help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 Korita, D.
Right arrow Articles by Fujii, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Korita, D.
Right arrow Articles by Fujii, S.
The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 11 5209-5219
Copyright © 2002 by The Endocrine Society

Original Article

Cyclic Mechanical Stretch Augments Prostacyclin Production in Cultured Human Uterine Myometrial Cells from Pregnant Women: Possible Involvement of Up-Regulation of Prostacyclin Synthase Expression

Daizo Korita, Norimasa Sagawa, Hiroaki Itoh, Shigeo Yura, Masahiro Yoshida, Kazuyo Kakui, Maki Takemura, Chieko Yokoyama, Tadashi Tanabe and Shingo Fujii

Department of Gynecology and Obstetrics (D.K., N.S., H.I., S.Y., M.Y., K.K., M.T., S.F.), Kyoto University Graduate School of Medicine, 606-8507 Kyoto, Japan; and Department of Pharmacology (C.Y., T.T.), National Cardiovascular Center Research Institute, 565-8565 Osaka, Japan

Address all correspondence and requests for reprints to: Norimasa Sagawa, M.D., Ph.D., Department of Gynecology and Obstetrics, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, 606-8507 Kyoto, Japan. E-mail: fetus{at}kuhp.kyoto-u.ac.jp.

Abstract

Prostacyclin (PGI2), a potent smooth muscle relaxant, is a major prostaglandin secreted from human myometrium. The concentrations of PGI2 metabolites in the maternal plasma were reported to be elevated during pregnancy, especially in labor. To clarify the mechanism in PGI2 secretion from the myometrium, we first investigated the protein expression of cytosolic phospholipase A2, cyclooxygenase (COX)-1, COX-2, and prostacyclin synthase (PGIS) in the human uterine myometrium at various gestational ages before labor. To elucidate the involvement of labor in the increase in PGI2 production during labor, we next examined the effect of labor-like cyclic mechanical stretch on PGI2 production by cultured human myometrial cells.

Pregnancy specifically increased COX-1 and PGIS protein expression in the myometrial tissues before labor (P < 0.01 for both). Cyclic mechanical stretch augmented PGIS promoter activity, via activation of activator protein-1 site, and PGIS mRNA and protein expression in cultured human myometrial cells and resulted in a 3.5-fold increase in the concentration of 6-keto-prostaglandin F1{alpha}, the stable metabolite of PGI2, in the culture medium (P < 0.05). However, stretch did not affect the levels of prostaglandin E2, prostaglandin F2{alpha}, or thromboxane A2 secreted into the same culture media. These results suggest that cyclic mechanical stretch during labor may contribute to the increase in the PGI2 concentration in the maternal plasma during parturition.

DURING PREGNANCY, THE uterine myometrium relaxes and expands to accommodate the growing fetus. However, when labor commences, the uterine myometrium contracts vigorously and cyclically to give birth to the fetus. These myometrial functions are characteristic of pregnancy and have been hypothesized to be related partly to profound changes in the local synthesis and metabolism of prostaglandins (PGs; Refs. 1, 2, 3, 4).

Prostaglandin E2 (PGE2), prostaglandin F2a (PGF2{alpha}; Refs. 1, 2, 3, 4) and thromboxane A2 (TXA2; Ref. 5) cause contraction of the human myometrium, whereas prostacyclin (PGI2) causes its relaxation (5, 6, 7). The plasma and/or amniotic fluid concentrations of PGE2 and PGF2{alpha} (1, 2, 3, 4) increase in labor. We previously reported that FP receptor, a specific receptor for PGF2{alpha}, and EP3 receptor, one of the isoforms of specific receptors for PGE2, were expressed in human pregnant myometrium and that the expression of these receptors was down-regulated in mid-gestation (8).

On the other hand, PGI2, known as a potent vasodilator (9), was reported to be a major PG secreted from human nonpregnant (10, 11), as well as pregnant (12, 13), myometrium, although the physiological roles of myometrial PGI2 during pregnancy are still controversial. PGI2 concentrations in the maternal plasma (14) and urine (15) and myometrial PGI2 production (16) were demonstrated to be elevated with progression of pregnancy before the onset of labor. After the onset of labor, the plasma PGI2 concentration was reported to increase further, accompanied by increases in PGE2 and PGF2{alpha} concentrations (17, 18).

Biosynthesis of PGI2 is catalyzed by sequential reactions of several enzymes. The primary precursor of series-2 PGs is arachidonic acid, which is liberated from membrane phospholipids by various phospholipases and diacylglycerol and/or monoacylglycerol lipases (19). Cytosolic phospholipase A2 (cPLA2) liberates arachidonic acid from the sn-2 position of phospholipids, and arachidonic acid is converted to prostaglandin G2 and subsequently to prostaglandin H2 by two isoforms of cyclooxygenase (COX), COX-1 and COX-2 (20). The resultant prostaglandin H2 is converted immediately into PGI2 by prostacyclin synthase (PGIS), whereas prostaglandin H2 is also converted into other PGs, such as PGE2, PGF2{alpha}, TXA2, etc., through catalytic reactions by the respective synthases (20). We previously reported the molecular cloning (16, 21) and promoter regions (22) of the PGIS gene. PGIS is reported to be expressed widely in the body, especially in vascular endothelial, vascular smooth muscle, and uterine myometrial cells (9, 13).

The comprehensive changes in cPLA2 expression in the human myometrium throughout gestation have not yet been fully assessed, although immunohistochemical expression of cPLA2 in the human pregnant uterus has been demonstrated (23). The pregnancy-associated changes in COX-1 and COX-2 expression in the human myometrium are still controversial, although COX-1 and COX-2 are reported to be distributed widely in the human pregnant myometrium (13). Several investigators reported that the expression of COX-2 (24, 25) or COX (13) in the myometrium was increased during pregnancy and/or in labor, whereas others demonstrated that labor did not affect COX-1 or COX-2 expression in either the upper or lower segments of the uterine myometrium (26). Thus, the changes in the entire enzymatic cascade of PGI2 synthesis in the human myometrium during pregnancy and in labor have not been fully clarified.

There have been several reports concerning the regulatory mechanism of PGI2 production in the myometrium. Both IL-1 (11) and oxytocin (27) treatment were reported to increase PGI2 production in cultured human myometrial cells. Wu et al. found in an in vivo study that estrogen enhanced COX-2 expression in the nonpregnant ovine myometrium (28) and that spontaneous labor increased PGIS expression in the pregnant ovine myometrium (29). However, the mechanism of regulation of PGI2 production in the human pregnant myometrium either before or after the onset of labor is not yet well understood.

In the present study, to elucidate the mechanism involved in the increase in PGI2 production in the human uterine myometrium during pregnancy and in labor, we first performed immunohistochemical and Western blot analyses of cPLA2, COX-1, COX-2, and PGIS protein expression in the upper segment of human myometrium obtained from nonpregnant women and pregnant women in the first, second, and third trimesters before labor onset.

Secondly, we developed a culture model of human myometrium in labor by applying stimulation of labor-like cyclic mechanical stretch to cultured human myometrial cells to examine the effect of labor on PGI2 synthesis in the myometrium. We measured the concentration of 6-keto-PGF1{alpha}, a stable metabolite of PGI2, in the medium of cultured human myometrial cells after stimulation by cyclic mechanical stretch. We further examined the effect of cyclic mechanical stretch on the expression of cPLA2, COX-1, COX-2, and PGIS and on the PGIS promoter activity in the presence or absence of several inhibitors of activator protein-1 (AP-1) or tyrosine kinases.

Materials and Methods

Reagents

All reagents were purchased from Sigma (St. Louis, MO), unless otherwise indicated, and were of analytical grade.

Collection of myometrial tissues

The upper part of the uterine myometrium was obtained at hysterectomy from premenopausal nonpregnant women (n = 5) and pregnant women in the first trimester (8–12 wk of gestation, n = 5), second trimester (16–21 wk of gestation, n = 3), and third trimester (37–38 wk of gestation, n = 5) with written informed consent. All of the patients in the first and second trimesters had received total hysterectomy for gynecological reasons such as uterine cervical cancer, ovarian cancer, or uterine myoma. Patients in the third trimester, before the onset of labor, underwent total hysterectomy after elective cesarean section. The tissues were snap-frozen in liquid nitrogen in blocks for protein and mRNA extraction and/or in optimal cutting temperature compound (Sakura Finetek Inc., Torrance, CA) for immunostaining and kept at -80 C. Uterine artery was isolated from the middle portion of uterine corpus for immunohistochemistry. Simultaneously, some tissues were immediately used for preparation of cultured human myometrial cells. This study was approved by the ethical committee on human research of Kyoto University Graduate School of Medicine (permission no. 90).

Preparation of cultured human uterine myometrial cells

Myometrial tissues obtained from nonpregnant women (n = 3) and pregnant women in the first trimester (7 and 8 wk of gestation; n = 2), second trimester (16, 20, and 21 wk of gestation; n = 3), and third trimester (37, 37, and 38 wk of gestation; n = 3), as mentioned above were used to establish cultured human uterine myometrial cells. Collected tissues were immediately placed in DMEM (Life Technologies, Grand Island, NY) with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 µg/ml). The tissues were rinsed several times with the medium and minced into approximately 0.5-mm pieces. Then, they were placed into tubes containing DMEM and 0.2% collagenase (Wako Pure Chemical Industries Ltd., Osaka, Japan) and incubated for 4 h at 37 C with continuous mixing. The medium was filtered through a 40-µm cell strainer, diluted in an equal volume of DMEM, and then centrifuged at 1000 rpm for 10 min. The supernatant was discarded, and the cell pellet was resuspended in DMEM with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 µg/ml). Ten milliliters of DMEM containing 3 x 104 cells/ml were placed in a 10-cm collagen-coated culture plate. Cells were maintained in a humidified 5% CO2/95% air atmosphere at 37 C. When the cells in each plate reached subconfluency, they were frozen and stored in liquid nitrogen as first passage cells until they were used. Third passage cells were used in the experiments as cultured human myometrial cells. Immunofluorescence studies of the cultured human myometrial cells at the third passage showed 99% and 70% positive staining for vimentin and {alpha}-smooth muscle actin, respectively, with less than 1% positive staining for cytokeratin (data not shown).

Immunohistochemistry of cPLA2, COX-1, COX-2, and PGIS

After a 1-h incubation with primary antibodies at room temperature, staining was detected using an avidin-biotin-peroxidase method kit (ELITE ABC, Vector Laboratories Inc., Burlingame, CA) with 3,3'-diaminobenzidine. The monoclonal antibodies used were an antibody raised against human cPLA2 (1:100 dilution, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and an antibody raised against sheep COX-1 and having cross-reactivity for human COX-1 (1:100 dilution, Cayman Chemical Co., Ann Arbor, MI). The polyclonal antibodies used were an antibody raised against human COX-2 (1:200 dilution, Santa Cruz Biotechnology, Inc.) and an antibody raised against bovine PGIS and having cross reactivity for human PGIS (1:100 dilution, Cayman Chemical Co.).

Western blot analysis of cPLA2, COX-1, COX-2, and PGIS

Protein extraction, SDS-PAGE, and immunoblotting were performed as previously described (30). Human term placenta was used as a positive control for cPLA2, COX-2, and PGIS. The ovine seminal vesicle (Cayman Chemical Co.) was used as a positive control for COX-1. For the immunoblots, the same antibodies were used as for immunohistochemistry. The dilutions of the antibodies used for Western blot analysis were 1:1000 for cPLA2, COX-1, and PGIS and 1:500 for COX-2.

RT-PCR analysis of cPLA2, COX-1, COX-2, PGIS, and glyceraldehyde-3-phosphate dehydrogenase (G3PDH)

Total RNA was extracted from cultured human uterine myometrial cells as previously described (31). After RT of 5 µg total RNA using oligo (dT) primer (Promega Corp., Madison, WI) and SUPERSCRIPT II RT (Life Technologies, Inc., Rockville, MD), the resulting single-stranded cDNA was subjected to PCR. The forward and reverse primers used were: cPLA2 (32), forward, 5'-GAGCTGATGTTTGCAGATTGGGTTG-3', and reverse, 5'-GTCACTCAAAGGAGACAGTGGATAAGA-3'; COX-1 (33), forward, 5'-GCTGGGAGTCTTTCTCCAACGTGAG-3', and reverse, 5'-GGCAATGCGGTTGCGGTATTGGAAC-3'; COX-2 (34), forward, 5'-TTCAAATGAGATTGTGGGAAAATTGCT-3', and reverse, 5'-AGATCATCTCTGCCTGAGTATCTTT-3'; PGIS (16), forward, 5'-AGGAG AAGCACGGTGACATC-3', and reverse, 5'-GCAGCGCCTCAATTCCGTAA-3'. Forward and reverse primers for the human G3PDH coding region were purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA). The programs used for amplification of cPLA2, COX-1, and COX-2 cDNA were 28 cycles of 94 C for 30 sec, 58.8 C for 60 sec, and 72 C for 60 sec; whereas that used for PGIS was 26 cycles of 94 C for 30 sec, 58.8 C for 60 sec, and 72 C for 60 sec. Each program was designed considering the linear correlation between PCR cycle number and logarithm of the density of PCR products (see Fig. 5AGo). The final products were extended to full length by incubation at 72 C for 7 min.



View larger version (32K):
[in this window]
[in a new window]
  		Figure 5. RT-PCR analysis of cPLA2, COX-1, COX-2, and PGIS mRNA expression in the cultured human myometrial cells from second trimester pregnant women with or without the 4- to 24-h stimulation by cyclic mechanical stretch (B). RT-PCR protocols described in Materials and Methods were determined considering the linear correlation range between logarithmic density of RT-PCR products and cycle numbers applied (A). The 510-, 722-, 305-, 387-, and 452-bp bands represent RT-PCR products of cPLA2, COX-1, COX-2, PGIS, and G3PDH mRNA, respectively. A shaded square indicates an apparent up-regulation of PGIS mRNA observed after 8-h stimulation of cyclic mechanical stretch. Data were representative of three independent experiments, using two different cultured human myometrial cells obtained from second trimester pregnant women.

 
Protocol for stretching experiment

Cultured human myometrial cells of the second passage were dispersed and seeded in 6-well silicone elastic-bottomed culture plates with collagen type I coating (Bioflex, Flexcell International Co., McKeesport, PA) and used in the experiments as the cells of the third passage. When the cells reached confluence, the medium was replaced with fresh medium without serum. The stimulation by cyclic mechanical stretch (repetition of 15-sec release and 45-sec stretch; -12 kPa, 14% elongation) was applied to the cells for 24 h using a computer-operated, vacuum-driven stretch device (Flexcell Strain Unit FX-3000; Flexcell International Co.) as previously described (35, 36). The reagents used were curcumin (3–20 µM), a specific inhibitor of AP-1 site (37, 38); herbimycin A (2–50 nM), a cSrc family tyrosine kinase inhibitor (39); and genistein (10–1000 nM), a general tyrosine kinase inhibitor (40). In the studies of the regulatory mechanism of stretch-induced augmentation of PGI2 (see Figs. 5–9GoGoGoGoGo), the cultured human myometrial cells of second trimester pregnant women were used. This is because the availability of the cells from the third trimester pregnant women was limited and the responses of the cells of second trimester pregnant women to the stimulation of cyclic mechanical stretch were qualitatively the same as those of the third trimester cells (see Fig. 4Go).



View larger version (28K):
[in this window]
[in a new window]
  		Figure 6. Western blot analysis (20 µg/lane) of cPLA2 (A), COX-1 (B), and PGIS (C) protein expression in cultured human myometrial cells obtained from second trimester pregnant women with (black columns) or without (white columns) stimulation by cyclic mechanical stretch for 24 h. The 110-kDa, 70-kDa, and 57-kDa bands represent cPLA2, COX-1, and PGIS protein expression, respectively. On the other hand, the COX-2 protein expression in the cultured human myometrial cells was below the limit of Western blot detection (data not shown). The expression of each protein was expressed as mean and SEM of AUs based on quantitative densitometric analysis of proteins from quadruplicate wells. Data were representative of three independent experiments using two different cultured human myometrial cells of second trimester pregnant women. The statistical significance of differences between the stretch and no-stretch groups was assessed by the Mann-Whitney U test. NS, Not significant.

 


View larger version (25K):
[in this window]
[in a new window]
  		Figure 7. The effects of cyclic mechanical stretch on PGIS promoter activities in the cultured human myometrial cells obtained from the second trimester pregnant women. Values are expressed as the means and SEM of PGIS promoter activities based on the analysis of triplicate wells with (black columns) or without (white columns) stimulation by cyclic mechanical stretch, as AUA, as described in Materials and Methods. Data are representative of three independent experiments, using two different cultured human myometrial cell preparations obtained from second trimester pregnant women. The statistical significance was assessed by the Mann-Whitney U test.

 


View larger version (23K):
[in this window]
[in a new window]
  		Figure 8. The effect of curcumin, an AP-1 inhibitor, on PGIS protein expression (A) and effects of curcumin (B), herbimycin A (C), a cSrc family tyrosine kinase inhibitor, and genistein (D), a general tyrosine kinase inhibitor, on 6-keto-PGF1{alpha} secretion in cultured human myometrial cells with or without stimulation by cyclic mechanical stretch. Values are expressed as means and SEM of 6-keto-PGF1{alpha} concentrations in the culture media from triplicate wells with (black columns) or without (white columns) stimulation by cyclic mechanical stretch. Data are representative of three independent experiments using two different preparations of cultured human myometrial cells obtained from second trimester pregnant women. The statistical significance was assessed by ANOVA, followed by Fisher’s protected least significant difference test.

 


View larger version (20K):
[in this window]
[in a new window]
  		Figure 9. The effect of 20 µM curcumin, an AP-1 inhibitor, on the PGIS promoter activity of the cultured human myometrial cells obtained from second trimester pregnant women. Values are expressed as the means and SEM of PGIS promoter activities based on the analysis of triplicate wells with (black columns) or without (white columns) stimulation by cyclic mechanical stretch for 4 h, as AUA, as described in Materials and Methods. Data are representative of three independent experiments using two different preparations of cultured human myometrial cells obtained from second trimester pregnant women. The statistical significance was assessed by ANOVA, followed by Fisher’s protected least significant difference test.

 


View larger version (26K):
[in this window]
[in a new window]
  		Figure 4. The effect of cyclic mechanical stretch on the secretion of 6-keto-PGF1{alpha} (A), PGE2 (B), PGF2{alpha} (C), and TXB2 (D) from cultured human myometrial cells. The cells were prepared from uterine myometrium from nonpregnant (NP) as well as pregnant women in the second trimester (2nd) and third trimester (3rd), as described in Materials and Methods. Values were indicated as the means and SEM of PG concentrations in the culture media from triplicate wells after 24 h incubation with (black columns) or without (white columns) stimulation with cyclic mechanical stretch. Data were representative data from 10 independent experiments, using a total of 9 cultured human myometrial cells obtained from the 3 nonpregnant, 3 second trimester, and 3 third trimester pregnant women. The statistical significance was assessed by Mann-Whitney U test. NS, Not significant.

 
The cycle of stretch has been determined to mimic stimulation of active labor (35, 36). The force of vacuum extraction was determined to cause maximal augmentation of PGI2 secretion (data not shown), because it was technically difficult in this in vitro culture model to estimate correlation between the force of vacuum extraction and that of active labor. After 4, 6, 8, 12, and/or 24 h of incubation, the media and cells were collected and were kept at -20 C and at -80 C, respectively. The 6-keto-PGF1{alpha}, PGE2, PGF2{alpha}, and TXB2 levels in the culture media were measured by ELISA (Cayman Chemical Co.). Inter- and intra-assay variations of these ELISA were all less than 10%. The mRNA and/or protein expression of cPLA2, COX-1, COX-2, and PGIS in the cells was assessed by RT-PCR and/or Western blot analysis.

No significant changes were observed in either the cell number or total protein content of the cells after stimulation by cyclic mechanical stretch for 24 h (data not shown).

Human umbilical artery smooth muscle cells, prepared by the explant method, and human coronary artery smooth muscle cells (Takara Shuzo Co. Ltd., Kyoto, Japan) were subjected to the same experiments for comparison.

Reporter gene analysis of PGIS

PGIS promoter (-3034/-10)/luciferase vector was prepared as previously reported (22). One microgram per milliliter PGIS promoter/luciferase vector and 10 ng/ml pRL-SV40/Renilla (sea pansy) luciferase vector (Toyo Ink Mfg. Co. Ltd., Tokyo, Japan) were transfected into confluent cultured human myometrial cells of the third passage for 3 h using Lipofectamine Plus Reagent (Invitrogen Co., Carlsbad, CA) according to the manufacturer’s manual. After preincubation for 12 h with fresh DMEM, stimulation by cyclic mechanical stretch was applied to the transfected cells. The cells were harvested using lysis buffer (100 µl/well; Toyo Ink Mfg. Co. Ltd.) after 3, 4, 6, 8, 12, and 24 h incubation with or without stimulation by cyclic mechanical stretch. PGIS promoter activities were estimated by measuring PGIS promoter luciferase activities/pRL-SV40 Renilla luciferase activities using a picagene dual sea pansy assay system kit (Toyo Ink Mfg. Co. Ltd.). The relative PGIS promoter activity was expressed as arbitrary units of activity (AUA).

Statistical analysis

Values were expressed as the means ± SEM. Statistical significance was assessed by the Mann-Whitney U test for comparison of two groups or ANOVA, followed by Fisher’s protected least significant difference test for comparison of more than three groups. P values less than 0.05 were regarded as significant.

Results

Protein expression of cPLA2, COX-1, COX-2, and PGIS in the myometrium from nonpregnant and pregnant women

Positive staining for cPLA2, COX-1, COX-2, and PGIS was detected in myometrial stromal cells (Fig. 1Go, A, C, E, and G) as well as vascular smooth muscle and endothelial cells (Fig. 1Go, B, D, F, and H) in the myometrium from third trimester pregnant women. The staining for COX-2 and PGIS in the endothelial cells was rather weak. Similar broad distributions of cPLA2, COX-1, COX-2, and PGIS in the stromal and vascular tissues were also observed in the myometrium from nonpregnant, first trimester, and second trimester pregnant women (data not shown). Negative controls using normal mouse IgG (Fig. 1Go, I and J) and normal rabbit serum (data not shown) showed greatly reduced staining.



View larger version (57K):
[in this window]
[in a new window]
  		Figure 1. Immunohistochemical detection of cPLA2 (A and B), COX-1 (C and D), COX-2 (E and F), and PGIS (G and H) in uterine myometrium obtained from a third trimester pregnant woman. Panels A, C, E, G, and I show myometrial stroma (original magnification was x200), whereas panels B, D, F, H, and J show uterine arterial wall (original magnification was x400). Positive staining is brown. Negative controls using mouse IgG (I and J) showed greatly reduced staining.

 
Western blot analysis of cPLA2, COX-1, COX-2, and PGIS detected 110-kDa, 70-kDa, 72-kDa, and 57-kDa bands in the nonpregnant as well as pregnant myometrium in the first and third trimesters (Fig. 2Go), and these molecular sizes were identical to those in corresponding positive control tissues (placenta for cPLA2, COX-2, and PGIS; ovine seminal vesicle for COX-1). The cPLA2 expression in the myometrium from third trimester pregnant women was 168 ± 74 arbitrary units (AU; mean ± SEM; n = 5), which was slightly higher than that in the myometrium from first trimester and nonpregnant women [100 ± 21 AU (n = 5), and 76 ± 17 AU (n = 5), respectively], but these differences were not significant (Fig. 2AGo). The COX-1 expression in the myometrium of third trimester pregnant women was 127 ± 27 AU (n = 5), which was significantly higher than that of nonpregnant women (30 ± 5 AU; n = 5; P < 0.05; Fig. 2BGo), but not that of first trimester pregnant women (100 ± 33 AU; n = 5). The COX-2 expression in the myometrium of third trimester pregnant women was 114 ± 32 AU (n = 5), which was slightly higher than that of first trimester and nonpregnant women (100 ± 26 AU, n = 5; and 82 ± 33 AU, n = 5, respectively), but the differences were not significant (Fig. 2CGo). The PGIS expression in the myometrium of third trimester pregnant women was 180 ± 21 AU (n = 5), which was significantly higher than that of first trimester and nonpregnant women (100 ± 23 AU, n = 5; P < 0.05; and 26 ± 6 AU, n = 5; P < 0.01, respectively; Fig. 2DGo).



View larger version (38K):
[in this window]
[in a new window]
  		Figure 2. Western blot analysis (20 µg/lane) of cPLA2 (A), COX-1 (B), COX-2 (C), and PGIS (D) protein expression in the myometrium from nonpregnant (white columns; n = 5) as well as pregnant women in the first trimester (shaded columns; n = 5) and third trimester (black columns; n = 5). The 110-kDa, 70-kDa, 72-kDa, and 57-kDa bands in each figure correspond to cPLA2, COX-1, COX-2, and PGIS protein expression, respectively, and are representative bands among five samples of each group. The expression of each protein was expressed as the mean and SEM in AUs based on quantitative densitometric analysis of three different blots using four identical internal controls of the first trimester myometrial tissues. Statistical significance was assessed by ANOVA, followed by Fisher’s protected least significant difference test.

 
The effect of cyclic mechanical stretch for 24 h on the concentrations of 6-keto-PGF1{alpha}, PGE2, PGF2{alpha}, and TXB2 in the media of cultured human myometrial cells

After 24-h incubation in the absence of stretch, the concentrations of 6-keto-PGF1{alpha}, PGE2, and PGF2{alpha} in the culture media of cultured human myometrial cells established from third trimester pregnant women were significantly higher than those in the culture media of myometrial cells obtained from nonpregnant, first trimester, and second trimester women (P < 0.01 for all comparisons; Fig. 3Go, A, B, and C). The concentration of TXB2, a stable metabolite of TXA2, in the culture media of myometrial cells from third trimester pregnant women was similar to that for nonpregnant women, but was significantly higher than the levels of first and second trimester pregnant women (P < 0.01 for each; Fig. 3DGo). The TXB2 concentrations in the culture media of the myometrial cells from first and second trimester pregnant women were significantly lower than that in the culture media of cells obtained from nonpregnant women.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 3. Basal secretion of 6-keto-PGF1{alpha} (A), PGE2 (B), PGF2{alpha} (C), and TXB2 (D) after incubation of cultured human myometrial cells for 24 h. The cells were prepared from the uterine myometrium of nonpregnant women (NP; white columns) as well as pregnant women at the first trimester (1st; shaded columns), second trimester (2nd; hatched columns), and third trimester (3rd; black columns), as described in Materials and Methods. Values are expressed as means and SEM of the eicosanoid concentrations in the culture media from triplicate wells, and are representative data from 10 independent experiments, in which a total of 11 cultured human myometrial cell preparations from 3 nonpregnant, 2 first trimester, 3 second trimester, and 3 third trimester pregnant women were used. The statistical significance was assessed by ANOVA, followed by Fisher’s protected least significant difference test.

 
We next examined the effects of cyclic mechanical stretch on the concentrations of 6-keto-PGF1{alpha}, PGE2, PGF2{alpha}, and TXB2 in the culture media of myometrial cells. The 6-keto-PGF1{alpha} concentration in the media from the cells of third trimester pregnant women after cyclic mechanical stretch for 24 h was 2565 ± 416 pg/ml (n = 3), which was significantly higher than that without stimulation (1205 ± 91 pg/ml, n = 3; P < 0.05; Fig. 4Go). In a similar manner, stimulation by cyclic mechanical stretch significantly augmented 6-keto-PGF1{alpha} secretion from cultured human myometrial cells from nonpregnant as well as second and third trimester pregnant women (Fig. 4AGo). We did not perform stretch experiments using cultured human myometrial cells from the first trimester pregnant women due to the limited availability of the tissues. On the other hand, cyclic mechanical stretch for 24 h did not alter PGE2 (Fig. 4BGo), PGF2{alpha} (Fig. 4CGo), or TXB2 (Fig. 4DGo) secretion from cultured myometrial cells from nonpregnant women or second or third trimester pregnant women.

The effect of cyclic mechanical stretch on the concentrations of 6-keto-PGF1{alpha}, PGE2, PGF2{alpha}, and TXB2 in the culture media of human umbilical artery smooth muscle cells and human coronary artery smooth muscle cells

To examine whether such a stretch-induced augmentation of 6-keto-PGF1{alpha} is specific to uterine myometrium, similar experiments were performed using cultured human umbilical artery smooth muscle cells or cultured human coronary artery smooth muscle cells. After 24 h of incubation with cyclic mechanical stretch, concentrations of 6-keto-PGF1{alpha}, PGE2, PGF2{alpha}, or TXB2 in the culture media of human umbilical artery smooth muscle cells (n = 4) were 434 ± 48 pg/ml, 170 ± 22 pg/ml, 77 ± 9 pg/ml, and 39 ± 9 pg/ml, respectively, which were similar to those without stretch (n = 4; 386 ± 32 pg/ml, 161 ± 19 pg/ml, 72 ± 4 pg/ml, and 31 ± 4 pg/ml, respectively).

After 24 h of incubation with cyclic mechanical stretch, concentrations of 6-keto-PGF1{alpha}, PGE2, PGF2{alpha}, or TXB2 in the culture media of human coronary artery smooth muscle cells (n = 4) were 196 ± 21 pg/ml, 188 ± 20 pg/ml, 81 ± 13 pg/ml, and 36 ± 11 pg/ml, respectively, which were similar to those without stretch (n = 4; 183 ± 21 pg/ml, 177 ± 16 pg/ml, 84 ± 17 pg/ml, and 35 ± 5 pg/ml, respectively).

RT-PCR and Western blot analysis of cPLA2, COX-1, COX-2, and PGIS expression in cultured human myometrial cells after stimulation with cyclic mechanical stretch

The cultured human myometrial cells from second trimester pregnant women were used in the following RT-PCR and Western blot analysis. RT-PCR analysis showed apparent increase of PGIS mRNA after 8 h of stimulation by cyclic mechanical stretch, but not after 4, 6, 12, or 24 h of stimulation (Fig. 5Go). On the other hand, none of cPLA2, COX-1, COX-2, or G3PDH mRNA expression levels were altered by the same stimulation throughout the time period examined (Fig. 5Go).

Western blot analysis demonstrated that the PGIS protein expression after 24 h of stimulation by cyclic mechanical stretch was 26 ± 7 AU (n = 4), which was significantly higher than that without the stimulation (8 ± 1 AU, n = 4; P < 0.01; Fig. 6CGo). On the other hand, the levels of cPLA2 and COX-1 expression in the cultured human myometrial cells after 24 h of stimulation by cyclic mechanical stretch were 43 ± 5 AU and 13 ± 2 AU (n = 4 for both), which were similar to those without the stimulation (33 ± 5 and 13 ± 3 AU, respectively; n = 4 for both; Fig. 6Go, A and B).

COX-2 protein expression in the cultured human myometrial cells was below the limit of Western blot detection (data not shown), although COX-2 mRNA expression was detected by RT-PCR (Fig. 5Go).

Promoter activities of PGIS in the cultured human myometrial cells of second trimester pregnant women after 3-, 4-, and 6-h stimulation with cyclic mechanical stretch were 0.24 ± 0.04 AUA, 0.40 ± 0.10 AUA, and 0.43 ± 0.07 AUA, which were significantly higher than those at the corresponding incubation times without the stretch stimulation (0.08 ± 0.04 AUA, 0.13 ± 0.04 AUA, and 0.27 ± 0.05 AUA; n = 5 and P < 0.05 for each; Fig. 7Go). In contrast, such augmentation of the promoter activity of PGIS was not observed after 8-, 12-, or 24-h stimulation.

The effect of curcumin, herbimycin A, and genistein on cyclic mechanical stretch-augmented 6-keto-PGF1{alpha} secretion and PGIS protein expression in the cultured human myometrial cells

Cotreatment with 20 µM curcumin significantly suppressed stretch-augmented PGIS protein expression, although it did not affect PGIS protein expression in the absence of cyclic mechanical stretch (Fig. 8AGo). 6-Keto-PGF1{alpha} concentrations in the media after cyclic mechanical stretch for 24 h under the treatment with 3 µM, 5 µM, 10 µM, 15 µM, and 20 µM curcumin were 430 ± 24 µM, 369 ± 32 µM, 290 ± 19 µM, 208 ± 14 µM (P < 0.05), and 149 ± 12 µM (P < 0.01), respectively (n = 3 for each), indicating a dose-dependent inhibition of stretch-mediated augmentation. The 6-keto-PGF1{alpha} concentrations with stretch and 15 µM or 20 µM curcumin were significantly lower than those without cotreatment with curcumin (427 ± 70 µM; n = 3; Fig. 8BGo). On the other hand, the 6-keto-PGF1{alpha} concentration was not altered by curcumin treatment in the absence of stretch (Fig. 8BGo). By contrast, treatment with herbimycin A or genistein did not alter the 6-keto-PGF1{alpha} concentration in the culture medium with stimulation by cyclic mechanical stretch (Fig. 8Go, C and D).

After 4 h of stimulation by cyclic mechanical stretch in the presence of 20 µM curcumin, the promoter activity of PGIS in the cultured human myometrial cells of second trimester pregnant women was 0.34 ± 0.01 AUA, which was significantly lower than that in the absence of curcumin treatment (0.57 ± 0.08 AUA; P < 0.05; Fig. 9Go). In contrast, the PGIS promoter activity was not affected by 20 µM curcumin treatment in the absence of stretch (Fig. 9Go).

Discussion

In the present study, positive staining of cPLA2, COX-1, COX-2, and PGIS was widely detected in the human pregnant myometrium (Fig. 1Go). Western blot analysis revealed that pregnancy significantly up-regulated COX-1 and PGIS protein expression in the myometrium (Fig. 2Go, B and D). The pregnancy-associated increase in COX-1 expression in the human myometrium observed in this study is consistent with reports about rat myometrium (41, 42), but not with several reports that COX-1 expression is rather stable during the course of human pregnancy (24, 25, 26). At present, we have no clear explanation for this discrepancy, but can only speculate that COX-1 expression might be affected by the method of tissue collection. We collected the upper part of human myometrial tissues after cesarean hysterectomy in the third trimester or after hysterectomy in the first or second trimester of gestation. PGIS expression in the pregnant myometrium was approximately 7-fold higher than that in nonpregnant myometrium (Fig. 2DGo), which is consistent with the report of Moonen et al. (43). The expression of cPLA2 and COX-2 was ubiquitous and relatively stable. The significant elevation of COX-1 expression and relatively stable expression of cPLA2 and COX-2 indicated that pregnancy up-regulates the production of prostaglandin H2, which is an unstable but common substrate of various PG synthases, including PGIS. Moreover, the increase of PGIS expression (7-fold) during pregnancy was much more marked than those of cPLA2, COX-1, and COX-2 (Fig. 2Go). All of these findings suggest that the enhancement of PGIS expression is the major alteration in the various enzymes involved in PGI2 synthesis in the course of pregnancy before the onset of labor. These findings are compatible with the report of local up-regulation of PGI2 synthesis in the conceptus region of the rat uterus (44).

The physiological role of the pregnancy-associated augmentation of myometrial PGI2 synthesis is not well understood. During normal pregnancy to accommodate the growing fetus, the uterine myometrium is dramatically distended and does not contract, a condition constituting so-called uterine quiescence. Because PGI2 is a potent relaxant of uterine myometrium (5, 6, 7), it is plausible that up-regulated PGI2 production may contribute in part to the maintenance of uterine quiescence. Another possible role of myometrial PGI2 production during pregnancy is that myometrial PGI2 may affect the uterine vasculature and contribute at least partly to maintenance of the uterine blood flow, because PGI2 is also a potent vasodilator (9). In fact, uterine blood flow is dramatically elevated during pregnancy (45). Further in vitro as well as in vivo studies will be necessary to clarify physiological roles of myometrial PGI2 production during pregnancy before the onset of labor.

In the present study, we prepared cultured human myometrial cells. The major PG secreted from cultured human myometrial cells was PGI2, a finding compatible with a previous report on the analysis of tissue concentrations of PGs (46). In addition, the basal secretion of PGI2, PGE2, and PGF2{alpha} from myometrial cells cultured for 24 h was increasingly elevated with the progression of gestational age at the collection of the myometrial tissues (Fig. 3Go). Such a pregnancy-related character of PGI2 production in the cultured human myometrial cells is not consistent with the report on ovine model (47). In contrast, the basal secretion of TXA2 from the cells of first and second trimester pregnant women was low compared with that from the cells of nonpregnant women or third trimester pregnant women (Fig. 3DGo). This finding is relevant to a previous report showing that the maternal plasma TXA2 concentration decreased transiently in the middle of gestation (14). Therefore, the cultured human myometrial cells at least partly retain the pregnancy-related character of PG production and are a useful model with which to investigate gestation-associated alterations in the PG production in the human myometrium.

Because it was rather difficult to obtain the upper part of human myometrial tissues after the onset of labor, we investigated the regulatory mechanism of myometrial PGI2 production during labor by mimicking labor with labor-like cyclic mechanical stretching of cultured human myometrial cells, as an in vitro model of labor. Cyclic mechanical stretch significantly augmented 6-keto-PGF1{alpha} secretion by 2.1- to 3.9-fold, but not PGE2, PGF2{alpha}, or TXB2 secretion, from the cells of nonpregnant (Fig. 4AGo), second trimester (Fig. 4BGo), and third trimester (Fig. 4CGo) pregnant women, indicating that mechanical stretch selectively up-regulates PGI2 secretion from cultured human myometrial cells. On the other hand, treatment with 10 ng/ml IL-1{alpha} significantly elevated 6-keto-PGF1{alpha}, PGE2, PGF2{alpha}, and TXA2 secretion from cultured human myometrial cells (Korita, D., H. Itoh, N. Sagawa, unpublished observation), indicating that the cultured myometrial cells preserve the potential to increase secretion of these PGs in response to stimuli other than cyclic mechanical stretch.

By contrast, the cyclic mechanical stretch did not affect the secretion of any PGs from human umbilical arterial smooth muscle cells or from human coronary artery smooth muscle cells, although these cells responded to the stimulation in the IL-1{alpha} (10 ng/ml) significantly and increased the secretion of 6-keto-PGF1{alpha}, PGE2, PGF2{alpha}, and TXA2 (data not shown), suggesting the possibility that stretch-induced augmentation of PGI2 secretion is specific for human uterine myometrial cells.

The promoter activity (3–6 h; Fig. 7Go), mRNA expression (8 h; Fig. 5Go), and protein expression (24 h; Fig. 6CGo) of PGIS were sequentially augmented by stimulation with cyclic mechanical stretch. These results indicate that cyclic mechanical stretch specifically up-regulates PGIS expression at the transcriptional level. These in vitro findings are compatible with the report on labor-induced augmentation of PGIS in ovine myometrium in vivo (29).

We further investigated part of the regulatory mechanism of stretch-induced up-regulation of PGIS expression in cultured human myometrial cells, in view of previous reports regarding cyclic mechanotransduction by AP-1 (48, 49, 50, 51) and tyrosine phosphorylation (52, 53, 54). The PGIS promoter (-3034/-10)/luciferase vector used has one AP-1 site (-2882/-2876; Ref. 22). Treatment with curcumin, a specific inhibitor of AP-1 site (37, 38, 47, 48, 49, 50), dose-dependently and significantly suppressed stretch-induced PGI2 secretion from cultured human myometrial cells (Fig. 8BGo). Moreover, the curcumin treatment suppressed stretch-augmented PGIS protein expression (Fig. 8AGo) as well as PGIS promoter activity (Fig. 9Go), indicating that cyclic mechanical stretch stimulates PGIS expression at the transcriptional level via activation of AP-1 site. Such stretch-associated activation of AP-1 site was also reported for heparin binding-epidermal growth factor expression in rat bladder smooth muscle cells (49), monocyte chemotactic protein-1 expression in human umbilical vein endothelial cells (50), and fibronectin expression in rat aorta vascular smooth muscle cells (51). There have been several reports on the involvement of tyrosine phosphorylation in cyclic mechanotransduction (39, 40, 52, 53, 54). In the present study, herbimycin A, a cSrc family tyrosine kinase inhibitor (Fig. 8CGo), as well as genistein, a general tyrosine kinase inhibitor (Fig. 8DGo), did not affect the stretch-induced augmentation of PGI2 secretion from cultured human myometrial cells, indicating that tyrosine phosphorylation may not be involved in this phenomenon in the human myometrial cells. Taken together, our findings indicate that cyclic mechanical stretch elevates PGI2 secretion, at least partly by activation of AP-1 site without involvement of tyrosine phosphorylation.

To our knowledge, this is the first report of a possible contribution of mechanical stretch to specific augmentation of PGIS expression at the transcriptional level and a consequent elevation of PGI2 production in cultured human myometrial cells. Only a slight induction of PGIS expression by mechanical stretch was reported in bovine aortic endothelial cells (55), and the involvement of stretch in the regulation of connexin 43 (56), oxytocin receptor (57), and PGF2{alpha} receptor (58) in the rat myometrium was found by in vivo studies.

In this study, we applied an in vitro culture model with cyclic mechanical stretch to mimic at least partly the myometrial cyclic contraction and relaxation during labor. However, cyclic stretch and relaxation is only one of the characteristic features of myometrial cells in labor. During active labor, myometrial cells themselves contract and relax repeatedly. Moreover, the present study does not exclude the possibilities that various factors other than cyclic stretch, such as oxytocin, PGE2, PGF2{alpha}, etc., affect pregnant myometrium in labor (4). Nevertheless, the present study revealed that cyclic stretch by exogenous mechanical force augmented PGIS promoter activities via stimulation of AP-1 site in cultured human myometrial cells. The possible involvement of AP-1 site in the regulation of stretch-associated signal transduction was also demonstrated in various cultured cells prepared not only from physiologically contractile tissues, vascular smooth muscle cells (51), bladder smooth muscle cells (49), and cardiocytes (59) but also from noncontractile tissues, endothelial cells (48), osteoblastic cells (60), and renal mesangial cells (61), in all of which exogenous cyclic mechanical stretch was applied to the cells by the same Flexcell Strain unit as we used in the present study. Taken together, it is plausible that application of cyclic mechanical stretch to cultured human myometrial cells mimics, at least partly, the labor-associated mechanical stimulation to the myometrial cells. Further investigation using improved experimental model systems in vitro as well as in vivo are necessary to draw an entire picture of the regulatory mechanism of myometrial PGI2 production during labor.

Physiological roles of possible stretch-induced augmentation of myometrial PGI2 synthesis have not been well clarified yet. Nevertheless, we speculate that myometrial PGI2 might protect myometrial cells themselves against extremely strong contraction during labor, because PGI2 relaxes pregnant human myometrium (12, 13). We also conjecture that PGI2, as a potent vasodilator (9), might dilate the uterine vasculature and maintain the oxygen supply to the fetoplacental unit during the active uterine contraction. Another possibility might be the protection of uterine circulation from hypercoagulation under the intermittent occlusion of blood flow during labor, because PGI2 is a potent anticoagulant (62). However, further in vivo studies are obviously necessary to elucidate the physiological roles of myometrial PGI2 production in labor.

In summary, the present study revealed firstly that pregnancy increases PGI2 production in the human myometrium via enhancing COX-1 as well as PGIS protein expression before the onset of labor. Using a culture model, we secondly demonstrated that cyclic mechanical stretch specifically up-regulates myometrial PGIS expression via activation of AP-1 site and elevates PGI2 production in cultured human myometrial cells, suggesting a possibility that the increased myometrial PGI2 production after the onset of labor is related partly to cyclic distension of the myometrium during labor.

Acknowledgments

We thank Dr. Kazuwa Nakao, Dr. Yoshihiko Saito, and Dr. Masaki Harada (Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine) for their kind technical support concerning the experiment of cyclic mechanical stretch. We also acknowledge Ms. Akiko Kishimoto and Ms. Akiko Abe for secretarial and technical assistance for this work.

Footnotes

This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Sports of Japan (Grants 12877262, 13470352, 13557139, and 13671707), a grant from the Ministry of Health and Welfare, Japan, and grants from the Smoking Research Foundation and the Kanzawa Medical Research Foundation, Japan.

Results from this work were presented in part at the 33rd Annual Meeting of the Society for the Study of Reproduction, Madison, Wisconsin, 2000; and at the 48th and 49th Annual Meetings of the Society for Gynecologic Investigation, Toronto, Canada, 2001, and Los Angeles, California, 2002, respectively.

Abbreviations: AP-1, Activator protein-1; AU, arbitrary units; AUA, arbitrary unit(s) of activity; COX, cyclooxygenase; cPLA2, cytosolic phospholipase A2; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; PG, prostaglandin; PGE2, prostaglandin E2; PGF2{alpha}, prostaglandin F2a; PGI2, prostacyclin; PGIS, prostacyclin synthase; TXA2, thromboxane A2.

Received April 19, 2002.

Accepted July 22, 2002.

References

  1. Okazaki T, Casey ML, Okita JR, MacDonald PC, Johnston JM 1981 Initiation of human parturition. XII. Biosynthesis and metabolism of prostaglandins in human fetal membranes and uterine decidua. Am J Obstet Gynecol 139:373–381[Medline]
  2. Challis JR, Lye SJ, Gibb W 1997 Prostaglandins and parturition. Ann N Y Acad Sci 828:254–267[Medline]
  3. Gibb W 1998 The role of prostaglandins in human parturition. Ann Med 30:235–241[Medline]
  4. Cunningham FG, Gant NF, Leveno KJ, Gilstrap III LC, Hauth JC, Wenstrom KD, eds. 2001 Parturition. In: Williams obstetrics, 21st Ed. Norwalk, CT: Appleton & Lange; 251–290
  5. Wilhelmsson L, Wikland M, Wiqvist N 1981 PGH2, TXA2 and PGI2 have potent and differentiated actions on human uterine contractility. Prostaglandins 21:277–286[CrossRef][Medline]
  6. Lumsden MA, Baird DT 1986 The effect of intrauterine administration of prostacyclin on the contractility of the nonpregnant uterus in vivo. Prostaglandins 31:1011–1022[CrossRef][Medline]
  7. Dyal R, Crankshaw DJ 1988 The effects of some synthetic prostanoids on the contractility of the human lower uterine segment in vitro. Am J Obstet Gynecol 158:281–285[Medline]
  8. Matsumoto T, Sagawa N, Yoshida M, Mori T, Tanaka I, Mukoyama M, Kotani M, Nakao K 1997 The prostaglandin E2 and F2{alpha} receptor genes are expressed in human myometrium and are down-regulated during pregnancy. Biochem Biophys Res Commun 238:838–841[CrossRef][Medline]
  9. Magness RR, Shideman CR, Habermehl DA, Sullivan JA, Bird IM 2000 Endothelial vasodilator production by uterine and systemic arteries. V. Effects of ovariectomy, the ovarian cycle, and pregnancy on prostacyclin synthase expression. Prostaglandins Other Lipid Mediat 60:103–118[CrossRef][Medline]
  10. Abel MH, Kelly RW 1979 Differential production of prostaglandins within the human uterus. Prostaglandins 18:821–828[CrossRef][Medline]
  11. Todd HM, Dundoo VL, Gerber WR, Cwiak CA, Baldassare JJ, Hertelendy F 1996 Effect of cytokines on prostaglandin E2 and prostacyclin production in primary cultures of human myometrial cells. J Matern Fetal Med 5:161–167[CrossRef][Medline]
  12. Christensen NJ, Green K 1983 Bioconversion of arachidonic acid in human pregnant reproductive tissues. Biochem Med 30:162–180[CrossRef][Medline]
  13. Moonen P, Klok G, Keirse MJ 1986 Distribution of prostaglandin endoperoxide synthase and prostacyclin synthase in the late pregnant uterus. Br J Obstet Gynaecol 93:255–259[Medline]
  14. Wang YP, Walsh SW, Guo JD, Zhang JY 1991 Maternal levels of prostacyclin, thromboxane, vitamin E and lipid peroxides throughout normal pregnancy. Am J Obstet Gynecol 165:1690–1694[Medline]
  15. Goodman RP, Killam AP, Brash AR, Branch RA 1982 Prostacyclin production during pregnancy: comparison of production during normal pregnancy and pregnancy complicated by hypertension. Am J Obstet Gynecol 142:817–822[Medline]
  16. Miyata A, Hara S, Yokoyama C, Inoue H, Ullrich V, Tanabe T 1994 Molecular cloning and expression of human prostacyclin synthase. Biochem Biophys Res Commun 200:1728–1734[CrossRef][Medline]
  17. Ylikorkala O, Makarainen L, Viinikka L 1981 Prostacyclin production increases during human parturition. Br J Obstet Gynaecol 88:513–516[Medline]
  18. Davidson BJ, Murray RD, Challis JR, Valenzuela GJ 1987 Estrogen, progesterone, prolactin, prostaglandin E2, prostaglandin F2{alpha}, 13, 14-dihydro-15-keto-prostaglandin F2{alpha} and 6-keto-prostaglandin F1{alpha} gradients across the uterus in women in labor and not in labor. Am J Obstet Gynecol 157:54–58[Medline]
  19. Okazaki T, Sagawa N, Okita JR, Bleasdale JE, MacDonald PC, Johnston JM 1981 Diacylglycerol metabolism and arachidonic acid release in human fetal membranes and decidua vera. J Biol Chem 256:7316–7321[Abstract/Free Full Text]
  20. Ganong WF 2001 Energy balance, metabolism, nutrition. In: Ganong WF, ed. Review of medical physiology, 20th Ed. New York: McGraw-Hill; 271–306
  21. Hara S, Miyata A, Yokoyama C, Inoue H, Brugger R, Lottspeich F, Ullrich V, Tanabe T 1994 Isolation and molecular cloning of prostacyclin synthase from bovine endothelial cells. J Biol Chem 269:19897–19903[Abstract/Free Full Text]
  22. Yokoyama C, Yabuki T, Inoue H, Tone Y, Hara S, Hatae T, Nagata M, Takahashi EI, Tanabe T 1996 Human gene encoding prostacyclin synthase (PTGIS): genomic organization, chromosomal localization, and promoter activity. Genomics 36:296–304[CrossRef][Medline]
  23. Skannal DG, Eis AL, Brockman D, Siddiqi TA, Myatt L 1997 Immunohistochemical localization of phospholipase A2 isoforms in human myometrium during pregnancy and parturition. Am J Obstet Gynecol 176:878–882[CrossRef][Medline]
  24. Zuo J, Lei ZM, Rao CV, Pietrantoni M, Cook VD 1994 Differential cyclooxygenase-1 and -2 gene expression in human myometria from preterm and term deliveries. J Clin Endocrinol Metab 79:894–899[Abstract]
  25. Slater DM, Dennes WJ, Campa JS, Poston L, Bennett PR 1999 Expression of cyclooxygenase types-1 and -2 in human myometrium throughout pregnancy. Mol Hum Reprod 5:880–884[Abstract/Free Full Text]
  26. Sparey C, Robson SC, Bailey J, Lyall F, Europe-Finner GN 1999 The differential expression of myometrial connexin-43, cyclooxygenase-1 and -2, and Gs {alpha} proteins in the upper and lower segments of the human uterus during pregnancy and labor. J Clin Endocrinol Metab 84:1705–1710[Abstract/Free Full Text]
  27. Molnar M, Rigo Jr J, Romero R, Hertelendy F 1999 Oxytocin activates mitogen-activated protein kinase and up-regulates cyclooxygenase-2 and prostaglandin production in human myometrial cells. Am J Obstet Gynecol 181:42–49[CrossRef][Medline]
  28. Wu WX, Ma XH, Zhang Q, Buchwalder L, Nathanielsz PW 1997 Regulation of prostaglandin endoperoxide H synthase 1 and 2 by estradiol and progesterone in nonpregnant ovine myometrium and endometrium in vivo. Endocrinology 138:4005–4012[Abstract/Free Full Text]
  29. Wu WX, Ma XH, Nathanielsz PW 1999 Changes in prostacyclin synthase in pregnant sheep myometrium, endometrium, and placenta at spontaneous term labor and regulation by estradiol and progesterone. Am J Obstet Gynecol 180:744–749[CrossRef][Medline]
  30. Itoh H, Bird IM, Nakao K, Magness RR 1998 Pregnancy increases soluble and particulate guanylate cyclases and decreases the clearance receptor of natriuretic peptides in ovine uterine, but not systemic, arteries. Endocrinology 139:3329–3341[Abstract/Free Full Text]
  31. Masuzaki H, Ogawa Y, Sagawa N, Hosoda K, Matsumoto T, Mise H, Nishimura H, Yoshimasa Y, Tanaka I, Mori T, Nakao K 1997 Nonadipose tissue production of leptin: leptin as a novel placenta-derived hormone in humans. Nat Med 3:952–953[CrossRef][Medline]
  32. Sharp JD, White DL, Chiou XG, Goodson T, Gamboa GC, McClure D, Burgett S, Hoskins J, Skatrud PL, Sportsman JR 1991 Molecular cloning and expression of human Ca2+ -sensitive cytosolic phospholipase A2. J Biol Chem 266:14850–14853[Abstract/Free Full Text]
  33. Yokoyama C, Tanabe T 1989 Cloning of human gene encoding prostaglandin endoperoxide synthase and primary structure of the enzyme. Biochem Biophys Res Commun 165:888–894[CrossRef][Medline]
  34. Jones DA, Carlton DP, McIntyre TM, Zimmerman GA, Prescott SM 1993 Molecular cloning of human prostaglandin endoperoxide synthase type II and demonstration of expression in response to cytokines. J Biol Chem 268:9049–9054[Abstract/Free Full Text]
  35. Yoshida M, Sagawa N, Itoh H, Yura S, Takemura M, Wada Y, Sato T, Ito A, Fujii S 2002 Prostaglandin F2{alpha}, cytokines, and cyclic mechanical stretch augment matrix metalloproteinase-1 secretion from cultured human uterine cervical fibroblast cells. Mol Hum Reprod 8:681–687[Abstract/Free Full Text]
  36. Terakawa K, Itoh H, Sagawa N, Yura S, Yoshida M, Korita D, Kakui K, Fujii S 2002 Site-specific augmentation of amnion cyclooxygenase-2 and decidua vera phospholipase-A2 expression in labor: possible contribution of mechanical stretch and interleukin-1 to amnion prostaglandin synthesis. J Soc Gynecol Investig 9:67–74[CrossRef]
  37. Bierhaus A, Zhang Y, Quehenberger P, Luther T, Haase M, Muller M, Mackman N, Ziegler R, Nawroth PP 1997 The dietary pigment curcumin reduces endothelial tissue factor gene expression by inhibiting binding of AP-1 to the DNA and activation of NF-{kappa}B. Thromb Haemost 77:772–782[Medline]
  38. Huang TS, Lee SC, Lin JK 1991 Suppression of c-Jun/AP-1 activation by an inhibitor of tumor promotion in mouse fibroblast cells. Proc Natl Acad Sci USA 88:5292–5296[Abstract/Free Full Text]
  39. Uehara Y, Fukazawa H, Murakami Y, Mizuno S 1989 Irreversible inhibition of v-src tyrosine kinase activity by herbimycin A and its abrogation by sulfhydryl compounds. Biochem Biophys Res Commun 163:803–809[CrossRef][Medline]
  40. Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N, Shibuya M, Fukami Y 1987 Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem 262:5592–5595[Abstract/Free Full Text]
  41. Dong YL, Gangula PR, Fang L, Yallampalli C 1996 Differential expression of cyclooxygenase-1 and -2 proteins in rat uterus and cervix during the estrous cycle, pregnancy, labor and in myometrial cells. Prostaglandins 52:13–34[CrossRef][Medline]
  42. Myatt L, Langdon G, Brockman DE 1994 Identification and changes in concentrations of prostaglandin H synthase (PGHS) isoforms in rat myometrium at parturition. Prostaglandins 48:285–296[CrossRef][Medline]
  43. Moonen P, Klok G, Keirse MJ 1984 Increase in concentrations of prostaglandin endoperoxide synthase and prostacyclin synthase in human myometrium in late pregnancy. Prostaglandins 28:309–321[CrossRef][Medline]
  44. Kawano M, Mori N 1988 The significance of prostacyclin produced by pregnant rat myometrium: the relationship between myometrial prostacyclin producing activity and passive stretch of myometrium by growing conceptus. Prostaglandins 35:305–325[CrossRef][Medline]
  45. Magness RR, Zheng J 1996 Maternal cardiovascular alternation during pregnancy. In: Gluckman PD, Heymann MA, eds. Pediatrics and perinatology, the scientific basis, 2nd Ed. London, Arnold; 762–772
  46. Bamford DS, Jogee M, Williams KI 1980 Prostacyclin formation by the pregnant human myometrium. Br J Obstet Gynaecol 87:215–218[Medline]
  47. Ipson MA, Rosenfeld CR, Magness RR, Kamm KE 1996 Alterations in myometrial stress during ovine pregnancy and the puerperium. Am J Physiol 271:R446–R454
  48. Du W, Mills I, Sumpio BE 1995 Cyclic strain causes heterogeneous induction of transcription factors, AP-1, CRE binding protein and NF-{kappa}B, in endothelial cells: species and vascular bed diversity. J Biomech 28:1485–1491[CrossRef][Medline]
  49. Park JM, Adam RM, Peters CA, Guthrie PD, Sun Z, Klagsbrun M, Freeman MR 1999 AP-1 mediates stretch-induced expression of HB-EGF in bladder smooth muscle cells. Am J Physiol 277:C294–C301
  50. Wung BS, Cheng JJ, Hsieh HJ, Shyy YJ, Wang DL 1997 Cyclic strain-induced monocyte chemotactic protein-1 gene expression in endothelial cells involves reactive oxygen species activation of activator protein 1. Circ Res 81:1–7[Abstract/Free Full Text]
  51. Tamura K, Chen YE, Lopez-Ilasaca M, Daviet L, Tamura N, Ishigami T, Akishita M, Takasaki I, Tokita Y, Pratt RE, Horiuchi M, Dzau VJ, Umemura S 2000 Molecular mechanism of fibronectin gene activation by cyclic stretch in vascular smooth muscle cells. J Biol Chem 275:34619–34627[Abstract/Free Full Text]
  52. Kimura M, Obara K, Sasase T, Ishikawa T, Tanabe Y, Nakayama K 2000 Specific inhibition of stretch-induced increase in L-type calcium channel currents by herbimycin A in canine basilar arterial myocytes. Br J Pharmacol 130:923–931[CrossRef][Medline]
  53. Joki N, Kaname S, Hirakata M, Hori Y, Yamaguchi T, Fujita T, Katoh T, Kurokawa K 2000 Tyrosine-kinase dependent TGF-ß and extracellular matrix expression by mechanical stretch in vascular smooth muscle cells. Hypertens Res 23:91–99[Medline]
  54. Loufrani L, Lehoux S, Tedgui A, Levy BI, Henrion D 1999 Stretch induces mitogen-activated protein kinase activation and myogenic tone through two distinct pathways. Arterioscler Thromb Vasc Biol 19:2878–2883[Abstract/Free Full Text]
  55. Segurola Jr RJ, Oluwole B, Mills I, Yokoyama C, Tanabe T, Kito H, Nakajima N, Sumpio BE 1997 Cyclic strain is a weak inducer of prostacyclin synthase expression in bovine aortic endothelial cells. J Surg Res 69:135–138[CrossRef][Medline]
  56. Cowan DB, Lye SJ, Langille BL 1998 Regulation of vascular connexin 43 gene expression by mechanical loads. Circ Res 82:786–793[Abstract/Free Full Text]
  57. Ou CW, Chen ZQ, Qi S, Lye SJ 1998 Increased expression of the rat myometrial oxytocin receptor messenger ribonucleic acid during labor requires both mechanical and hormonal signals. Biol Reprod 59:1055–1061[Abstract/Free Full Text]
  58. Ou CW, Chen ZQ, Qi S, Lye SJ 2000 Expression and regulation of the messenger ribonucleic acid encoding the prostaglandin F2{alpha} receptor in the rat myometrium during pregnancy and labor. Am J Obstet Gynecol 182:919–925[CrossRef][Medline]
  59. Komuro I, Kudo S, Yamazaki T, Zou Y, Shiojima I, Yazaki Y 1996 Mechanical stretch activates the stress-activated protein kinases in cardiac myocytes. FASEB J 10:631–636[Abstract]
  60. Peverali FA, Basdra EK, Papavassiliou AG 2001 Stretch-mediated activation of selective MAPK subtypes and potentiation of AP-1 binding in human osteoblastic cells. Mol Med 7:68–78[Medline]
  61. Ingram AJ, Ly H, Thai K, Kang M, Scholey JW 1999 Activation of mesangial cell signaling cascades in response to mechanical strain. Kidney Int 55:476–485[CrossRef][Medline]
  62. Buczko W, Matys T, Kucharewicz I, Chabielska E 1999 The role of endothelium in antithrombotic effect of the renin-angiotensin system blockade. J Physiol Pharmacol 50:499–507[Medline]



This article has been cited by other articles:


Home page
 Mol Hum ReprodHome page
A. Dalrymple, K. Mahn, L. Poston, E. Songu-Mize, and R.M. Tribe
Mechanical stretch regulates TRPC expression and calcium entry in human myometrial smooth muscle cells
Mol. Hum. Reprod., March 1, 2007; 13(3): 171 - 179*.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
S.R. Sooranna, P.L. Grigsby, N. Engineer, Z. Liang, K. Sun, L. Myatt, and M.R. Johnson
Myometrial prostaglandin E2 synthetic enzyme mRNA expression: spatial and temporal variations with pregnancy and labour
Mol. Hum. Reprod., October 1, 2006; 12(10): 625 - 631.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
I. B. Copland, D. Reynaud, C. Pace-Asciak, and M. Post
Mechanotransduction of stretch-induced prostanoid release by fetal lung epithelial cells
Am J Physiol Lung Cell Mol Physiol, September 1, 2006; 291(3): L487 - L495.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
C. Hermenegildo, P. J. Oviedo, M. A. Garcia-Perez, J. J. Tarin, and A. Cano
Effects of Phytoestrogens Genistein and Daidzein on Prostacyclin Production by Human Endothelial Cells
J. Pharmacol. Exp. Ther., November 1, 2005; 315(2): 722 - 728.
[Abstract] [Full Text] [PDF]

Home page
 Annals of Clinical & Laboratory ScienceHome page
E. Fosslien
Cardiovascular Complications of Non-Steroidal Anti-Inflammatory Drugs
Ann. Clin. Lab. Sci., October 1, 2005; 35(4): 347 - 385.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
M. Takemura, H. Itoh, N. Sagawa, S. Yura, D. Korita, K. Kakui, M. Kawamura, N. Hirota, H. Maeda, and S. Fujii
Cyclic mechanical stretch augments hyaluronan production in cultured human uterine cervical fibroblast cells
Mol. Hum. Reprod., September 1, 2005; 11(9): 659 - 665.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
V. Terzidou, S. R. Sooranna, L. U. Kim, S. Thornton, P. R. Bennett, and M. R. Johnson
Mechanical Stretch Up-Regulates the Human Oxytocin Receptor in Primary Human Uterine Myocytes
J. Clin. Endocrinol. Metab., January 1, 2005; 90(1): 237 - 246.
[Abstract] [Full Text] [PDF]

Home page
 Reproductive SciencesHome page
D. Korita, H. Itoh, N. Sagawa, S. Yura, M. Yoshida, K. Kakui, M. Takemura, and S. Fujii
17{beta}-Estradiol Up-Regulates Prostacyclin Production in Cultured Human Uterine Myometrial Cells Via Augmentation of Both Cyclooxygenase-1 and Prostacyclin Synthase Expression
Reproductive Sciences, October 1, 2004; 11(7): 457 - 464.
[Abstract] [PDF]

Home page
 Mol Hum ReprodHome page
M. Takemura, H. Itoh, N. Sagawa, S. Yura, D. Korita, K. Kakui, N. Hirota, and S. Fujii
Cyclic mechanical stretch augments both interleukin-8 and monocyte chemotactic protein-3 production in the cultured human uterine cervical fibroblast cells
Mol. Hum. Reprod., August 1, 2004; 10(8): 573 - 580.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
T. D. WARNER and J. A. MITCHELL
Cyclooxygenases: new forms, new inhibitors, and lessons from the clinic
FASEB J, May 1, 2004; 18(7): 790 - 804.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
K. Kakui, H. Itoh, N. Sagawa, S. Yura, D. Korita, M. Takemura, Y. Miyamaoto, Y. Saito, K. Nakao, and S. Fujii
Augmented endothelial nitric oxide synthase (eNOS) protein expression in human pregnant myometrium: possible involvement of eNOS promoter activation by estrogen via both estrogen receptor (ER){alpha} and ER{beta}
Mol. Hum. Reprod., February 1, 2004; 10(2): 115 - 122.
[Abstract] [Full Text] [PDF]

Home page
 Reproductive SciencesHome page
S. M. Yellon, A. M. Mackler, and M. A. Kirby
The Role of Leukocyte Traffic and Activation in Parturition
Reproductive Sciences, September 1, 2003; 10(6): 323 - 338.
[Abstract] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 Korita, D.
Right arrow Articles by Fujii, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Korita, D.
Right arrow Articles by Fujii, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals