Increased MMPs expression and decreased contraction in the rat … · 2017-06-09 · Increased MMPs expression and decreased contraction in the rat myometrium during pregnancy and

Increased MMPs expression and decreased contraction in the rat myometriumduring pregnancy and in response to prolonged stretch and sex hormones

Zongzhi Yin, Alaa A. Sada, Ossama M. Reslan, Neha Narula, and Raouf A. KhalilVascular Surgery Research Laboratory, Division of Vascular and Endovascular Surgery, Brigham and Women’s Hospital,and Harvard Medical School, Boston, Massachusetts

Submitted 31 October 2011; accepted in final form 8 April 2012

Yin Z, Sada AA, Reslan OM, Narula N, Khalil RA. IncreasedMMPs expression and decreased contraction in the rat myometriumduring pregnancy and in response to prolonged stretch and sexhormones. Am J Physiol Endocrinol Metab 303: E55–E70, 2012. Firstpublished April 10, 2012; doi:10.1152/ajpendo.00553.2011.—Normalpregnancy is associated with uterine relaxation to accommodate thestretch imposed by the growing fetus; however, the mechanismsunderlying the relationship between pregnancy-associated uterinestretch and uterine relaxation are unclear. We hypothesized thatincreased uterine stretch during pregnancy is associated with upregu-lation of matrix metalloproteinases (MMPs), which in turn causeinhibition of myometrium contraction and promote uterine relaxation.Uteri from virgin, midpregnant (day 12), and late-pregnant rats (day19) were isolated, and myometrium strips were prepared for measure-ment of isometric contraction and MMP expression and activity usingRT-PCR, Western blot analysis, and gelatin zymography. Oxytocincaused concentration-dependent contraction of myometrium stripsthat was reduced in mid- and late-pregnant rats compared with virginrats. Pretreatment with the MMP inhibitors SB-3CT (MMP-2/MMP-9Inhibitor IV), BB-94 (batimastat), or Ro-28–2653 (cipemastat) en-hanced contraction in myometrium of pregnant rats. RT-PCR, West-ern blot analysis, and gelatin zymography demonstrated increasedmRNA expression, protein amount, and activity of MMP-2 andMMP-9 in myometrium of late-pregnant�midpregnant�virgin rats.Prolonged stretch of myometrium strips of virgin rats under 8 g basaltension for 18 h was associated with reduced contraction and en-hanced expression and activity of MMP-2 and MMP-9, which werereversed by MMP inhibitors. Concomitant treatment of stretchedmyometrium of virgin rats with 17�-estradiol (E2), progesterone (P4),or E2�P4 was associated with further reduction in contraction andincreased MMP expression and activity. MMP-2 and MMP-9 causedsignificant reduction of oxytocin-induced contraction of myometriumof virgin rat. Thus, normal pregnancy is associated with reducedmyometrium contraction and increased MMPs expression and activ-ity. The results are consistent with the possibility that myometriumstretch and concomitant increase in sex hormones during pregnancyare associated with increased expression/activity of specific MMPs,which in turn inhibit uterine contraction and promote uterine relax-ation.

estrogen; progesterone; matrix metalloproteinases; uterus

THE UTERUS IS SENSITIVE TO various mechanical and chemicalstimuli, and its response varies in the nonpregnant and preg-nant state and during parturition. The nonpregnant uteruscontracts in response to stretch, but during pregnancy mecha-nisms are in play to stabilize the excitability of the myometrialmuscles and maintain mechanical quiescence to allow suffi-

cient time for the development of the fetus before it is deliveredduring labor (43). The uterus dramatically expands in volumeduring pregnancy. Hypertrophy and distension of the pregnantuterus provide sufficient space for the developing and growingfetus. At term, other changes occur to counteract the factorsthat maintain uterine quiescence, increase electrical excitabilityof myometrial muscles, and coordinate contractions betweenuterine smooth muscle bundles and thereby ensure successfulparturition. The mechanisms that facilitate uterine quiescenceduring pregnancy and enhance contractility at term are notfully understood (7).

Several steroid and neurohypophysial hormonal changesoccur during the menstrual cycle, pregnancy, and parturition,and could influence the uterine structure and mechanical prop-erties. Plasma 17�-estradiol (E2) levels increase during theproliferative phase of the menstrual cycle. A decrease inplasma E2 is associated with endometrial shedding, and anincrease in plasma progesterone (P4) occurs during the lutealphase of the menstrual cycle (30). Plasma E2 and P4 markedlyincrease during pregnancy (52) and could contribute to thepregnancy-associated uterine relaxation (15, 45). Changes inneurohypophysial hormones such as oxytocin also affect uter-ine contraction. Oxytocin is a nonapeptide produced largely bythe hypothalamus-pituitary and other tissues including thecorpus luteum (65, 66), adrenal medulla (1), and placenta (16).In the nonpregnant state, plasma levels of oxytocin are rela-tively low and do not appear to change during the menstrualcycle. During pregnancy, plasma oxytocin levels demonstrateprogressive increases starting at week 12 of gestation (60).Oxytocin is important for cervical dilation before birth andcauses uterine contractions during the second and third stagesof labor.

In addition to the role of steroid and neurohypophysialhormones, uterine enzymes and proteins could modulate itsstructure and function. Matrix metalloproteinases (MMPs) area group of zinc-dependent proteases that degrade the extracel-lular matrix (49). MMP-2 (gelatinase A), MMP-9 (gelatinaseB), and MMP-7 are expressed in the uterus and could play arole in the endometrial tissue remodeling during normal estrousand menstrual cycles and pregnancy (53, 62, 70), as well as inthe endometrial changes associated with menstrual disordersand endometriosis (35, 58). MMP-2 and -9 have been localizedin the bovine endometrium and myometrium during pregnancy(25, 53, 62) and in uterine natural killer cells in early humanpregnancy (41). Also, trophoblast- and vascular smooth mus-cle-derived MMP-12 mediate elastolysis and uterine spiralartery remodeling during pregnancy (22). MMP-2 and MMP-9could also play a role in degradation of the uterine proteins andremodeling of the cervical extracellular matrix (33), and in-creased MMP-2 expression may precede the final ripening

Address for reprint requests and other correspondence: R. A. Khalil, HarvardMedical School, Brigham and Women’s Hospital, Division of Vascular Surgery,75 Francis St., Boston, MA 02115 (e-mail: [email protected]).

Am J Physiol Endocrinol Metab 303: E55–E70, 2012.First published April 10, 2012; doi:10.1152/ajpendo.00553.2011.

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process and collagen denaturation of the cervix in late preg-nancy (63).

MMPs are regulated by several factors including mechanicalstretch and sex hormones. MMP-2 expression increases inmechanically stretched skeletal muscle fibers (39). Also, wehave recently shown that protracted increases in vein walltension are associated with increased MMP-2 and -9 expres-sion and decreased contraction of rat inferior vena cava (48, 50,51). Other studies have suggested that uterine tissue remodel-ing and endometrium shedding during menstruation involveE2-induced changes in MMPs activity (54, 56, 70). Progestinsmay also regulate important factors for the establishment andmaintenance of endometrial lesions partly by affecting MMPsexpression (40). However, the interrelationship between myo-metrium stretch, sex hormones, and MMPs expression in theuterine relaxation during pregnancy is unclear.

The objective of this study was to test the hypothesis thatuterine stretch during pregnancy is associated with increasedexpression of MMPs, which in turn inhibit myometrium con-traction and promote uterine relaxation. We used the rat uterusto investigate whether: 1) different stages of pregnancy areassociated with decreased myometrium contraction and en-hanced MMPs expression/activity, 2) prolonged myometriumstretch is associated with decreased contraction and increasedMMP expression/activity, 3) sex hormones augment the effectsof prolonged stretch on uterine relaxation and MMP expres-sion, and 4) specific MMPs inhibit myometrium contraction.

MATERIALS AND METHODS

Animals and Tissue Preparation

Virgin, midpregnant (12 days of gestation, mid-Preg) and late-pregnant (19 days of gestation, late-Preg) Sprague-Dawley rats (12 wkof age, 250 to 350 g weight) were purchased from Charles RiverLaboratories (Wilmington, MA). The rats were housed in the animalfacility and maintained on ad libitum standard rat chow and tap waterin 12:12-h light-dark cycle. All experiments on virgin rats wereconducted during estrus to control for reproductive cycle and endo-crine confounders. The estrus cycle was determined by taking avaginal smear with a Pasteur pipette daily in the morning (36). Anestrus smear primarily consisted of anucleated cornified squamouscells (68), and this was confirmed prior to all experimentations. On theday of experiment, the rats were euthanized by inhalation of CO2.Death was judged by cessation of breathing and heart beats. Theabdominal cavity was opened, and the uterus was rapidly excised,placed in Krebs solution, and carefully dissected and cleaned ofconnective tissue under microscopic visualization. The virgin uteruswas cut open, and the pregnant uterus was cut open and the placentaeand pups were removed. The uterus was then portioned along itslongitudinal axis into 7-mm long, 3-mm wide strips. We did notattempt to separate the circular muscle layer from the longitudinalmuscle or to remove the endometrium lining from the uterine strip.Experiments were performed on two to four uterine strips from eachrat, and cumulative data from four to six rats were collected. Allprocedures followed the guidelines of the Institutional Animal Careand Use Committee at Harvard Medical School.

Isometric contraction. Using silk string, one tie was made 1 mmfrom each end of the myometrium strip, leaving a functional 5-mm-long strip between two string loops. Each myometrium strip wassuspended in a water-jacketed tissue bath filled with 50 ml Krebssolution bubbled with 95% O2/5% CO2 at 37°C. One end of theuterine strip was attached to a fixed glass hook at the bottom of thetissue bath, and the other end was connected to a Grass forcetransducer (model FT03; Astro-Med, West Warwick, RI). Changes in

isometric contraction were recorded on Grass polygraph (model 7D;Astro-Med).

To construct basal tension-contraction relationship, uterine strips ofvirgin rats were stretched under increasing basal tension 0.5, 1, 2, 3,4, 5, 6, 7, and 8 g, maintained under each basal tension for 30 min andthen stimulated with 96 mM KCl. These experiments demonstratedmaximal KCl contraction at 2 g basal tension and significant decreasein contraction at 8 g basal tension.

Uterine strips of virgin and pregnant rats were stretched undercontrol of 2 g basal tension for 30 min. To determine the controlcontraction properties, uterine strips were stimulated twice with 96mM KCl, and each control KCl contraction was followed by threewashes in Krebs, 10 min each. The uterine strips were then stimulatedwith increasing concentrations of oxytocin (10�11 to 10�7 M) and thecontractile response was recorded. To correct for differences in themyometrium thickness, contraction measurements were normalized tothe weight of the myometrium strip and presented as gram permilligram tissue weight. The contractile response at each oxytocinconcentration was also presented as percentage of maximum oxytocincontraction. The individual oxytocin concentration-response curveswere analyzed using a nonlinear regression curve (best-fit sigmoidaldose-response curve; Sigmaplot), and the effective concentration thatproduced half of the maximal contraction was presented as pEC50

(�log M).We have previously demonstrated that prolonged increases in rat

inferior vena cava wall tension for 18 to 24 h was associated withdecreased contraction (31, 50). To demonstrate the effect of prolongedstretch on myometrium contraction, uterine strips of virgin rats weresubjected to either control (2 g basal tension) or high (8 g tension) for18 h in tissue culture medium. On the next day, the bathing solutionwas changed to Krebs solution, and oxytocin contraction was mea-sured. To determine the effects of sex hormones, uterine strips ofvirgin rats were treated with E2 (10�6 M), P4 (10�5 M), or both, andtheir effects on uterine contraction were measured.

To determine whether the changes in uterine contraction associatedwith pregnancy or prolonged stretch involve changes in MMP activ-ity, contraction experiments were repeated in the presence of theMMP inhibitors SB-3CT (MMP-2/MMP-9 inhibitor IV), BB-94 (ba-timastat), or Ro-28–2653 (cipemastat). SB-3CT is a potent, selective,slow-binding and mechanism-based inhibitor of the gelatinasesMMP-2 (Ki � 13.9 nM) and MMP-9 (Ki � 600 nM) (5). Theinteraction of SB-3CT with MMP-2 and -9 follows a slow-bindinginhibition similar to that observed with TIMP-1 and TIMP-2, andultimately results in covalent modification of the enzyme in the activesite (42). SB-3CT directly binds to the catalytic zinc ion of MMP-2and changes the conformational environment around the active sitezinc ion back to that of the proenzyme (26). BB-94 is a potent broadspectrum MMP inhibitor (IC50 � 3, 4, 4, 6, and 20 nM for MMP-1,-2, -9, -7 and -3, respectively) (13, 64). Other studies have reportedBB-94 IC50 values of 25, 32, 67, 27, 23, 19, and 29 nM for MMP-1,-2, -3, -8, -9, -14 (membrane-type 1-MMP, MT-1-MMP), and -16(MT-3-MMP), respectively (21). Ro-28–2653 {5-biphenyl-4-yl-5-[4-(4-nitro-phenyl)-piperazin-1-yl]-pyrimidine-2,4,6-trione} is a newzinc chelator of MMPs, with higher selectivity for MMP-2, -9, -14,and -8 (IC50 � 10, 12, 10, and 12 nM, respectively), compared withMMP-3 and -1 (IC50 � 1200 and 16000 nM, respectively) (21, 28,34). We have previously shown that these MMP inhibitors at 10�6 Mconcentration reverse the inhibitory effects of MMP-2 on vein con-traction (48). Therefore, the present experiments were conductedusing these inhibitors at 10�6 M concentration. In the initial contrac-tion experiments, SB-3CT was more effective than BB-94 and Ro-28–2653 in causing contraction in uterine segments of pregnant rats.Therefore, further experiments to study the effects of sex hormonesand stretch on uterine contraction and MMP expression/activity wereconducted using only two MMP inhibitors: SB-3CT as a potentinhibitor and Ro-28–2653 for comparison.

E56 MMPs EXPRESSION AND MYOMETRIUM RELAXATION DURING PREGNANCY

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Also, to test the direct effects of MMPs, uterine strips of virgin ratswere precontracted with oxytocin (10�7 M), MMP-2 or MMP-9 (1�g/ml) was added, and the effect on uterine contraction was mea-sured.

Real-time RT-PCR. RNA was isolated from the uterine strips usingRNeasy Fibrous Tissue Mini Kit (QIAGEN, Valencia, CA). TotalRNA (1 �g) was used for RT to synthesize single-strand complimen-tary DNA (cDNA) in 15- to 33-�l reaction mixture following theinstructions for the First-Strand cDNA Synthesis Kit (AmershamBiosciences, Pittsburgh, PA). Next, 2 �l of the cDNA dilution (1:5 forMMP-2, MMP-7, and MMP-9 and 1:25 for �-actin) of the RT productwas applied to 20 �l of RT-PCR reaction mixture. Quantification ofgene expression was performed using a real-time RT-PCR machine(model Mx4000; Multiplex Quantitative PCR System, Stratagene, LaJolla, CA), published oligonucleotide primers for MMP-2, MMP-7,and MMP-9 (Integrated DNA Technologies, Coralville, Iowa), andiQSYBR-Green Supermix, which employs the fluorescein compoundSYBR-Green for amplicon detection (Bio-Rad, Hercules, CA). �-Ac-tin primer was included in the RT-PCR reaction as internal standardto normalize the results (31).

The following primers were used. MMP-2: forward, 5=-CATCGCT-GCACCATCGCCCATCATC-3=; reverse, 5=-CCCAGGGTCCA-CAGCTCATCATCATCAAAG-3=. MMP-7: forward, 5=-TGGGT-CTGGGTCACTCTTCT-3=; reverse, 5=-CACAGCTTGTTCCTC-TTTCC-3=. MMP-9: forward, 5=-GAAGACTTGCCGCGAGACCT-GATCGATG-3=; reverse, 5=-GCACCAGCGATAACCATCCGAGC-GAC-3=. �-Actin: forward, 5=-GACACCAGGGAGTGATGGTT-3=;reverse, 5=-GTTAGCAAGGTCGGATGCTC-3=.

PCR was carried out with one cycle for 10 min at 95°C and then 40cycles of 30 s of denaturation at 95°C, 45 s of annealing at 59°C, and30 s of extension at 72°C, followed by 1 min of final extension at95°C. The number of PCR cycles varied according to the expressionlevel of the target gene. An appropriate primer concentration andnumber of cycles was determined to ensure that the PCR is takingplace in the linear range and thereby guarantees a proportionalrelationship between input RNA and the cycles readout. The relativegene expression was calculated by comparison of cycle thresholdswith the housekeeping gene �-actin.

Western blot analysis. The uterus was homogenized in a buffercontaining 20 mM 3-[N-morpholino] propane sulfonic acid, 4% SDS,10% glycerol, 2.3 mg dithiothreitol, 1.2 mM EDTA, 0.02% BSA, 5.5M leupeptin, 5.5 M pepstatin, 2.15 M aprotinin and 20 M 4-(2-aminoethyl)-benzenesulfonyl fluoride, using a 2-ml tight-fitting ho-mogenizer (Kontes Glass). The homogenate was centrifuged at 10,000g for 2 min. The supernatant was collected, and protein concentrationwas determined using a protein assay kit (Bio-Rad). Tissue homoge-nate was subjected to electrophoresis on 8% SDS polyacrylamide geland then transferred electrophoretically to nitrocellulose membranes(Bio-Rad). The membranes were incubated in 5% dried nonfat milk inPBS-Tween buffer for 1 h, and then in the antibody solution contain-ing MMP-2, MMP-7, or MMP-9 (1:500) rabbit polyclonal antibody(Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C for 24 h. �-Actinwas used as an internal control and detected by a monoclonal anti-actin antibody (1:500,000; Sigma). The nitrocellulose membraneswere washed in PBS-Tween five times for 15 min each and thenincubated in horseradish peroxidase-conjugated secondary antibody(1:1,000) for 1.5 h. The membrane blots were washed with PBS-Tween and visualized with enhanced chemiluminescence (ECL)Western blotting detection reagent (GE Healthcare Bio-Sciences,Piscataway, NJ), and the reactive bands corresponding to MMP-2,MMP-7, and MMP-9 were analyzed by optical densitometry andImageJ software (National Institutes of Health, Bethesda, MD). Thedensitometry values represented the pixel intensity normalized to�-actin to correct for loading.

Gelatin zymography. The uterus homogenate (without dithiothrei-tol) was subjected to electrophoresis on 8% SDS polyacrylamide gelcontaining 0.1% gelatin (Sigma). The gel was then incubated in a

zymogram renaturing buffer containing 2.5% Triton X-100 (Sigma)with gentle agitation for 30 min at room temperature. The gel was thenequilibrated in a zymogram developing buffer containing 50 mMTris-base, 0.2 M NaCl, 5 mM CaCl2, 0.02% Brij35 (Fisher Scientific),and 1 �M ZnCl2 (Sigma) for 30 min at room temperature and thenincubated in the zymogram developing buffer at 37°C for 24 h. Thegel was stained with 0.5% coomassie blue R-250 (Sigma) for 30 minand then destained with an appropriate coomassie R-250 destainingsolution (methanol/acetic acid/water � 50:10:40). Areas correspond-ing to MMP-2 and MMP-9 activity appeared as clear bands against adark blue background. The clear bands were analyzed by opticaldensitometry and ImageJ software, and the integrated protease activitydensity was recorded (pixel intensity mm2) and then normalized toactin intensity.

Solutions and drugs. Normal Krebs solution contained (in mM):120 NaCl, 5.9 KCl , 25 NaHCO3, 1.2 NaH2PO4, 11.5 dextrose (FisherScientific, Fair Lawn, NJ), 2.5 CaCl2 (BDH Laboratory Supplies,Poole, England), 1.2 MgCl2 (Sigma). Krebs solution was bubbledwith 95% O2/5% CO2 for 30 min at an adjusted pH 7.4. High KClsolution (96 mM) was prepared as normal Krebs but with equimolarsubstitution of NaCl with KCl. Stock solution of oxytocin (10�3 M;Sigma) was prepared in deionized water. Stock solution (10�2 M) ofMMP-2/MMP-9 inhibitor IV (SB-3CT; EMD Millipore, Billerica,MA), Ro-28–2653 {5-biphenyl-4-yl-5-[4-(-nitro-phenyl)-piperazin-1-yl]-pyrimidine-2,4,6-trione; Roche Diagnostics, Pharma Research.Penzberg, Germany}, and BB-94 (batimastat; Tocris Bioscience,Minneapolis, MN) was prepared in DMSO. The final concentration ofDMSO in the experimental solution was 0.1%. The tissue culturemedium used to incubate the uterus overnight was composed of MEMsupplemented with penicillin, streptomycin, and amphotericin B(Gibco/Invitrogen, Grand Island, NY). All other chemicals were ofreagent grade or better.

Statistical analysis. Cumulative data from uterine strips of 4 to 6rats were presented as means � SE. The data were first analyzed usingANOVA. When a statistical difference was observed, the data werefurther analyzed using Student-Newman-Keuls post hoc test for mul-tiple comparisons. Student’s t-test for paired and unpaired data wasused for comparison of two means. Differences were consideredstatistically significant at P 0.05.

RESULTS

In uterine strips of virgin rats, oxytocin caused a contractileresponse that had two components: 1) a steady increase incontraction and 2) an additional variable spontaneous phasiccontractile response (Fig. 1A). For consistency, we focused onthe steady component of oxytocin-induced contraction. Oxy-tocin caused concentration-dependent contraction that reacheda maximum of 1.81 � 0.13 g at 10�7 M. The oxytocin-inducedcontraction was significantly reduced in uterine strips of mid-Preg rats (maximum, 0.82 � 0.15 g) and late-Preg rats (max-imum, 1.20 � 0.13 g) compared with virgin rats (Fig. 1, A andB). Measuring the weight of the uterine strips at the end of thecontraction experiments demonstrated that the uterine stripweight was in late-Preg � mid-Preg � virgin rats (Fig. 1C).When the oxytocin response was normalized to the uterine stripweight, the oxytocin-induced contraction was reduced in uter-ine strips of mid-Preg (maximum, 0.04 � 0.01 g/mg tissue wt)and further reduced in late-Preg rats (maximum, 0.03 � 0.00g/mg) compared with virgin uterus (maximum, 0.14 � 0.01g/mg) (Fig. 1D). When the oxytocin response was measured aspercentage of maximum contraction and the EC50 was calcu-lated, oxytocin was equally potent in uterine strips of virgin(pEC50, 6.36 � 0.12), mid-Preg (6.4 � 0.1), and late-Preg rats(6.34 � 0.09) (Fig. 1E).

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In uterine strips of virgin rats precontracted with oxytocin,addition of MMP-2/MMP-9 inhibitor IV (SB-3CT, 10�6 M),BB-94 (10�6 M), or Ro-28–2653 (10�6 M) did not cause anysignificant change in contraction. In contrast, in uterine stripsof mid-Preg and late-Preg rats, addition of the MMP inhibitors,particularly the MMP-2/MMP-9 inhibitor IV (SB-3CT), en-hanced uterine contraction (Fig. 2). Similarly, pretreatment ofuterine strips of virgin rats with SB-3CT, BB-94, or Ro-28–2653 for 30 min did not significantly change oxytocin contrac-tion. In contrast, pretreatment with MMP inhibitors, particu-larly SB-3CT, enhanced oxytocin contraction in uterine stripsof mid-Preg rats. Also, pretreatment with SB-3CT, BB-94, orRo-28–2653 significantly enhanced oxytocin contraction inuterine strips of late-Preg rats (Fig. 3).

RT-PCR analysis revealed expression of MMP-2, MMP-9,and MMP-7 mRNA in uterine strips of virgin rats (Fig. 4).

MMP-2 and -9 mRNA expression was significantly enhancedin uterine strips of mid-Preg and late-Preg rats compared withvirgin rats (Fig. 4, A and B). In contrast, MMP-7 mRNAexpression was significantly reduced in mid-Preg and late-Pregrats compared with virgin rats (Fig. 4C).

Western blot analysis revealed prominent bands correspond-ing to pro-MMP-2 (72 kDa), MMP-2 (63 kDa), MMP-9 (92kDa), and MMP-7 (28 kDa) in uterine strips of virgin rats(Fig. 5). The protein amount of MMP-2 and MMP-9 wassignificantly enhanced in mid-Preg and late-Preg rats comparedwith virgin rats (Fig. 5, A and B). In contrast, MMP-7 proteinwas slightly reduced in mid-Preg rats and significantly reducedin late-Preg rats compared with virgin rats (Fig. 5C). In parallelWestern blot experiments, the effect of pretreating the uterinestrips of virgin, mid-Preg, and late-Preg rats with the MMPinhibitor SB-3CT (10�6 M) on MMPs protein amount was

Fig. 1. Oxytocin-induced contraction. Uterinestrips from virgin, mid-Preg (12d-Preg), andlate-Preg rats (19d-Preg) were incubated innormal Krebs solution. The uterine stripswere stimulated with oxytocin (10�11 to 10�7

M) and the contractile response was recorded(A). At the end of the experiment, the uterinetissue weight was recorded (C). Cumulativecontraction data from uterine strips of differ-ent rats were presented in grams (B), grams/milligram tissue weight (D) and % of maxi-mal contraction (E). Preg, pregnant; 12d, 12days. Data represent means � SE, n � 4 to 6.*Significantly different (P 0.05).




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tested (Fig. 5C). Western blot analysis demonstrated that theoptical density of the pro-MMP-2, MMP-2, MMP-9, andMMP-7 immunoreactive bands in uterine strips of virgin,mid-Preg, and late-Preg rats were not significantly different inuterine strips nontreated or concomitantly pretreated with theMMP inhibitor SB-3CT.

Gelatin zymography analysis revealed prominent digestedgelatin bands corresponding to pro-MMP-2 and MMP-2, and aless prominent band corresponding to MMP-9 in uterine tissuehomogenate from virgin rats (Fig. 6). The intensity of pro-MMP2, MMP-2, and MMP-9 digested gelatin bands weresignificantly enhanced in uterine strips of mid-Preg and late-Preg rats compared with virgin rats (Fig. 6, A, C, and E). Theintensity of the pro-MMP-2, MMP-2, and MMP-9 digestedgelatin bands was significantly reduced in uterine strips treatedwith the MMP inhibitor SB-3CT (Fig. 6, B, D, and F) com-pared with control nontreated uterine strips of virgin, mid-Preg,and late-Preg rats.

To determine whether the observed changes in oxytocincontraction and MMP expression/activity in the pregnantuterus are related to uterine tissue stretch, experiments wereconducted to determine the effects of uterine tissue stretch onuterine contraction and MMP expression/activity. Our workinghypothesis predicts that the decreased uterine contraction dur-ing pregnancy are related to increased MMPs expression partly

due to prolonged uterine stretch caused by the developing fetusand partly by the pregnancy-associated increase in sex hor-mones. To dissect these components, we tested the effects ofprolonged stretch alone and sex hormones alone comparedwith combined stretch and sex hormones in virgin uterus,which would roughly mimic the conditions during pregnancy.Our rationale for not testing the effects of stretch on thepregnant uterus is that the pregnant myometrium has alreadybeen stretched and MMPs have already been overexpressed invivo. If this is the case, then one would predict that stretchingthe pregnant uterus should not cause any further increases inMMPs expression/activity or reduction in contraction, and thisshould be further examined in future experiments.

In uterine strips of virgin rat, the magnitude of KCl (96mM)-induced contraction, dependent on the initial basal ten-sion applied to the tissue, showed progressive increases withincreasing basal tension and reached a maximum at 2 g basaltension (Fig. 7). The KCl-induced contraction remained steadyat 3 and 4 g basal tension, significantly decreased at 5 and 6 gbasal tension, and remained at reduced levels at 7 and 8 g basaltension (Fig. 7).

We tested the effect of uterine tissue stretch on oxytocincontraction. In uterine strips of virgin rats stretched under 8 gbasal tension for 30 min, oxytocin contraction was significantlyreduced to a maximum of 0.04 � 0.02 g/mg tissue compared

Fig. 2. Effect of matrix metalloproteinase (MMP) inhibitors on oxytocin-induced contraction. Uterine strips of virgin (A), mid-Preg (B), and late-Preg rats (C)were precontracted with oxytocin 10�7 M. Once oxytocin contraction reached steady state the tissues were treated with SB-3CT (MMP-2/MMP-9 inhibitor IV),BB-94, or Ro-28–2653 (10�6 M) or the vehicle (DMSO), and the effect on oxytocin contraction was observed. Data represent means � SE, n � 4 to 6.*Significantly different (P 0.05).




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with the contraction of control uterine strips under 2 g basaltension (0.14 � 0.01 g/mg tissue). In virgin uterine stripssubjected to prolonged 18-h stretch under 8 g basal tension, theoxytocin contraction was significantly reduced (Fig. 8A). Inuterine strips of virgin rats stretched under 8 g basal tension for18 h and treated with the MMP inhibitor SB-3CT or Ro-28–2653, the oxytocin contraction was significantly enhanced to

levels similar to oxytocin contraction in uterine strips undercontrol 2 g basal tension (Fig. 8A).

To test the effects of sex hormones on uterine contraction,uterine strips of virgin rats were incubated under 2 g tension for18 h in the presence of E2 (10�6 M), P4 (10�5 M), or E2�P4.Oxytocin contraction was reduced in virgin uterus treated withE2 (Fig. 8B), P4 (Fig. 8C), or E2�P4 (Fig. 8D) compared withvirgin uterus without hormone treatment (Fig. 8A). In uterinestrips under 2 g tension and treated with E2, SB-3CT caused noeffect and Ro-28–2653 caused insignificant enhancement ofcontraction (Fig. 8B). Also, in uterine strips under 2 g tensionand treated with P4, SB-3CT caused no effect and Ro-28–2653caused slight enhancement of contraction that was not statis-tically different from that in uterine strips treated with P4 aloneor with P4�SB-3CT (Fig. 8C). In uterine strips under 2 gtension and treated with E2�P4, both SB-3CT and Ro-28–

Fig. 4. MMPs mRNA expression. Uterine tissue homogenate from virgin,mid-Preg, and late-Preg rats were prepared for real-time RT-PCR. The mRNAexpression of MMP-2 (A), MMP-9 (B), and MMP-7 (C) was measured andnormalized to the housekeeping gene for actin. Data represent means � SE,n � 4 to 6. *Significantly greater (P 0.05) than corresponding measurementin virgin rats. §Significantly less (P 0.05) than corresponding measurementsin virgin rats.

Fig. 3. Effect of MMP inhibitors on oxytocin-induced contraction. Uterinestrips of virgin (A), mid-Preg (B), and late-Preg rats (C) were pretreated withSB-3CT, BB-94, or Ro-28–2653 (10�6 M) for 30 min. The uterine strips werestimulated with oxytocin (10�11 to 10�7 M), and the contractile response wasrecorded and presented (in g/mg tissue wt). Data represent means � SE, n �4 to 6. *Significantly different (P 0.05).




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2653 caused significant enhancement of contraction and theRo-28–2653 response was not significantly different from thatof SB-3CT (Fig. 8D). In virgin uterus stretched under 8 g basaltension and treated with E2, P4, or E2�P4 for 18 h, theoxytocin contraction was further reduced. In virgin uterusexposed to prolonged 18-h stretch under 8 g basal tension andtreated with E2, P4, or E2�P4, concomitant treatment with theMMP inhibitor SB-3CT or Ro-28–2653 significantly enhancedoxytocin-induced contraction to levels approaching those ob-served in control virgin uterus (Fig. 8).

RT-PCR analysis revealed an increase in MMP-2 andMMP-9 mRNA expression in virgin uterus stretched under 8 gbasal tension for 18 h compared with control uterine stripsunder 2 g basal tension (Fig. 9, A and B). MMP-2 and MMP-9mRNA expression was slightly enhanced in virgin uterustreated with E2, P4, or E2�P4, and further enhanced in virginuterus stretched under 8 g for 18 h and treated with E2, P4, orE2�P4 (Fig. 9, A and B). In contrast, MMP-7 mRNA wasreduced in virgin uterus exposed to prolonged stretch (Fig. 9C).In uterine strips under 2 g basal tension, prolonged treatmentwith E2, P4, or E2�P4 caused an enhancement of MMP-7expression (Fig. 9C). However, in uterine strips exposed toprolonged 18-h stretch under 8 g basal tension, the enhancingeffects of E2, P4, or E2�P4 appear to be prevented, and thesex hormones did not cause any significant change in MMP-7mRNA expression (Fig. 9C).

Western blot analysis revealed that the protein amount ofpro-MMP-2 was not significantly changed in virgin uterusunder 8 g of stretch for 18 h compared with control uterinestrips under 2 g basal tension (Fig. 10A). However, the proteinamount of both MMP-2 and MMP-9 was significantly en-hanced in virgin uterus under prolonged stretch compared withcontrol uterine strips (Fig. 10, A and B). The protein amount ofpro-MMP-2, MMP-2, and MMP-9 was slightly but signifi-cantly enhanced in virgin uterus under 2 g basal tension and

treated with E2, P4, or E2�P4 (Fig. 10, A and B). MMP-2 andMMP-9 protein was further enhanced in virgin uterine stripsstretched under 8 g for 18 h and concomitantly treated with E2,P4, or E2�P4 (Fig. 10, A and B). The protein amount ofMMP-7 was not significantly different in virgin uterus underprolonged stretch compared with control uterine strips (Fig.10C). In uterine strips under 2 g basal tension, prolongedtreatment with E2, P4, or E2�P4 caused some enhancement ofMMP-7 protein (Fig. 10C). However, in uterine strips exposedto prolonged 18-h stretch under 8 g basal tension, the enhanc-ing effects of E2, P4, or E2�P4 were prevented, and sexhormones did not cause significant change in the proteinamount of MMP-7 (Fig. 10C). In parallel Western blot analy-sis, the effect of pretreating the uterine strips with the MMPinhibitor SB-3CT (10�6 M) on MMPs protein amount wastested (Fig. 10). Western blot analysis demonstrated that theoptical density of the pro-MMP-2, MMP-2, MMP-9, andMMP-7 immunoreactive bands in uterine strips exposed toprolonged stretch with or without sex hormones were notsignificantly different in uterine strips nontreated or concomi-tantly pretreated with the MMP inhibitor SB-3CT.

Gelatin zymography analysis revealed that pro-MMP-2,MMP-2, and MMP-9 protease activity was significantly en-hanced in virgin uterine strips under high 8 g tension for 18 hcompared with uterine strips under 2 g basal tension (Fig. 11,A, C, and E). Pro-MMP-2, MMP-2, and MMP-9 activity wassignificantly enhanced in virgin uterus under 2 g basal tensionand treated with combined E2�P4, and further enhanced inuterine strips stretched under 8 g for 18 h and treated witheither E2, P4, or E2�P4 (Fig. 11, A, C, and E). The increasedpro-MMP-2, MMP-2, and MMP-9 gelatinase activity in uterinestrips exposed to prolonged stretch and treated with sex hor-mones was reversed in tissues concomitantly treated with theMMP inhibitor SB-3CT (Fig. 11, B, D, and F).

Fig. 5. MMPs protein amount. Uterine stripsfrom virgin, mid-Preg, and late-Preg rats,were either nontreated (�) or pretreated (�)with the MMP inhibitor SB-3CT (10�6 M) for30 min and then homogenized in preparationfor Western blot analysis. MMPs were de-tected using antibodies to MMP-2 (1:500; A),MMP-9 (1:500; B), and MMP-7 (1:500; C).The intensity of the immunoreactive bandswas analyzed using optical densitometry andnormalized to the housekeeping protein actin.Data represent means � SE, n � 4 to 6.*Significantly different (P 0.05) from cor-responding measurement in virgin rats. §Sig-nificantly less (P 0.05) than correspondingmeasurements in virgin rats.




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The direct effects of MMPs on oxytocin contraction werealso tested. In uterine strips of virgin rats, oxytocin (10�7 M)caused rapid contraction that was maintained at steady state forat least 30 min (Fig. 12A). In uterine strips of virgin ratsprecontracted with oxytocin (10�7 M), MMP-2 (1 �g/ml)caused slow but progressive inhibition of contraction thatreached 42.9 � 6.8% in 30 min (Fig. 12, B and D). Similarly,MMP-9 (1 �g/ml) caused slow and progressive relaxation ofoxytocin contraction that reached 33.8 � 4.6% in 30 min (Fig.12, C and D).

DISCUSSION

The main findings of the present study are 1) normal pregnancyis associated with decreased myometrium contraction and en-hanced MMP-2 and MMP-9 expression and activity, 2) prolongedincreases in myometrium basal tension are associated with de-creased myometrium contraction and increased MMP expression/activity, 3) E2 and P4 cause further reduction in contraction andenhancement of MMPs expression/activity in uterine strips ex-posed to prolonged stretch, and 4) MMP-2 and MMP-9 causeinhibition of myometrium contraction.

During normal pregnancy, the balance between uterine con-traction and relaxation is tightly regulated to maintain healthyand full-term pregnancy. The mechanisms responsible formaintaining the uterus in a quiescent relaxed state are impor-tant, as changes in these mechanisms could cause pretermuterine contraction with untoward outcome to the pregnancyand premature newborn. Consistent with the concept of uterinequiescence during the course of pregnancy, we found that theoxytocin contraction was reduced in mid-Preg and late-Preg ratcompared with virgin rats. Some studies have shown thatoxytocin contraction of midterm and late-Preg uterus is greaterthan that in nonpregnant uterus (2), which is opposite from thepresent findings. This is likely because during late pregnancythe uterus is bigger and often thicker, and studies often mea-sure uterine contraction in absolute grams. In effect, when wemeasured the uterine contraction in grams, the contraction inlate-Preg uterus was greater than that in mid-Preg uterus, butless than that in virgin uterus. On the other hand, when theuterine contraction was normalized to the uterine tissue weight,the contraction in late-Preg uterus was smaller than that inmid-Preg uterus and far less than that in the virgin uterus.

Fig. 6. MMPs activity. Uterine strips isolatedfrom virgin, mid-Preg, and late-Preg rats wereeither nontreated (A, C, and E) or pretreatedwith MMP-2/MMP-9 inhibitor IV (SB-3CT,10�6 M) (B, D, and F). The uterine tissue wasthen homogenized and prepared for gelatinzymography analysis. Digested bands corre-sponding to pro-MMP-2 (A and B), MMP-2(C and D), and MMP-9 (E and F) were mea-sured and presented (pixel intensity mm2

normalized to actin intensity). W/O, without.Data represent means � SE, n � 4 to 6.*Significantly different (P 0.05) from cor-responding measurement in virgin rats. #Sig-nificantly different (P 0.05) from corre-sponding measurements in the absence ofMMP inhibitor.




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These observations are consistent with the concept that uterinecontraction is reduced during pregnancy.

Previous studies have shown that the sensitivity to oxytocinwas reduced in rat mid-Preg uterus compared with nonpregnantuterus (2). This is different from the present observation thatoxytocin EC50 was not different in mid-Preg and late-Preg ratscompared with virgin rats. The causes of the differences in theoxytocin response is not clear, but could be related to thesource of oxytocin or the orientation of the longitudinal andcircular muscle in the uterine strip. Nevertheless, the presentobservations suggest that the decreased uterine contractionduring pregnancy may not be due to reduction in the sensitivity

to oxytocin, but rather due to potential changes in postreceptorsignaling pathways and uterine smooth muscle contractionmechanisms.

We tested whether the pregnancy-associated reduction inuterine contraction reflect changes in MMP expression/activ-ity. MMPs is a family of more than 28 proteases includingcollagenases, gelatinases, stromelysins, matrilysins, and mem-brane-type MMPs with different tissue expression, distribution,and substrate specificity (49). Previous studies have shown thatduring normal gestation MMP-1, -2, -3, -7, and -9 are found inthe amniotic fluid and fetal membranes. MMP-2 and MMP-3are expressed constitutively, while MMP-9 is barely detectableuntil labor. At labor, MMP-9 is the major MMP responsible forgelatinolytic activity in the membranes, while MMP-2 is dom-inant in the decidua. We have previously shown that prolongedvein wall stretch is associated with decreased venous contrac-tion and increased expression of MMP-2 and MMP-9 (50).Therefore, the present study focused on examining the preg-nancy-associated changes in the expression and effects ofMMP-2 and MMP-9. In search for the potential mechanismsinvolved in the changes in uterine contraction during preg-nancy, we observed that the reduced contraction in the mid-Preg and late-Preg rat uterus was reversed in the presence ofMMP inhibitors, supporting a potential role of MMPs in thepregnancy-associated reduction in uterine contraction. The roleof MMPs in the reduced uterine contraction during mid- andlate gestation is further supported by the observation that themRNA expression and protein amount of MMP-2 and MMP-9were significantly enhanced in mid-Preg and late-Preg ratuterus. Also, MMP-2 and MMP-9 activity, as measured by

Fig. 7. Basal tension-contraction relationship in the rat uterus. Uterine strips ofvirgin rats were stretched under increasing basal tension 0.5, 1, 2, 3, 4, 5, 6, 7,and 8 g for 30 min, stimulated with 96 mM KCl, and the contractile responsewas then recorded and presented (in g/mg tissue wt). Data represent means �SE, n � 4 to 6. *Significantly different (P 0.05) from correspondingmeasurement under 2 g basal tension.

Fig. 8. Effect of prolonged stretch and sexhormones on uterine contraction. Uterinestrips from virgin rats were stretched undercontrol (2 g) or high (8 g) basal tension for 18h in the absence or presence of the MMPinhibitor MMP-2/MMP-9 inhibitor IV (SB-3CT) or Ro-28–2653 (A). In other experi-ments, uterine strips under control (2 g) orhigh (8 g) tension were concomitantly treatedwith 17�-estradiol (E2; 10�6 M) (B), proges-terone (P4; 10�5 M) (C), or E2�P4 (D) andeither in the absence or presence of MMPinhibitors. The uterine strips were stimulatedwith oxytocin (10�11 to 10�7 M), and thecontractile response was recorded and pre-sented (in g/mg tissue wt). Data representmeans � SE, n � 4 to 6. *Significantlydifferent (P 0.05) from correspondingmeasurement in uterine strips under controlbasal tension. #Significantly different (P 0.05) from corresponding measurements inthe absence of MMP inhibitor. †Response toRo-28-2653 was not significantly differentfrom that of SB-3CT.




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gelatin zymography, was enhanced in mid-Preg and late-Pregrat uterus compared with virgin rat uterus. We should note thatin mid-Preg uterus, only SB-3CT, but not BB-94 or Ro-28–2653, enhanced oxytocin contraction (Fig. 3B), whereas allthree inhibitors enhanced contraction in late-Preg uterus (Fig.3C). This can be explained by the changes in uterine MMPactivity during the course of pregnancy. In mid-Preg uterusmoderate increases in MMP expression/activity occur, andtherefore the potent inhibitor SB-3CT potentiates the contrac-tion. Further increases in MMP expression/activity occur onlate-Preg, and therefore all inhibitors with different potenciesshow measurable increases in uterine contraction. However, ascontrol experiments, it is important to determine whether thepregnancy-associated changes in MMPs expression/activity arespecific to MMP-2 and -9, or involve other MMPs. Our

rationale to examine the expression of MMP-7 (matrilysin-1)was that MMP-7 is known to be expressed in the uterus and isoften termed uterine MMP (49). Also, MMP-7 changes duringgestation, but does not appear to play a major role in labor (67).Importantly, MMP-7 expression did not increase during preg-nancy, supporting that the increased MMPs expression is spe-cific to MMP-2 and -9. Instead, MMP-7 showed a pregnancy-associated decrease in expression. Although the causes of thisdecrease are unclear at the present time, because MMPs mayhave common gene transcription pathways it is possible thatthe increase in MMP-2 and -9 expression will take away fromthe common MMPs transcription pool and lead to a decrease inMMP-7 expression. The decrease in MMP-7 expression alsosuggests that MMP-7 may function via a mechanism differentfrom that induced by MMP-2 and -9. In this respect, whetherMMP-7 or MMP-7 inhibitors affect uterine contraction shouldbe examined in future studies.

As different stages of pregnancy are associated with differ-ent degrees of uterine stretch, the present observations sug-gested a relationship between uterine stretch, MMP expression,and decreased contraction. To support this concept it is impor-tant to demonstrate that uterine stretch leads to increased MMPexpression/activity and that MMPs cause reduction in uterinecontraction.

Previous studies have demonstrated that mechanical stretchof skeletal muscle and microvessels is associated with de-creased contraction and enhanced MMP expression (39). Also,we have previously shown that prolonged stretch of rat inferiorvena cava is associated with decreased vein contraction andincreased MMP-2 and MMP-9 expression (50). In the presentstudy, we found that prolonged stretch of virgin uterus wasassociated with decreased contraction. The decreased contrac-tion in virgin uterus exposed to prolonged stretch appeared toinvolve upregulation of MMP expression/activity because thedecreased uterine contraction in stretched virgin uterus wasreversed in tissues treated with MMP inhibitors. Also, thedecreased uterine contraction in stretched virgin uterus wasassociated with increased mRNA expression, protein amountand enzymatic activity of MMP-2 and MMP-9. Importantly,the stretch-induced changes in MMPs were specific to MMP-2and MMP-9, and were not observed with other MMPs such asMMP-7. The present observations in rat uterus are consistentwith previous reports in skeletal muscle and blood vessels (39,50) and support the relationship among mechanical stretch,MMP tissue expression, and decreased tissue contraction.

During pregnancy the uterus is exposed to not only mechan-ical stretch caused by progressive fetal growth, but also hor-monal changes that could influence the myometrium structureand mechanical properties. Pregnancy is associated with in-creased plasma levels of E2 and P4 (52). Endometrial cellularconcentrations of both E2 and P4 are positively correlated withthe plasma levels of E2 during the proliferative phase of themenstrual cycle, and a large concentration of P4 receptorscharacterizes the early pregnancy endometrium (30). Studieshave shown that E2 causes relaxation of smooth muscle ofblood vessels, including rat aorta and uterine artery (11, 55), aswell as the uterus (45). Studies have also shown that P4 inhibitscontraction of rat blood vessels and human uterus (11, 15).Consistent with previous reports, we found that E2 and P4decreased contraction of uterine strips of virgin rats. Also, in

Fig. 9. Effect of prolonged stretch and sex hormones on uterine MMP mRNAexpression. Uterine strips from virgin rats were stretched under control (2 g) orhigh (8 g) basal tension for 18 h, concomitantly treated with E2, P4, or E2�P4,and the uterine tissue homogenate was prepared for real-time RT-PCR. ThemRNA expression of MMP-2 (A), MMP-9 (B), and MMP-7 (C) was measuredand normalized to the housekeeping gene for actin. Data represent means �SE, n � 4 to 6. *Significantly different (P 0.05) from correspondingmeasurements in uterine strips under control basal tension.




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uterine strips exposed to prolonged stretch, sex hormonesfurther reduced uterine contraction.

MMP-9 is one of the major MMPs in the uterus thatmodulates uterine biology during various reproductive pro-cesses. In mouse uterus, gelatin zymography revealed that E2alone or in combination with P4 increased MMP-9 activation,whereas Northern blot analysis showed that E2 decreasedMMP-9 steady-state mRNA expression. In contrast, uterineMMP-2 expression and activity were not affected by steroidaltreatment. Collectively, these data suggest that E2 and P4regulate uterine MMPs expression and activity via a complexmechanism and may play a role in uterine tissue remodeling(70). However, little is known on whether the effects of E2 andP4 on uterine relaxation involve MMPs. The present studydemonstrates that concomitant treatment of stretched virginuterus with E2, P4, or E2�P4 caused greater reduction ofuterine contraction. The reduced contraction in the sex hor-mone-treated uterus was reversed in the presence of MMPinhibitors. Also, E2 and P4 treatment of stretched virgin uteruswas associated with greater increases in MMP-2 and MMP-9mRNA expression, protein amount, and enzymatic activity.

Together, these observations suggest a synergistic relationshipbetween mechanical stretch and sex hormones in the reduceduterine contraction and MMP expression/activity. The mecha-nisms via which E2 enhances uterine MMP expression/activityare unclear, but E2 has been shown to stimulate MMP produc-tion and activity via ER-mediated MEK/ERK (MAPK) (23).Also, while some studies suggest anti-inflammatory effects ofE2 (14, 37, 61), other studies suggest E2-mediated increase ininflammatory cytokines such as TNF-� and IL-6, which couldin turn increase MMPs expression/activity (3, 10, 23, 29, 70).

The questions remain of how mechanical stretch causesincreases in MMPs expression and how MMPs cause uterinesmooth muscle relaxation. Hypoxia-inducible factors (HIFs)are nuclear transcriptional factors that regulate genes involvedin oxygen homeostasis (57). In addition to the regulation ofHIF by oxygen, hormones, cytokines, metallic ions, and me-chanical stretch may induce HIF expression (20, 57). HIF-1�and HIF-2� mRNA and protein are increased in skeletalmuscle fibers exposed to stretch (38, 39). Also, HIF-1� isoverexpressed in rat cardiac and aortic vascular smooth musclecells exposed to mechanical stretch (8, 24). The mechanism of

Fig. 10. Effect of prolonged stretch and sex hormones on uterine MMPs protein amount. Uterine strips from virgin rats were stretched under control (2 g) orhigh (8 g) basal tension for 18 h and concomitantly treated with E2, P4, or E2�P4, in the absence (�) or presence (�) of MMP-2/MMP-9 inhibitor IV (SB-3CT,10�6 M), and the uterine tissue homogenate was prepared for Western blot analysis. MMPs were detected using antibodies to MMP-2 (1:500; A), MMP-9 (1:500;B), and MMP-7 (1:500; C). Representative MMP-2 and MMP-7 blots on the right side have the same actin because the membrane was reacted first with MMP-2antibody, stripped, reacted with MMP-7 antibody, and then stripped and reacted with anti-actin antibody. The intensity of the immunoreactive bands was analyzedusing optical densitometry and normalized to the housekeeping protein actin. Data represent means � SE, n � 4 to 6. *Significantly different (P 0.05) fromcorresponding measurement in uterine strips under control basal tension.




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HIF regulation by mechanical stretch is unclear but mayinvolve phosphatidylinositol 3-kinase and MAPK (8, 20, 38).Studies have shown that the expression and activity of MMP-2and MMP-9 are regulated by HIF (19, 27). Also, mechanicalstretch is associated with increased HIF-1� expression, andHIF could increase MMP expression (31). Studies have shownthat HIF expression may increase during pregnancy (44) andthat estrogen stimulates uterine HIF-2�, while P4 primarilyupregulates uterine HIF-1� expression (12). Whether HIFrepresents a link between myometrial stretch and MMPs ex-pression remains to be examined.

The effects of MMPs are generally thought to involvedegradation of the extracellular matrix components and exten-sive tissue remodeling. The relatively rapid effects of MMPinhibitors and MMPs on uterine contraction suggest that theseeffects may not be solely due to tissue remodeling. We havepreviously shown that MMP-2 and -9 could have additionalinhibitory effects on vascular smooth muscle contraction in theabsence of detectable tissue degradation (9, 48). Also, ourprevious experiments on rat inferior vena cava have suggestedthat MMP-2 may cause membrane hyperpolarization and re-duction in Ca2� influx (48, 51). In the present study, treatmentof uterine strips with MMP-2 or MMP-9 caused significant

relaxation of oxytocin-induced contraction. These observationsare consistent with our previous reports in vascular tissues (9,48, 51) and point to inhibitory effects of MMPs on themechanisms of uterine smooth muscle contraction. WhetherMMPs affect the plasma membrane channels or other mecha-nisms of uterine smooth muscle contraction remains to beexamined in future studies.

Consistent with previous reports in the rat uterus (2, 47), thepresent study demonstrates that oxytocin causes a steady in-crease in contraction and an additional spontaneous phasiccontractile response. The frequency and amplitude of oxytocin-induced phasic contraction appear to be different among virginuteri and in pregnant vs. virgin uteri. Although the presentstudy focused on the mechanisms underlying the reduction inthe steady component of oxytocin contraction during preg-nancy, that should not minimize the importance of examiningthe causes of the different frequency and amplitude of oxyto-cin-induced phasic contraction among virgin uteri and themechanisms of the pregnancy-associated changes in the phasicuterine contraction. Studies in human myometrial cells haveshown that oxytocin causes an initial [Ca2�]i transient fol-lowed by uniform relatively low-frequency [Ca2�]i oscillations(4, 6, 69). The oxytocin-induced [Ca2�]i oscillations in myo-

Fig. 11. Effect of prolonged stretch and sex hormones on uterine MMPs activity. Uterine strips from virgin rats were stretched under control (2 g) or high (8g) basal tension for 18 h and concomitantly treated with E2, P4, or E2�P4, in the absence (A, C, and E) or presence of MMP-2/MMP-9 inhibitor IV (SB-3CT,10�6 M; B, D, and F), and the uterine tissue homogenate was prepared for gelatin zymography analysis. Digested bands corresponding to pro-MMP-2 (A andB), MMP-2 (C and D), and MMP-9 (E and F) were measured and presented (as pixel intensity mm2 normalized to actin intensity). Data represent means �SE, n � 4 to 6. *Significantly different (P 0.05) from corresponding measurements in uterine strips under control basal tension. #Significantly different(P 0.05) from corresponding measurements in the absence of MMP inhibitor.




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metrial cells are attenuated by caffeine and the voltage-depen-dent Ca2� channel antagonist verapamil and blocked by theinorganic Ca2� antagonist La3� and the Ca2�-ATPase inhibi-tor 2.5-di-tert-butylhydroquinone (18). Similarly, in rat uterinesegments, oxytocin induced simultaneous [Ca2�]i oscillationsand phasic contractions that were inhibited by the Ca2� chan-nel blocker nifedipine (47). Collectively, these studies suggestthat the oxytocin-induced [Ca2�]i oscillations and phasic uter-ine contraction are mediated by intracellular Ca2� release frominositol 1,4,5-trisphosphate-sensitive Ca2� stores combinedwith voltage-dependent and capacitative Ca2� influx. Interest-ingly, we have previously reported that MMP-2 may causehyperpolarization and reduction in Ca2� influx in rat inferiorvena cava (48, 51), a smooth muscle preparation that alsodemonstrates phasic contraction. These observations make itimportant to investigate the pregnancy-associated changes andthe effects of MMPs on the frequency and amplitude of phasicuterine contractions and whether the changes in uterine con-traction involve parallel changes in [Ca2�]i and Ca2� regula-tory mechanisms. These questions should be examined infuture experiments.

One of the limitations of the present study is that uterinecontraction and MMP expression were measured at only twotime points of pregnancy, namely day 12 and day 19 of

gestations, and the progressive changes during the course ofpregnancy and reversal of these uterine changes in the post-partum period need to be examined. Likewise, the stretchexperiments were limited to 8 g basal tension for 18 h, and theprogressive effects of different levels of basal tension for moreextended time periods would be more analogous to the pro-gressive uterine stretch imposed by the growing fetus over thecourse of pregnancy. The present study also tested the effectsof oxytocin and whether the uterine response to other contrac-tile agonists, such as prostaglandins, are affected during preg-nancy and whether uterine stretch should be examined. Also,while the present study suggests a role of MMP-2 and MMP-9in the observed changes in uterine contraction during preg-nancy and uterine tissue stretch, the results should not mini-mize the possibility of involvement of other MMPs.

Perspectives

Myometrium activity is tightly regulated during pregnancy.At the first and midtrimester, myometrium relaxation is neededto accommodate fetal growth. As fetal growth nears its com-pletion during late pregnancy the uterine activity may bestabilized before the activity starts to increase in preparationfor delivery. However, little is known regarding the mecha-

Fig. 12. Effects of MMPs on uterine contraction. Uterine strips of virgin rats were precontracted with oxytocin (10�7 M), and the steady-state contraction wasrecorded. Either the vehicle H2O (A), MMP-2 (B), or MMP-9 (1 �g/ml; C) was added, and the changes in oxytocin-induced contraction were measured.Cumulative data from uterine strips of different rats were presented as %relaxation of oxytocin contraction (D). Data represent means � SE, n � 6. *Significantlydifferent (P 0.05).




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nisms controlling the balance between myometrium contrac-tion and relaxation during pregnancy. The present study dem-onstrates a relationship between uterine stretch, MMPs expres-sion, and uterine relaxation during gestation. The study alsodemonstrates a role of sex hormones in promoting the effectsof uterine stretch on MMPs expression and uterine relaxation.These findings may have clinical relevance as a disturbance inthe balance of MMPs, and the endogenous tissue inhibitors ofMMPs (TIMPs) could disturb uterine activity and lead topremature labor. The imbalance between MMPs and TIMPsmay be further aggravated by changes in the sex hormonelevels or their uterine receptors.

Preterm delivery complicates 10% to 15% of all pregnanciesand is a leading cause of perinatal morbidity and death (17),but the mechanisms involved are not completely understood.Importantly, studies on samples of amniochorion and amnioticfluid collected from women undergoing cesarean delivery be-fore term, with either premature rupture of membranes or withpreterm labor with no rupture of membranes, demonstratedalterations in mRNA expression and bioactivity of MMP-2,MMP-9, MT1-MMP, and TIMP-2 in prematurely rupturedmembranes compared with preterm labor membranes. Also,enzyme-linked immunosorbent assays demonstrated altera-tions in the amniotic fluid concentrations of immunoreactiveand bioactive MMP-2 and MMP-9, as well as TIMP-2 in fluidsobtained from the premature rupture of membranes groupcompared with the preterm labor group (17). Other studieshave shown that plasma P4 levels are lower in some pretermdelivery patients compared with normal pregnant women. Forexample, P4 concentration was �30% lower at 28 to 34 wkgestation in women who delivered prematurely than in womenwho delivered at term (59). Studies have also suggested thatprogestin supplementation may be a promising approach toboth preventing initiation of preterm labor and treating it onceit is already established (32). Whether changes in MMPs andsex hormone levels/activity could interfere with uterine relax-ation and trigger uterus contraction and preterm delivery needsto be further examined. Also, whether the potential beneficialeffects of P4 in preterm labor are mediated via modulation ofMMP expression/activity warrant further investigation.

ACKNOWLEDGMENTS

We thank Apoorva Kumar and Yuanyuan Zhang for their assistance in theanalysis of the Western blots and contraction data.

GRANTS

R. A. Khalil was partly supported by National Heart, Lung, and BloodInstitute Grants HL-65998, HL-98724 and The Eunice Kennedy ShriverNational Institute of Child Health and Human Development Grant HD-60702.Z. Yin was a visiting scholar from Tongji Hospital, Huazhong University ofScience and Technology, Wuhan, Hubei Province, P. R. China, and a recipientof a scholarship from the China Scholarship Council.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: Z.Y., O.M.R., and R.A.K. conception and design ofresearch; Z.Y., A.A.S., O.M.R., and N.N. performed experiments; Z.Y.,A.A.S., N.N., and R.A.K. analyzed data; Z.Y. and R.A.K. interpreted results ofexperiments; Z.Y. and R.A.K. prepared figures; Z.Y., A.A.S., and R.A.K.edited and revised manuscript; Z.Y. and R.A.K. approved final version ofmanuscript; R.A.K. drafted manuscript.

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