help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ohmichi, M.
Right arrow Articles by Murata, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ohmichi, M.
Right arrow Articles by Murata, Y.
Endocrinology Vol. 138, No. 8 3103-3111
Copyright © 1997 by The Endocrine Society

ARTICLES

Role of Mitogen-Activated Protein Kinase Pathway in Prostaglandin F2{alpha}-Induced Rat Puerperal Uterine Contraction

Masahide Ohmichi, Koji Koike, Akiko Kimura, Kanji Masuhara, Hiromasa Ikegami, Yoshihide Ikebuchi, Tohru Kanzaki, Kazushige Touhara, Motoyoshi Sakaue, Yuzuru Kobayashi, Masuo Akabane, Akira Miyake and Yuji Murata

Department of Obstetrics and Gynecology, Osaka University Medical School (M.O., K.K., A.K., K.M., H.I., T.K., A.M., Y.M.), 2–2, Yamadaoka, Suita-shi, Osaka 565; Department of Biochemistry, Institute for Brain Research, Faculty of Medicine, The University of Tokyo (K.T.), 7–3-1 Hongo, Bunkyo-ku, Tokyo 113; Second Department of Internal Medicine, Kobe University School of Medicine (M.S.),Chuo-ku, Kobe 650; and Kissei Pharmaceutical Company Limited (Y.K., M.A.), 43665–1, Kashiwabara, Hotaka, Minamiazumi, Nagano 399–83, Japan

Address all correspondence and requests for reprints to: Masahide Ohmichi, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka 565, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study, prostaglandin (PG) F2{alpha} was found to activate mitogen-activated protein (MAP) kinase and MAP kinase kinase (MEK) in cultured rat puerperal uterine myometrial cells. PGF2{alpha} stimulation also led to an increase in phosphorylation of raf-1, son of sevenless (SOS), and Shc. Furthermore, we examined the mechanism by which PGF2{alpha} induced MAP kinase phosphorylation. Both pertussis toxin (10 ng/ml), which inactivates Gi/Go proteins, and expression of a peptide derived from the carboxyl terminus of the ß-adrenergic receptor kinase 1 (ßARK1), which specifically blocks signaling mediated by the ß{gamma} subunits of G proteins, blocked the PGF2{alpha}-induced activation of MAP kinase. Ritodrine (1 µM), which is known to relax uterine muscle contraction, attenuated PGF2{alpha}-induced tyrosine phosphorylation of MAP kinase. Moreover, to examine the role of MAP kinase pathway in uterine contraction, an inhibitor of MEK activity, PD098059, was used. Although MEK inhibitor had no effect on PGF2{alpha}-induced calcium mobilization, this inhibitor partially inhibited PGF2{alpha}-induced uterine contraction. These results provide evidence that PGF2{alpha} stimulates the MAP kinase signaling pathway in cultured rat puerperal uterine myometrial cells through Gß{gamma} protein, suggesting that this new pathway may play an important role in the biological action of PGF2{alpha} on these cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT HAS BEEN proposed that uterine contractions are initiated by a multitude of biological pathways. It is accepted that prostaglandin (PG) F2{alpha} and PGE2 can act pharmacologically as uterine contractants at any stage of gestation when administered to pregnant women (1, 2). On the other hand, PGF2{alpha} has been reported to induce mitogenic responses in some cultured cells, including NIH-3T3 cells (3, 4). It was clarified that the PGF2{alpha} receptor coupled to phospholipase C via a pertussis toxin (PTX)-insensitive GTP-binding protein, probably Gq, and that this pathway is responsible for the proliferation of NIH-3T3 cells (4, 5). Mitogen-activated protein (MAP) kinase is activated during growth or differentiation by a variety of stimuli (6), thereby playing a key role in the kinase cascade originating from receptor activation (7). A pathway leading from the tyrosine kinase receptor to MAP kinase activation has been elucidated; ligand-receptor interaction causes formation of the ras-GTP complex, which in turn activates a kinase cascade comprising p74raf-1 (8), MAP kinase kinase, and MAP kinase. Recently, it was also reported that PGF2{alpha} stimulates formation of p21ras-GTP complex and MAP kinase in NIH-3T3 cells via Gq protein-coupled pathway (9).

Recently, two genes that encode proteins that have src homology 2 (SH2) domains were identified that are thought to reside upstream of p21ras activation by tyrosine kinases (10). One gene, Shc, encodes two overlapping proteins of 46 and 52 kDa (11). Another Shc protein of 66 kDa is thought to be encoded by a related gene (11). Shc proteins can associate with tyrosine-phosphorylated receptors and are themselves phosphorylated on tyrosine in response to growth factors (11). The second gene encodes an abundant 23-kDa polypeptide known as growth factor receptor-bound protein 2 (Grb2) (12, 13, 14). This protein contains SH2 and SH3 domains, although its state of phosphorylation does not appear to be increased by growth factors (12, 14). The association of Shc with Grb2 has been implicated in activation of the ras pathway by tyrosine kinases via association with son of sevenless (SOS), the ras nucleotide exchange factor (10, 15, 16, 17, 18, 19).

Oxytocin is also a well known uterine contractant that we identified as stimulating MAP kinase activity in cultured human puerperal uterine myometrial cells (20). Taken together, these concepts led us to examine the effect of PGF2{alpha} on the MAP kinase pathway in cultured rat puerperal myometrial cells. Moreover, we examined the effect of a specific inhibitor of MAP kinase kinase (MEK), which is thought to be an invaluable tool that will help elucidate the role of the MAP kinase cascade in a variety of biological settings (21, 22, 23), on PGF2{alpha}-induced uterine contraction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Enhanced chemiluminescence (ECL) Western blotting detection reagents were obtained from Amersham Corp. (Arlington Heights, IL). [{gamma}-32P]ATP (3000 Ci/mmol) was obtained from New England Nuclear (Bannockburn, IL). PGF2{alpha} and PGE2 were donated by Ono Pharmaceuticals (Osaka, Japan). Antiphosphotyrosine (PY20), mouse monoclonal anti-MAP kinase, anti-Shc, and anti-SOS1 antisera were obtained from Upstate Biotechnology (Lake Placid, NY). Erk1 rabbit polyclonal anti-MAP kinase antiserum and monoclonal antibody 9E10 to the Myc epitope were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-raf-1 antiserum was developed as described previously (24). Anti-MEK antiserum was the generous gift of Dr. Kunliang Guan (25). Pregnant Wister female rats were obtained from Nihon Dobutu Co. (Osaka, Japan).

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 (26). The tissues were cut into 1–2 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 cloth. The cells were maintained at 37 C in an atmosphere of 95% air and 5% CO2 in RPMI 1640 medium containing 10% FBS supplemented with penicillin (200 U/ml) and streptomycin (200 µg/ml). They were used for experiments after 5 days.

Construction of expression plasmids
Myc-tagged p42mapk expression plasmid (pEXV-Erk2-tag) was obtained from C.J. Marshall (Institute of Cancer Research, London, UK) (27). The ßARKct peptide-encoding minigene, containing complementary DNA encoding the carboxyl-terminal 195 amino acids of ßARK1, was prepared as described previously (28, 29).

Assay of MAP kinase activity
Cells were incubated in the absence of serum overnight and then treated with various materials. 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 µM sodium orthovanadate, 100 mM NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) (30). 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 Labs., Richmond, CA). Erk1 rabbit polyclonal antibody was bound to protein A-Sepharose beads, and 300 µg 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 µl kinase assay buffer containing 10 µg myelin basic protein and 40 µM [{gamma}-32P]ATP (1 µCi) as described previously (31). The kinase reaction was allowed to proceed at room temperature for 5 min and stopped by the addition of Laemmli SDS sample buffer (32). Reaction products were resolved on 15% SDS-PAGE.

Assay of 42-kDa MAP kinase activity using a transient expression system
Rat puerperal uterine myometrial cells cultured in 10-cm diameter dishes were transfected with Myc-tagged p42mapk expression plasmid (1 µg of pEXV-Erk2-tag) in combination with 9 µg pRK or pRK-ßARK1 using LipofectAMINE (Life Technologies, Gaithersburg, MD). At 72 h after transfection, serum-deprived cells were incubated with 1 µM PGF2{alpha} for 5 min, and expressed Myc-tagged p42mapk was immunoprecipitated with 1 µg 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 ß-gal staining after transfection of a ß-gal containing expression plasmid.

Assay of MEK activity
Cells (150 mm dishes) were serum deprived for 16 h. After treatments, cells were lysed in HNTG buffer, and lysates were immunoprecipitated with anti-MEK antiserum (1:200 dilution) for 2 h at 4 C. This antiserum precipitates both MEK1 and MEK2. Immunoprecipitates were mixed with protein A-Sepharose beads for 30 min, and the beads were washed twice with 1 ml HNTG buffer. The sample was then resuspended in 100 µl reaction buffer, containing 20 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM MnCl2, 1 mM EGTA. Reactions were initiated by the addition of 10 µCi [{gamma}-32P]ATP (50 µM) and 10 µg of a glutathione S-transferose (GST)-fusion protein containing p44MAPK with a lysine to alanine mutation at position 71 (MAPK/KA) (21). This mutation eliminates kinase activity of MAPK, so only kinase activity attributed to the added MEK remains. After a 15-min incubation at 25 C, reactions were stopped with 20 µl Laemli sample buffer, and phospho-MAPK/KA was detected by SDS-PAGE followed by autoradiography.

Assay of raf-1 kinase activity
This was assayed as described (24). Briefly, after hormonal treatment, cells were lysed in 1 mL HNTG buffer. Lysates were precleared with rabbit IgG-agarose and precipitated with anti-raf-1 antiserum. Immunoprecipitates were resuspended in 20 µl reaction buffer, and 2 µl of 20 µM [{gamma}-32P]ATP (10 µCi) was added. Reactions were stopped with Laemmli sample buffer, and equal amounts of protein were electrophoresed on 8% SDS-PAGE, followed by autoradiography.

Immunoblots
For analysis of tyrosine phosphorylation of MAP kinase, cells were grown in 60-mm dishes. After treatment, the cells were washed, and then 100 µl 1% SDS was added. Lysates were heated for 5 min at 100 C and diluted 1:10 with ice-cold HNTG buffer, followed by incubation with anti-MAP kinase antiserum. Immune complexes were precipitated with protein A-Sepharose, and the isolated proteins were analyzed by electrophoresis on 8% SDS-PAGE. Transfer to nitrocellulose, immunoblotting with antiphosphotyrosine antiserum, and washing were performed as described elsewhere (30). In some experiments, 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 x g for 10 min. Supernatants were incubated for 1 h with the indicated antiserum. Immunocomplexes were precipitated with protein A-Sepharose and washed three times with HNTG buffer, and samples were resolved on 8% SDS-PAGE, followed by immunoblotting with the indicated antiserum.

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 Locke-Ringer 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 the spontaneous contractions. The contractions were measured isometrically using a mechano-electric transducer (NEC San-ei, Tokyo, Japan) coupled to a potentiometric pen-recorder (NEC San-ei). The initial tension was set at approximately 1.0 g. In the absence of spontaneous contractions, 1 µM of PGF2{alpha} 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.

Measurement of intracellular Ca2+ concentration ([Ca2+]i)
[Ca2+]i was monitored with a digital imaging fluorescence microscope using a Ca2+-sensitive fluorescent dye, fura-2AM, as described previously (33, 34). Briefly, uterine cells were incubated at 37 C for 60 min in medium 199 containing 5 mmol/L fura-2AM. The cells were then rinsed with HBSS and placed on the microscope stage. [Ca2+]i was measured at 100-msec intervals with a digital imaging microscopic system M-500 (Scholar Tech Corp., Osaka, Japan). The ratio of the intensities of fluorescent emission at 510 nm with excitation at 340 and 380 nm was determined as [Ca2+]i. The excitation beam was targeted on the cells, and emission images were recorded on video film and analyzed with a computer. The effects of PGF2{alpha} or oxytocin with 100 µM MEK inhibitor on the [Ca2+]i in 10 cells were determined.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PGF2{alpha} stimulation of MAP kinase activity
Cultured rat puerperal uterine myometrial cells were treated with 1 µM PGF2{alpha}, PGE2, or oxytocin for 5 min. Cell lysates were immunoprecipitated with anti-MAP kinase antibody and assayed for MAP kinase by examining the incorporation of 32P into myelin basic protein (MBP), followed by SDS-PAGE and autoradiography (Fig. 1AGo). PGF2{alpha}, PGE2, and oxytocin each produced a marked increase in this kinase activity compared with the control cells.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 1. PGF2{alpha} stimulation of MAP kinase activity (A) and tyrosine phosphorylation of MAP kinase (B). A, Cells were grown in 150-mm dishes and treated with 1 µM PGF2{alpha}, 1 µM PGE2, or 1 µM oxytocin for 5 min. Lysates of cells were subsequently immunoprecipitated with an anti-MAP kinase antiserum, and immunoprecipitates were incubated with [{gamma}-32P]ATP in presence of MBP, as described in Materials and Methods. After reactions were stopped with Laemmli sample buffer, SDS-PAGE and autoradiography were performed. Experiments were repeated three times with essentially identical results. B, Cells were grown in 60-mm dishes and treated with PGF2{alpha} at increasing concentrations for 5 min each. Cells were then harvested and lysed in 100 µl 1% SDS. Cell lysates were diluted with HNTG buffer and centrifuged. Supernatant was precipitated with an anti-MAP kinase antiserum, and immunoprecipitated MAP kinase was subjected to SDS-PAGE, followed by immunoblotting with antiphosphotyrosine antiserum. Experiments were repeated three times with essentially identical results.

 
Mitogenic stimuli activate MAP kinase by increasing tyrosine and serine or threonine phosphorylation of the protein, due to the activity of the dual specificity of MEK (35). Therefore, the hormone-dependent phosphorylation of the predominant form of MAP kinase was evaluated by antiphosphotyrosine Western blotting of the anti-MAP kinase immunoprecipitates (Fig. 1BGo). Increasing concentrations of PGF2{alpha} were added to cultured rat myometrial cells for 5 min, followed by lysis and evaluation of tyrosine phosphorylation of MAP kinase. Tyrosine phosphorylation of MAP kinase was detected after treatment with as little as 1 nM PGF2{alpha}; the phosphorylation was increased up to 1 µM.

PGF2{alpha} stimulation of MEK activity
MAP kinase was phosphorylated and activated by an immediately upstream activating kinase, MEK. Cells were treated with 1 µM PGF2{alpha}, PGE2, or oxytocin for 5 min. Cell lysates were immunoprecipitated with anti-MEK antibody and assayed for MEK activity by examining the incorporation of 32P into GST-ERK fusion protein (Fig. 2AGo). Both PGF2{alpha} and oxytocin produced a marked increase in this activity, whereas the effect of PGE2 appeared to be less marked.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 2. PGF2{alpha} stimulation of MEK activity (A) and phosphorylation of raf-1 kinase. A, Cells were grown in 150-mm dishes and treated with 1 µM PGF2{alpha}, 1 µM PGE2, or 1 µM oxytocin for 5 min. Lysates of cells were subsequently immunoprecipitated with an anti-MEK antiserum, and immunoprecipitates were incubated with [{gamma}-32P]ATP in presence of GST-ERK fusion protein, as described in Materials and Methods. After reactions were stopped with Laemmli sample buffer, SDS-PAGE and autoradiography were performed. Experiments were repeated three times with essentially identical results. B, Cells were grown in 150-mm dishes and treated with 1 µM PGF2{alpha} for 5 min. Lysates were immunoprecipitated with anti-raf-1 antiserum. Immunoprecipitates were incubated with 2 µl 20 µM [{gamma}-32P]ATP (10 µCi) for 5 min in kinase buffer at 24 C. After reactions were stopped with Laemmli sample buffer, SDS-PAGE and autoradiography were performed. Each treatment was performed in triplicate, and the pattern of raf-1 phosphorylation from a representative set is shown.

 
PGF2{alpha} stimulation raf-1 phosphorylation
The proto-oncogene c-raf, encoding a serine/threonine kinase, has been reported to be a direct activator of MEK. Cells were treated with 1 µM of PGF2{alpha} for 5 min. Cell lysates were immunoprecipitated with anti-raf-1 kinase antibody and assayed for autophosphorylation of raf-1 kinase by examining the incorporation of 32P into its protein. PGF2{alpha} produced a marked increased in the autophosphorylation of raf-1 kinase (Fig. 2BGo)

PGF2{alpha} stimulation of SOS and Shc 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 (36). In the examination of the effect of PGF2{alpha} on SOS phosphorylation, cells were treated with 1 µM PGF2{alpha} for the indicated times, 10 nM epidermal growth factor (EGF), 1 µM PGE2, and 1 µM oxytocin for 5 min (Fig. 3Go). PGF2{alpha} (lanes 3–8), PGE2 (lanes 9), oxytocin (lane 10), and EGF (lane 1) stimulation resulted in a significant retardation of mobility of SOS on SDS-PAGE, reflecting SOS phosphorylation. This occurred first within 1 min of stimulation and was maximal by 30 min, decreasing to the control level by 6 h.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 3. Effects of PGF2{alpha}, PGE2, oxytocin, or EGF on SOS phosphorylation. Cells were grown in 150-mm dishes and treated with 1 µM PGF2{alpha} for indicated times (lanes 3–8), 10 nM EGF (lane 1), 1 µM PGE2 (lane 9), or 1 µM oxytocin (lane 10) for 5 min. Lysates were immunoprecipitated with anti-SOS antiserum, and immunoprecipitates were subjected to SDS-PAGE, followed by immunoblotting with anti-SOS antiserum. Experiments were repeated three times with essentially identical results.

 
Moreover, we measured tyrosine phosphorylation of Shc after stimulation with PGF2{alpha} or EGF (Fig. 4Go). Shc exists in three isoforms of 46, 52, and 66 kDa. All three Shc proteins were phosphorylated by EGF (lane 2), whereas pp52Shc and pp46Shc were clearly phosphorylated by PGF2{alpha} (lane 3).



View larger version (37K):
[in this window]
[in a new window]
  		Figure 4. PGF2{alpha} and EGF stimulation of phosphorylation of Shc. Cells were grown in 150-mm dishes and treated with 1 µM PGF2{alpha} (lane 3) or 10 nM EGF (lane 2) for 5 min. Lysates were immunoprecipitated with anti-Shc antiserum, and immunoprecipitates were subjected to SDS-PAGE, followed by immunoblotting with anti-phosphotyrosine antiserum. Experiments were repeated 3 times with essentially identical results.

 
Gß{gamma}-mediated PGF2{alpha}-induced MAP kinase activation
The structure of the PGF2{alpha} receptor represents seven transmembrane domains typical of G protein-coupled receptors (37). To determine what type of G protein couples to this receptor, we preincubated cells for 4 h with 100 ng/ml PTX, followed by incubation with 1 µM PGF2{alpha} (Fig. 5AGo). PTX at 100 ng/ml almost completely blocked the PGF2{alpha}-induced MAP kinase activation. It was reported that the carboxyl terminus of the ß-adrenergic receptor kinase, containing its Gß{gamma}-binding domain, is a cellular Gß{gamma} antagonist capable of specifically distinguishing G{alpha}- and Gß{gamma}-mediated processes (29, 30). To examine the effect of the Gß{gamma} subunit-sequestrant ßARKct peptide on PGF2{alpha}-induced exogenous MAP kinase activity, a Myc-tagged p42mapk expression plasmid was used to distinguish exogenous MAP kinase from endogenous MAP kinase. We transfected cells with pRK or pRK-ßARK1 together with Myc-tagged p42mapk expression plasmid (pEXV-Erk2-tag) and after 72 h stimulated them with 1 µM of PGF2{alpha} for 5 min (Fig. 5BGo). 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 Myc-tagged p42mapk expression plasmid. Transfection of pRK-ßARK1 attenuated the PGF2{alpha}-induced MAP kinase activation. These results suggest that PGF2{alpha} induces MAP kinase activation mediated by Gß{gamma}.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 5. Gß{gamma}-mediated PGF2{alpha}-induced MAP kinase activation. A, Cells, grown in 150-mm dishes, were pretreated with 100 ng/ml PTX for 4 h (lanes 2 and 4), followed by treatment with 1 µM PGF2{alpha} (lanes 3 and 4) for 5 min. Lysates of cells were subsequently immunoprecipitated with an anti-MAP kinase antiserum, and immunoprecipitates were incubated with [{gamma}-32P]ATP in presence of MBP, as described in Materials and Methods. After reactions were stopped with Laemmli sample buffer, SDS-PAGE and autoradiography were performed. Experiments were repeated three times with essentially identical results. B, Cells were transfected with pRK (lanes 1 and 2) or pRK-ßARK1 (lanes 3 and 4) together with Myc-tagged p42mapk expression plasmid (pEXV-Erk2-tag) and, after 72 h, were stimulated with 1 µM PGF2{alpha} (lanes 2 and 4). Autoradiogram of MAP kinase activity of immunoprecipitates with antibody to Myc epitope by 32P-labeled MBP is shown at lower part of figure. Coomassie blue-stained 32P-labeled MBP was excised from gels, and incorporated radioactivity was measured by Cerenkov counting. Bar diagram of quantitation of MAP kinase activity is shown at upper part of figure. Values shown represent mean ± SE from at least three separate experiments.

 
Ritodrine attenuation of PGF2{alpha}-induced tyrosine phosphorylation of MAP kinase
Ritodrine is an agent well known to relax the uterine muscle contraction (38). To explore the role of MAP kinase in the physiological function of PGF2{alpha}, we evaluated the effect of ritodrine on PGF2{alpha}-induced tyrosine phosphorylation of MAP kinase (Fig. 6Go). Pretreatment of cells for 10 min with 1 µM ritodrine attenuated both PGF2{alpha}- and oxytocin-induced tyrosine phosphorylation of MAP kinase, suggesting that MAP kinase might be involved in PGF2{alpha}-induced uterine contraction.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 6. Effect of pretreatment with ritodrine on PGF2{alpha}-induced tyrosine phosphorylation of MAP kinase. Cells, grown in 60-mm dishes, were pretreated with 1 µM ritodrine for 10 min (lanes 2, 4, and 6), followed by treatment with 1 µM PGF2{alpha} (lanes 3 and 4) or 1 µM oxytocin (lanes 5 and 6) for 5 min. Cells were then harvested and lysed in 100 µl 1% SDS. Cell lysates were diluted with HNTG buffer and centrifuged. Supernatant was precipitated with an anti-MAP kinase antiserum, and immunoprecipitated MAP kinase was subjected to SDS-PAGE, followed by immunoblotting with antiphosphotyrosine antiserum. Experiments were repeated three times with essentially identical results.

 
Effect of MEK inhibitor on PGF2{alpha}-induced contraction of rat pregnant uterine smooth muscle
In the examination of the role of MAP kinase pathway in PGF2{alpha}-induced 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 (21, 22, 23). MEK inhibitor (100 µM) completely attenuated the PGF2{alpha}-induced tyrosine phosphorylation of MAP kinase (Fig. 7AGo). Rat puerperal uterine smooth muscle strips were stretched to optimal length, and active force was measured after treatment with PGF2{alpha}. Treatment of uterine strips with PGF2{alpha} resulted in contraction. A solution of 1% dimethylsulfoxide had no effect on PGF2{alpha}-induced uterine contraction, whereas 100 µM MEK inhibitor significantly inhibited the PGF2{alpha}-induced uterine contraction (Fig. 7BGo).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 7. Effect of MEK inhibitor on PGF2{alpha}-induced uterine contraction. A, Cells were grown in 60-mm dishes, then pretreated with 100 µM MEK inhibitor for 15 min (lanes 2 and 4), followed by treatment with 1 µM PGF2{alpha} (lanes 3 and 4) for 5 min. Cells were then harvested and lysed in 100 µl 1% SDS. Cell lysates were diluted with HNTG buffer and centrifuged. Supernatant was precipitated with an anti-MAP kinase antiserum, and immunoprecipitated MAP kinase was subjected to SDS-PAGE, followed by immunoblotting with antiphosphotyrosine antiserum. Experiments were repeated three times with essentially identical results. B, Isolated uterine tissues were suspended at 26 C in modified Locke-Ringer solution aerated with 95% O2 and 5% CO2 and weighted with 1 g. Activity of uterine tissues was measured with a pressure transducer and a rectigram. Either 1% dimethylsulfoxide or 100 µM MEK inhibitor was added to tissues before treatment with PGF2{alpha} for 30 min, and percent changes of PGF2{alpha}-induced uterine activity were compared (n = 5).

 
It was reported that PGF2{alpha} elevated the [Ca2+]i in puerperal uterine myometrial cells (39, 40). To examine why MEK inhibitor does not inhibit PGF2{alpha}-induced uterine contraction completely, we evaluated the effect of MEK inhibitor on PGF2{alpha}-induced Ca2+ mobilization in rat puerperal uterine myometrial cells using a digital imaging fluorescence microscope (Fig. 8Go). The [Ca2+]i was elevated by PGF2{alpha} (first peak) or oxytocin (second peak) and even the pretreatment with 100 µM MEK inhibitor, as has been found previously (41).



View larger version (8K):
[in this window]
[in a new window]
  		Figure 8. Effect of MEK inhibitor on PGF2{alpha}-induced [Ca2+]i mobilization. Both 1 µM of PGF2{alpha}-induced (first changing) and oxytocin-induced (second changing) [Ca2+]i changing in rat puerperal uterine myometrial cells were measured by digital imaging fluorescence microscope after treatment with 100 µM MEK inhibitor for 30 min.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Considerable evidence now indicates that the family of MAP kinases play an important role in the control of cellular growth (42). This enzyme is activated in a variety of cell types and in response to numerous growth factors whose receptors are structurally unrelated, suggesting the existence of distinct pathways that converge at this site of regulation. Although the activation of this enzyme by tyrosine kinase receptors was established early (43, 44), it is now believed that G protein-linked receptors, such as bombesin (45), endothelin (46, 47), thrombin (48), angiotensin-II (49), thyrotropin-releasing hormone (50), GnRH (31), and oxytocin (20), as well as direct G protein activation by fluoroaluminate (51), can result in the activation of MAP kinase. The distinct pathways of Gi- and Gq-mediated MAP kinase activation were reported (29). In the case of Gi-coupled receptors like thrombin (48) and oxytocin (20), activation appears to be PTX sensitive and PKC independent. In addition, Gi-mediated MAP kinase activation is initiated by a tyrosine phosphorylation event and proceeds by a pathway common to tyrosine-kinase receptors, involving a series of SH2- and SH3-dependent protein-protein interactions among tyrosine-phosphorylated receptors, Shc, Grb2, and SOS, resulting in 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 a subsequent PKC-mediated stimulation of MAP kinase (45). We report here that PGF2{alpha}, presumably working through Gq-coupled receptors in NIH-3T3 cells (9), also stimulates MAP kinase in cultured rat puerperal uterine myometrial cells. In this study, both cell pretreatment with PTX and expression of ßARK1 blocked the PGF2{alpha}-induced MAP kinase activation, suggesting that PGF2{alpha} coupling to PTX-sensitive G protein (Gi) may be responsible for the activation of MAP kinase in cultured rat puerperal uterine myometrial cells. However, the inhibitory effect of PTX on PGF2{alpha}-induced MAP kinase activation was not complete. Therefore, there is still a possibility that PGF2{alpha} also couples to Gq protein.

Like TRH (50), PGF2{alpha} did induce acute changes in tyrosine phosphorylation. We did detect phosphorylation of two of the proteins encoded by Shc gene. Moreover, PGF2{alpha} stimulated the phosphorylation of SOS, the ras nucleotide exchange factor, reflected by an apparent shift in mobility on SDS-PAGE, as well as increased activity of the raf-1 kinase, presumably activated as a consequence of ras stimulation (8). Following this event, the direct upstream activator, MEK and MAP kinase were activated by PGF2{alpha}. The demonstration of a G protein-coupled receptor linked to tyrosine phosphorylation of Shc indicates an expanding role for this important protein.

As for the role that MAP kinase might play in eliciting physiological response to PGF2{alpha}, the function of PGF2{alpha} has been investigated in relation to cell growth in NIH-3T3 cells (3, 4) and uterine contraction (1, 2). Pretreatment of cells with ritodrine, which is well known to block the uterine contraction, attenuated both the oxytocin- (20) and the PGF2{alpha}-induced phosphorylation of MAP kinase. In addition, the time frame of PGF2{alpha}-induced uterine contractions, which occurred within 3 min and declined thereafter, appeared to be similar to the PGF2{alpha}-induced activation of MAP kinase and MEK, suggesting the role of MAP kinase pathway in PGF2{alpha}-induced uterine contraction. Although an inhibitor of MEK activity, PD098059, completely attenuated the PGF2{alpha}-induced tyrosine phosphorylation of MAP kinase, partial inhibition of this compound in PGF2{alpha}-induced uterine contraction was detected. We detected that this compound also partially blocked oxytocin-induced uterine muscle contraction (41). This compound had no effect on Ca2+ mobilization in cultured human puerperal uterine myometrial cells (41), suggesting that PGF2{alpha}-induced uterine contraction might be both dependent on and independent of Ca2+ mobilization. The potential relationships between these pathways are shown in the scheme in Fig. 9Go.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 9. Proposed scheme of signal transduction pathway in PGF2{alpha}-induced uterine contraction. Binding of PGF2{alpha} to its seven-membrane spanning receptor results in rapid intracellular signal transduction, including PTX-insensitive Gq protein-stimulated phosphatidylinositol (PtdIns) metabolism via phospholipase C-ß (PLC-ß), protein kinase C (PKC) activation, intracellular calcium (Ca2+) mobilization, calmodulin-calcium-dependent protein kinase (calmodulin-Ca2+ kinases) activation, and myosin light-chain kinase (MLCK) activation. PGF2{alpha} also activates tyrosine phosphorylation of Shc, leading to sequential activation of SOS-Ras-Raf-1-MEK-MAP kinase cascade via PTX-sensitive Gi protein. These early signaling events are hypothesized to lead to nuclear protooncogene transcriptional activation (c-fos, c-jun, c-myc) and produce cellular responses, including uterine contraction. Data presented are indicates with an *.

 
In conclusion, tyrosine phosphorylation of Shc induced by PGF2{alpha} is among the earliest detectable events in this pathway, followed by a signaling cascade that proceeds through SOS, raf-1, and MEK to MAP kinase activation mediated by Gß{gamma}. This pathway might have some role in PGF2{alpha}-induced uterine contraction


   Acknowledgments
 
We thank Dr. Alan R. Saltiel for the generous gift of the MEK inhibitor (PD098059).

Received February 21, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Keirse MJNC, Mitchell MD, Turnbull AC 1977 Changes in prostaglandin F and 13,14-dihydro-15-keto-prostaglandin F concentrations in amniotic fluid at the onset of and during labour. Br J Obstet Gynecol 84:743–746[Medline]
  2. Dray F, Frydman R 1976 Primary prostaglandins in amniotic fluid in pregnancy and spontaneous labour. Am J Obstet Gynecol 126:13–19[Medline]
  3. DeAsua LJ, Clingan D, Rudland PS 1975 Initiation of cell proliferation in cultured mouse fibroblasts by prostaglandin F2 alpha. Proc Natl Acad Sci USA 72:2724–2728[Abstract/Free Full Text]
  4. Nakao A, Watanabe T, Taniguchi S, Nakamura M, Honda Z, Shimizu T, Kurokawa K 1993 Characterization of prostaglandin F2 alpha receptor of mouse 3T3 fibroblasts and its functional expression in Xenopus laevis oocytes. J Cell Physiol 155:257–264[CrossRef][Medline]
  5. Watanabe T, Nakao A, Emerling D, Hashimoto Y, Tsukamoto K, Horie Y, Konoshita M, Kurokawa K 1994 Prostaglandin F2 alpha enhances tyrosine phosphorylation and DNA synthesis through phospholipase C-coupled receptor via Ca(2+)-dependent intracellular pathway in NIH-3T3 cells. J Biol Chem 269:17619–17625[Abstract/Free Full Text]
  6. Ray LB, Sturgill TW 1987 Rapid stimulation by insulin of a serine/threonine kinase in 3T3–L1 adipocytes that phosphorylate microtubule-associated protein 2 in vitro. Proc Natl Acad Sci USA 84:1502–1506[Abstract/Free Full Text]
  7. Nishida E, Gotoh Y 1993 The MAP kinase cascade is essential for diverse signal transduction pathways. Trends Biochem Sci 18:128–131[CrossRef][Medline]
  8. Wood KW, Sarnecki C, Roberts TM, Blenis J 1992 ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, Raf-1, and RSK. Cell 68:1041–1050[CrossRef][Medline]
  9. Watanabe T, Waga I, Honda Z, Kurokawa K, Shimizu T 1995 Prostaglandin F2 alpha stimulates formation of p21ras-GTP complex and mitogen-activated protein kinase in NIH-3T3 cells via Gq-protein-coupled pathway. J Biol Chem 270:8984–8990[Abstract/Free Full Text]
  10. Rozakis-Adcock M, McGlade J, Mbamalu G, Pelicci G, Daly R, Li W, Batzer A, Thomas S, Brugge J, Pelicci PG, Schlessinger J, Pawson T 1992 Association of the Shc and Grb2/Sem5 SH2-containing proteins is implicated in activation of the Ras pathways by tyrosine kinases. Nature 360:689–692[CrossRef][Medline]
  11. Pelicci G, Lanfrancone L, Grignani F, McGlade J, Carallo F, Forni G, Nicoletti I, Grignani F, Pawson T, Pelicci PG 1992 A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction. Cell 70:93–104[CrossRef][Medline]
  12. Clark SG, Stern MJ, Horvitz HR 1992 C.elegant cell-signalling gene sem-5 encodes a protein with SH2 and SH3 domains. Nature 356:340–344[CrossRef][Medline]
  13. Lowenstein EJ, Daly RJ, Batzer AG, Li W, Margolis B, Lammers R, Ullrich A, Skolnik EY, Bar-Sagi D, Schlessinger J 1992 The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell 70:431–442[CrossRef][Medline]
  14. Matsuoka K, Shibata M, Yamakawa A, Takenawa T 1992 Cloning of ASH, a ubiquitous protein composed of one Src homology region (SH)2 and two SH3 domains, from human and rat cDNA libraries. Proc Natl Acad Sci USA 89:9015–9019[Abstract/Free Full Text]
  15. Oliver JP, Raabe T, Henkemeyer M, Dickson B, Mbamalu G, Margolis B, Schlessinger J, Hafen E, Pawson T 1993 A Drosophila SH2-SH3 adaptor protein implicated in coupling the sevenless tyrosine kinase to an activator of Ras guanine exchange, Sos. Cell 73:179–191[CrossRef][Medline]
  16. Egan SE, Giddings BW, Brooks MW, Buday L, Sizeland AM, Weinberg RA 1993 Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation. Nature 363:45–51[CrossRef][Medline]
  17. Rozakis-Adcock M, Fernley R, Wade J, Pawson T, Bowtell D 1993 The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1. Nature 363:83–85[CrossRef][Medline]
  18. Li N, Batzer A, Daly R, Yajnik V, Skolnik E, Chardin P, Bar-Sagi D, Margolis B, Schlessinger J 1993 Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling. Nature 363:85–88[CrossRef][Medline]
  19. Gale NW, Kaplan S, Lowenstein EJ, Schlessinger J, Bar-Sagi D 1993 Grb2 mediates the EGF-dependent activation of guanine nucleotide exchange of Ras. Nature 363:88–92[CrossRef][Medline]
  20. Ohmichi M, Koike K, Nohara A, Kanda Y, Sakamoto Y, Zhang ZX, Hirota K, Miyake A 1995 Oxytocin stimulates mitogen-activated protein kinase activity in cultured human puerperal uterine myometrial cells. Endocrinology 136:2082–2087[Abstract]
  21. Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR 1995 A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci USA 92:7686–7689[Abstract/Free Full Text]
  22. Pang L, Sawada T, Decker SJ, Saltiel AR 1996 Inhibition of MAP kinase kinase blocks the differentiation of PC-12 cells induced by NGF. J Biol Chem 270:13585–13588[Abstract/Free Full Text]
  23. Lazar DF, Wiese RJ, Brady MJ, Mastick CC, Waters SB, Yamauchi K, Pessin JE, Cuartrecasas P, Saltiel AR 1996 Mitogen-activated protein kinase kinase inhibitor does not block the stimulation of glucose utilization by insulin. J Biol Chem 270:20801–20807[Abstract/Free Full Text]
  24. Ohmichi M, Pang L, Decker SJ, Saltiel AR 1992 Nerve growth factor stimulates the activities of the raf-1 and the mitogen-activated protein kinases via the trk protooncogene. J Biol Chem 267:14604–14610[Abstract/Free Full Text]
  25. Zheng C-F, Ohmichi M, Saltiel AR, Guan K-L 1994 Growth factor induced MEK activation is primarily mediated by an activator different from c-raf. Biochemistry 33:5595–5599[CrossRef][Medline]
  26. Palmberg L, Thyberg J 1986 Uterine smooth muscle cells in primary culture. Alterations in fine structure, cytoskeletal organization and growth characteristics. Cell Tissue Res 246:253–262[CrossRef][Medline]
  27. Howe LR, Leevers SJ, Gomez N, Nakielny S, Cohen P, Marshall CJ 1992 Activation of the MAP kinase pathway by the protein kinase raf. Cell 71:335–342[CrossRef][Medline]
  28. Koch WJ, Hawes BE, Allen LF, Lefkowitz RJ 1994 Direct evidence that Gi-coupled receptor stimulation of mitogen-activated protein kinase is mediated by Gß{gamma} activation of p21ras. Proc Natl Acad Sci USA 91:12706–12710[Abstract/Free Full Text]
  29. Hawes BE, van Biesen T, Koch WJ, Luttrell LM, Lefkowitz RJ 1995 Distinct pathway of Gi- and Gq-mediated mitogen-activated protein kinase activation. J Biol Chem 270:17148–17153[Abstract/Free Full Text]
  30. Ohmichi M, Matuoka K, Takenawa T, Saltiel AR 1994 Growth factors differentially stimulate the phosphorylation of Shc proteins and their association with Grb2 in PC-12 pheochromocytoma cells. J Biol Chem 269:1143–1148[Abstract/Free Full Text]
  31. Sundaresan S, Colin IM, Pestell RG, Jameson JL 1996 Stimulation of mitogen-activated protein kinase by gonadotropin-releasing hormone: evidence for the involvement of protein kinase C. Endocrinology 137:304–311[Abstract]
  32. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685[CrossRef][Medline]
  33. Masumoto N, Tasaka K, Kasahara K, Miyake A, Tanizawa O 1991 Purification of gonadotropes and intracellular free calcium. J Biol Chem 26:6485–6488
  34. Tasaka K, Masumoto N, Miyake A, Tanizawa O 1991 Direct measurement of intracellular free calcium in cultured human puerperal myometrial cells stimulated by oxytocin: effects of extracellular calcium and calcium channel blockers. Obstet Gynecol 77:101–106[Abstract/Free Full Text]
  35. Kosako H, Gotoh Y, Matsuda S, Ishikawa W, Nishida E 1992 Xenopus MAP kinase activator is a serine/threonine/tyrosine kinase activated by threonine phosphorylation. EMBO J 11:2903–2908[Medline]
  36. van Biesen T, Hawes BE, Luttrell DK, Krueger KM, Touhara K, Porfiri E, Sakaue M, Luttrell LM, Lefkowitz RJ 1995 Receptor-tyrosine-kinase- and Gß{gamma}-mediated MAP kinase activation by a common signalling pathway. Nature 376:781–784[CrossRef][Medline]
  37. Sugimoto Y, Hasumoto K, Namba T, Irie A, Katsuyama M, Negishi M, Kakizuka A, Narumiya S 1994 Cloning and expression of a cDNA for mouse prostaglandin F receptor. J Biol Chem 269:1356–1360[Abstract/Free Full Text]
  38. Beall MH, Edgar BW, Paul RH, Smith-Wallaie T 1985 A comparison of ritodrine, terbutaline, and magnesium sulfate for the suppression of preterm labour. Am J Obstet Gynecol 153:854–859[Medline]
  39. Rubanyi G, Csapo I 1977 The effect of prostaglandin F2alpha on the activator calcium of the uterus. Life Sci 20:2061–2074[CrossRef][Medline]
  40. Huszar G, Roberts JM 1982 Biochemistry and pharmacology of the myometrium and labor: regulation at the cellular and molecular levels. Am J Obstet Gynecol 142:225–237[Medline]
  41. Nohara A, Ohmichi M, Koike K, Masumoto N, Kobayashi M, Akahane M, Ikegami H, Hirota K, Miyake A, Murata Y 1996 The role of mitogen-activated protein kinase in oxytocin-induced contraction of uterine smooth muscle in pregnant rat. Biochem Biophys Res Commun 229:938–944[CrossRef][Medline]
  42. Thomas G 1992 MAP kinase by any other name smells just as sweet. Cell 68:3–6[CrossRef][Medline]
  43. Ahn NG, Weiel JE, Chan CD, Krebs EG 1990 Identification of multiple epidermal growth factor-stimulated protein serine/threonine kinases from Swiss 3T3 cells. J Biol Chem 265:11487–11494[Abstract/Free Full Text]
  44. Tsao H, Aletta JM, Greene LA 1990 Nerve growth factor and fibroblast growth factor selectively activate a protein kinase that phosphorylates high molecular weight microtubule-associated proteins. Detection, partial purification and characterization in PC12 cells. J Biol Chem 265:15471–15480[Abstract/Free Full Text]
  45. Pang L, Decker SJ, Saltiel AR 1993 Bombesin and epidermal growth factor stimulate the mitogen-activated protein kinase through different pathways in Swiss 3T3 cells. Biochem J 289:283–287
  46. Cazaubon S, Parker PJ, Strosberg AD, Couraud P-O 1993 Endothelins stimulate tyrosine phosphorylation and activity of p42/mitogen-activated protein kinase in astrocytes. Biochem J 293:381–386
  47. Wang Y, Simonson MS, Pouyssegur J, Dunn MJ 1992 Endothelin rapidly stimulates mitogen-activated protein kinase activity in rat mesangial cells. Biochem J 287:589–594
  48. Vouret-Craviari V, Van-Obberghen-Schilling E, Scimeca JC, Van-Obberghen E, Pouyssegur P 1993 Differential activation of p44 mapk (ERK1) by alpha-thrombin and thrombin-receptor peptide agonist. Biochem J 289:209–214
  49. Duff JL, Berk BC, Corson MA 1992 Angiotensin II stimulates the pp44 and pp42 mitogen-activated protein kinases in cultured rat aortic smooth muscle cells. Biochem Biophys Res Commun 187:257–264
  50. Ohmichi M, Sawada T, Kanda Y, Koike K, Hirota K, Miyake A, Saltiel AR 1994 Thyrotropin-releasing hormone stimulates MAP kinase activity in GH3 cells by divergent pathways. J Biol Chem 269:3783–3788[Abstract/Free Full Text]
  51. Anderson NG, Kilgour E, Sturgill TW 1991 Activation of mitogen-activated protein-tyrosine kinase in BC3H1 myocytes by fluoroaluminate. J Biol Chem 266:10131–10135[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
I. Garcia-Verdugo, D. Leiber, P. Robin, E. Billon-Denis, R. Chaby, and Z. Tanfin
Direct Interaction of Surfactant Protein A with Myometrial Binding Sites: Signaling and Modulation by Bacterial Lipopolysaccharide
Biol Reprod, April 1, 2007; 76(4): 681 - 691.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
M. Mewe, C. K. Bauer, J. R. Schwarz, and R. Middendorff
Mechanisms Regulating Spontaneous Contractions in the Bovine Epididymal Duct
Biol Reprod, October 1, 2006; 75(4): 651 - 659.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
A. Alfranca, M. A. Iniguez, M. Fresno, and J. M. Redondo
Prostanoid signal transduction and gene expression in the endothelium: Role in cardiovascular diseases
Cardiovasc Res, June 1, 2006; 70(3): 446 - 456.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
M. Breuiller-Fouche and G. Germain
Gene and protein expression in the myometrium in pregnancy and labor.
Reproduction, May 1, 2006; 131(5): 837 - 850.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
E. W. Hillhouse and D. K. Grammatopoulos
The Molecular Mechanisms Underlying the Regulation of the Biological Activity of Corticotropin-Releasing Hormone Receptors: Implications for Physiology and Pathophysiology
Endocr. Rev., May 1, 2006; 27(3): 260 - 286.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol SciHome page
K. Brant and R. Loch Caruso
Late-Gestation Rat Myometrial Cells Express Multiple Isoforms of Phospholipase A2 That Mediate PCB 50-Induced Release of Arachidonic Acid with Coincident Prostaglandin Production
Toxicol. Sci., November 1, 2005; 88(1): 222 - 230.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
P. Florio, M. Torricelli, L. Galleri, G. De Falco, E. Leucci, G. Calonaci, E. Picciolini, G. Ambrosini, E. A. Linton, and F. Petraglia
High Fetal Urocortin Levels at Term and Preterm Labor
J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5361 - 5365.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
S. Husain, F. Jafri, and C. E. Crosson
Acute Effects of PGF2{alpha} on MMP-2 Secretion from Human Ciliary Muscle Cells: A PKC- and ERK-Dependent Process
Invest. Ophthalmol. Vis. Sci., May 1, 2005; 46(5): 1706 - 1713.
[Abstract] [Full Text] [PDF]

Home page
 Reproductive SciencesHome page
H. A. Otun, M. W.J. MacDougall, J. Bailey, G. N. Europe-Finner, and S. C. Robson
Spatial and Temporal Expression of the Myometrial Mitogen-Activated Protein Kinases p38 and ERK1/2 in the Human Uterus During Pregnancy and Labor
Reproductive Sciences, April 1, 2005; 12(3): 185 - 190.
[Abstract] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
Y. Li, H.-D. Je, S. Malek, and K. G. Morgan
Role of ERK1/2 in uterine contractility and preterm labor in rats
Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2004; 287(2): R328 - R335.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
N. Papadopoulou, J. Chen, H. S. Randeva, M. A. Levine, E. W. Hillhouse, and D. K. Grammatopoulos
Protein Kinase A-Induced Negative Regulation of the Corticotropin-Releasing Hormone R1{alpha} Receptor-Extracellularly Regulated Kinase Signal Transduction Pathway: The Critical Role of Ser301 for Signaling Switch and Selectivity
Mol. Endocrinol., March 1, 2004; 18(3): 624 - 639.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
S. Mabuchi, M. Ohmichi, A. Kimura, Y. Ikebuchi, K. Hisamoto, E. Arimoto-Ishida, Y. Nishio, K. Takahashi, K. Tasaka, and Y. Murata
Tamoxifen Inhibits Cell Proliferation via Mitogen-Activated Protein Kinase Cascades in Human Ovarian Cancer Cell Lines in a Manner Not Dependent on the Expression of Estrogen Receptor or the Sensitivity to Cisplatin
Endocrinology, March 1, 2004; 145(3): 1302 - 1313.
[Abstract] [Full Text]