Environmental Health Perspectives Volume 108, Supplement 5, October 2000
Comparing Regulation of the connexin43 Gene by Estrogen in Uterine Leiomyoma and Pregnancy Myometrium
Janet Andersen
Center for Biotechnology, SUNY at Stony Brook, Stony Brook, New York, USA
Abstract
Classical estrogen receptor (ER)-
is expressed in human myometrial and leiomyoma tissues from nonpregnant women. A comparison of these tissues shows that leiomyomas overexpress ER-
compared to normal myometrium. It was hypothesized that overexpression of ER-
in leiomyomas may account for observed overexpression of pregnancy-associated genes that are regulated by estrogen. For this reason, regulation of the labor-associated gene
connexin43 (
cx43) was compared in primary cultures of myometrial and leiomyoma cells. It was shown that a DNA element called activating protein (AP)-1 in the
cx43 promoter is necessary for induction of
cx43 gene transcription in primary uterine smooth muscle cells after activation of cellular protein kinases. However, estrogen did not induce myometrial
cx43 gene transcription
in vitro; instead, it inhibited AP-1 induction of
cx43 expression. This is likely because the myometrial and leiomyoma cells begin to express the novel ER-ß upon culturing, and agonist-bound ER-ß is known to inhibit AP-1 activity. Interestingly, ER-ß is the predominant ER in myometrial tissue from pregnant women at term. Results from an examination of pregnancy myometrial tissue support the concept that AP-1 activity is involved in the induction of myometrial
cx43 expression at term and that suppression of ER-ß expression is needed for this induction. As pregnancy myometrium expresses primarily ER-ß, and nonpregnancy leiomyomas express primarily ER-
, AP-1 activity is predicted to be suppressed in pregnancy myometrium and elevated in leiomyomas under the influence of estrogen. This may be important in understanding tumor pathology, as AP-1 activity is associated with cell growth.
Key words: AP-1 activity,
connexin43, estrogen receptor, myometrium, pregnancy, uterine leiomyomas. --
Environ Health Perspect 108(suppl 5):811-815 (2000).
http://ehpnet1.niehs.nih.gov/docs/2000/suppl-5/811-815andersen/abstract.html
This article is based on a presentation at the conference on Women's Health and the Environment: The Next Century--Advances in Uterine Leiomyoma Research held 7-8 October 1999 in Research Triangle Park, North Carolina, USA.
Address correspondence to J. Andersen, Center for Biotechnology, Psychology A Bldg., 3rd Floor, SUNY at Stony Brook, Stony Brook, NY 11794-2580 USA. Telephone: (631) 632-8521. Fax: (631) 632-8577. E-mail: jandersen@notes.cc.sunysb.edu
This work was supported by a grant from the National Institutes of Health (HD 30482) and by the Department of Obstetrics and Gynecology at SUNY Stony Brook. I wish to thank my collaborators R.L. Barbieri, P. Brink, R.A. Garfield, R. Miksicek, and A. Royek for their contributions to these studies. I also thank those in my research laboratory whose work contributed significantly to these studies: E. Geimonen, V. DyReyes, and J.J. Wu.
Received 23 February 2000; accepted 11 May 2000.
The literature suggests that uterine leiomyomas and pregnancy myometrium have similar regulation of gene expression for certain genes. Thus, comparing the regulation of a gene of interest in uterine leiomyomas with that in nonpregnancy and pregnancy myometrium may help to decipher the pathobiology of the tumors. Several genes, such as the ones for insulin-like growth factor 1 and its receptor, have elevated expression in leiomyomas and have high expression in normal myometrium during pregnancy (
1-11). The noted genes are also estrogen-targeted genes in reproductive tissues. We felt that characterizing the estrogen regulation of one of these genes might contribute to understanding the pathobiology of leiomyomas, especially since the tumors are believed to be dependent on estrogen for growth. This article is a summary of our studies comparing the gene regulation of uterine leiomyomas and term myometrium. Details of the methods and results are published elsewhere (
9-16).
Uterine smooth-muscle tumors were shown in several studies to overexpress estrogen receptor (ER) (
7,12,17-19). Detailed analysis of autologous myometrial and leiomyoma tissues from cycling women showed that uterine leiomyomas overexpress classical ER-
throughout the menstrual cycle but especially during follicular phase when estrogen is unopposed by progesterone (Figure 1) (
12). In contrast, the levels of ER-
in myometrial tissue depend on the timing of the menstrual cycle, which is low at the beginning of the cycle, peaking at midluteal phase, and dropping off at the end of the cycle (Figure 1). The tumors appear to have lost a regulation that cycles the levels of myometrial ER-
during the month, and thus leiomyomas maintain a continually high classical response to estrogen.
|
Figure 1. Relative levels of ER- in autologous myometrial and leiomyoma tissue extracts versus reported days from last menstrual cycle. Squares ( ) mark the relative levels of ER- in myometrial tissue; circles ( ) mark ER- levels in leiomyoma tissue. Tissues from same cycle day are from the same woman. Data were derived from immunoblot analysis using monoclonal antibody specific for ER-. There is a correlation (n = 8; r = 0.9046; p = 0.002) in the increase in myometrial ER- levels during follicular and early luteal phases with days from last menstrual period. In contrast, leiomyoma ER- levels remain elevated during follicular phase (12).
|
Our goal was to understand differences in the estrogen regulation of gene expression in uterine leiomyomas, nonpregnancy myometrium, and pregnancy myometrium from humans. Regulation of the
connexin43 (
cx43) gene expression was chosen for study because it is expressed in leiomyomas from nonpregnant women (
9), it was believed to be regulated by estrogen, and because of the important role of connexin43 (Cx43) gap junctions in myometrial function at term. Cx43 gap junctions form intracellular communication channels in term myometrium, and these are important to myometrial function during labor by coordinating smooth muscle contractions. Increased
cx43 expression is associated with the onset of labor, and the intracellular communication provided by Cx43 gap junctions is believed to help initiate labor (
20-22). Nonpregnancy myometrium does not express the
cx43 gene (
22,23). The role of Cx43 gap junctions in leiomyoma pathobiology is unknown.
A system was developed for culturing human uterine smooth muscle cells to be used as a tool to study leiomyoma gene expression (9). The regulation of the cx43 gene expression was characterized using the primary cultures. Also using this in vitro system, the responses of autologous cultures of myometrial and leiomyoma cells to estrogen and progesterone were compared (12,13). Transient expression assays using progesterone-responsive vectors indicated that primary cultures of human myometrial and leiomyoma cells respond similarly to progestin (13). The effects of progestin on the expression of the cx43 gene in myometrial and leiomyoma primary cells were also remarkably similar (13). Regulation by progesterone is not discussed further in this article.
Upon culturing, human myometrial and leiomyoma cells from nonpregnant women begin to express the cx43 gene and have high levels of Cx43 protein in their cell membrane just like term myometrium (9). This can be observed by indirect immunofluorescence using anti-Cx43 antibodies (Figure 2). The white dots along the cell membrane are Cx43 gap junction proteins. The Cx43 gap junctions formed are open and allow for the transfer of small molecules between the cultured cells (9,24,25).
|
Figure 2. The detection of Cx43 protein in the cultured myometrial and leiomyoma cells by immunofluorescence. Large clusters of the Cx43 protein (seen as white dots along the cell membrane) are detected in the leiomyoma cells (A) and in the myometrial cells (B); C shows the no primary antibody control.
|
Even though cx43 expression was reported to be regulated by estrogen, no complete estrogen response elements (EREs) were found in the sequence of over 5,000 base pairs in the 5´-flanking region of the human cx43 gene promoter (14). At best, six sequences that were similar to half-EREs were noted. Transient expression assays, however, using the 5´-flanking region containing the half-EREs failed to show ER-mediated transactivation upon addition of estrogen (14). Nuclear runoff transcription assays also failed to show that estrogen could induce transcription of the cx43 gene in the human primary cells (14). The conclusion was that estrogen did not directly induce transcription of the cx43 gene in human myometrial and leiomyoma cultured cells.
The 5´-flanking region of the cx43 gene promoter contains DNA sequences for activating protein (AP)-1, AP-2, and promoter-specific transcription factor (SP-1) regulatory elements that are potential regulators of cx43 expression. Using the primary tissue culture system, the AP-1 site proximal to transcription start was analyzed for its role in regulation of the human cx43 gene (14).
The AP-1 site is a DNA element that binds activated transcription factors from the Jun and Fos families (26). Activated Jun and Fos proteins mediate transcriptional regulation through AP-1 sites (27). The general term used to describe this is AP-1 activity. AP-1 activity is associated with cell growth and occurs after stimulation of cellular protein kinases such as protein kinase C (PKC) (26,27).
After human primary myometrial cells were treated with the phorbol ester 12-O-tetradecanolyl-13-acetate (TPA), which activates PKC (28), cx43 expression increased significantly within 6 hr (Figure 3) (14). The increase in Cx43 protein levels was preceded by significant increases in c-Jun and c-Fos levels, suggesting that AP-1 activity is involved in the induction of cx43 expression in the uterine smooth muscle cultured cells (14). Nuclear runoff assays, transient expression assays, and site-specific mutagenesis techniques showed that TPA-induced cx43 expression was due to increased transcription of the gene, and that the proximal AP-1 site in the 5´-flanking region of the promoter was necessary for the induced expression (14).
|
Figure 3. Analysis of cx43, c-Fos, c-Jun protein levels in myometrial primary culture after treatment with TPA and/or estrogen: Autoradiograph of a representative immunoblot showing the relative levels of Cx43, c-Jun, c-Fos, and desmin proteins in the cell lysates of myometrial primary culture cells after treatment with reagent vehicles (0), 100 ng/mL TPA for 3 hr (TPA), 10 nM ethynyl estradiol for 48 hr (eE2), or both treatments (Both) [see Geimonen et al. (15) for method details]. Two forms of c-Jun protein are detected at 46 kDa (activated form) and at 39 kDa in TPA-treated cells. Controls and treated cells were harvested at the same time points. Cx43 protein, c-Jun, and c-Fos levels were normalized to the levels of desmin, a muscle-specific intermediate filament. Reproduced from Geimonen et al. (15) with permission of The Endocrine Society.
|
In addition, relatively high levels of Cx43, c-Jun, and c-Fos proteins were observed on immunoblots of a lysate from myometrial tissue from a woman who needed a hysterectomy after delivery (15). This suggested that the activity observed in vitro was relevant to what happens in vivo at term. So, after patient consent, 10 pieces of myometrial tissue were obtained from pregnant women undergoing elective and indicated caesarian section (Cs). Each sample was assigned a number in the order of being obtained. Table 1 lists the samples and describes the labor profiles of the women from whom tissue was obtained. These tissue samples were analyzed for Cx43, c-Jun, and c-Fos expressions by observing the relative levels of Cx43, c-Jun and c-Fos proteins by immunoblot (15). It was observed that elevated levels of Cx43 protein correlated with elevated levels of c-Jun, and c-Fos proteins in the term tissue (15). The tissue samples with the highest Cx43 levels were from women in active labor. Elevated levels of c-Jun and c-Fos were found to coordinate with elevated levels of Cx43 protein in pregnancy myometrial tissue from women undergoing elective and indicated Cs. We concluded that the AP-1 site in the cx43 promoter functions in the induction of gene transcription in primary cultures of uterine smooth muscle cells upon activation of c-Jun and c-Fos and that increased AP-1 activity is associated with the induction of myometrial cx43 expression at term in humans (15).
Our attention then turned to understanding the role of estrogen in the regulation of cx43 expression in human myometrium at term. Estrogens are found to be stimulatory to the initiation of labor in animals that experience progesterone withdrawal prior to parturition (29-31). However, in animals such as guinea pigs that do not have preparturition progesterone withdrawal, estrogen is inhibitory to labor initiation (31). Humans also do not experience preparturition progesterone withdrawal.
We found that estrogen inhibited induction of cx43 expression through AP-1 activity in the human myometrial primary cells without inhibiting expression of c-Fos and c-Jun (Figure 3) (15). These results came at about the same time that a second ER was discovered--ER-ß (32,33). ER-ß was first cloned from rat prostate and ovarian tissues (34). Its function in transactivation has been characterized in several studies. ER-ß was found to bind ligand with a similar affinity to ER- (32,33,35). Ligand-bound ER- and ER-ß display similar activities at canonical EREs in DNA promoters (36) and can form heterodimers (33,35). Of interest to the cx43 results was a study from Paech et al. (35), who characterized the influence of ER- and ER-ß with AP-1 activity using a variety of ligands. They showed that agonist-bound ER- stimulates AP-1 activity, whereas agonist-bound ER-ß inhibits AP-1 activity when mediated by c-Jun and c-Fos. The influence is believed to occur through protein-protein interactions between the receptor and the AP-1 proteins. In addition, Paech et al. also found that antagonist-bound ER- and ER-ß both stimulate AP-1 activity. Because AP-1 activity can be a principal regulator of cell growth, these findings help understand how estrogen stimulates growth in some estrogen-targeted tissues but not in others. The outcome is dependent on which ER is expressed. These findings also help understand why ligands such as tamoxifen can be a partial agonist, as both ERs stimulate AP-1 activity when they are bound by tamoxifen (35).
Using immunoblot and immunohistochemical methods, the relative levels of progesterone receptor (PR), ER-, and ER-ß were analyzed in the same myometrial tissue analyzed for Cx43, c-Jun, and c-Fos levels. PR was detected in the term tissue at elevated levels compared to that in nonpregnancy myometrial and leiomyoma tissues (15).
Examination of Cs tissue for ER-ß expression compared to nonpregnancy tissue revealed two things. First, there was a dramatic switch in ER subtype in the myometrium during pregnancy from ER-, to ER-ß (Figure 4). ER- is primarily expressed in uterine smooth-muscle tissue (myometrial and leiomyoma) from nonpregnant women, whereas ER-ß is primarily expressed in myometrial tissue from women at term (Figure 4) (16).
|
Figure 4. Analysis of ER- and ER-ß protein levels in uterine smooth muscle: Representative immunoblots showing ER- (67 kDa) and ER-ß (54-55 kDa) protein levels (as marked) in nonpregnancy and term myometrial tissue (Table 1). One hundred micrograms of protein lysate were loaded for the each tissue. Mf, lysate from nonpregnancy myometrial tissue derived from a woman in follicular phase; Lf, lysate from leiomyoma tissue from the same woman as Mf; Ml, lysate from nonpregnancy myometrial tissue derived from a woman in luteal phase; Ll, lysate from leiomyoma tissue from the same woman as Ml. Lanes 6, 10, 4, 7 each contain term myometrial lysate Cs tissue samples. The Cs numbers correspond with the numbers in Table 1, which gives the labor profile for each woman. ER-ß is not seen in lane 7 because it contains myometrial lysate from a woman who had been in labor, and its levels diminish during labor. Lane Pp contains postpartum myometrial tissue lysate; lane H contains HeLa cell lysate; lane B contains lysate from the breast cancer cell line MCF-7; lane m contains lysate from primary myometrial cultured cells derived from luteal-phase tissue. Dashes mark the two ER-ß isoforms detected in myometrial cultured cells (lane m). Reproduced from Wu et al. (16) with permission of the European Journal of Endocrinology.
|
Second, it was observed that as ER-ß levels decreased, the labor-associated Cx43 protein levels increased (Figure 5) (16). The inverse relationship between ER-ß and Cx43 levels suggests that in term myometrium, ER-ß is inhibitory to the induction of cx43 expression. Perhaps estrogen's dual regulation of AP-1 activity is the key to understanding the regulation of other pregnancy- and labor-associated genes that have AP-1 sites in their promoters [compare (37)]. We conclude that estrogen, like progesterone, is involved in pregnancy maintenance in humans.
|
Figure 5. ER-ß levels are inverse to connexin43 protein levels in term myometrial tissues. (A) Representative immunoblot analysis showing relative levels of ER-ß (54-55 kDa) in myometrial tissues and cultured cells. Each myometrial tissue sample contains 100 µg of protein lysate. Lanes 1-9 contain lysates from termed Cs myometrial tissue. (ER-ß is not detected in lane 10 because of technical difficulties but was observed in other blots [compare with Figure 4].) The numbers designate the Cs sample number listed in Table 1. Note that samples 5 and 7 came from women in labor. Lane H contains lysate from HeLa cells; lane B contains lysate from the MCF-7 breast cancer cell line; lane Pp contains lysate from postpartum myometrial tissue; lane m contains lysate from primary myometrial cultured cell. (B) Bar graph depicting the relative levels of Cx43 protein in the term myometrial tissues shown in A. Reproduced from Wu et al. (16) with permission of the European Journal of Endocrinology.
|
Both ER- and ER-ß are expressed in the cultured uterine smooth muscle cells. It was observed that TPA and estrogen treatments altered the relative levels of each receptor (16). Estrogen treatment elevated the levels of ER-ß, whereas TPA treatment decreased the levels of ER-. Treatment with both TPA and estrogen changed the ratio of ER- and ER-ß significantly from that found in the vehicle-treated control sample to favor ER-ß (16) (Figure 6). We concluded that estrogen inhibited TPA induction of cx43 expression in primary myometrial cells because the change of ER-ß/ER- ratio was sufficient to inhibit the AP-1 activity necessary for TPA induction of cx43 expression.
|
Figure 6. Ratio of ER- and ER-ß is altered in primary smooth-muscle cells treated with estrogen and/or TPA: Representative immunoblot showing the relative levels of ER- and ER-ß (as marked) in cultured smooth-muscle cells (derived from luteal-phase tissue) that were treated with reagent vehicle (0); treated with TPA (TPA) for 3 hr, pretreated with 10 nM eE2 (E) for 48 hr, or pretreated with eE2 and then treated with TPA (TPA/E) (see Figure 3 for Cx43 results). Note that TPA treatment suppresses ER- expression, whereas estrogen treatment induces ER-ß expression. Compare the change in ratio of ER- to ER-ß in the vehicle-treated versus the TPA/estrogen-treated cultures. Reproduced from Wu et al. (16) with permission of the European Journal of Endocrinology.
|
Two isoforms of ER-ß are found in relatively equal levels in primary myometrial cells (Figure 4, lane m) (16). The slower moving isoform predominates in leiomyoma primary cells. The slower moving ER-ß is also found in term myometrial tissue from unlaboring women. Appearance of the faster moving form is associated with decreased ER-ß levels and increased Cx43 levels. The two isoforms may have different functions.
Little ER-ß expression is detected by immunoblot analysis of nonpregnancy myometrial and leiomyoma tissues compared to the term myometrial tissue (Figure 4) (16,38). ER-ß protein levels were reanalyzed across the menstrual cycle in a number of nonpregnancy myometrial and leiomyomas tissues from cycling women that had been previously used to measure the relative levels of ER- (Figure 1). The relative low levels of ER-ß protein for these samples required using more protein for each sample than usual and having long exposures for the autoradiographs. It was found that myometrial and leiomyoma tissues from nonpregnant women may have similar patterns of expression of ER-ß (Figure 7). Its highest expression in these tissues is at the beginning of the menstrual cycle. Its expression is lowest at early midluteal phase. There may be some myometrial/leiomyoma differences in ER-ß expression at the end of the menstrual cycle. However, the impact of ER-ß in the regulation of myometrial/leiomyoma gene expression in cycling women is probably negligible given its low levels.
Figure 7. Relative levels of ER-ß protein in human uterine smooth-muscle tissues from nonpregnant cycling women. Very low levels of ER-ß protein are detected in nonpregnancy tissues (Figure 4); therefore, 200 µg of protein were loaded for each sample to enhance detection of ER-ß in these tissues. M designates the immunoblot results from myometrial tissue and L designates the results from leiomyoma tissue. Numbers at the top of each lane are the reported days from the last menstrual period before surgery. The asterisk marks the follicular/luteal phase border as determined by endometrial biopsy for each sample; endometrial determination was proliferative to the right of the asterisk and secretory to the left of the asterisk.
Our work provides a detailed understanding of the regulation of cx43 expression in human myometrium at term and what we have learned about estrogen-regulated gene expression in leiomyomas. Leiomyomas have elevated expression of several genes that are regulated by estrogen and that have elevated myometrial expression some time during pregnancy. However, leiomyomas express primarily ER-, whereas term myometrium expresses primarily ER-ß. The characterized difference between ER- and ER-ß is their influence on AP-1 activity and not in their transactivation activities from EREs. This indicates that leiomyomas, which express primarily ER-, have a different estrogen regulation of AP-1 activity than term myometrium, which expresses primarily ER-ß. Leiomyomas then appear to overexpress the cx43 gene through a regulation that differs from that in term myometrium. Perhaps leiomyomas have elevated cx43 expression through ER--increased AP-1 activity.
A second interesting conclusion from these studies stems from the observed similarities between primary myometrial cultures taken from nonpregnant woman and term myometrium itself. Primary myometrial cultures are similar to term myometrium in a number of ways. The primary cultured cells have high levels of PR-like term myometrium (13,15). Cx43 protein is not detectable in nonpregnancy myometrial tissue but is in primary cultured cells derived from this tissue (9). The cultured cells also have high expression of other labor-associated genes such as thrombospondin-1 (39). They also contain labor-associated oxytocin receptors and respond to oxytocin with transient calcium oscillations not distinguishable from those of cells derived from pregnancy tissue (40). Although the cultured cells express ER-, they also express ER-ß like term myometrium (16). Treatment of the primary cells can alter the ratio of ER- and ER-ß in favor of ER-ß and allows for suppression of AP-1 activity (16). Leiomyoma cells appear to have the same isoform of ER-ß found in term myometrium before labor onset. The reasons for the similarities between cultured uterine smooth-muscle cells and pregnancy myometrium are not known; however, these similarities make the primary cells a useful in vitro tool to study the gene regulation of cx43 and ER-ß.
REFERENCES AND NOTES
1. Chandrasekhar Y, Heiner J, Osuamkpe C, Nagamani M. Insulin-like growth factor I and II binding in human myometrium and Leiomyomas. Am J Obstet Gynecol 166:64-69 (1992).
2. Brandon DD, Bethers CL, Strawn ET, Novy MJ, Burry KA, Harrington MS, Erickson TE, Warner C, Keenan EJ, Clinton GM. Progesterone receptor messenger ribonucleic acid and protein are overexpressed in human uterine leiomyomas. Am J Obstet Gynecol 169:78-85 (1993).
3. Fujita M. Histological and biochemical studies of collagen in human uterine leiomyomas [English Abstract]. Hokkaido Igaku Zasshi 60:602-615 (1985).
4. Kapur S, Tamada H, Dey SK, Andrews GK. Expression of Insulin-Like growth factor-1 (IGF-1) and its receptor in the peri-implantation mouse uterus, and cell-specific regulations of IGF-1 gene expression by estradiol and progesterone. Biol Reprod 46: 208-219 (l992).
5. Padayachi T, Pegoraro RJ, Rom L, Joubert SM. Enzyme immunoassay of oestrogen and progesterone receptors in uterine and intrauterine tissue during human pregnancy and labour. J Steroid Biochem Mol Biol 37:509-511 (1990).
6. Pastore G, Dicola L, Dollahon N, Gardner R. Effect of estriol on the structure and organization of collagen in the lamina propria of the immature rat uterus. Biol Reprod 47:83-91 (1992).
7. Rein MS, Friedman AJ, Stuart JM, MacLaughlin DT. Fibroid and myometrial steroid receptors in women treated with gonadotropin-releasing hormone agonist leuprolide acetate. Fertil Steril 53:1018-1023 (1990).
8. Tabb T, Thilander G, Grover A, Hertzberg E, Garfield R. An immunochemical and immunocytologic study of the increase in myometrial gap junctions (and connexin 43) in rats and humans during pregnancy. Am J Obstet Gynecol 167:559-567 (1992).
9. Andersen J, Grine EA, Eng C L-Y, Zhao K, Barbieri RL, Chumas J, Brink P. Expression of connexin43 in human myometrium and leiomyomas. Am J Obstet Gynecol 169:1266-1276 (1993).
10. Thiede M, Harm S, Hasson D, Gardner R. In vivo regulation of parathyroid hormone-related peptide messenger ribonucleic acid in the rat uterus by 17 beta-estradiol. Endocrinology 128: 2317-2323 (1991).
11. Weir EC, Goad DI, Daifotis AG, Burtis WJ, Dreyer BE, Nowak RA. Relative overexpression of the parathyroid hormone-related peptide gene in human leiomyomas. Baillieres Clin Endocrinol Metab 78:784-789 (1994).
12. Andersen J, DyReyes V, Barbieri RL, Coachman D, Miksicek R. Leiomyoma primary cultures have elevated transcriptional response to estrogen compared to autologous myometrial cultures. J Soc Gynecol Investig 2:542-551 (1995).
13. Zhao K, Kuperman L, Geimonen E, Andersen J. Progestin represses human Connexin43 gene expression similarly in primary cultures of myometrial and uterine leiomyomas. Biol Reprod 54:607-615 (1996).
14. Geimonen E, Jiang W, Ali M, Fishmann G, Garfield RE, Andersen J. Activation of protein kinase C in human uterine smooth muscle induces connexin43 gene transcription through an AP-1 site in the promoter sequence. J Biol Chem 271:23667-23674 (1996).
15. Geimonen E, Boylston E, Royek A, Andersen J. Elevated Connexin43 expression in term Human myometrium correlates with elevated c-Jun expression and is independent of myometrial estrogen receptors. J Clin Endocrinol Metab 83: 1177-1185 (1998).
16. Wu JJ, Geimonen E, Andersen J. Increased expression of estrogen receptor b in human uterine smooth muscle at term. Eur J Endocrinol 142:92-99 (2000).
17. Green S, Walter P, Kumar V, Krust A, Bornert JM, Argos P, Chambon P. Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature 320:134-139 (1986).
18. Lessey BA, Killam AP, Metzger DA, Haney AF, Greene GL, McCarty KS. Immunohistochemical analysis of human uterine estrogen and progesterone receptors throughout the menstrual cycle. J Clin Endocrinol Metab 67:334-340 (1988).
19. Navarro D, Cabrera JJ, Falcón O, Jiménez P, Ruiz A, Chirino R, López A, Rivero JF, Díaz-Chico JC, Díaz-Chico BN. Monoclonal antibody characterization of progesterone receptors, estrogen receptors and the stress-responsive protein of 27 kDa (SRP27) in human uterine leiomyoma. J Steroid Biochem 34:491-498 (1989).
20. Balducci J, Risek B, Gilula NB, Hand A, Egan JFX, Vintzileos AM. Gap junction formation in human myometrium: a key to preterm labor? Am J Obstet Gynecol 168:1609-1615 (1993).
21. Garfield RE, Puri CP, Csapo AI. Endocrine, structural and functional changes in the uterus during premature labor. Am J Obstet Gynecol 142:21-27 (1982).
22. Garfield RE, Sims S, Daniel EE. Gap junctions: their presence and necessity in myometrium during parturition. Science 198: 958-959 (1977).
23. Risek B, Guthrie S, Kumar N, Gilula NB. Modulation of gap junction transcripts and protein expression during pregnancy in the Rat. J Cell Biol 110:269-282 (1990).
24. Goodenough DA, Goliger JA, Paul DL. Connexins, connexons, and intercellular communication. Annu Rev Biochem 65: 475-502 (1996).
25. Garfield RE. Cell-to-cell communication in smooth muscle. In: Calcium and Contractility: Smooth Muscle, Chap 6 (Grover AK, Daniel EE, eds). Clifton, NJ:Humana Press, 1985;143-173.
26. Karin M. The regulation of AP-1 activity by mitogen-activated protein kinases. J Biol Chem 270:16483-16486 (1995).
27. Karin M, Liu ZG, Zandi E. AP-1 function and regulation. Curr Opin Cell Biol 9:240-246 (1997).
28. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature 308:693-698 (1984).
29. MacKenzie LW, Garfield RE. Effects of 17ß-estradiol on myometrial gap junctions and pregnancy in the rat. Can J Physiol Pharmacol 64:462-466 (1986).
30. Claire-Wathes DC, Porter DG. Effect of uterine distension and oestrogen treatment on gap junction formation in myometrium of rat. J Reprod Fertil 65:497-505 (1982).
31. Chwalisz K. The use of progesterone antagonists for cervical ripening and as an adjunct to labor and delivery. Hum Reprod 9(suppl 1):131-161 (1994).
32. Mosselman S, Polman J, Kijkema R. ER beta: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49-53 (1996).
33. Ogawa S, Inoue S, Watnabe T, Hiroi H, Orimo A, Hosoi T, Ouchi Y, Muramatsu M. The complete primary structure of human estrogen receptor b (hERb) and its heterodimerization with ERa in vivo and in vitro. Biochem Biophys Res Commun 243: 122-126 (1998).
34. Kuiper GJM, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson J. Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:5925-5930 (1996).
35. Paech K, Webb, P, Kuiper GGJM, Nilsson S, Gustafsson J, Kushner PJ, Scanlan TS. Differential ligand activation of estrogen receptors ER and ERß at AP-1 sites. Science 277: 1508-1510 (1997).
36. Pace P, Taylor J, Suntharalingam S, Coombes RC, Ali S. Human estrogen receptor b binds DNA in a manner similar to and dimerizes with estrogen receptor a. J Biol Chem 272: 25832-25838 (1997).
37. Umayahara Y, Kawamori R, Watada H, Imano E, Iwama N, Morishima T, Yamasaki Y, Kajimoto Y, Kamada T. Estrogen regulation of the insulin-like growth factor I gene transcription involves an AP-1 enhancer. J Biol Chem 269: 16433-16442 (1994).
38. Pedeutour F, Quade BJ, Weremowicz, Dal Cin P, Ali S, Morton CC. Localization and expression of the human estrogen receptor beta in uterine leiomyomata. Genes Chromosomes Cancer 23: 361-366 (1998).
39. Morimoto T, Head JR, MacDonald PC, Casey ML. Thrombospondin-1 expression in human myometrium before and during pregnancy, before and during labor and in human myometrial cells in culture. Biol Reprod 59:862-870 (1998).
40. Burghardt RC, Barhoumi, Sandborn BM, Andersen J. Oxytocin-induced Ca2+ responses in human myometrial cells. Biol Reprod 60:777-782 (1999).
Last Updated: October 2, 2000
