparindecreases AP-1 binding in part by decreasing Jun Bexpression. Heparin probably affects the posttransla-tional phosphorylation of Jun B. We hypothesize thatheparin inhibits the kinases that phosphorylate Jun B. Itwill be important to identify the kinases that phosphor-ylate Jun B in vivo, to determine the actual phosphor-ylation sites in Jun B, and to examine the effect ofheparin on phosphorylation of these sites.

AcknowledgmentsThis study was supported by National Institutes of Health

grants HL-18645, HL-30946, and RR-00166. We thank DrRony Seger for discussion, Dr Robert Rosenberg for therecombinant heparinase, and Trevina Wang for technicalassistance.

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Y P Au, G Dobrowolska, D R Morris and A W Clowesmodification of Jun B.

Heparin decreases activator protein-1 binding to DNA in part by posttranslational

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