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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-0663
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 7 4151-4156
Copyright © 2005 by The Endocrine Society

Up-Regulation of Gap Junctional Intercellular Communication and Connexin43 Expression by Retinoic Acid in Human Endometrial Stromal Cells

Prasong Tanmahasamut and Neil Sidell

Division of Research, Department of Gynecology and Obstetrics, Emory University School of Medicine, Atlanta, Georgia 30322

Address all correspondence and requests for reprints to: Neil Sidell, Ph.D., Department of Gynecology and Obstetrics, Emory University School of Medicine, 1639 Pierce Drive, Atlanta, Georgia 30322. E-mail: nsidell{at}emory.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: Gap junctions, made up of connexins (Cxs), play fundamental roles in coordinating a number of cellular processes through their ability to directly regulate cell-cell communication. Cx43 is the most widely expressed Cx in the endometrium and is known to be important in a variety of physiological and pathological processes in this tissue.

Objective: In this study, we investigated the ability of the retinoid, all-trans-retinoic acid (RA), to regulate Cx43 expression in human endometrial stromal cells.

Design: Primary endometrial stromal cells obtained from patients undegoing surgery for infertility workup were treated in vitro with RA and control compounds for different time periods, up to 48 h. Cx43 mRNA and protein levels, protein phosphorylation, and gap junctional intercellular communication (GJIC) were analyzed.

Results: Treatment of the cells with RA showed a dose-dependent increase in Cx43 expression at both the mRNA and protein levels. In addition, RA induced a relative decrease in the phosphorylated species of Cx43 while causing a corresponding increase in the nonphosphorylated form. Concomitant with these changes, RA-treated cells demonstrated up to a 250% enhancement of GJIC as assessed by dye transfer experiments. Augmentation of GJIC and alterations of Cx43 expression were observed over the same range of RA concentrations. Treatment of cells with the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate increased the phosphorylated species of Cx43 and correspondingly inhibited GJIC.

Conclusions: Phosphorylation of Cx43 is inversely related to GJIC in endometrial stromal cells. Retinoids increase GJIC in endomentrial stromal cells through upregulation of Cx43 expression while inducing a decrease in the phosphorylated species of the protein. The data suggest a novel mechanism by which retinoids can influence endometrial cell biology.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CONNEXIN (Cx) PROTEINS are the major, if not only, component of gap junctions. So far, about 20 family members of Cx proteins have been identified ranging in size from 26–56 kDa identified from genomic sequences (1). These Cxs are differentially expressed in a variety of tissues, which is generally believed to reflect cell-specific regulation of gap junctional coupling and functional demands for gap junctions in different cell types. Cx43 is the most widely expressed Cx (2) and has been the subject of intense investigation because its expression is frequently decreased or aberrantly expressed in a variety of pathological conditions, including cancer. In this regard, restoration of gap junctional intercellular communication (GJIC) by induction or transfection of Cx43 can reverse the transformed phenotype of certain cancer types, including those derived from breast, cervix, and muscle (3, 4, 5). Although many tumor-promoting agents, oncogenes, and growth factors inhibit Cx43 (2, 6), antineoplastic agents such as retinoids and vitamin D have been shown to up-regulate Cx43 (7, 8, 9). Concurrent with modulation of GJIC, several growth factors and cytokines also alter the phosphorylation status of Cx43 (2). These observations led to the demonstration that increased phosphorylation of Cx43 mediated by two serine/threonine protein kinase families, protein kinase C (PKC) (10) and MAPK (11), is causally linked with disruption of GJIC.

The main Cxs expressed in endometrium are Cx43 and Cx26, with the major Cxs expressed in endometrial stromal and epithelial cells being Cx43 and Cx26, respectively (12). Recent studies have suggested that gap junction protein in endometrial stromal cells play a regulatory role in maintaining normal levels of GJIC in the epithelial cells (13). Thus, it appears that the expression of Cx43 in endometrial stromal cells functions in the maintenance and regulation of endometrial gap junction proteins in both cell populations. This expression of Cx43 is a dynamic process. Levels of Cx43 in endometrium increase during the follicular phase under the influence of estrogen and are reduced at the luteal phase of the menstrual cycle in response to progesterone (14). These cycle-associated patterns of Cx43 expression in endometrium point to a physiological role of Cx43 in the implantation process; Cx43 expression is known to be dramatically reduced during the implantation window, which reduces cell-to-cell communication (14). This process may facilitate the invasion of the trophectoderm through the stromal cells and, in addition, promote angiogenesis (15, 16). This role of Cx43 in reproduction is consistent with its demonstrated function in malignant progression where forced expression of Cx43 has been shown to inhibit tumor cell growth and invasive potential and to retard neovascularization (3, 4, 5, 17).

Retinoic acid (RA) and other retinoids have been shown to up-regulate GJIC and Cx43 levels in a variety of normal and malignant cell types (18). This activity, along with the well-characterized functions played by Cxs in embryonic implantation and development, suggest a possible link between the known teratogenic effects of RA (19) and its modulation of Cx activity. These facts have prompted us to assess the regulatory properties of RA on Cx43 expression in human endometrial stromal cells. The results indicate that RA can stimulate GJIC in this cell type by modulating the levels and phosphorylation profile of Cx43. This finding may have important implications in understanding some of the known action of retinoids in reproductive biology.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

The primary endometrial stromal cells were obtained from patients undergoing surgery for infertility workup, according to protocols approved by the Institutional Review Board, Emory University School of Medicine (Atlanta, GA). Separation of endometrial and stromal cells from eutopic endometrial tissue was performed using the procedure developed by Ryan et al. (20) and Hornung et al. (21). Cells were grown and maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM glutamine, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 1 µg/ml Fungizone (complete medium).

Treatment of cells

All-trans-RA and 12-O-tetradecanoylphorbol-13-acetate (TPA) were obtained from Sigma Chemical Co. (St. Louis, MO). RA was diluted in dimethyl sulfoxide (DMSO) to a stock concentration of 50 mM. TPA was diluted in DMSO to a stock of 50 µM. RA and TPA were diluted to the indicated concentration in complete medium for the experiments. The final concentration of DMSO as solvent was always less than 0.1%. Treatment of the cells with vehicle control or RA was for the time periods and concentrations as described in figure legends. Experiments in which treatment with TPA was performed were at a final concentration of 50 nM for the last 30 min of culturing.

RNA isolation, RT-PCR, and real-time PCR

Total RNA was isolated from cells using TRI-reagent (Sigma Chemical Co.) following the provider’s protocol. Total RNA extracts were frozen at –80 C until analyzed. cDNA was synthesized from mRNA samples and subsequently used as template for conventional and real-time PCR assays. Analysis of amplicons was visualized on 1% agarose gel containing 0.2 µg/µl ethidium bromide. A 100-bp ladder (Promega, Madison, WI) was used as a size standard. Primers for amplifications were made by Sigma-Genosys, and the sequences were as follows: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense, 5'-CCATGGAGAAGGCTGGG-3'; GAPDH antisense, 5'-CAAAGTTGTCATGGATGAC-3' (approximately 200-bp amplicon); Cx43 sense, 5'-TACCATGCGACCAGTGGTGCGCT-3'; and Cx43 antisense, 5'-GAATTCTGGTTATCATCGTCGGGGAA-3' (approximately 290-bp amplicon).

For real-time PCR, we used the Qiagen Master Mix kit and followed the vendor guidelines, with some modifications. A total reaction volume of 25 µl contained 12.5 µl Master Mix, 2 µl 25 mM MgCl2, and 0.25 µl 25x SyBr Green (BioWhittaker Molecular Applications, Rockland, ME). For Cx43, 5 µl cDNA was used, and 3 µl was used for GAPDH control. Samples were processed using the Cephied Smart Cycler software (Cephied Systems, Sunnyvale, CA) under the following conditions: one denaturation cycle of 95 C for 30 sec followed by 32 amplification cycles of 95 C for 10 sec, 60 C for 15 sec, and 72 C for 40 sec.

Melt curve analysis of each sample was supplemented with agarose gel electrophoresis of randomly selected samples to confirm the success of reactions. Fluorescence spectra were recorded during the annealing phase of the reaction. Second derivative analysis of the amplification curves was done to arrive at the threshold cycles for each sample. The following formula was used to arrive at the fold increase Cx43 mRNA level for each sample:


where C = cycle threshold for Cx43 or GAPDH mRNA detection in control samples, and T = cycle threshold for Cx43 or GAPDH mRNA detection in treatment samples.

Western-blot analysis

Western-blot analysis was performed on whole-cell extracts that were obtained by direct dissolution of cell pellets in sample buffer (1 mM NaHCO3, 0.2 M phenylmethylsulfonylfluoride, 0.1 M NaVO4, and 1 M NaF), followed by protein determination using a bicinchoninic acid protein assay kit (Sigma Chemical Co.). Protein (20 µg) from cells treated with medium (control), RA, or TPA was loaded on the 12% SDS-PAGE gel, then transferred to nitrocellulose membrane and blocked with 5% skim milk in PBS. For treatment with alkaline phosphatase, 20 µg lysate protein was treated with 40 U calf intestine alkaline phosphatase (Roche Diagnostics, Indianapolis, IN) for 60 min at 37 C. Total Cx43 was detected using the rabbit polyclonal anti-Cx43 antibody (Zymed, South San Francisco, CA), then incubated with the secondary antibody linked to horseradish peroxidase. The immunoreactive bands were visualized by the Enhanced Chemiluminescence System (Amersham Biosciences AB, Uppsala, Sweden). Blots were washed, reprobed with an anti-ß-actin antibody, and developed in an identical manner for assessing ß-actin protein levels to ensure even loading.

Scrape loading (SL)/dye transfer (DT)

Levels of GJIC in control and treated cultures were determined using the SL/DT technique (22), using a fluorescent dye, Lucifer Yellow (LY) (Molecular Probes, Eugene, OR). Primary endometrial stromal cells, cultured as described above, were washed thoroughly with PBS. SL was performed applying three cuts on cell monolayer with a razor blade, and then the LY was added to the cells. The dye was rinsed away after 5 min. Cells were washed three times with PBS, fixed with 4% paraformaldehyde, and cells stained with LY were detected by fluorescence emission with an inverted fluorescent microscope equipped with a camera. Cells that received the LY from the scrape-loaded cells were considered communicating. The numbers of communicating cells in the untreated and treated cultures were counted. GJIC was expressed as percentage of the control.

Statistical analysis

All of the experiments have a minimum of three determinations. The data were expressed as mean ± SEM. The data in some figures are from a representative experiment, which was qualitatively similar in the replicate experiments. Statistical significance (P < 0.05) was determined with Student’s t test (two-tailed) between an individual experimental group and the corresponding control condition set as 100% (one-sample t test).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RA up-regulates Cx43 mRNA expression

We have examined the effects of RA on Cx43 mRNA levels in primary endometrial stromal cells using RT-PCR. Figure 1Go shows stimulation by RA of Cx43 mRNA after 2 d of culture; treatment with 1 and 10 µM RA resulted in a greater than 2-fold enhancement of Cx43 mRNA levels as quantified by real-time RT-PCR (Fig. 1BGo). No significant effects were seen with 0.1 µM RA. Time course studies demonstrated that significant enhancement of Cx43 mRNA by 10 µM RA was evident as early as 12 h of treatment (Fig. 1CGo). In contrast, RA had no effect on the growth, morphology, or viability of the stromal cells even at the highest concentration tested (10 µM).



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  		FIG. 1. Effect of RA on Cx43 mRNA expression. Total RNA was isolated from endometrial stromal cells that were treated with RA under different conditions. A, Result of a representative experiment as assessed by RT-PCR of cultures that were treated with different concentrations of RA for 48 h. Lane 1, Control; lane 2, RA 0.1 µM; lane 3, RA 1 µM; lane 4, RA 10 µM. B and C, Results of 48-h treatment with different concentrations of RA (B) and with 10 µM RA for different time periods (C) represent fold-increased Cx43 mRNA levels that were quantified by real-time RT-PCR as described in Materials and Methods. Columns represent the mean (±SEM) of at least three determinations for each treatment condition. *, Significant difference when compared with vehicle control, P < 0.05.

 
Effects of RA on Cx43 protein levels

Using Western blotting, we investigated the effects of RA on Cx43 protein expression and phosphorylation status. Figure 2Go indicates that endometrial stromal cells exhibited three clearly distinct immunoreactive bands (41, 42, and 45 kDa; designated P0, P1, and P2, respectively). The lower strong band (P0) corresponds to the nonphosphorylated form of Cx43 (lane 1) as shown by the fact that it aligned with Cx43 dephosphorylated by alkaline phosphatase (lane 2). After treatment with RA for 2 d, lane 3 shows a change in the relative levels of the Cx43 species; P0 is enhanced, whereas P1 and P2 show relative decreases compared with control cultures. Further treatment of lysate with alkaline phosphatase confirmed that the enhanced lower (P0) band induced by RA was due to dephosphorylation of P1 and P2 (lane 4). Densitometric analysis of total Cx43 protein expression (P0 + P1 + P2) demonstrated that RA up-regulated Cx43 protein levels approximately 1.2-fold at the same concentrations showing enhancement of mRNA expression (Fig. 2BGo).



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  		FIG. 2. Effect of RA on Cx43 protein and phosphorylation. A, Lysate of endometrial stromal cells that had been treated with RA (10 µM) or vehicle control as indicated for 48 h were incubated with and without alkaline phosphatase and analyzed for Cx43 expression by Western blotting. B, Data obtained by densitometry and representing the mean (±SEM) of Cx43/actin band densities of at least three determinations for each RA concentration. *, Significant difference when compared with vehicle control, P < 0.05.

 
RA enhances GJIC

To determine the extent to which Cx43 expression and phosphorylation status is related to GJIC in endometrial stromal cells, we performed SL/DT assays using the gap junction permeable fluorescent dye LY. As assessed by this technique, serum-starved cells were communication-competent and transferred LY to numerous cells distant to the wound edge. The presence of RA in the cell medium caused up to a 250% increase in the number of cells that showed LY fluorescence (Fig. 3AGo). This effect was observed over the same range of RA concentrations as demonstrated for RA-induced increases of Cx43 mRNA and protein levels (Fig. 3BGo).



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  		FIG. 3. RA increases GJIC. A, Endometrial stromal cells were treated with RA (10 µM) or vehicle control for 48 h, after which GJIC was assessed by the SL/DT technique as described in Materials and Methods. B, Mean (±SEM) of the number of cells that show positive dye staining of at least three determinations for each RA concentration. Values are expressed relative to those obtained after treatment with vehicle control. *, Significant difference when compared with vehicle control, P < 0.05.

 
TPA-induced phosphorylation of Cx43 antagonizes the effects of RA

Reports by a number of investigators have demonstrated that treatment of cells by the PKC activator TPA stimulates phosphorylation of Cx43, which reduces its ability to function in GJIC (23, 24, 25). Because we have shown that RA decreased the phosphorylated species of Cx43 and increased GJIC, we were interested in whether TPA could antagonize these RA-induced effects. As shown in Fig. 4Go, treatment with RA alone resulted in a single strong band at 41 kDa, corresponding to the nonphosphorylated form of Cx43 (lane 2). As expected, culturing with TPA alone showed a modest change in phosphorylation as exemplified by the appearance of higher order phosphorylated species (lane 4). Together, TPA inhibited the dephosphorylation of Cx43 induced by the RA treatment (lane 3).



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  		FIG. 4. Antagonistic effects of TPA on RA-induced dephosphorylation of Cx43. Endometrial stromal cells were cultured with vehicle control or RA for 48 h and TPA (50 nM) for the last 30 min of treatment, after which time Western-blot analysis was performed. Lane 1, Control; lane 2, RA 10 µM; lane 3, RA 10 µM + TPA 50 nM; lane 4, TPA 50 nM.

 
To assess whether changes in the phosphorylation patterns after RA ± TPA treatment were associated with alterations in GJIC, SL/DT experiments were performed. Figure 5Go shows that TPA completely reversed RA enhancement of GJIC. TPA treatment by itself resulted in a reduction of GJIC in comparison with control cultures and was quantitatively similar to values obtained wtih RA/TPA-treated cells.



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  		FIG. 5. TPA antagonizes RA-mediated enhancement of GJIC. Endometrial stromal cells were treated with vehicle control or RA (10 µM) for 48 h and TPA (50 nM) for the last 30 min of culturing as indicated. GJIC was then assessed by the SL/DT technique. The graph represents the mean (±SEM) of the number of cells that show positive dye staining of three independent experiments. Results are expressed relative to those obtained after treatment with vehicle control. *, Significant difference when compared with vehicle control, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Direct cell-cell communication mediated by Cxs has been implicated in coordinating proliferation and differentiation processes, probably mediated through exchange of second messengers such as cAMP and inositol 1,4,5-triphosphate, as well as Ca2+ (26, 27). Correspondingly, loss of cell-cell communication through inappropriate expression of Cxs may be responsible for dedifferentiation processes leading to tumorigenesis. A report by Regidor et al. (28) has shown chronically decreased levels of Cx43 in endometriotic stromal cells, comparable in expression with their normal counterpart during the implantation window portion of the reproductive cycle (14). These and other results (29, 30) suggest that Cx43 plays a key role in the maintenance of normal human endometrium and that its aberrant expression may be involved in the development or progression of endometriosis. Taken together, these reports suggest the possibility that pharmacological manipulation of Cx43 expression may have therapeutic potential in some disease states.

Several studies have demonstrated the ability of RA to modulate Cx43 expression, with the nature of the regulation (up or down) depending on the cell type (31, 32, 33, 34). For example, retinoids have been shown to increase Cx43 mRNA and protein levels in certain human cancer cell types, mouse fibroblasts, and rat liver cells, whereas they decrease Cx43 expression and GJIC in p19 embryonal carcinoma cells and human pluripotential teratocarcinoma cells (32, 33, 34). Although the mechanism(s) responsible for these divergent effects is unknown, it is speculated that they may involve the influence of cell-type-specific transcriptional regulators (e.g. coactivators or corepressors). In the present work, we have demonstrated that RA up-regulated Cx43 mRNA, protein expression, and resulting GJIC in human endometrial stromal cells. The ability of this cell type to serve as RA-responsive target tissue is supported by studies showing the presence in these cells of RA nuclear receptors (i.e. RARs and RXRs) (35, 36) and by our previous work demonstrating RA-induced modulation of IL-6 production (37). In addition to increasing Cx43 mRNA and protein levels, our data showed that RA altered the phosphorylated status of Cx43 protein such that there was a decrease in the phosphorylated (P1 and P2) species accompanied by an increase in the nonphosphorylated (P0) form. Treatment with alkaline phosphatase resulted in the total disappearance of the slower migrating bands, confirming that these bands corresponded to the phosphorylated forms of Cx43. Because phosphorylation of Cx43 is believed to be causally linked with disruption of GJIC (10, 11), our results demonstrate, for the first time, that RA can enhance GJIC through this posttranslational mechanism of action. This effect may be distinctive to endometrial stromal cells because retinoid-induced dephosphorylation of Cx43 has heretofore not been detected in other cell types (38, 39, 40).

To confirm the cause-and-effect relationship between Cx43 phosphorylation status and GJIC, we treated the cells with TPA in a manner previously shown to stimulate phosphorylation of Cx43 in other cell systems (23, 24, 25, 41). Results showed that TPA induced phosphorylation of Cx43 in the stromal cells and antagonized the dephosphorylation effects of RA. Correspondingly, SL/DT experiments indicated that TPA inhibited GJIC activity, both by itself and in the presence of RA. These findings suggest that RA’s dephosphorylation effects on Cx43 may be through its ability to inhibit PKC activity. RA has been shown to antagonize the effects of TPA and inhibit PKC in a number of cell systems (42, 43, 44), although stimulation of PKC by RA has also been described (45). In addition to PKC, MAPK is another kinase family that has been connected to phosphorylation of Cx43 occurring together with decreased GJIC (11). Because inhibition by RA of MAPK has also been demonstrated (44), the kinase signaling pathway(s) involved in the phosphorylation effects of RA on Cx43 remains to be elucidated.

Evidence suggests that retinoid signaling may be an important part of the process of embryo implantation; endogenously produced RA and its nuclear receptors have been detected in both the implanting blastocyst and in decidual cells at the site of implantation (46, 47). With regard to the latter, in vitro studies have shown that RA can specifically suppress endometrial decidualization (48). The decidua, derived from stromal cells, control the invasive nature of the trophoblast (49). Our finding that RA can modulate GJIC expression in stromal cells combined with the importance of cell-to-cell communication in regulating embryo implantation provide compelling new information regarding a possible mechanism by which retinoids can influence this process. Although RA has long been known to be a potent teratogen, recent evidence demonstrates that pharmacological (micromolar) concentrations of RA is embryotoxic at the early postimplantation stage of development (50). This observation is consistent with the possibility that RA at such concentrations may reduce effective trophoblast invasive capacity through increased GJIC. As such, our results suggest that RA inhibition of blastocyst implantation through this mechanism of action is partly responsible for embryotoxicity after systemic retinoid administration (50, 51).

Taken together, our findings provide the first evidence that RA regulates Cx43 expression in human endometrial stromal cells. RA treatment of the cells induced an increase in the protein, mRNA levels, and relative abundance of the nonphosphorylated species of Cx43, resulting in enhanced GJIC. These novel findings suggest a heretofore unknown role of retinoids in endometrial physiology and pregnancy.


   Acknowledgments
 
The authors acknowledge the excellent technical assistance of Lijuan Hao during the course of these studies.


   Footnotes
 
This work was supported by National Institutes of Health Grant CA85589 and by a Siriraj Hospital Foundation grant from the Faculty of Medicine Siriraj Hospital, Mahidol University (Bangkok, Thailand).

First Published Online April 5, 2005

Abbreviations: Cx, Connexin; DMSO, dimethyl sulfoxide; DT, dye transfer; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GJIC, gap junctional intercellular communication; LY, Lucifer Yellow; PKC, protein kinase C; RA, retinoic acid; SL, scrape loading; TPA, 12-O-tetradecanoylphorbol-13-acetate.

Received April 20, 2004.

Accepted March 29, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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