Effect of Proteasome Pathway on Initiationof Mouse Labor Induced byAntiprogesterone

INTRODUCTION

The physiological basis of the initiation of labor hasnot been fully elucidated. Progesterone withdrawalinduced by luteolysis in rodents or alteration of steroidhormone metabolism in ewes plays a crucial role in theinitiation of labor. However, progesterone withdrawalat parturition is not observed in humans or higherprimates.1 During the course of labor in mammals,various contraction-associated molecules includingoxytocin receptor (OTR), prostaglandin (PG) F2a

receptor (FP), cyclooxygenases (COX), PGE2 andPGF2a, are up-regulated in the pregnant uterus, fetalmembranes and placenta.2 Similar up-regulation isalso observed in a rodent preterm birth model inducedby ethanol,3 by progesterone antagonists,4 or bybacterial infection.5 To date, there is no evidence that

withdrawal of progesterone directly controls theexpression of the genes relevant to these molecules.

However, experiments using DNA microarrays haverevealed that more genes are suppressed than activatedat labor in the uteri of rodents and humans.6–8 Wepostulated that degradation of proteins whose genesare inactivated might also play an important role in theinitiation of labor. Recent studies of the ubiquitin-proteasome pathway have shown that lysosome-inde-pendent proteolysis is extensively involved in theregulation of various biological processes such as:progression of the cell cycle, oncogenesis, transcrip-tion, development and growth.9 Lactacystin, isolatedfrom Streptomyces lactacystinaeus, is a potent inhibitorof proteasome activity and is effective both in vitro andin vivo.10 To investigate the importance of proteasomefunction in the initiation of labor, we administered

American Journal of Reproductive ImmunologyAJRI 2004; 52: 317–322Copyright � Blackwell Munksgaard, 2004

Kimura T, Nakamura H, Ogita K, Koyama S, Tomiie M, Yoshida S,Tsutsui T, Shimoya K, Koyama M, Murata Y. Effect of proteasomepathway on initiation of mouse labor induced by antiprogesterone.AJRI 2004; 52:317–322 � Blackwell Munksgaard, 2004

PROBLEM:Various kinds of contraction-associated molecules are up-regulated at the initiation of labor. However, expression profiling hasrevealed that many molecules are also down-regulated. The effect ofdown-regulation of molecules by protein degradation on parturition isnot known.METHODS OF STUDY:We administered lactacystin, a specificproteasome inhibitor, to mouse preterm birth model induced byantiprogesterone RU486 on day 16.0 post-coitus. NF-kappaB activity,and the levels of transcripts for oxytocin receptor, prostaglandin F2a

receptor (FP), cyclooxygenase-1, -2, and interleukin-1b in the uteruswere examined by electrophoretic mobility shift assay and semi-quantitative reverse transcriptase-polymerase chain reaction,respectively.RESULTS:Administration of lactacystin significantly prolonged thetime until the delivery of the first pup. FP mRNA level was solelyelevated by RU486 treatment, and lactacystin significantly suppressedthis up-regulation.CONCLUSIONS:Proteolysis by proteasomes in the uterus regulatesthe initiation of labor, at least in part, via control of contraction-associated molecules such as FP.

Tadashi Kimura, Hitomi Nakamura,Kazuhide Ogita, Shinsuke Koyama,Mari Tomiie, Susumu Yoshida,Tateki Tsutsui, Koichiro Shimoya,Masayasu Koyama, Yuji MurataDivision of Obstetrics and Gynecology, Department ofSpecific Organ Regulation, Osaka University GraduateSchool of Medicine, Osaka, Japan

Key words: Antiprogesterone, initiation of parturition,lactacystin, mouse, preterm birth, proteasome

Address reprint requests to Tadashi Kimura, Division ofObstetrics and Gynecology, Department of Specific OrganRegulation, Osaka University Graduate School of Medi-cine, 2-2, Yamadaoka, Suita, Osaka 5650871, Japan.E-mail: tadashi@gyne.med.osaka-u.ac.jp

Submitted August 2, 2004;revised October 7, 2004;accepted October 7, 2004.

AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY VOL. 52, 2004

lactacystin to preterm labor model mice induced byantiprogesterone RU 486 and observed the time courseof their parturition. We also examined the expressionprofile of contraction-associated molecules (COX-1,COX-2, OTR, FP) and immunomodulators [NF-jB,interleukin (IL)-1b], to determine the effects of pro-teasome inhibition on their regulation.

MATERIALS AND METHODS

AnimalsFemale ICRmice (SLC, Shizuoka, Japan) in estrus werebred at age 8–10 weeks with male ICR mice. Themorning when vaginal plugging was observed wasdesignated as day 0.5 post-coitus (p.c.), and the success-fully bred females were separated from males. At21:00 hr on day 16.0 p.c., pregnant mice were injectedsubcutaneously with 150 lg (350 lL) of RU486 (mife-pristone, Sigma, Tokyo, Japan) or vehicle. RU486 wasfirst dissolved to a concentration of 10)2 m in absoluteethanol, and then diluted in 0.9% sodium chloride.Forty or 80 lg per mouse of lactacystin (Kyowa MedixCo. Ltd, Tokyo, Japan) at a concentration of 0.1 mg/mL or vehicle (0.9% saline) was injected intraperiton-eally simultaneously.11 The period required for initi-ation of parturition was determined as the time fromdrug administration until the dam delivered the firstpup, and was checked every hour. All animal experi-ments were performed according to the appropriateguidelines for animal use approved by the InstitutionalAnimal Care and Use Committee of Osaka UniversityGraduate School of Medicine.

Nuclear Extract PreparationMice were killed 6 or 12 hr after RU486 + lactacystintreatment or vehicle treatment by administration of anexcess of anesthetic. The uteri were removed, separatedfrom fetus and placenta, and snap frozen in liquidnitrogen after washing the mucosal and serosal surfacewith normal saline. Nuclear proteins from uterine tissuewere prepared as described previously.12Approximatelyhalf of each frozen uterus was used and the pelletednuclei were resuspended in 200 lL of nuclear extractionbuffer. The nuclear extract was aliquoted, snap frozen inliquid nitrogen and stored at )80�C. Protein concentra-tion was determined using a Bio-Rad protein assay kit(Bio-Rad Laboratories, Inc., Hercules, CA, USA)according to the manufacturer’s instructions.

Electrophoretic Mobility Shift AssayFor electrophoretic mobility shift assay (EMSA), senseand antisense oligonucleotides corresponding to the jBconsensus sequence (Table I) were synthesized byHokkaido System Science (Sapporo, Japan), annealed

and labeled with a-32P dCTP (Amersham Biosciences,Co., Piscataway, NJ, USA) by the Klenow fill-inreaction. Labeled probe was purified by SephadexG-50 (Amersham) spun column fractionation. Thebinding reaction was performed in 20 lL with approxi-mately 1 ng (10,000 cpm) of labeled NF-jB probe atroom temperature (22�C) for 30 min. Then the mixturewas electrophoresed at 4�C on a 5% polyacrylamidegel made with 0.5X TBE buffer. To determine thebinding specificity, an excess (·10–1000-fold) of coldNF-jB probe was added 10 min before the addition oflabeled NF-jB probe. EMSA was performed fornuclear extracts from four animals of each condition,and the signal intensities of shifted bands wereanalyzed using a Fuji BAS 2500 image analyzer (FujiPhoto Film Co., Ltd, Tokyo, Japan).

Reverse Transcriptase-Polymerase Chain ReactionTotal RNA was extracted from approximately half of auterus using TRIZOL� (Invitrogen, Tokyo, Japan),according to the manufacturer’s instructions. Single-stranded cDNA was synthesized from 3 lg of totalRNA using 50 ng of random primers (Invitrogen) and200 U of SuperScriptTMII Reverse Transcriptase (In-vitrogen). One-tenth of the first-strand cDNA productwas subjected to PCR. PCR on complementary DNAwas performed with primers purchased from HokkaidoSystem Science using rTaq DNA polymerase (Toyobo,Osaka, Japan) to test the expression levels of IL-1b, FP,OTR, COX-1, and COX-2 transcripts. The PCR primersequences and annealing temperatures used are listed inTable I. Primers for IL-1b and G3PDHwere purchasedfrom R&D (Minneapolis, MN, USA) and Toyobo,respectively. After 30 cycles of PCR (which was beforeamplification reached a plateau), the products weresubjected to electrophoresis in a 2% agarose gel andvisualized by ethidium bromide staining. PCR wasperformed for four samples for each condition, and themRNA levels were analyzed using the NIH imagesoftware program (http://rsb.info.nih.gov/nih-image/)and normalized against the level of G3PDHmRNA. Asa negative control, the same procedure without addi-tion of reverse transcriptase was performed for eachprimer set.

Statistical Evaluation of ResultsStatistical analysis was performed using the Mann–Whitney U-test, and differences with P-value <0.05were considered significant.

RESULTS

All animals delivered pups 16–18 hr after administra-tion of RU486 on day 16.0 p.c. In the lactacystin

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administration group, all dams delivered their pupsafter 18 hr. There were significant differences of theperiod required until the delivery of the first pupbetween the RU486 treatment group and theRU486 + lactacystin 40 or 80 lg treatment groups.Although the period until the dam’s first pup deliveryin the 80 lg lactacystin treatment group appeared tobe longer than the period in the 40 lg treatment group,the difference did not reach the level of statisticalsignificance (Fig. 1).

The levels of NF-jB activity determined by EMSAafter 6 and 12 hr of the treatment were similar in thecontrol, RU486-treated, and RU486 + lactacystin-treated mice. The specificity of shifted signals wasdetermined by addition of an excess cold probes asdescribed previously (Fig. 2A,B).12 NF-jB activity was

TABLE I. Sequences of EMSA probes and PCR primers

For EMSA probes

For labeled NF-jB probe 5¢-AGTTGAGGGGACTTTCCCAGG-3¢5¢-GCCTGGGAAAGTCCCCTCAACT-3¢

For cold NF-jB probe 5¢- AGTTGAGGGGACTTTCCCAGGC-3¢5¢-GCCTGGGAAAGTCCCCTCAACT-3¢

For PCR primers Annealing temp. (�C) cDNA size (bp)

OTR 5¢-GAAGGATCTGGCGCGTTT-3¢5¢-CCACATCTGCACGAAGAAGA-3¢

58 428

COX-1 5¢-AGGAGATGGCTGCTGAGTTGG-3¢5¢-AATCTGACTTTCTGAGTTGCC-3¢

58 600

COX-2 5¢-ACACACTCTATCACTGGCACC-3¢5¢-TTCAGGGAGAAGCGTTTGC-3¢

60 273

IL-1b R&D RDP-174–025 55 587

FP 5¢-GCTCTTGGTGTTTCCTTCTCG-3¢5¢-TGCTTGCTGGCTCTCCTTCTC-3¢

58 446

40 g lactacystin i.p. (n = 4)

80 g lactacystin i.p. (n = 6)

RU486 s.c. (n = 6)

• RU486(16.0d) s.c.+ lactacystin:

• Control:

vehicle s.c. (n = 6)

Per

cent

age

00 15 20 25 30

50

100

0Time (hr)

Fig. 1. Time course of parturition after RU486 � lactacystin treat-

ment. Mice were treated at 21:00 hr on day 16 p.c. with 150 lg of

RU486 (n ¼ 6), 150 lg of RU486 + 40 lg of lactacystin (n ¼ 4),

150 lg of RU486 + 80 lg of lactacystin (n ¼ 6), or vehicle (n ¼ 6).

The time of the delivery of the first pup was observed by checking

every hour. Lactacystin administration (40 and 80 lg) significantlydelayed the delivery compared with the RU486 treatment group (log-

rank test, P < 0.05). There was no statistical difference between the

40 and 80 lg lactacystin treatment groups.

A

Con

trol

+ x

1000

col

d

Con

trol

+ x

100

cold

Con

trol

+ x

10 c

old

Con

trol

cont

rol

RU

486

RU

486+

lact

acys

tin

cont

rol

RU

486

RU

486+

lact

acys

tin

Fre

e P

6H 12H

B

0

0.5

1

1.5

NF

-B

activ

ity(A

rbitr

ary

units

)

control RU486RU486

+lactacystin

control RU486RU486

+lactacystin

6H 12H

Fig. 2. NF-jB activity at 6 and 12 hr after administration of

RU486 � lactacystin. Nuclear protein extracted from the whole

uterus was examined by EMSA. In the control uterus obtained at the

same gestational period, significant NF-jB activity was detected and

the levels were similar after 6 and 12 hr of treatment with

RU486 � lactacystin. The specificity of the signals was determined

by the addition of an excess of cold probe (A). Quantitative analysis

of five animals in each group (B).

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relatively high even in the control uterus, and both p50and p65 proteins were localized mainly in the endome-trial layer in the uterus (data not shown).We examined the levels of transcripts for contrac-

tion-associated molecules by semiquantitative reversetranscriptase-polymerase chain reaction (RT-PCR).After 6 hr of RU486 treatment, OTR mRNA wassignificantly up-regulated, whilst the levels of IL-1band FP mRNAs were suppressed. However, there wereno differences of these levels between RU486-treatedand RU486 + lactacystin-treated groups. FP expres-sion was significantly up-regulated after 12 hr ofRU486 treatment (approximately 15-fold). Simulta-neous administration of lactacystin significantly sup-pressed RU486-induced FP up-regulation. Nosignificant difference was observed in the expressionlevel of other molecules after 12 hr of treatment(Table II, Fig. 3).

DISCUSSION

To our knowledge, this is the first report indicatingthat aberration of the proteolytic pathway mediated bythe proteasome leads to a delay of initiation ofparturition. As we did observe neither the prolongedlabor nor dystocia after lactacystin treatment, protea-some-related metabolism mainly affected the mechan-ism of preparation for or initiation of labor.Progesterone plays an essential role in blockingmyometrial excitatory responsiveness and promotingmaintenance of pregnancy. Physiological luteolysis,removal of the ovaries or administration of progester-one antagonists leads to increased uterine activity andinduction of labor in most mammals.1 Our mousepreterm birth model, which is induced by RU486 onday 16.0 p.c. might mimic the process of normalparturition or infection-free spontaneous preterm birthrather than preterm birth associated with infection.However, the final events of uterine activation and up-

regulation of contraction-associated molecules appearto be similar between normal birth and pathologicalpreterm birth. Our model modulating the RU486-induced birth process will provide important insightsinto the physiology and pathophysiology of partur-ition. The day 16.0 p.c. is almost equivalent to30 weeks of gestation in human. This period is criticalfor fetal lung maturation and elongation of latentperiod until delivery might give benefit to respiratorymanagement of the newborn. The molecular target oflactacystin in the proteasome is of great interestbecause it will become a treatment target for prevent-ing preterm birth, which is still a leading cause ofneonatal mortality and morbidity.13 It is reported thatone of the major targets of lactacystin is the NF-jB-IjB system. Proteasome inhibitors decrease the degra-dation of IjB, and prevent the activation of NF-jB bynormally effective stimuli. Such proteasome inhibitor-induced NF-jB suppression is observed in many kindsof cancer cells.14 However in an exceptional cell line,NF-jB is activated after proteasome inhibitor treat-ment.15 NF-jB is reported to be as one of the keymodulators regulating contraction-associated mole-cules in the myometrium, choriondecidua, placentaand amnion of humans.16–19 NF-jB is reported toshare the progesterone receptor-binding element ongenes and lead to functional progesterone withdrawalin humans.20 However, in our experiments, weobserved a relatively high level of NFjB activity incontrol mice not in labor (Fig. 2) and no significantchange of NF-jB activity after RU486 treatment orRU486 + lactacystin treatment. These results indicatethat in mice NF-jB activation did not occur afterartificial progesterone withdrawal, at least within 6 hrbefore labor and NF-jB was not a molecular target oflactacystin in antiprogesterone-induced preterm birth.

The effect of lactacystin treatment on the delay oflabor initiation was limited to approximately 6 hr. Thedose we used (approximately 1–2 mg/kg, i.p.) waschosen based on the dose which prevented ischemic

TABLE II. The Expression Profile of Contraction-Associated Molecules (COX-1, COX-2, OTR, FP) and Immunomodulator

(IL-1b) by Semi-Quantitative RT-PCR

6 H 12 H

Control RU486 RU486 + lactacystin Control RU486 RU486 + lactacystin

OTR 1.0 � 0.50 1.36 � 0.10** 1.42 � 0.03** 1.0 � 0.09 1.49 � 0.09* 1.21 � 0.08

COX-1 1.0 � 0.09 0.95 � 0.09 0.97 � 0.05 1.0 � 0.06 0.99 � 0.09 0.83 � 0.02

COX-2 1.0 � 0.07 0.81 � 0.08 1.77 � 0.40* 1.0 � 0.19 1.48 � 0.11* 1.40 � 0.09

IL-1b 1.0 � 0.10 0.47 � 0.07** 0.65 � 0.05* 1.0 � 0.09 1.15 � 0.09 0.99 � 0.10

FP 1.0 � 0.06 0.34 � 0.09** 0.50 � 0.17* 1.0 � 0.26 15.19 � 1.22** 6.86 � 1.45**

*P<0.05; **p<0.01.

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acute renal failure when administered 1 hr beforeocclusion of the renal artery in rats.21 We tested theeffects of lactacystin treatment administered 3 hrbefore RU486 administration, or at the same time asRU486 or at the same time and thereafter by continu-ous infusion. There was no difference in the effect onthe time course of parturition among these groups(data not shown). Lactacystin is a potent and selectiveprotease inhibitor that does not affect other protein-ases examined so far.22 The time course of proteasomeinhibition by lactacystin in vivo was not characterizedin this study, but it is known that the effect ofbortezomib (PS-341), a peptide-derived proteasomeinhibitor reaches a peak at 6 hr after injection.23 Thecascade-like chain reaction toward parturition inducedby progesterone withdrawal might overcome the effectof proteasome inhibition after several hours.

In our experiments, mRNA levels of IL-1b and FPwere suppressed after 6 hr of RU486 treatment, andlactacystin had no effect on this suppression. Themechanism of this suppression is totally unknown.RU486-induced FP mRNA up-regulation after 12 hrwas significantly suppressed by lactacystin treatment.The levels of OTR and COX-2 mRNAs appeared to beelevated, but lactacystin showed no effect on theirexpression. In a mouse preterm birth model afterovariectomy on day 17 p.c., it has been reported thatmRNAs encoding three major contraction-associated

proteins (OTR, FP, and COX-2) are significantly up-regulated.24 The trend of up-regulation of these con-traction associated molecules appeared to be similar inRU486-induced and ovariectomy-induced pretermbirth. The molecular mechanism of FP up-regulationat the transcriptional level in the uterus at parturitionwas also not elucidated. From the gene structure, noputative steroid hormones-responsive elements wereidentified.25,26 The fact that lactacystin suppressed FPmRNA in RU486-treated animals suggests that thetranscription factor involved in the suppression of FPgene is degraded by the proteasome.

In conclusion, we demonstrated the importance ofthe proteolytic pathway for determination of thetiming of labor. Our observations suggest that func-tional modulation of the proteasome might become anovel target for regulation of labor initiation, ofwhich abnormality leads to preterm birth, overtermpregnancy, and hypersensitivity of uterine contrac-tion.

AcknowledgementsWe especially thank Ms Satomi Okamoto for technicalassistance. This work was supported in part by Grants-in-Aid for Scientific Research (Nos. 15390505,15209054, 15591746 and 16390476) from the Ministryof Education, Science and Culture of Japan (Tokyo,Japan).

6 H

control RU486RU486

+Lactacystin

12 H

control RU486RU486

+Lactacystin R

T (

-)

IL-1

FP

OTR

COX-1

COX-2

G3PDH

IL-1

FP

OTR

COX-1

COX-2

G3PDH

Fig. 3. Semi-quantitative RT-PCR. The

levels of expression of mRNAs for OTR,

COX-1, COX-2, IL-1b, and FP were deter-

mined by RT-PCR. The PCR products were

subjected to electrophoresis in a 2% agarose

gel and visualized by ethidium bromide

staining. PCR was performed for four

samples for each condition, and the mRNA

levels were analyzed using the NIH image

software program. The ratio of target

mRNA/G3PDH mRNA observed in the

control uterus was assigned the arbitrary

value of 1.0 unit.

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