Differential expression and regulation of connexin-43 and cell-cell ...

0013-7227/95/$03.00/O Endocrinology Copyright 0 1995 by The Endocrine

Vol. 136, No. 12 Printed in U.S.A. Society

Differential Expression and Regulation of Connexin-43 and Cell-Cell Coupling in Myocytes from the Circular and Longitudinal Layers of Bovine Myometrium” F. DOUALLA-BELL,

S. J. LYE?,

F. LABRIE,

AND

M. A. FORTIER

Unite’ de Recherche en Ontogtkie et Reproduction (F.D.-B., M.A.F.), Laboratoire d%ndocrinologie Mokulaire (F.L.), Le Centre Hospitalier de l’llniversitk Laval, Quebec, Quebec; and the Program in Development and Fetal Health, Samuel Lunenfeld Research Institute, Mount Sinai Hospital (S. J.L.), and the Department of Obstetrics and Gynecology, University of Toronto (S.J.L.), Toronto, Ontario, Canada ABSTRACT

Cx-43 mRNA was decreased after treatment with 0.1 PM EM-139 (1.37 2 0.25, Cx-43 over 18s RNA) compared to that in untreated cells (2.38 t 0.46, Cx-43 over 18s RNA), representing a 40% inhibition. In parallel, cell-cell coupling and the amount of Cx-43 protein were also reduced after antiestrogen treatment. In contrast, treatment of cells from the longitudinal layer with the antiestrogen did not significantly affect the level of Cx-43 mRNA, protein, or cell-cell coupling. These data demonstrate that Cx-43 protein and mRNA are expressed and regulated differentially in myocytes from the circular and longitudinal layers of bovine myometrium. Furthermore, the circular myometrial layer may represent a preferential target for estrogen regulation of the biochemical and mechanical processes controlling contractility. (Endocrinology 136: 5322-5328, 1995)

The expression and localization of the gap junction protein connexin-43 (Cx-43) as well as functional coupling were studied in myocytes from the two layers of the bovine myometrium: the circular and the longitudinal layers. Intercellular communication (measured by Lucifer yellow dye transfer through gap junctions) was more intense in the circular than in the longitudinal layer of the bovine myometrium. The circular layer also exhibited a greater degree of punctuate immunofluorescence to Cx-43. Myocytes from the circular layer expressed more Cx-43 messenger RNA (mRNA, 2.38 ? 0.46, Cx-43 over 18s RNA) than the longitudinal layer (1.46 ? 0.48, Cx-43 over 18s RNA; P < 0.05). The modulation of Cx-43 expression by sex steroids in the two myometrial layers was tested using a pure steroidal antiestrogen, EM-139. In myocytes from the circular layer, the level of

G

Al’ JUNCTIONS are intercellular channels that allow the exchange of low mol wt metabolites, ions, and second messengersbetween adjacent cells. Each channel or connexon is composed of a hexamer of connexin proteins. The connexins comprise a multigene family of gap junction proteins whose expression is regulated in both a temporal and tissue-specific fashion. Within the myometrium, the major gap junction protein is connexin-43 (Cx-43) (1). Myometrial gap junctions contribute to the mechanical and electrical coupling in this tissue (2) and enable the myometrium to develop the synchronous intense contractions required during labor (3). The expression of myometrial gap junctions (4) and Cx-43 transcript (5,6) hasbeen correlated with a decrease in the progesterone/estrogen ratio in viva. Although the Cx-43 gene is responsive to estrogen (71, the precise mechanism by which this steroid acts to regulate gap junction formation at the cellular level in the myometrium is unknown. The effects of estrogen have been difficult to demonstrate Received May 5,1995. Address requests for reprints to: Dr. Michel A. Fortier, Unite de Recherche en Ontogenie et Reproduction, Le Centre Hospitalier de l’Universit6 Laval, 2705 boulevard Laurier, Quebec, Quebec, Canada GlV 4G2. * This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (to M.A.F.), a studentship from Lava1 University (to F.D.-B.), and a Medical Research Council Group Grant in Development and Fetal Health (to S.J.L.). t Career Scientist of the Ontario Ministry of Health.

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in z&o in primary cultures of myocytes (8). The mechanism responsible for this phenomenon is unknown, but perhaps includes a loss of some in viva inhibitory regulatory mechanisms,an increased sensitivity of cultured cells to the basal estrogenic activity present in culture medium, or a loss of estrogen receptor expression during culture (9). The latter hypothesis is unlikely, as we have demonstrated that the pure antiestrogen, EM-139, is able to influence several aspects of myocyte function/expression in our cell culture system (10). The pure antiestrogenic properties of this new classof antiestrogen (ll), a steroidal derivative, have been described on the uterus (10, 12). There is increasing evidence to suggest that the circular and longitudinal layers of the myometrium are functionally and morphologically distinct. The heterogeneity of the smooth muscle cellsfrom the circular and longitudinal layers has been described at the histological (131,biochemical (1416), and pharmacological (17,18) levels. We have shown that this heterogeneity is maintained in cultured myocytes isolated from the two layers of bovine myometrium (10). The bovine is particularly well suited as a model for our studies; not only it is possible to obtain slaughterhouse tissues at a definite time of the estrous cycle (191,but the different layers of the uterus can also be separated mechanically before enzyme isolation of the myocytes. In the present study we investigated the possibility that Cx-43 may be differentially expressed within the two layers of the myometrium and that the responsivenessof this gene

EXPRESSION

OF Cx-43 ON MYOMETRIAL

to estrogen might also be dependent upon the layer from which the cells are derived. Cx-43 expression and gap junctional intercellular communication were measuredby a combination of Northern and Western blotting, immunofluorescence, and Lucifer yellow dye transfer in cells or tissue derived from the circular or longitudinal muscle layers of the bovine myometrium. The steroid sensitivity of these parameters was studied with the use of the antiestrogen, EM-139. Materials Myometrial

and Methods

tissue and cell preparation

Bovine uteri were collected at local slaughterhouses, and myometrial cells were prepared as previously described (10). The stage of the estrous cvcle at which the uteri were collected was estimated bv examination of ovarian morphology (19). To prepare cells, uterine horns were asepticallv removed and washed in sterile Ca’+- and ME’+free Hanks’ Balanced Salt Solution (HBSS) containing amphocyclin?500 U/ml), buffered with HEPES (20 mM) and sodium bicarbonate, and saturated with 5% C02-95% 0, at pH 7.4. Circular and longitudinal myometrial layers were separated sequentially by dissection. A portion of the tissue sample was frozen for subsequent extraction of total RNA. The remainder was transferred to HBSS containing trypsin (0.3%) and incubated at room temperature for 3 h. At the end of the incubation period, myometrial tissues were washed twice with 20 ml HBSS before transfer to the second digestion solution containing trypsin (0.03%), collagenase (0.064%), and deoxyribonuclease I (0.016%). The mixture was incubated for 1 h in a freshly gassed enzyme solutions in a shaking water bath (37 Cl. After incubation, the fragments were filtered through a nylon mesh. Proteolytic enzymes were neutralized by addition of 10% calf bovine serum. The filtrated cells were then centrifuged at 800 X g for 12 min, washed twice in 30 ml HBSS, and cultured in RPM1 1640 supplemented with 10% steroid-stripped fetal calf serum (FBS-DC). The average estradiol concentration in the medium supplemented with serum was 5 PM (8). Cells were cultured in the presence or absence of EM-139 (a gift from Dr. Fernand Labrie, Endocrinologie Moleculaire, University of Laval, Quebec, Canada) for the entire experiment. Medium with or without EM-139 was changed every 2 days. Cells were maintained at 37 C in an atmosphere of 5% CO, in humidified air. When they had reached confluency, the medium was changed to RPM1 containing no phenol red or serum (with or without EM-139). The dose and duration of exposure to EM-139 used in the current study represent the optimal time and dosage from our previous studies in which we examined the effects of this antiestrogen on prostaglandin production by the circular and longitudinal layers of the myometrium (10). I

Scrape-loading

and dye transfer

Cells were rinsed with PBS before addition of the fluorescent dye mixture. Two milliliters of 0.05% Lucifer yellow dissolved in PBS were added to the cell culture at room temperature, and the cells were subsequently scrape-loaded using the back of a scalpel blade, as described by El-Fouly and collaborators (201. Cells were incubated with the dye solution for 2 min, then rinsed with PBS to remove detached cells and background fluorescence. Cells were examined under an epifluorescence phase microscope, and photomicrographs were taken using the same exposure parameters for all fields and cell types. Quantitative estimation of intercellular communication through gap junctions was analyzed by Bioimage-Visage 110s (Millipore Corp., Ann Arbor, MI) using ZD-GEL software.

Northern

LAYERS

bond-N, catalog no. RPN.303N, Amersham Corp., Oakville, Canada) in 20 X SSC (3 M NaCl and 0.3 M Na citrate). The membrane was exposed to UV light for 3 min, prehybridized in a buffer containing 50% formamide, 6X SSPE (900 AM NaCl, 60 mM NaH,PO, . H,O, 6mM EDTA, pH 7.4),5 X Denhardt’s solution (500 mg Ficoll, 500 mg polyvinylpyrrolidone, 500 mg BSA; fraction V in 50 ml H,O), and 0.5% sodium dodecyl sulfate (SDS) and then hybridized in the same solution with the Cx-43 urobe (a gift from Dr. David Paul, Deuartment of Anatomv and Cell B&logy, H&ard Medical School, Boston, MA) for 18 h at 42 C. The probe was labeled with [a-32PJdeoxy-CTP to specific activity of lo* cpm/pg using random priming (Multiprime DNA labeling system, Amersham). After different washes with a final stringency of 0.1 X SSC at 65 C, the membrane was placed under x-ray film (Eastman Kodak, Rochester, NY) for a 48-h exposure period. For consecutive hybridization with the ribosomal subunit 18Sspecific probe, the Cx-43 probe was removed from the membrane by immersion in a boiling solution of 0.1% SDS (wt/vol) and cooling to room temperature. The relative intensities of the resulting autoradiographic images for Cx-43 messenger RNA (mRNA) and 18s ribosomal RNA (rRNA) were analyzed by BioimageVisage 110s (Millipore Corp., Ann Harbor, MI) using whole band analysis software.

Western blot analysis Cells were rinsed with PBS and lysed in a homogenization buffer [50 mM Tris (pH 8.3),150 mM NaCl, 50 mM ZnCl,, 50 mM O-phosphate, 2 mM EDTA, 1 mM NazVO,, 2 mM phenylmethylsulfonylfluoride, 1% Nonidet P-40,0.1% SDS, and 0.1% sodium deoxycholatel. Cell lysate was centrifuged at 10,000 X g for 20 min at 4 C. The supernatant was collected, and the protein content was determined using the method of Bradford (22). Protein (100 pg) was precipitated with ethanol 75% (vol/ vol), then lyophylized and resuspended in the sample buffer (1% SDS, 10% glycerol, and 50 m&r Tris, pH 6.8). The samples were boiled at 100 C for 2 min, then loaded on a 12% acrylamide gel and transferred to a polyvinylidene difluoride membrane (Millipore) using a semidry transfer method (Trans Blot SD, Bio-Rad Laboratories, Richmond, CA) for 30 min at 20 V. The membrane was blocked with 3% BSA in a 25 mM Tris-HCl and 156 mM NaCl, pH 7.5, buffer (TBS). The membrane was incubated with the anti-Cx-43 polyclonal antibody (prepared by S.J.L.) and used at 1:lOO dilution in TBS buffer containing 0.1% Tween 20. An alkaline phosphatase-labeled goat antirabbit antibody was used to visualize the protein-Cx-43 antibody complex.

Immunofluorescence

of Cx-43

Cells at confluency were fixed in 2% paraformaldehyde for 10 min and washed with PBS containing 0.05% Tween 20 (PBS-T). Nonspecific binding was prevented by the use of a blocking solution (1% BSA, 5% normal goat serum, 0.05% Tween 20, and 0.3% Triton X-100 in PBS) at room temperature for 30 min. Cells were then incubated with the primary antibody against Cx-43 (1:lOO dilution) overnight at 4 C. Cells were washed three times with PBS-T and incubated in the presence of a goat antirabbit immunoglobulin G (1:50 dilution), conjugated with fluorescein isothiocyanate (Jackson Immunoresearch Laboratory, Bar Harbor, ME), for 1 h at 4 C. After three washes with PBS-T, cells were fixed with one drop of 0.02% phenylenemide in a 1:l dilution of 0.2 M sodium carbonate and glycerol and examined under a fluorescence microscope.

Data analysis Statistically using unpaired

significant Student’s

differences between groups were analyzed t test. P < 0.05 was considered significant.

Results

blot analysis

Myocytes from the circular and longitudinal layers at confluency were washed with HBSS, pH 7.4, and total RNA was isolated according to a modification of the method of Chomczynski and Sacchi (21). Total RNA (20 Fg/lane) was subjected to electrophoresis (3 h, 75 V) on 1.2-6% agarose-formaldehyde gels. The RNA was visualized with ethidium bromide before transfer onto a nylon membrane of neutral charge (Hy-

5323

Immunofluorescence

of Cx-43

The pattern of Cx-43 protein in the two smooth muscle layers of the bovine myometrium was evaluated using double antibody immunofluorescence (Fig. 1). The polyclonal antibody against Cx-43 revealed characteristic punctuate

EXPRESSION

5324

OF Cx-43 ON MYOMETRIAL

LAYERS

Endo. 1995 Vol 136 . No 12

FIG. 1. Morphology of myocytes from the circular (A) and longitudinal (B) layers of bovine myometrium at midcycle (magnification, X 125; phase contrast). Immunofluorescence staining of Cx-43 in myocytes from the circular (C) and longitudinal (D) layers of bovine myometrium is shown. Anti-Cx-43 polyclonal antibody preferentially stains the myocytes from the circular layer with characteristic punctuate staining.

_ 0 I

I

Tissues

b

,.

185-W

c

d

..

i

T

d

Myocytes

FIG. 2. Cx-43 mRNA levels in the tissues and myocytes obtained from the circular and longitudinal layers of bovine myometrium. Expression of Cx-43 mRNA was measured as described in Materials and Methods and analyzed relative to that of the 1% ribosomal RNA. Values represent the mean -C SEM of three separate experiments.

in myocytes derived from the circular and longitudinal layers of bovine myometrium. Although numerous Cx-43-positive spots were found in myocytes from the circular layer, only a few positive spots were detected in the longitudinal layer.

staining

Northern

a

Circular Layer Longitudinal Layer

blot analysis

The expression of Cx-43 mRNA was quantified in tissues (in viva) and confluent myocytes (in vitro) from the circular and longitudinal layers of bovine myometrium (Fig. 2). A Northern blot of total myocyte RNA using the rat Cx-43 complementary DNA probe revealed a single 3.0-kilobase mRNA transcript similar to that reported for other species (Fig. 3). In vitro, the ratio of Cx-43 mRNA normalized to the hybridization signal for the 1% rRNA was significantly (P