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Endocrinology Vol. 146, No. 4 2048-2054
Copyright © 2005 by The Endocrine Society

Differential Activation of the Connexin 43 Promoter by Dimers of Activator Protein-1 Transcription Factors in Myometrial Cells

Jennifer A. Mitchell and Stephen J. Lye

Samuel Lunenfeld Research Institute (J.A.M., S.L.), Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5; and Institute of Medical Science (J.A.M., S.L.), and Departments of Obstetrics and Gynecology and Physiology (S.L.), University of Toronto, Toronto, Ontario, Canada M5S 1A1

Address all correspondence and requests for reprints to: Dr. Stephen J. Lye, Samuel Lunenfeld Research Institute and Mount Sinai Hospital, 600 University Avenue, Suite 982, Toronto, Ontario, Canada M5G 1X5. E-mail: lye{at}mshri.on.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The expression of activator protein-1 (AP-1) transcription factors is increased in the myometrium at term and may therefore regulate the expression of genes, such as connexin 43 (Cx43), required for the onset of labor. The region upstream of the mouse, rat, and human Cx43 genes contains two consensus AP-1 binding sequences, a proximal AP-1, located close to the TATA box, and a distal AP-1, 1 kb upstream. A transient transfection system was developed in which Syrian hamster myometrial cells were transfected with Cx43 promoter-luciferase constructs in combination with expression vectors for the AP-1 family. Transfection with c-Jun or JunB had no effect on transcription from the Cx43 promoter, whereas transfection with JunD or combinations of Jun and Fos family members led to significant increases in transcription. Deletion of the distal AP-1 site did not abrogate transcription driven by Fos/Jun, whereas a 2-bp mutation in the proximal AP-1 site significantly reduced pCx43 transactivation by AP-1 dimers. Dimers comprising Fos/Jun proteins conferred greater transcriptional activity than Jun dimmers, with Fra-2/JunB combination conferring greatest activity. These data suggest that increased expression of Fos family members in the myometrium at term drives the increase in Cx43 transcription and expression during labor. Because expression of Fra-2 increases earlier than other Fos family members and confers the highest transcriptional drive to the Cx43 promoter, our data suggest that Fra-2 is a central component in the regulation of Cx43 expression during labor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MEMBERS OF THE activator protein-1 (AP-1) transcription factor family appear to be involved in a wide range of cellular processes including cell proliferation, survival, differentiation, apoptosis, and oncogenic transformation. AP-1 proteins bind to the consensus AP-1 site (TGAG/CTCA) as either homodimers of Jun proteins or heterodimers of Fos and Jun proteins but not as homodimers of Fos proteins (reviewed in Ref. 1). Although these proteins bind the same DNA consensus element, they possess distinct biological functions as illustrated by the results of targeted gene disruption studies, which have revealed that whereas c-Jun, JunB, and Fra-1 are essential during mouse embryonic development, JunD, c-Fos, and FosB are required only postnatally (reviewed in Ref. 2). Their distinct functions can be attributed to the differential tissue-specific and inducible expression patterns of each AP-1 gene and the distinct dimerization, DNA binding, and trans-activating capabilities of each protein. In addition, AP-1 proteins have been shown to interact with several other groups of transcription factors including cAMP response element-binding protein/activating transcription factor and Maf proteins as well as the glucocorticoid and estrogen receptors, increasing the complexity of AP-1 function (reviewed in Ref. 3).

The gap Junction protein connexin 43 (Cx43) is thought to play a critical role in the onset of labor by allowing for an increase in myometrial muscle cell coupling. Before the onset of labor, there is a dramatic increase in both mRNA and protein levels of Cx43 in rat myometrium (4). This increase in expression has been shown to be regulated by both mechanical and hormonal stimuli during pregnancy. Specifically, expression of Cx43 is induced by estrogen and inhibited by progesterone only in the gravid horn of pregnant animals (4, 5). The Cx43 promoter of the mouse, rat, and human genes contains several putative transcription factor binding sites including AP-1, AP-2, cAMP response element, Ets, and specificity protein-1 consensus sites, several sequences resembling half the palidromic estrogen response element and progesterone response element sequences as well as an activator and a repressor site that have been functionally characterized in the mouse gene (6, 7, 8). There are two consensus AP-1 sites in the Cx43 promoter region, both of which are conserved in the mouse, rat, and human sequences. The distal AP-1 site is located 1 kb upstream of the transcription start point and has not been functionally characterized, whereas the proximal AP-1 site, located from 44–50 bp upstream of the transcription start point, has been shown to bind c-Jun in EMSAs and mediate 12-O-tetradecanoylphorbol-13-acetate responsiveness of primary human myometrial cells (8, 9).

The AP-1 binding sites in the Cx43 promoter are of particular interest in the regulation of Cx43 in the pregnant myometrium because we have shown that the expression of several AP-1 family transcription factors is dramatically increased in the myometrium before the onset of labor (10). Furthermore, this expression was shown to be regulated by both mechanical and hormonal stimuli, in a manner similar to Cx43, suggesting AP-1 proteins are involved in Cx43 expression in the pregnant myometrium. Importantly, the gestational expression profiles of individual family members are quite distinct. Expression of c-Jun and JunD remains constant throughout gestation, Fra-2 expression increases before the onset of labor on d21, whereas JunB and other members of the Fos family exhibit a dramatic increase in expression only on the day of labor (d 23). Moreover, our in vitro studies indicate that not all members of the Fos/Jun family are responsive to mechanical stretch (11), raising the question as to which members of this family of transcription factors regulate Cx43 expression within the myometrium. Therefore, in this study we investigated the ability of different Fos/Jun dimers to transactivate the Cx43 promoter through its AP-1 consensus sites in a myometrial cell line. Our experiments revealed that Fos/Jun heterodimers increase transcription through the proximal AP-1 site within the Cx43 promoter. Moreover, the magnitude of this induction was variable depending on the specific Fos and Jun proteins present.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Vector constructs
Construction of the pCx1686-Luc and pCx300-Luc vectors was initially described by Mitchell and Lye (12). The pCx300(AP-1)M-Luc construct was derived from pCx300-Luc by mutation of the AP-1 site, located at –50 to –44, from TGAGTCA to TGAGTTG using the QuikChange site-directed mutagenesis kit (Stratagene, Cedar Creek, TX). Mutation of this site was confirmed by sequencing.

The AP-1 expression vectors were constructed by cloning of the coding sequences into pcDNA3.1 (Invitrogen, Burlington, Ontario, Canada). The coding region for human c-Jun (a generous gift from Dr. J. R. Woodgett, Ontario Cancer Institute, Toronto, Ontario, Canada) was excised from the pMT2 vector by EcoRI digest and cloned into PCR2.1 (Invitrogen) at the EcoRI site. The orientation was determined by digest with PstI, and the correct orientation was excised by digest with HindIII/XhoI and cloned into pcDNA3.1 at the HindIII/XhoI sites. The coding region for mouse JunB was generated by RT-PCR of mRNA from laboring mouse myometrium using Taq polymerase (MBI Fermentas, Flamborough, Ontario, Canada) and the following primers: JunB(cds)-U, 5'-CTT AAG CTT GAC CTT TCC CAG ATC GCC CA-3' and JunB(cds)-L, 5'-TCA GAA TTC GGG GTG TCC GTA TGG GGC AA-3' to amplify the JunB coding region with the addition of a 5' HindIII and 3' EcoRI site. The digested amplicon was then inserted into the pcDNA3.1 vector at the HindIII/EcoRI sites. The coding region for mouse JunD (a generous gift from Dr. Woodgett) was excised from the pMT2 vector by EcoRI digest, cloned into pcDNA3.1 at the EcoRI site, and the orientation checked by double digest with AccI/XhoI. The coding region for human c-Fos (Dr. Woodgett) was excised from the pBK28 vector by first digesting with EcoRI, a blunt end generated with the Klenow fragment (MBI Fermentas), and then digested with XhoI to obtain a blunt/XhoI insert.

The vector was prepared in a similar manner by first digesting with ApaI, blunting, and then digesting with XhoI. The c-Fos coding region was then ligated into the pcDNA3.1 vector at XhoI/ApaI(blunt). The coding region for rat Fra-1 was generated by RT-PCR of mRNA from laboring rat myometrium using the following primers: Fra-1(cds)-U, 5'-CCG GGA TCC ACC CTA CCG AAC ATC CAG CCC AG-3', and Fra-1(cds)-L, 5'-GGG GAA TTC GAT GAC AAC GGG TAG CAC CTG CA-3' to amplify the Fra-1 coding region with the addition of a 5' BamHI and 3' EcoRI site. The digested amplicon was then inserted into the pcDNA3.1 vector at the BamHI/EcoRI sites. The coding region for rat Fra-2 was generated by RT-PCR of mRNA from laboring mouse myometrium using the following primers: Fra-2(cds)-U, 5'-GCG GGA TCC AAA ACC ACC CTG TTT CCT CT-3', and Fra-2(cds)-L, 5'-GCG GAA TTC TTA CAG GGC TAG AAG TGT GG-3' to amplify the Fra-2 coding region with the addition of a 5' BamHI and 3' EcoRI site. The digested amplicon was then inserted into the pcDNA3.1 vector at the BamHI/EcoRI sites. The coding region for mouse FosB (ATCC 63118) was cloned into pcDNA3.1 at the EcoRI and XbaI sites. All expression vector sequences were confirmed by sequencing.

Transfection experiments
On d 1, Syrian hamster myometrial (SHM) (13) cells were seeded at 100,000 cells/well in a 24-well plate (Sarstedt, Newton, NC) to ensure cells were in the log phase of growth when transfected. On d 2 the cells were transferred to serum and phenol red-free DMEM (Sigma, St. Louis, MO) before transfection. Cells were transfected using 5–10 µl ExGen500 cationic polymer (MBI Fermentas) in the presence of 0.4 µg luciferase construct, 0.1 µg pRSVßgal vector (containing Escherichia coli lacZ gene under the Rous sarcoma virus promoter), and a total of 0.01–0.5 µg of the AP-1 vectors (a range of 10,000,000 to 200,000 cells/µg). A total of 0.1 µg or less of the AP-1 vectors was used in combination with 5 µl ExGen500 reagent, whereas amounts of AP-1 vectors greater than 0.1 µg were used in combination with 10 µl ExGen500 reagent. Unless otherwise indicated, a total 0.1 µg AP-1 vectors were used in the transfection. The DNA-reagent complex was applied to the cells, and plates were centrifuged for 5 min at 1500 rpm to allow the complex to adhere to the surface of the cell membrane. The cells were incubated with the complex for 3 h at 37 C after which the cells were transferred to fresh DMEM (serum and phenol red free).

After 48 h incubation, the medium was removed, and the cells were washed with PBS (Mg2+ and Ca2+ free) and harvested in 100 µl of reporter lysis buffer (Promega, Madison, WI). Luciferase activity in 10 µl of cell lysate was determined using 25 µl of luciferin reagent (Promega) by measuring the luminescence for 5 sec in a luminometer (MicroLumat Plus LB 96V, EG&G Berthold, Fisher Scientific, Nepean, Ontario, Canada). Transfection efficiency was determined by assay for ß-galactosidase activity according to the method described by Hall et al. (14) and used to normalize luciferase activity. Results shown are an average of at least three independent experiments, each of which was performed in triplicate, with error bars representing the SE (SEM).

Western analysis
SHM cells were seeded at 500,000 cells/well in 6-well plates and transfected with 1 µg of AP-1 expression vector (500,000 cells/µg, a per-cell amount in the same range as the two highest doses used in the transfection experiments detailed above) by using 10 µl of ExGen transfection reagent as described above and harvested in 200 µl radioimmunoprecipitation assay lysis buffer 24 h after transfection. Cells were scraped from the plate on ice in radioimmunoprecipitation assay lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% (vol/vol) Triton X-100, 1% (vol/vol) sodium deoxycholate, and 0.1% (wt/vol) sodium dodecyl sulfate (SDS), supplemented with 100 µM sodium orthovanadate and protease inhibitor cocktail tablets (Complete; Roche, Québec, Canada). Samples were spun at 12,000 x g for 15 min at 4 C, and the supernatant was transferred to a fresh tube to obtain a crude protein lysate. Protein concentrations were determined using the protein assay buffer (Bio-Rad Laboratories, Hercules, CA). Protein samples (20 µg) were suspended in Laemmli buffer, heated at 95 C for 5 min, and resolved by electrophoresis on a 10% SDS-polyacrylamide gel. Proteins were transferred onto polyvinylidene difluoride membrane (Millipore, Bedford, MA) in 48 mM Tris-HCl, 39 mM glycine, and 0.037% (wt/vol) SDS (pH 8.3) for 1.5 h at 300 mA at 4 C. The expression of each of the AP-1 proteins was verified by Western analysis as described by Oldenhof et al. (15), using the following primary antibodies: c-Jun (SC-45), JunB (SC-46x), JunD (SC-74x), c-Fos (SC-52x), Fra-1 (SC-605x), Fra-2 (SC-604), and FosB (SC-7203x, all from Santa Cruz Biotechnology Inc., Santa Cruz, CA).

Statistical analysis
Statistical analysis was carried out using SigmaStat (version 1.01, Jandel Corp., San Rafael, CA) with the level of significance for comparison set at P < 0.05. Transfection data were subjected to one- or two-way ANOVA using the Student’s-Newman-Keul’s method for pairwise comparison to determine differences between groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of AP-1 proteins in SHM cells
SHM cells were transiently transfected for 24 h with each of the AP-1 expression vectors to verify that proteins were expressed in SHM cells before the 48-h harvest time. Protein expression was assessed by Western analysis using antibodies specific to each member of the AP-1 family (Fig. 1Go). Proteins of the appropriate size were expressed by each of the AP-1 family expression vectors at levels dramatically higher than the basal levels of AP-1 proteins present in SHM cells transfected with empty expression vector (pcDNA3.1). With the exception of c-Jun, there were no AP-1 proteins detected except in the cells transfected with the appropriate expression vector. Basal levels of c-Jun were present in all pools of cells; however, the highest levels were detected in cells transfected with c-Jun expression vector. Higher levels of c-Jun protein were also repeatedly detected in cells transfected with JunD expression vector. More than one band was detected for all of the AP-1 proteins, depending on the exposure time, suggesting that these proteins were phosphorylated in SHM cells.



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  		FIG. 1. Transfected AP-1 proteins are expressed in SHM cells. SHM cells were transiently transfected for 24 h with the expression vectors for the AP-1 proteins as indicated. Parallel Western analysis was performed using the appropriate primary antibody to detect the expression of transfected proteins.

 
The effect of AP-1 protein transfection on the activity Cx43 promoter constructs in SHM cells
Whereas transfection with either c-Jun or c-Fos alone did not alter transcription from the Cx43 promoter (data not shown), transfection of c-Fos and c-Jun in combination with the Cx43 promoter-luciferase constructs, pCx1686-Luc and Cx300-Luc, lead to an increase in the level of transcription from the Cx43 promoter, determined by measuring luciferase activity (Fig. 2Go). Increasing the amount of AP-1 protein transfected further increased the level of transcription from the Cx43 promoter. Although the basal activity of pCx1686-Luc was higher than that of pCx300-Luc as previously reported by Chen et al. (6) (data not shown), there was no difference observed in the induction of these two constructs by c-Fos/c-Jun dimers, indicating that although the sequences upstream of the –300 position play a role in basal promoter activity, they are not involved in the response of the Cx43 promoter to c-Fos/c-Jun dimers (Fig. 2Go). In contrast, a 2-bp mutation of the proximal AP-1 site resulted in a significant reduction in the levels of luciferase activity, suggesting that the proximal AP-1 site is involved in the response of the Cx43 promoter to c-Fos/c-Jun dimers (P < 0.005, two-way ANOVA). Because there was no difference between the induction of pCx1686-Luc and pCx300-Luc by c-Fos/c-Jun, only the pCx300-Luc construct was used in the following experiments. A total of 0.1 µg of AP-1 expression vectors was used in the following experiments.



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  		FIG. 2. Induction of Cx43 promoter constructs by increasing amounts of transfected c-Fos/c-Jun dimers. SHM cells were transfected with either pCx1686-Luc (black bars), pCx300-Luc (dark gray bars), or pCx300(AP-1)M-Luc (light gray bars) and increasing amounts of either pcDNA3.1 alone (white bar) or a 1:1 mixture of c-Fos and c-Jun expression vectors for 48 h. The relative luciferase to ß-galactosidase activity is represented as the fold induction over cells transfected with the appropriate amount of control vector (pcDNA3.1). Values are an average of four independent experiments (with each experiment performed in triplicate) represented as mean ± SEM. A significant increase over the pCx300(AP-1)M-Luc transfected cells is indicated by ** (P < 0.005) or *** (P < 0.001).

 
All combinations of a Fos expression vector cotransfected with a Jun expression vector led to a significant increase in transcription from the Cx43 promoter (P < 0.05, one-way ANOVA), whereas transfection with only Jun expression vectors (with the exception of JunD) did not increase transcription from the Cx43 promoter (Fig. 3Go). From the Jun family, only expression of JunD protein alone significantly increased transcription from the pCx300-Luc promoter construct (P < 0.001, one-way ANOVA). When c-Jun or JunB was added in combination with JunD, the induction of transcription from this construct was not observed, although half the amount of JunD was used in these transfections to maintain the same total amount of expression vector.



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  		FIG. 3. The magnitude of the induction of the Cx43 promoter varies with cotransfection of different dimers of the AP-1 family of transcription factors. SHM cells were cotransfected with pCx300-Luc and different combinations of Jun and Fos proteins for 48 h. The relative luciferase to ß-galactosidase activity is represented as the fold induction over cells transfect-ed with the control vector (pcDNA3.1). Values are an average of three to four independent experiments (with each experiment performed in triplicate) represented as mean ± SEM. A significant increase over the control levels is indicated by * (P < 0.05), ** (P < 0.005), or *** (P < 0.001). Significant differences within eachgroup of dimers are indicated by different letters (P < 0.05). Differences between the induction of pCx300-Luc by dimers containing JunB are indicated ({Delta}{Delta}{Delta}) (P < 0.001), whereas differences between the induction of pCx300-Luc by dimers containing JunD are indicated ({circ}) (P < 0.05).

 
Expression of combinations of Fos and Jun proteins led to varied induction of the Cx43 promoter, depending on both the Fos and Jun protein expressed (Fig. 3Go). In the case of transfection with the c-Fos expression vector, the presence of JunD generated the highest activation of pCx300-Luc, c-Jun gave an intermediate level of activation, and transfection of JunB led to significantly lower activation of pCx300-Luc (P < 0.05, one-way ANOVA). A similar pattern was observed in cells transfected with Fra-1 expression vector; however, no significant differences were detected. In the case of transfection with the Fra-2 expression vector, an opposite pattern was observed in which expression of JunB generated the highest activation of pCx300-Luc, expression of c-Jun led to an intermediate activation, and expression of JunD gave significantly lower levels (P < 0.05, one-way ANOVA). Similar to Fra-1, no significant difference was detected in the induction of the Cx43 promoter for FosB transfected in conjunction with different Jun proteins. The presence of a particular Fos protein also modifies the magnitude of the induction of the Cx43 promoter. When the JunB protein was transfected in combination with the Fos proteins, significantly higher promoter activity was induced by Fra-2 (6.1-fold over control) and FosB (5.1-fold over control), compared with both Fra-1 and c-Fos (P < 0.001, one-way ANOVA). When JunD was transfected in combination with the Fos proteins, significantly higher promoter activity was detected only with FosB (5.3-fold over control, P < 0.05, one-way ANOVA). In the case of c-Jun, no difference was observed between the Fos proteins.

A 2-bp mutation introduced into the proximal AP-1 site in the Cx43 promoter significantly reduced the induction of the Cx43 promoter by most combinations of Fos and Jun proteins, consistent with the idea that this effect was mediated through the proximal AP-1 site (Fig. 4Go). Specifically, all combinations of c-Fos/Jun, Fra-1/c-Jun, Fra-1/JunD, Fra-2/JunB, FosB/JunB, and FosB/JunD mutation of the proximal AP-1 site led to a significant reduction in induced transcription from the Cx43 promoter, indicating that the effect of these proteins is through the proximal AP-1 site (P < 0.05, two-way ANOVA). Interestingly, a significant increase in luciferase activity from the mutated pCx300(AP-1)M-Luc was detected for all combinations of Fra-2/Jun and FosB/Jun, compared with the control vector (P < 0.001, two-way ANOVA).



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  		FIG. 4. Effect of mutating the proximal AP-1 binding site on the induction of the Cx43 promoter by different dimers of the AP-1 family of transcription factors. SHM cells were cotransfected with either pCx300-Luc (dark gray bars) or pCx300(AP-1)M-Luc (light gray bars) and different combinations of Jun and Fos proteins for 48 h. The relative luciferase to ß-galactosidase activity is represented as the fold induction over cells transfected with the control vector (pcDNA3.1). Values are an average of three to four independent experiments (with each experiment performed in triplicate) represented as mean ± SEM. A significant increase over the control levels is indicated by * (P < 0.05), ** (P < 0.005), or *** (P < 0.001). A significant difference between pCx300-Luc and pCx300(AP-1)M-Luc is indicated by {Delta} (P < 0.05), {Delta}{Delta} (P < 0.005), or {Delta}{Delta}{Delta} (P < 0.001).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies have shown that there is a strong correlation between the expression of Cx43 and AP-1 transcription factors in the myometrium during labor, specifically, both endocrine and mechanical signals increase expression of AP-1 transcription factors and of Cx43 (4, 5, 10, 16). In this study transient transfection experiments revealed that transcription from the Cx43 promoter was most strongly stimulated by the expression of Fos/Jun heterodimers in myometrial cells. Taken together these data suggest that AP-1 transcription factors might integrate these mechanical and hormonal signals to regulate Cx43 transcription and possibly the transcription of other genes containing AP-1 binding sites (e.g. oxytocin receptor, extracellular matrix components) that are required to initiate labor (17, 18, 19, 20, 21, 22, 23).

SHM cells were transiently transfected with expression vectors for the Fos and Jun proteins in the 18 different combinations in which stable dimers are formed. Importantly, we found that, in general, dimers comprising Jun family proteins do not strongly activate the Cx43 promoter, whereas dimers containing both Jun and Fos family proteins conferred up to a 6-fold higher activity of the Cx43 promoter. This is consistent with our previous data showing expression of c-Jun and JunD within the rat myometrium throughout pregnancy but no expression of Cx43 until near term (10). During late gestation, however, as expression of Fos family genes increases, both c-Jun and JunD may form complexes with the Fos proteins and positively influence Cx43 expression (Fig. 5Go).



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  		FIG. 5. Model for the involvement of AP-1 transcription factors in the expression of Cx43 and the onset of labor. A, Early in pregnancy only c-Jun and JunD are expressed in the myometrium leading only to the formation of Jun family homo- and heterodimers (dark circles), which are weak transcriptional activators at the Cx43 promoter, leading to little or no transcription of Cx43 in the myometrium. B, Before the onset of labor, there is an increase in Fra-2 expression in the myometrium, which drives the preferential formation of Fos/Jun family heterodimers (dark/light circles) containing Fra-2, which confer increased transcriptional activity at the Cx43 promoter. In addition, active ERK present in the myometrium can phosphorylate (P) Fra-2 and potentially further increase the transcriptional activity of Fra-2/Jun heterodimers, leading to the initial increase in Cx43 expression before the onset of labor. C, During labor maximal expression of all members of the Fos family leads to the highest transcriptional activity and maximal expression of Cx43 during labor.

 
The effect of c-Fos/c-Jun heterodimers on the Cx43 promoter appeared to be through only the proximal AP-1 site in the Cx43 promoter because deletion of the sequences upstream of the –300 position, containing the distal AP-1 consensus sequence, did not affect the response, whereas mutation of the proximal AP-1 site significantly reduced the effect of c-Fos/c-Jun transfection. Interestingly, the sequences surrounding the proximal AP-1 site in the Cx43 promoter are completely conserved between the mouse rat and human genes for at least 5 nucleotides (nt) on either side. This may be functionally significant in the regulation of Cx43 because nt up to one helical turn (10 nt) form the center of the AP-1 site have been shown to affect the orientation of the AP-1 dimer (24). Furthermore, nucleotides flaking consensus AP-1 sites have been shown to affect the binding of specific AP-1 dimers (25).

Although in many promoters consensus AP-1 sites are strongly activated by dimers of the Jun family, it has been shown that the sequences flanking a consensus AP-1 site can strongly affect the binding of particular AP-1 complexes. Studies by Ryseck and Bravo (25) investigating the ability of different AP-1 dimers to bind several gene specific and artificial promoters revealed that the sequences flanking consensus AP-1 sites influenced the binding of specific dimers. These differences appeared to be more dramatic in the case of Jun/Jun binding than Fos/Jun. Furthermore, the presence of a Fos protein often conferred strong binding to a site that was not bound by Jun/Jun complexes (25). Therefore, we speculate that the residues flanking the proximal Cx43 AP-1 site confer weaker binding of the Cx43 promoter by Jun/Jun dimers, preventing expression of Cx43 throughout gestation. Because the flanking sequences appeared to have less of an effect on the binding of Fos/Jun dimers, the Cx43 promoter may be strongly bound by Fos/Jun dimers during late gestation leading to the increased expression of Cx43.

Our data showed that the degree of transactivation of the Cx43 promoter was dependent on the specific Fos and Jun proteins present. c-Fos and Fra-1 displayed a dependency on the specific Jun partner, with c-Jun and JunD conferring increased activity, compared with JunB. This is in agreement with previous experiments investigating the ability of AP-1 dimers to bind an oligo containing the consensus AP-1 site in which complexes of both c-Fos and Fra-1 with JunB bound with lower affinity to the AP-1 consensus sequence than complexes with c-Jun (25). Although it has been observed that the addition of Fra-2 can suppress the activation of the collagenase promoter by c-Jun, this is clearly not the case for the Cx43 promoter in which complexes of Fra-2/c-Jun were all more activating than c-Jun alone (26). Complexes containing Fra-2 conferred higher activation than those containing c-Fos or Fra-1, with the Fra-2/JunB combination conferring the highest transcriptional activation. Furthermore, although mutation of the proximal AP-1 site affected the activation of the Cx43 promoter by c-Fos and Fra-1 complexes, Fra-2-containing complexes appeared to be less dramatically affected. This may represent the ability of a Fra-2-containing dimer to bind to a functional AP-1 half-site. This possibility has not been examined to date because many of the experiments characterizing the binding affinities of various AP-1 dimers did not include Fra-2 in their studies. Furthermore, this effect may be specific to the Cx43 promoter because binding affinities of Fos/Jun dimers are affected by the sequences flanking the AP-1 site (25).

The ability of Fra-2-containing dimers to dramatically increase transcription from the Cx43 promoter is interesting in the context of the onset of labor in that the levels of Fra-2 mRNA were found to increase earlier in gestation than the other members of the AP-1 family (10). This earlier increase in Fra-2 expression, beginning on d 21 of gestation, may act as part of a switch-initiating expression of Cx43 in the myometrium through the formation of dimers with c-Jun and JunD (both of which are expressed at constant levels throughout gestation, Fig. 5Go). On d 21 there is also a significant increase in the activation of ERK1/2 in the pregnant rat myometrium (15). It has been shown that hyperphosphorylation of the Fra-2 protein by ERK greatly increases the transcriptional activity of Fra-2/c-Jun dimers. These activated dimers have been shown to stimulate expression of the Fra-2 gene through two AP-1 sites located in the proximal promoter region, generating a positive autoregulatory loop (27). In the myometrium before the onset of labor, this activated ERK1/2 may lead to the hyperphosphorylation of Fra-2, increasing the transcriptional activity of Fra-2/Jun dimers (Fig. 5Go). Taken together, these data suggest that Fra-2-containing dimers contribute to the increased expression of Cx43 and subsequently the synchronization of myometrial contractile activity required for effective labor.

Regulation of Cx43 mRNA expression during gestation almost certainly involves other transcription factors in addition to the Fos and Jun family members. AP-1 proteins have been shown to interact with other transcription factors including the cAMP response element-binding protein/activating transcription factor family and Maf proteins as well as glucocorticoid and estrogen receptors (28, 29, 30, 31). Furthermore, transfection with either c-Jun or specificity protein-1 (which is present in the human myometrium) increased transcription from the human Cx43 promoter, with cotransfection of these transcription factors having an additive effect (9).

In summary, we have conducted an analysis of the transcriptional control of Cx43 by all members of the Fos/Jun family of transcription factors. Our data suggest that the presence of Jun family members in the myometrium during pregnancy is not sufficient to induce transcription of Cx43, but the dramatic increase in expression of Fos family members (in particular Fra-2) near term likely contributes to the increased expression of Cx43, and possibly other AP-1-containing labor genes, which together contribute to the initiation and progression of labor. Our analysis of AP-1 activation at the Cx43 promoter also offers more general insights into the transactivating properties of this family. Our data suggest that the specific dimer complex is critical to the transcriptional potential at target genes. To define the role of these transcription factors in target gene expression, information on the dimers expressed in the particular tissue/cell type or following specific challenges as well as the action of those dimers on the individual AP-1 element in that target gene is required.


   Footnotes
 
This work was supported by a grant from the Canadian Institutes of Health Research (37775) and a Doctoral Research Award from the Canadian Institutes of Health Research (to J.A.M.).

First Published Online December 23, 2004

Abbreviations: AP-1, Activator protein-1; Cx43, connexin 43; nt, nucleotides; SDS, sodium dodecyl sulfate; SHM, Syrian hamster myometrial.

Received August 16, 2004.

Accepted for publication December 15, 2004.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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