Mitogen-Activated Protein Kinase Cascade Is ...

0013-7227/99/$03.00/0 Endocrinology Copyright © 1999 by The Endocrine Society

Vol. 140, No. 2 Printed in U.S.A.

Mitogen-Activated Protein Kinase Cascade Is Involved in Endothelin-1-Induced Rat Puerperal Uterine Contraction AKIKO KIMURA, MASAHIDE OHMICHI, TAKASHI TAKEDA, HIROHISA KURACHI, HIROMASA IKEGAMI, KOJI KOIKE, KANJI MASUHARA, JUN HAYAKAWA, TOHRU KANZAKI, MAMORU KOBAYASHI, MASUO AKABANE, MASAKI INOUE, AKIRA MIYAKE, AND YUJI MURATA Department of Obstetrics and Gynecology (A.K., M.O., T.T., H.K., H.I., K.M., J.H., T.K., A.M., Y.M.), Osaka University Medical School, Suita, Osaka 565, Japan; Department of Obstetrics and Gynecology (K.K., M.I.), Kanazawa University Medical School, Kanazawa, Ishikawa 920, Japan; and Kissei Pharmaceutical Company Ltd (M.K., M.A.), Minamiazumi, Nagano 399 – 83, Japan ABSTRACT The regulation of mitogen-activated protein (MAP) kinase by endothelin-1 (ET-1) in cultured rat puerperal uterine myometrial cells was investigated. ET-1 caused the rapid stimulation of MAP kinase activity. ET-1-induced MAP kinase activation is neither extracellular Ca21- nor intracellular Ca21-dependent. ET-1 stimulation also led to an increase in phosphorylation of son-of-sevenless (SOS), and transfection of dominant negative SOS attenuated the ET-1-induced MAP kinase activity. Phorbol-12-myristate 13-acetate (PMA) also induced the MAP kinase activity, but pretreatment of the cultured cells with PMA, to down-regulate protein kinase C (PKC), did not abolish the activation of MAP kinase by ET-1. In addition, down-regulation of PKC had no effect on ET-1-induced SOS phosphorylation. Pertussis toxin, which inactivates Gi/Go proteins, blocked the ET-1-induced MAP kinase activation but not the PMA-induced MAP kinase activation. The results suggested that MAP kinase is acutely activated by

ET-1 through a pertussis toxin-sensitive G protein and SOS, not through the PMA-sensitive PKC. In addition, although reverse-transcriptase PCR assays detected messenger RNA for both ET-1 receptor subtypes in cultured rat puerperal uterine myometrial cells, ET-1induced MAP kinase activity and uterine contraction were blocked by treatment with BQ485, an antagonist selective for an ET type A receptor (but not by BQ788, an ET type B receptor antagonist). Ritodrine, which is known to relax uterine muscle contraction, attenuated ET-1-induced MAP kinase activity. We further examined the role of MAP kinase pathway in uterine contraction using an inhibitor of MEK activity, PD098059. This inhibitor completely inhibited the ET-1-induced MAP kinase activation and partially, but significantly, inhibited the ET-1-induced uterine contraction. These results indicate that ET-1-induced MAP kinase signaling cascade may play an important role in the ET-1-induced uterine contraction. (Endocrinology 140: 722–731, 1999)

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HE ENDOTHELINS (ETs) are a family of 21 amino acid peptides; and ET-1, ET-2, and ET-3 were first demonstrated as vasoconstrictor peptides secreted by bovine aortic endothelial cells in culture (1). In addition to effecting vasoconstriction by binding to vascular smooth muscle, ET-1 exerts a potent contractile effect on human isolated myometrium from both nonpregnant (2) and term pregnant women (3). In pregnancy, the myometrial responsiveness to ET-1 increases by 20 –30%, compared with that of nonpregnant tissue (2). Furthermore, ETs are mitogenic, or comitogenic for fibroblasts, vascular smooth muscle, and other cells (4, 5). The actions of ET are mediated by binding to distinct cell-surface receptors. Two distinct ET receptor subtypes [ET type A (ETA) and ET type B (ETB)], which have seven transmembrane-spanning regions and belong to the superfamily of G protein-coupled receptors, have been cloned from complementary DNA (cDNA) libraries of various cell types (6 – 11). The ETA is selective for ET-1 and ET-2, whereas the ETB is nonselective for the three isoforms ET-1, ET-2, and ET-3 (6, 7). Although both ETA and ETB are present in human myometrium, the ETA subtype predominates, as in other smooth muscles (12). The ratio of ETA/ETB in the myometrium is

approximately 3/1 (13). In tissue bath experiments, only ETA mediated myometrial contractility (13), and myometrial cells in culture have been found to possess only ETA subtypes coupled to phospholipase C (14). Thus, the contractile effect of ET-1 in the myometrium is mediated by ETA, functionally coupled to phospholipase C, generating inositol triphosphate (14) and inducing an increase in cytosolic calcium followed by activation of myosin light chain kinase (15), which is essential for promoting uterine contractility (12). On the other hand, although the importance of ETB in the human myometrium is still poorly understood, they may allow the ETs to release PGs or nitric oxide in a paracrine fashion (16). The signal transduction pathways activated by ET-1 in myometrium have only been partially characterized. It was reported that ET-1 stimulates the mitogen-activated protein (MAP) kinase activity in rat glomerular mesangial cells (17). A family of serine/threonine kinases, comprised of p44 and p42 MAP kinases, which phosphorylate microtubule-associated protein-2 and myelin basic protein (MBP), have been identified as an important intermediary factor in converting extracellular signals into intracellular responses (18, 19). These kinases are activated through phosphorylation on both tyrosine and threonine residues of the kinase by diverse stimuli, including growth factors, hormones, osmotic shock, stress, and elevated temperature (20 –26). Both oxytocin (OT) and PG F2a are also well-known uterine contractants, which

Received June 24, 1998. Address all correspondence and requests for reprints to: Dr. Masahide Ohmichi, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka 565, Japan. E-mail: [email protected].

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ET-1 STIMULATES MAP KINASE CASCADE

we identified as stimulators of MAP kinase activity in cultured human and rat puerperal uterine myometrial cells (27–29). In addition, we identified that the specific MAP kinase kinase (MEK) inhibitor, PD098059, partly inhibited both OT- (28) and PG F2a- (29) induced pregnant rat uterine contraction. These observations led us to examine the effects of ET-1 on the MAP kinase cascade in cultured rat puerperal myometrial cells. Moreover, we examined the regulatory mechanism of ET-1-induced MAP kinase activity and the precise role of MAP kinase cascade in the ET-1-induced uterine contraction using the MEK inhibitor. Materials and Methods Materials ECL Western blotting detection reagents were obtained from Amersham Co. (Arlington Heights, IL). [g-32P]ATP (3000 Ci/mmol) was obtained from New England Nuclear (Bannockburn, IL). Anti-SOS1 antisera were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). ERK1 rabbit polyclonal anti-MAP kinase antiserum and monoclonal antibody 9E10 to the Myc epitope were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Pregnant Wistar rats were obtained from Nihon Dobutu Co. (Osaka, Japan).

Construction of expression plasmids An expression plasmid (SRa-dnSOS) encoding a mutant mSOS1 that lacks the guanine nucleotide exchange domain (amino acids 618 to 1036) of the wild-type protein, a kind gift from Dr. D. Bowtell (Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Australia), was constructed by removing a PstI-PstI fragment (1.49 kbp) from the mSOS1 cDNA (30, 31). Myc-tagged p42mapk expression plasmid (pEXV-Erk2-tag) was obtained from Dr. C. J. Marshall (Institute of Cancer Research, London, UK) (32).

Preparation of rat puerperal uterine myometrial cells Rats, at 21 days of pregnancy, were stunned and bled in the morning; the uterus was removed, and the fetuses were gently expelled. Cells were prepared by the modified method of Palmberg and Thyberg (33). The tissues were cut into 1- to 2-mm3 fragments and digested with 0.1% trypsin for 1 h at 37 C in calcium-magnesium (Ca-Mg)-free Hanks’ solution. The tissues were digested with 0.1% collagenase and 0.1% deoxyribonuclease for 30 min at 37 C in Ca-Mg- free Hanks’ solution. Cell aggregates were isolated by gentle pipetting. Nondispersed fragments were separated by filtration through gauze. The cells were maintained at 37 C under an atmosphere of 95% air-5% CO2 in RPMI1640 medium containing 10% FBS supplemented with penicillin (200 U/ml) and streptomycin (200 mg/ml). They were used for the following experiments after 5 days culture.

Assay of MAP kinase activity Cells were incubated, in the absence of serum, overnight and then treated with various reagents. They were then washed twice with PBS and lysed in ice-cold HNTG buffer (50 mm HEPES (pH 7.5), 150 mm NaCl, 10% glycerol, 1% Triton X-100, 1.5 mm MgCl2, 1 mm EDTA, 10 mm sodium pyrophosphate, 100 mm sodium orthovanadate, 100 mm NaF, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride) (34). The extracts were centrifuged to remove cellular debris, and the protein content of the supernatants was determined using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Inc., Richmond, CA). Erk1 rabbit polyclonal antibody was bound to protein A-Sepharose beads, and 300 mg protein from the lysate samples was immunoprecipitated at 4 C for 2 h. The immunoprecipitated products were washed once in HNTG buffer, twice in 0.5 m LiCl-0.1 m Tris (pH 8.0), and once in kinase assay buffer (25 mm HEPES (pH 7.2–7.4), 10 mm MgCl2, 10 mm MnCl2, and 1 mm dithiothreitol), and samples were resuspended in 30 ml kinase assay buffer containing 10 mg MBP and 40 mm [g-32P]ATP (1 mCi), as described previously (35). The kinase reaction was allowed to

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proceed at room temperature for 5 min and stopped by the addition of Laemmli SDS sample buffer (36). Reaction products were resolved by 15% SDS-PAGE.

Assay of 42-kDa MAP kinase activity using a transient expression system Rat puerperal uterine myometrial cells, cultured in 100-mm diameter dishes, were transfected with Myc-tagged p42mapk expression plasmid (1 mg pEXV-Erk2-tag) in combination with 9 mg SRa or SRa-dnSOS using LIPOFECTAMINE PLUS (Life Technologies, Gaithersburg, MD). At 72 h after transfection, serum-deprived cells were incubated with 100 nm ET-1 for 5 min, and expressed Myc-tagged p42mapk was immunoprecipitated with 1 mg antibody 9E10. The MAP kinase activity in the immunoprecipitate was measured as described above. The transfection efficiency of each experiment was 3–5%, as assessed by b-gal staining, after transfection of a b-gal containing expression plasmid (29).

Immunoblots For analysis of SOS phosphorylation, cells were grown in 100-mm dishes. After treatment, the cells were washed once with ice-cold PBS before the addition of 1 ml HNTG buffer. Lysates were centrifuged at 10,000 3 g for 10 min. Supernatants were incubated for 12 h with anti-SOS 1 antiserum. Immunocomplexes were precipitated with protein A-Sepharose and washed three times with HNTG buffer, and samples were resolved by 6% SDS-PAGE, followed by immunoblotting with anti-SOS 1 antiserum.

RT and PCR Total cellular RNA was isolated (37) using Tri-reagent (Molecular Research Center, Inc.), and 3-mg samples were reverse-transcribed using the Access RT-PCR System (Promega Corp., Madison, WI). PCR primers were synthesized, based on the published sequences for rat ETA (sense, 59TTCGTCATGGTACCCTTCGA-39; antisense, 59-GATACTCGTTCCATACATGG-39; 546 bp) (8) and rat ETB receptor (sense, 59-TTCACCTCAGCAGGATTCTG-39; antisense, 59-AGGTGTGGAAAGTTAGAACG-39; 475 bp) (6). The PCR conditions were optimized to ensure that the amplification was within the linear range, as described in detail (38). Amplification was carried out by 30 cycles, as follows: the initial cycle; 3 min at 94 C (for denaturation); 1 min at 54 C (for annealing); 3 min at 72 C (for extension); and the subsequent cycles of 15 sec at 94 C, 20 sec at 54 C, and 1 min at 72 C. PCR products were electrophoresed on a 2.0% agarose gel.

Measurement of uterine contractions Rats, at 21 days of pregnancy, were stunned and bled in the morning; the uterus was removed, and the fetuses were gently expelled. A uterine muscle strip (15 mm long, 5 mm wide) was longitudinally dissected and suspended vertically in a 10-ml chamber containing modified LockeRinger solution (the composition of which was as follows: NaCl, 154 mm; NaHCO3, 4.8 mm; KCl, 5.4 mm; CaCl2, 0.36 mm; MgCl2, 0.19 mm; KH2PO4, 0.15 mm; and glucose, 3.1 mm), gassed with 95% O2-5%CO2, and maintained at 26 C to suppress spontaneous contractions. The contractions were measured isometrically using a mechano-electric transducer (NEC San-ei, 45196A, Tokyo, Japan) coupled to a potentiometric pen-recorder (NEC San-ei, 8K-23). The initial tension was set at about 1.0 g. In the absence of spontaneous contractions, 3 3 10-8 m ET-1 was added to the chamber, and the effect of MEK inhibitor on uterine contractions was evaluated. Uterine activity was calculated, as the sum of the amplitudes of each contraction during 30 min; and the percent changes, before and after the drug application, were compared.

Statistics Statistical analysis was performed by Student’s t test, and P , 0.05 was considered significant. Data are expressed as the means 6 se.


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Results ET-1 stimulation of MAP kinase activity and phosphorylation

The dose dependence of ET-1-induced MAP kinase activity was evaluated. Cultured rat puerperal uterine myometrial cells were treated with the indicated concentrations of ET-1 or with 10 nm epidermal growth factor (EGF) for 5 min. Cell lysates were immunoprecipitated with anti-MAP kinase antibody and assayed for MAP kinase activity by examining the incorporation of 32P into MBP, followed by SDS-PAGE and autoradiography (Fig. 1A). Both ET-1 and EGF produced a marked increase in this kinase activity, compared with the control. MAP kinase activation was detected after treatment with 10 nm ET-1, and it increased up to 100 nm. The time course of ET-1-induced MAP kinase activity was also evaluated. Cultured rat myometrial cells were treated with 100 nm ET-1 for the indicated times or with 10 nm EGF for 5 min (Fig. 1B). ET-1 increased the kinase activity within 2.5 min and, with the maximum effect at 5 min, followed by a gradual decrease. ET-1 stimulation of SOS phosphorylation

Receptor tyrosine kinase-mediated mitogenic signaling involves a series of SH2- and SH3-dependent protein-protein interactions among tyrosine-phosphorylated receptor, Shc, Grb2, and SOS, resulting in p21ras and p74raf-1-dependent MAP kinase activation (39). To examine the effect of ET-1 on SOS phosphorylation, cells were treated with 100 nm ET-1 for the indicated times (Fig. 2). ET-1 stimulation resulted in a significant retardation in the mobility of SOS on SDS-PAGE, reflecting SOS phosphorylation. This occurred within 2.5 min of stimulation and was maximal at 10 –15 min, decreasing to the control level by 24 h. The time course showed a similar time frame of PG F2a-induced SOS phosphorylation (29). To examine whether or not MAP kinase activation by

FIG. 1. ET-1 stimulates MAP kinase activity. Cells were grown in 100-mm dishes. A, Cells were treated with the indicated concentrations of ET-1 for 5 min (lanes 3– 6) or with 10 nM EGF for 5 min (lane 1); B, cells were treated with 100 nM ET-1 for the indicated times (lanes 2–5) or with 10 nM EGF for 5 min (lane 6). Lysates of cells were subsequently immunoprecipitated with anti-MAP kinase antiserum, and the immunoprecipitates were incubated with [g-32P]ATP in the presence of MBP, as described in Materials and Methods. After the reactions were stopped, with the addition of Laemmli sample buffer, samples were subjected to SDS-PAGE and autoradiography. Experiments were repeated three times, with essentially identical results. I.P., Immunoprecipitation; C, Control.

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ET-1 is SOS dependent, these cells were transfected with a dominant negative SOS (dnSOS), which lacks the guanine nucleotide exchange domain of the wild-type protein. To examine the effect of dnSOS on ET-1-induced exogenous MAP kinase activity, a Myc-tagged p42mapk expression plasmid was used to distinguish exogenous MAP kinase from endogenos MAP kinase. We transfected cells with a vehicle (SRa) or SRa-dnSOS, together with Myc-tagged

FIG. 2. Effects of ET-1 on SOS phosphorylation. Cells were grown in 100-mm dishes and treated with 100 nM ET-1 for the indicated times (lanes 2– 8). Lysates were immunoprecipitated with anti-SOS antiserum, and the immunoprecipitates were subjected to SDS-PAGE, followed by immunoblotting with anti-SOS antiserum. Experiments were repeated three times, with essentially identical results. MW, Molecular weight. I.B., Immunoblot.

FIG. 3. SOS-mediated ET-1-induced MAP kinase activation. Cells were transfected with a vector (SRa, lanes 1 and 2) or dominant negative SOS expression vector (SRa-dnSOS, lanes 3 and 4), together with Myc-tagged p42mapk expression plasmid (pEXV-ERK2-tag). Seventy-two hours after transfection, cells were stimulated with 100 nM ET-1 for 5 min (lanes 2 and 4). Lysates of cells were subsequently immunoprecipitated with antibody to Myc epitope, and the immunoprecipitates were incubated with [g-32P]ATP in the presence of MBP, as described in Materials and Methods. After the reactions were stopped with Laemmli sample buffer, SDS-PAGE and autoradiography were performed. An autoradiogram of 32P-labeled MBP is shown at the lower panel. Relative densitometric units (R.D.U.) of the MBP bands are shown at the upper panel, with the density of the control bands set arbitrarily at 1.0 in SRa transfection. Values shown represent mean 6 SE from at least three separate experiments. **, Significant differences (P , 0.01).



p42mapk expression plasmid (pEXV-ERK2-tag), and stimulated them with 100 nm ET-1 for 5 min (Fig. 3). We measured the phosphorylation of MBP after incubation with immunoprecipitates prepared from cells with antibody to the Myc epitope, and the level of phosphorylation was normalized by the amount of Myc-tagged p42mapk. Thus, exogenous MAP kinase activity was measured via the introduction of a Myctagged p42mapk expression plasmid. Transfection of SRadnSOS significantly attenuated the ET-1-induced MAP kinase activation. These results suggest that ET-1 induces MAP kinase activation through SOS. Effects of down-regulation of protein kinase C (PKC) on ET1-induced MAP kinase activation and SOS phosphorylation

It was reported that PKC is also an important serine/ threonine kinase in ET postreceptor signaling (40). To explore the possible contribution of PKC to ET-1-induced MAP kinase activation, we used phorbol-12-myristate 13-acetate (PMA) for direct activation of PKC. PMA stimulated MAP kinase activity (Fig. 4, lane 2), as did ET-1. We pretreated the cells with PMA for 24 h before ET-1 treatment. The effect of pretreatment for the down-regulation of PKC was confirmed

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by the total loss of the acute effect of PMA (Fig. 4, lane 3). The MAP kinase activity induced by ET-1 was not attenuated by pretreatment with PMA (Fig. 4, lane 4), which suggests that activation by ET-1 may not be PKC-dependent. To examine whether ET-1-induced SOS phosphorylation is also PKCindependent, we pretreated the cultured cells with PMA for 24 h before ET-1 treatment (Fig. 5). Apparent retardation in the mobility of SOS induced by ET-1 was not inhibited by pretreatment with PMA, suggesting that ET-1-induced SOS phosphorylation may be PKC-independent. However, the results do not rule out the possibility that the MAP kinase activation by ET-1 may be mediated by some isoforms of PKC that are resistant to PMA-induced down-regulation. Involvement of pertussis toxin (PTX)-sensitive G protein in ET-1-induced MAP kinase activation

The ET-1 receptor has seven transmembrane domains typical for G protein-coupled receptors (6 –11). To determine which type of G-protein is involved in ET-1-induced MAP kinase activation, we preincubated cells for 4 h with 100 ng/ml PTX, followed by incubation with 100 nm ET-1 (Fig. 6A). PTX, at 100 ng/ml, completely blocked the ET-1-induced MAP kinase activation. These results suggest that ET-1 receptor couples to a PTX-sensitive G protein-coupled, followed by activation of MAP kinase. Moreover, to examine whether a PTX-sensitive G-protein is also involved in PMAinduced MAP kinase activation, we preincubated cells with PTX, followed by incubation with 1 mm PMA (Fig. 6B). PTX did not block the PMA-induced MAP kinase activation. Specific antagonist of ETA blocks ET-1-induced MAP kinase activation and contraction of rat pregnant uterine smooth muscle

To clarify which ET receptor is expressed in cultured rat puerperal uterine myometrial cells, RT-PCR for ETA and ETB was performed using RNA samples from myometrial cells. As shown in Fig. 7, products amplified by the ETA- and

FIG. 4. Effects of down-regulation of PKC on ET-1-induced MAP kinase activity. Cells were grown in 100-mm dishes and treated with (lanes 3 and 5) or without (lanes 1, 2, and 4) 1 mM PMA for 24 h and then with 1 mM PMA (lanes 2 and 3) or 100 nM ET-1 (lanes 4 and 5) for 5 min. Lysates of cells were subsequently immunoprecipitated with anti-MAP kinase antiserum, and the immunoprecipitates were incubated with [g-32P]ATP in the presence of MBP, as described in Materials and Methods. After the reactions were stopped, with Laemmli sample buffer, SDS-PAGE and autoradiography were performed. An autoradiogram of 32P-labeled MBP is shown at the lower panel. Relative densitometric units (R.D.U.) of the MBP bands are shown at the upper panel, with the density of the control bands set arbitrarily at 1.0. Values shown represent mean 6 SE from at least three separate experiments. **, Significant differences (P , 0.01).

FIG. 5. Effects of down-regulation of PKC on ET-1-induced SOS phosphorylation. Cells were grown in 100-mm dishes and treated with (lane 3) or without (lanes 1 and 2) 1 mM PMA for 24 h and then with 100 nM ET-1 (lanes 2 and 3) for 10 min. Lysates were immunoprecipitated with anti-SOS antiserum, and the immunoprecipitates were subjected to SDS-PAGE, followed by immunoblotting with anti-SOS antiserum. Experiments were repeated three times, with essentially identical results.


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FIG. 6. ET-1 induces MAP kinase activation through a PTX-sensitive G protein-coupled. A, Cells, grown in 100-mm dishes, were pretreated with 100 ng/ml PTX for 4 h (lanes 2 and 4), followed by treatment with 100 nM ET-1 (lanes 3 and 4) for 5 min; B; cells, grown in 100-mm dishes, were pretreated with 100 ng/ml PTX for 4 h (lanes 3), followed by treatment with 1 mM PMA (lanes 1 and 3) for 5 min. Lysates of cells were subsequently immunoprecipitated with anti-MAP kinase antiserum, and the immunoprecipitates were incubated with [g-32P]ATP in the presence of MBP, as described in Materials and Methods. After the reactions were stopped, with Laemmli sample buffer, samples were subjected to SDS-PAGE and autoradiography. Experiments were repeated three times, with essentially identical results.

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FIG. 8. ET-1 mediates MAP kinase activation through ETA. A and B, Cells grown in 100-mm dishes were pretreated with 100 nM BQ485 (A, lane 3) or 10 nM BQ788 (B, lane 3), followed by treatment with 100 nM ET-1 (lanes 2 and 3) for 5 min; C; cells were treated with 100 nM ET-1 (lane 2) or 100 nM ET-3 (lane 3) for 5 min. Lysates of cells were subsequently immunoprecipitated with anti-MAP kinase antiserum, and the immunoprecipitates were incubated with [g-32P]ATP in the presence of MBP, as described in Materials and Methods. After the reactions were stopped, with Laemmli sample buffer, SDS-PAGE and autoradiography were performed. Experiments were repeated three times, with essentially identical results.

uterine contraction dose-dependently (Fig. 9). These results suggest that both the induction of MAP kinase activation and the uterine contraction by ET-1 are mainly mediated by the ETA. Ritodrine attenuation of ET-1-induced MAP kinase activation FIG. 7. Expression of both ETA and ETB in cultured rat puerperal uterine myometrial cells. Total RNA samples were isolated from cultured myometrial cells. RT-PCR was performed under standard conditions, as described in Materials and Methods. The PCR products were resolved by electrophoresis on an agarose gel and stained with ethidium bromide.

ETB-specific primers, with the predicted sizes of 546 and 475 bp, respectively, were observed. The results showed the presence of both ETA and ETB messenger RNA (mRNA) in cultured rat puerperal uterine myometrial cells. To examine which receptor subtype mediates the MAP kinase activation by ET-1, we pretreated the cultured cells with BQ485 (an antagonist of ETA) or BQ788 (an antagonist of ETB) and exposed them to 100 nm ET-1 for 5 min. It has been reported that 100 nm BQ485 specifically blocks the ETA, but not the ETB, and that 10 nm BQ788 works as a specific inhibitor of the ETB (41). BQ485 remarkably suppressed the activation of MAP kinase induced by ET-1, whereas BQ788 had no inhibitory effect on ET-1-induced MAP kinase activation (Fig. 8, A and B). Moreover, the effect by ET-3, which has lower affinity for ETA than does ET-1, on the MAP kinase activity was less than that by ET-1 (Fig. 8C). We further examined which receptor subtype mediates ET-1-induced uterine contraction. Rat puerperal uterine smooth muscle strips were stretched to optimal length, and the active force by ET-1 was measured. Treatment of uterine strips with ET-1 resulted in a muscle contraction. Although BQ788 had no effect, BQ485 attenuated the ET-1-induced

Ritodrine is an agent well known to relax the uterine muscle contraction (42). To explore the role of MAP kinase in the physiological function of ET-1, we evaluated the effects of ritodrine on ET-1-induced MAP kinase activation (Fig. 10A). Pretreatment of cells for 10 min with 1 mm ritodrine attenuated ET-1-induced MAP kinase activation, suggesting that MAP kinase may be involved in ET-1-induced uterine contraction. Effects of ritodrine on other activators of MAP kinase were examined. Although ritodrine attenuated both EGF- and A23187- (data not shown) induced MAP kinase activation, this drug had no effect on PMA-induced MAP kinase activation (Fig. 10B). The pharmacological effect of ritodrine might be through elevation of intracellular cAMP (43, 44), and we previously reported that increasing cAMP by forskolin or (Bu)2cAMP attenuated OT-induced phosphorylation of MAP kinase (27). The effect of cAMP on the MAP kinase cascade is dependent on the cell types (45); it antagonizes the growth factor (46 –50) -activated MAP kinase in some cell types, whereas cAMP itself has a stimulative effect in other cells (51, 52). Thus, the mechanisms involved are likely to be complex, and it was reported that the cell typespecific actions of cAMP on MAP kinase depend on the expression of serine/threonine kinase B-Raf (53). The results showing the inhibitory effects of ritodrine on OT- (27), PG F2a- (29), ET-1-, EGF- (54, 55), and A23187- (56) induced MAP kinase activation suggest that ritodrine might attenuate the MAP kinase activity of the agents that act pharmacologically as uterine contractants.



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FIG. 10. Effects of pretreatment with ritodrine on ET-1-induced MAP kinase activation. Cells were grown in 100-mm dishes. A, Cells were pretreated with 1 mM ritodrine for 10 min (lane 3), followed by treatment with 100 nM ET-1 (lanes 2 and 3) for 5 min; B, cells were pretreated with 1 mM ritodrine for 10 min (lanes 3 and 5), followed by treatment with 10 nM EGF (lanes 2 and 3) or I mM PMA (lanes 4 and 5) for 5 min. Lysates of cells were subsequently immunoprecipitated with anti-MAP kinase antiserum, and the immunoprecipitates were incubated with [g-32P]ATP in the presence of MBP, as described in Materials and Methods. After the reactions were stopped, with Laemmli sample buffer, SDS-PAGE and autoradiography were performed. An autoradiogram of 32P-labeled MBP is shown at the lower panel. Relative densitometric units of the MBP bands are shown at the upper panel, with the density of the control bands set arbitrarily at 1.0. Values shown represent mean 6 SE from at least three separate experiments. **, Significant differences (P , 0.01). RT, Ritodrine. FIG. 9. ET-1 mediates uterine contraction through ETA. The isolated uterine tissues were suspended at 26 C in modified Locke-Ringer solution aerated with 95% O2-5% CO2 and weighed with 1 g. Activity of the uterus was measured with a pressure transducer and a rectigram. Either 10-9–10-6 M BQ485 or 10-9–10-6 M BQ788 was added to the tissues after treatment with ET-1.

Effects of MEK inhibitor on ET-1-induced contraction of rat pregnant uterine smooth muscle

To examine the role of MAP kinase pathway in ET-1induced uterine contraction, an inhibitor of MEK activity, PD098059, was used. This compound is relatively specific for MEK, with no inhibitory activity against a number of other serine/threonine and tyrosine kinases (57–59). MEK inhibitor (100 mm) completely attenuated the ET-1-induced MAP kinase activation (Fig. 11A). Rat puerperal uterine smooth muscle strips were stretched to optimal length, and active force was measured after treatment with ET-1. Treatment of uterine strips with ET-1 resulted in a contraction. A solution of 1% dimethyl sulfoxide had no effect on ET-1-induced uterine contraction, whereas 100 mm MEK inhibitor significantly inhibited the ET-1-induced uterine contraction (Fig. 11B). Figure 11C shows the dose-response relationship of MEK inhibitor and ET-1-induced uterine contraction: 100 mm MEK inhibitor partly, but significantly, inhibited ET-1induced uterine contraction.

Effect of Ca21 on ET-1-induced MAP kinase activation

To examine the reason why MEK inhibitor did not completely inhibit ET-1-induced uterine contraction, we evaluated the role of Ca21 in ET-1-induced MAP kinase activation (Fig. 12A). We pretreated cells with 2 mm EGTA for 5 min, to eliminate extracellular Ca21, or with 50 mm 1,2-bis(o-amino-phenoxy)ethane-N,N, N9, N9-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) for 20 min, to eliminate intracellular Ca21 (60). Neither pretreatment of EGTA nor BAPTA-AM attenuated ET-1-induced MAP kinase activation, although EGTA attenuated A23187-induced, and BAPTA-AM attenuated OT-induced, MAP kinase activation (Fig. 12B). In addition, the intracellular Ca21 concentration was elevated by ET-1, even with the pretreatment by 100 mm MEK inhibitor (data not shown), as we have found previously (28, 29). These results suggest that MAP kinase might be involved in Ca21independent uterine contraction induced by ET-1. Discussion

MAP kinase is activated in a variety of cell types and in response to numerous growth factors, the receptors for which are structurally unrelated, suggesting the existence of distinct pathways that converge at this site of regulation. The question remained as to how ET-1 stimulates the activation of MAP kinase. It is now generally accepted that the signal from ET-1 is transduced by a G protein that interacts with


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FIG. 11. Effects of MEK inhibitor on ET-1-induced uterine contraction. A, Cells, grown in 100-mm dishes, were pretreated with 100 mM MEK inhibitor for 15 min (lanes 4 – 6), followed by treatment with 100 nM ET-1 (lanes 2 and 6) or 1 mM PMA (lanes 3 and 5) for 5 min. Lysates of cells were subsequently immunoprecipitated with anti-MAP kinase antiserum, and the immunoprecipitates were incubated with [g-32P]ATP in the presence of MBP, as described in Materials and Methods. Experiments were repeated three times, with essentially identical results. B, The isolated uterine tissues were suspended at 26 C in modified Locke-Ringer solution aerated with 95% O2-5% CO2 and weighed with 1 g. Activity of the uterus was measured with a pressure transducer and a rectigram. Then 100 mM MEK inhibitor was added to the tissues after treatment with ET-1. C, The dose dependency of MEK inhibitor on ET-1-induced uterine activity in isolated pregnant rat uterus was determined by the Magnus method (n 5 5). Significant differences (vs. the control): **, P , 0.01; *, P , 0.05. MEKI, MEK inhibitor.

cell-surface receptors and phospholipase-C, resulting in the breakdown of phosphatidylinositol 4,5-bisphosphate, subsequently producing a second messenger, inositol 1,4,5triphosphate (14, 15). However, the identity of this G protein remained unclear. Distinct pathways of Gi- and Gq-mediated MAP kinase activation were reported (61). In the case of Gi-coupled receptors, such as thrombin (62), OT (27), and PG F2a (29), activation by these seems to be PTX-sensitive and PKC-independent. In addition, Gi-mediated MAP kinase activation is initiated by phosphatidylinositol 3-kinase activity, followed by a pathway common to tyrosine-kinase receptors (63). This involves a series of SH2- and SH3-dependent protein-protein interactions among tyrosine-phosphorylated receptors, Shc, Grb2, and SOS, resulting in a Ras-dependent MAP kinase activation. However, in the case of receptors that couple to Gq, such as bombesin, activation is thought to be secondary to stimulation of phosphatidylinositol 4,5,bisphosphate-PLC, leading to production of inositol phosphate and diacylglycerol, with subsequent PKC-mediated stimulation of MAP kinase (64). In this study, pretreatment of cells with PTX completely blocked the ET-1-induced activation of MAP kinase, suggesting that ET receptor couples

with the Gi or Go families of PTX-sensitive G-proteins. Our results are consistent with those observed in rat mesangial cells (65) and rat ventricular myocytes (66), where PTX suppressed ET-1-induced inositol phosphate production and the positive inotropic effect, respectively. However, contradictory findings have also been reported in rat myometrial tissue (67) and cultured vascular smooth muscle cells (68). The results in these studies show the PTX-insensitive coupling of ET-1 to phospholipase C. In addition, pretreatment of cells with PTX did not block the PMA-induced activation of MAP kinase (Fig. 6B). Thus, the role of a PTX-sensitive G protein-coupled(s), in transmitting ET-1-generated signals, has been controversial. Activation of MAP kinase is induced by phosphorylation of both threonine and tyrosine residues of the enzyme as a result of successive stimulation of Ras, MAP kinase kinase kinase, which may be Raf-1, MEK kinase, or an alternative kinase, and MEK (69, 70). PKCa activates Raf-1 by direct phosphorylation (71). PKC is also an important serine/threonine kinase in ET postreceptor signaling (40). We therefore analyzed the possible involvement of PKC, which is involved in many types of receptor-mediated activation of MAP ki-



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ETA and ETB mRNA in rat puerperal myometrial cells. It was reported that both human ETA and ETB were coupled to the MAP kinase cascade in ETA or ETB cDNA-transfected CHO cells (75), and stimulation of ETB with ET1 activated MAP kinase in rat astrocytes without the expression of ETA (76). However, in this study, ET-1-induced MAP kinase activation was significantly inhibited by the ETA-specific antagonist BQ123, but not by the ETB-specific antagonist BQ788. Moreover, the effect of ET-3, which has lower affinity for ETA than does ET-1, on MAP kinase activity was less than that by ET-1. In addition, although BQ788 had no effect, BQ485 attenuated the ET-1-induced uterine contraction dose-dependently (Fig. 9). These data suggest that ET-1 induced both MAP kinase activation and uterine contraction through ETA, although both receptors are expressed. Similar results were reported in ET-1-induced MAP kinase activa-

FIG. 12. Effect of Ca21 on ET-1-induced MAP kinase activation. Cells were grown in 100-mm dishes. A, Cells were pretreated with either 50 mM BAPTA-AM for 20 min (lane 3) or 2 mM EGTA for 5 min (lane 4), followed by treatment with 100 nM ET-1 (lanes 2– 4) for 5 min; B, cells were pretreated with either 2 mM EGTA for 5 min (lane 3) or 50 mM BAPTA-AM for 20 min (lane 5), followed by treatment with 1 mM A23187 (lanes 2 and 3) or 1 mM OT (lanes 4 and 5) for 5 min. Lysates of cells were subsequently immunoprecipitated with anti-MAP kinase antiserum, and the immunoprecipitates were incubated with [g-32P]ATP in the presence of MBP, as described in Materials and Methods. After the reactions were stopped, with Laemmli sample buffer, samples were subjected to SDS-PAGE and autoradiography. Autoradiogram of 32P-labeled MBP is shown at the lower panel. Relative densitometric units of the MBP bands are shown at the upper panel, with the density of the control bands set arbitrarily at 1.0. Values shown represent mean 6 SE from at least three separate experiments. **, Significant differences (P , 0.01). OXY, Oxytocin.

nase cascade (69, 70). Direct stimulation of PKC with PMA led to an activation of MAP kinase in the myometrial cells (Fig. 8). However, the ability of PMA to induce the activation of MAP kinase does not necessarily mean that the PKC pathway is involved in the MAP kinase signaling pathway, as in the case of norepinephrine-induced MAP kinase activation in adipocytes (72) and GT-1 GnRH neuronal cell lines (73). Apparent down-regulation of PKC by a prolonged incubation with PMA did not attenuate the stimulation of MAP kinase activity by ET-1. It is reported that MAP kinase activation by TRH is partly mediated by PKC and also by a Ras-dependent pathway (23). Moreover, like OT (27) and PG F2a (29), ET-1 stimulated the phosphorylation of SOS, the ras nucleotide exchange factor (Fig. 3). Down-regulation of PKC by a prolonged incubation with PMA had no effect on ET1-induced SOS phosphorylation (Fig. 5). Moreover, dominant negative SOS significantly inhibited ET-1-induced MAP kinase activation (Fig. 3). Thus, ET-1 stimulation of MAP kinase activity is likely to be mediated by SOS, not by PMAsensitive PKC. To identify the ET-1 receptors involved in MAP kinase activation, and presumably in uterine contraction, we characterized ET receptors in myometrial cells. Because it was reported that ETA and ETB are both present in human myometrium (14, 15, 74), RT-PCR confirmed the presence of both

FIG. 13. Proposed scheme of signal transduction pathway in ET-1induced uterine contraction. Binding of ET-1 to its seven-membrane spanning receptor (ETA) results in rapid intracellular signal transduction, including PTX-insensitive Gq protein-stimulated phosphatidylinositol (PtdIns) metabolism via phospholipase C-b (PLC-b), PKC activation, intracellular calcium (Ca21) mobilization, calmodulin-calcium-dependent protein kinase (calmodulin-Ca21 kinases) activation, and myosin light chain kinase (MLCK) activation. ET-1 also activates the tyrosine phosphorylation of Shc, leading to the sequential activation of the SOS-Ras-Raf-1-MEK-MAP kinase cascade via PTX-sensitive Gi protein. These early signaling events are hypothesized to lead the phosphorylation of cytoskeletal proteins (myosin and microtubule-associated protein-2) and produce a uterine contraction. *, Data presented in this study.


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tion in cardiomyocytes (77) and ET-1-induced immediate response gene expression in normal rat kidney cells (78). We examined a possible physiological role of ET-1-induced MAP kinase activation in the contractility of uterine muscle. Contraction of smooth muscle involves phosphorylation of the myosin light chain by Ca21/calmodulin-dependent myosin light chain kinase (79, 80). However, the involvement of Ca21-independent protein kinase in the Ca21-free contraction is also suggested (81, 82). As we identified in this report, ET-1-induced MAP kinase activation is Ca21-independent. A substrate of MAP kinase is microtubule-associated protein-2, which is one of the cytoskeletal proteins (83). Pretreatment of cells with ritodrine, which blocks the uterine contraction, attenuated the OT-(27), PG F2a- (29), and ET-1-induced MAP kinase activation. In addition, the specific inhibitor of MEK, PD 098059, which had no effect on Ca21 mobilization in cultured uterine myometrial cells (28, 29), partially inhibited OT- (27), PG F2a- (30), and ET-1-induced uterine muscle contraction. Moreover, we reported here that ETA, but not ETB, mediates myometrial contractility (12–14). Taken together, these observations suggest that MAP kinase also has some role in ET-1-induced uterine contraction, and ET-1-induced uterine contraction might be partly dependent on and partly independent of Ca21 mobilization. The potential relationships between these pathways are shown in the scheme in Fig. 13. In conclusion, the results presented in this paper suggest that MAP kinase is acutely activated by ET-1 through an ETA, a PTX-sensitive G protein, and SOS, not through the PMA-sensitive PKC. This new pathway might have some role in ET-1-induced uterine contraction. Acknowledgment We thank Dr. Motoyoshi Sakaue for the gift of SRa-dnSOS and Dr. Kazushige Touhara for the gift of pEXV-Erk2-tag.

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