Differential Up-Regulation of Gap Junction Connexin 26 Gene in Mammary and Uterine Tissues: The Role of Sp Transcription Factors

Zheng Jin Tu, Rahn Kollander and David T. Kiang

Breast Cancer Research Laboratory Department of Medicine University of Minnesota Medical School Minneapolis, Minnesota 55455


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The mRNA and protein expressions of connexin 26 (Cx26) in rat mammary gland and uterus can be up-regulated during pregnancy as well as by the administration of human CG (hCG). In the present study, we found that the time course and magnitude of Cx26 induction by hCG was different in these two tissues. The molecular mechanism underscoring this difference was therefore investigated. We had previously demonstrated that both Sp1 and Sp3 transcription factors play a functional role in Cx26 expression. By the electrophoretic mobility shift assay, nuclear extracts from both virgin mammary gland and uterus were capable of binding to a labeled oligonucleotide probe that contained the proximal GC box and formed three protein-DNA complexes (C1, C2, and C3). In the mammary gland, pregnancy enhanced the intensity of all three complexes, whereas in the uterine tissue there was a decrease in the C2 and C3 complexes and an emergence of a new major component, C4 complex. In the supershift study, the C1 complex could be supershifted only by an antibody against Sp1, whereas C2, C3, and C4 could all be supershifted by an antibody against Sp3, suggesting a potential presence of Sp3 isoforms of various sizes. We therefore conclude that the basal Sp profiles in virgin mammary gland and uterine tissue are similar. However, in response to pregnancy, the changes in Sp profile are tissue specific and may account for the temporal and quantitative differences between these two tissues in Cx26 induction.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Gap junctional intercellular communication serves a critical role in facilitating physiological function as well as in maintaining tissue homeostasis (1, 2, 3). Gap junctions are formed by the assembly of connexin (Cx) proteins (Ref. 4 , for review). The type and degree of Cx expression in each tissue is cell-, age-, and stage-dependent, and such regulation is tightly controlled (5, 6, 7). One of the well known physiological functions of Cx in the reproductive system is a reduction of Cx43 in uterine myometrium during pregnancy that is presumably responsible for maintaining uterine quiescence before parturition (8, 9). At term, however, Cx43 expression abruptly increases 5- to 9-fold to synchronize a simultaneous contractile activity during labor (9, 10).

It has been shown that Cx43 in mammary myoepithelial cells is up-regulated during parturition, resulting in coordinated contraction and therefore milk ejection (11). Consistent with this finding is our recent observation that the Cx43 expression was down-regulated during pseudopregnancy when induced by the administration of human CG (hCG) (12). One may therefore postulate that such Cx43 down-regulation in breast tissue may serve a similar physiological function in preventing a premature milk ejection before parturition. In mammary luminal epithelial cells, however, Cx26 is the major Cx. It is markedly up-regulated during pregnancy and lactation, presumably to facilitate and coordinate all mammary epithelial cells to pursue their intended physiological function, i.e. milk production (13, 14). This up-regulation of Cx26 in luminal epithelial cells could also be induced by daily administration of hCG (12).

During tumorigenesis, the transformed and neoplastic cells are frequently associated with a down-regulation of various Cx expressions and functions, so that the aberrant cells in their pursuit of autonomous growth will receive less influence from surrounding normal cells (15, 16, 17). A loss of intercellular communication and a down-regulation of Cx26 expression occurs in most breast cancer cells. Of interest, several recent reports support the contention that Cx26 is a candidate suppressor gene (5, 18, 19, 20, 21); when introduced into MCF-7 breast cancer cells by transfection, the Cx26 gene confers not only a reduction of tumor growth potential but also a restoration of cellular differentiation (20).

As important as it appears to be, our knowledge of transcriptional regulation on Cx in general and Cx26 in particular is surprisingly limited. Little is known of Cx26 regulation owing to a lack of information on its promoter region. We have recently mapped and characterized the basal promoter of the human Cx26 gene (22) as well as the rat Cx26 gene (GenBank accession number AF015311). The promoter regions in both species are extremely GC rich and highly conserved. The GC consensus boxes at the proximal promoter region and Sp transcription factors have been shown to play an important role in regulating the Cx26 expression (Z. J. Tu, Z. Gong, and D. T. Kiang, manuscripts in preparation).

Because Cx26 is a major Cx in mammary epithelial and uterine endometrial cells, we investigated the molecular mechanisms underscoring the up-regulation of the Cx26 gene during pregnancy, lactation, and hCG administration, particularly the transcriptional regulation at the promoter region of the rat Cx26 gene.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cx26 Up-Regulation in Pregnancy and Lactation
A marked up-regulation of Cx26 mRNA and protein was seen during pregnancy in both mammary epithelial and uterine endometrial cells. These changes are demonstrated by Northern blot analysis (Fig. 1AGo), in situ hybridization (Fig. 1BGo), and by fluorescent immunocytochemistry using a monoclonal antibody against Cx26 (Fig. 1CGo). During pregnancy, there was a 15-fold induction of steady-state Cx26 mRNA in uterine tissues, far greater than the 8-fold increase in the mammary glands. When rats reached the lactation and postlactation stages, the Cx26 expression in mammary gland remained elevated, whereas it was attenuated to below its basal level in uterine tissue (Fig. 1AGo). These data clearly demonstrated a differential tissue-specific response of the Cx26 gene at various physiological stages. Using the in situ hybridization technique, the enhanced Cx26 mRNA expression could be localized in the luminal epithelial cells of the lactating mammary gland (Fig. 1BGo). The induction of Cx26 protein in the mammary epithelial cells during pseudopregnancy were previously reported by our group (12). An enormous induction of Cx26 protein could also be demonstrated in the endometrial cells of the pregnant uterus by the fluorescent immunocytochemical staining (Fig. 1CGo).



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Figure 1. Up-Regulation of Rat Cx26 Expression in the Mammary Gland and Uterus

A, Northern blot analysis on the Cx26 mRNA expression in rat mammary gland and uterus at virgin (V), pregnant (P), lactating (L), and postlactating (PL) stages. The 28S and 18S rRNAs were used as loading controls. B, Cx26 mRNA expression in virgin (vm) and lactating (lm) rat mammary glands using the in situ hybridization technique with digoxigenin labeling. There was no labeling in the control when sense probe was used (magnification, 400x). C, Fluorescent immunocytochemical staining of Cx26 protein expression in virgin (v) and pregnant (p) rat uterus. Mouse IgG was used as control primary antibody (c) (magnification, 640x).

 
Differential hCG Effects on Cx26 Regulation in Different Tissues
The kinetics of Cx26 induction by the administration of hCG were further examined. During the hCG treatment, rats were killed at different time intervals. By Northern analysis, a 5-fold induction of Cx26 in the uterus was seen after 3 days of hCG administration (Fig. 2Go), whereas the induction lagged behind in mammary gland and took 5 days of hCG to reach a similar intensity (Fig. 2BGo). In the uterine tissue, Cx26 induction reached its maximum level (a 18-fold increment) by day 8 and then declined to near the basal level by day 21, a time point equivalent to natural parturition. In the mammary gland, however, the effect on Cx26 induction reached and maintained its maximal level (a near 11-fold increment) from day 8 through day 21 of hCG administration. Therefore, regardless of whether it is natural pregnancy (Fig. 1Go) or hCG-induced pseudopregnancy (Fig. 2Go), the results consistently show that the time course and the magnitude for Cx26 induction between these two tissues are different.



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Figure 2. The Kinetics of Cx26 Induction by hCG, a Comparison between Mammary Gland and Uterus

A, Northern blot analysis of rat Cx26 expression in the mammary gland and uterus. The rats were given hCG ip at a dose of 100 U daily up to 21 days. The 28S and 18S rRNAs were used as loading control. B, Relative Cx26 mRNA expressions during hCG treatment in comparison with the pretreatment levels (day 0) in the rat uteri (closed circles) and mammary glands (open circles).

 
Binding of Transcription Factors to the Cx26 Promoter
Because the promoter region of Cx26 is GC rich and our previous experiments revealed that the proximal GC box is essential for the basal function of human and rat Cx26, we decided to use a gel mobility shift assay to analyze and compare the nuclear protein-binding profiles of this promoter region. The physical structure of the rat Cx26 promoter region is shown in Fig. 3Go. There are four major transcription start sites at four consecutive nucleotides. A TATA-like box is located at -39 and a GC box at -136, relative to the first transcription start site; both are highly conserved when compared with mouse and human Cx26.



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Figure 3. Genomic Structure of the Rat Cx26 Promoter Region in Comparison with Those of Mouse and Human

The GC and TATA-like boxes are identified with rectangles. Gaps (dotted lines) are introduced to maximize the alignment. The dashed lines represent aligned identical bases. The arrows denote the major transcription start sites: four for rat Cx26 (rCx26), two for mouse Cx26 (mCx26), and a single site for human Cx26 (hCx26).

 
In the gel mobility shift assay, the labeled probe used was a rat Cx26 promoter oligonucleotide (-144 to -123) encompassing the critical proximal GC box (Table 1Go). Proteins in nuclear extracts of the virgin mammary gland and uterine tissue bound to this labeled probe and formed three complexes designated C1, C2, and C3. Their intensities were C2 > C3 ~ C1 (Fig. 4Go). In the mammary gland, all of these three complexes proportionally increased in abundance during pregnancy and lactation and then returned to their basal level at the postlactation stage. The uterine tissue had a quite different response to pregnancy; the increase in the C1 complex was similar to that of mammary gland during pregnancy; however, there was a marked decrease in the C2 and C3 bands, and a new C4 complex emerged to become the dominant component. It appears that the C2/C3 components were markedly switched to a C4 complex during pregnancy, a unique response seen in uterine tissues but not in mammary glands.


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Table 1. The Oligonucleotides Used in the EMSAs

 


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Figure 4. EMSAs on Nuclear Proteins Extracted from the Mammary Gland and Uterus in Virgin (V), Pregnant (P), Lactating (L), and Postlactating (PL) Rats

A double-stranded oligonucleotide encompassing the promoter GC box was used as the probe (sequence shown in Table 1Go). Four protein-DNA complexes were observed and designated as C1, C2, C3, and C4.

 
Differential Expression of Sp among Tissues during Pregnancy
Since the labeled probe contained a potential binding site for Sp transcription factors, we performed a competitive assay using wild-type or mutated self-oligonucleotides, as well as wild-type or mutated Sp1 consensus oligonucleotides as competitors (Table 1Go). The nuclear extracts from pregnant mammary gland again formed three complexes with the DNA probe (Fig. 5Go). These bindings could be effectively competed by wild-type self-oligonucleotide or Sp1 consensus oligonucleotide, but not by the two mutated ones. Similar results were also seen in pregnant uterus. Here, the additional C4 complex was also efficiently suppressed by wild-type self- and Sp1 oligonucleotides. Therefore, the major transcription factors that bind to the Cx26 promoter region belong to the Sp family.



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Figure 5. Competitive Suppression of Nuclear Protein Binding to the Labeled Probe

The nuclear extracts were either from pregnant mammary glands or pregnant uteri. The competitors were either nonlabeled GC, or consensus Sp1 oligonucleotides, or their mutated counterparts (mGC and mSp1). The sequences of the labeled probe and its competitors are listed in Table 1Go.

 
To further delineate the type of Sp transcription factors involved in the binding, as well as the dynamic changes of Sp during pregnancy, we performed supershift assays using antibodies against Sp1, Sp2, and Sp3 (Fig. 6Go). The antibody against Sp2 did not supershift any of these complexes. However, the C1 complex was clearly supershifted by Sp1 but not by Sp3 antibody in both pregnant mammary gland and uterus. On the contrary, C2, C3, and C4 could be supershifted by an antibody against Sp3, suggesting that these three complexes could contain Sp3 isoforms of various sizes, with C4 having the smallest Sp3. In comparing the gel mobility shift pattern between virgin and pregnant uterus, there was a nearly total switch in dominant Sp3 complexes, from C2 and C3 in virgin uterus to C4 during pregnancy.



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Figure 6. Supershift Analysis of Protein-DNA Complexes Using Polyclonal Antibodies (Ab) against Sp1, Sp2, and Sp3 Transcription Factors

The nuclear extracts were from rat mammary glands or uteri. The protein-DNA binding complexes, C1 to C4, were designated as in Fig. 4Go. The two supershift bands are identified as S1 and S2.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Gap junctional intercellular communication plays a pivotal role in developmental functions. There are dynamic changes in the types as well as in quantities of connexins in the reproductive system. Our primary interest was to examine the molecular mechanism underscoring the Cx26 up-regulation in mammary gland and uterus. We have found that Cx26 was up-regulated in both tissues, but there are differences in time course as well as in quantity between these two tissues in response to pregnancy and lactation.

Transcriptional regulation has been shown to serve as the primary control of connexin gene expression (23, 24, 25, 26). By reporter gene analysis, the basal promoter regions of mouse Cx43 and rat Cx32 have been mapped and characterized. An activator and a repressor have been identified in the 5'-flanking region of the mouse Cx43 gene (25), and a liver-specific binding complex has been demonstrated to be an essential component of rat Cx32 basal promoter (24). Although GC-like sequences have been described in these connexin gene promoters, they are not Sp1 binding sites (24, 25).

In our study, Sp1 and Sp3 are clearly involved in the induction of Cx26 during pregnancy and lactation when the mammary gland is fully differentiated. It is worth noting that similar phenomenon also occurred in p21Cip1/WAF1, another tumor suppressor gene intimately related to terminal differentiation and growth arrest. Sp1 and Sp3 activate the p21 promoter, but only Sp3 overexpression enhances promoter inducibility during keratinocyte differentiation (27). In our study, Sp3 is also the dominant transcription factor that bound to the promoter of Cx26. It forms C2, C3, and C4 protein-DNA complexes at the site of GC box and activates the transcription of Cx26 during pregnancy.

Sp3 is a bifunctional transcription regulator. It is capable of acting either as a repressor or an activator in a promoter-dependent manner (Ref. 27 and references therein). When Sp3 binds to a single site of the promoter, the repressor activity is lost. Therefore, the positive or negative regulation is determined by the number of binding sites present in the promoter region. Activation or repression of Sp3 has been studied in promoters with various numbers of functional GC boxes, e.g. the H4 histone gene with a single functional GC box, the thymidine kinase promoter with multiple GC boxes but only one functional, and the dihydrofolate reductase promoter that contains multiple functional GC boxes. Only the dihydrofolate reductase promoter displays Sp3 repression of Sp1 activation (28), whereas in the former two promoters with only one functional GC box, the primary function of Sp3 acts as an activator. Although the human Cx26 promoter has two GC boxes, only one of them is capable of Sp binding. Therefore, with both human and rat Cx26 promoters having only one functional GC box, one would predict that Sp3 in this case would serve as an activator. Indeed, during pregnancy, there is not only an increase in Sp3 quantity but also a qualitative shift in Sp3 complexes in association with a marked induction of Cx26.

In the present study, there is a switch of Sp3 complex from C2/C3 to C4 in the rat uterus during pregnancy. It is possible that through protein-protein interactions Sp3 may form different complexes with various cofactors. An alternative explanation is the presence of Sp3 isoforms. It has been shown that these isoforms are not generated by proteolysis, but rather via the mechanism of different internal translational initiation within Sp3 mRNA (29). Most recently, independent activation and repressor domains of Sp3 have been characterized (30). The repressor activity has been mapped to the 5'-end of the DNA-binding domain. At this moment, we can only speculate that the Sp3 in the C4 complex may lose the 5'-repressor domain and result in a robust activation of the Cx26 gene. Among these three Sp3 complexes, the C4 may exert the strongest activating function, and its abundant presence in uterus, but not in mammary gland, during pregnancy may account for the temporal and quantitative differences between these two tissues in Cx26 induction.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals
Female Sprague Dawley rats were purchased from Harlan Farm (Indianapolis, IN). The pregnant rats were 15–18 days postconception. The lactating rats were approximately 7 days postpartum with litters. The postlactation rats were those whose litters had been removed 7 days previously. For the hCG experiment, 90-day-old female rats received hCG (Profasi from Serono Laboratory, Norwell, MA) 100 U/day ip for 21 days. The dosage chosen (100 U/day) was based on our previous experience (12) as well as dosages described in the literature (31).

The rats were housed at a constant temperature (22 C) and in an imposed diurnal cycle with the light period from 0600 to 1800 h. Food (rodent pellets, Harlan Tek-Lad, Madison, WI) and water were available ad libitum. Mammary and uterine tissues were dissected out and frozen immediately in liquid nitrogen. They were stored in liquid nitrogen until used for Northern blot analysis, in situ hybridization, and immunocytochemistry staining.

RNA Isolation and Northern Blot Analysis
Total RNA was isolated from rat mammary glands and uteri by the method of Chomczynski and Sacchi (32) using phenol and guanidine isothiocyanate. RNA (20 µg/lane) was loaded on a 1.5% agarose gel, separated by electrophoresis, and transferred to a positively charged nylon membrane (Ambion, Austin, TX). For hybridization, 32P-labeled antisense RNA probes were generated using rat Cx26 cDNA (provided by David Paul, Harvard University). The hybridization was performed at 65 C overnight, and washing was done according to the manufacturer’s instructions (Ambion). After washing, autoradiography was performed with intensifying screens at -70 C. The amounts of 28S and 18S RNA were determined as loading controls.

In Situ Hybridization Analysis for Cx26 mRNA Expression
Six-micron cryosections of the frozen tissues were fixed in 3.7% formaldehyde in PBS, pH 7.2, for 1 h. The slides were washed with diethyl pyrocarbonate-treated PBS and treated with 1 µg/µl proteinase K for 8 min at 37 C. After dehydration and delipidation with a series of ethanol and chloroform washes, the slides were rehydrated with an ethanol and 2xsaline sodium citrate series and acetylated with 0.25% acetic anhydride, 0.1 M triethanolamine, pH 8.0, for 10 min. Hybridization was carried out overnight with digoxigenin (Boehringer Mannheim, Indianapolis, IN)-labeled antisense transcripts from a Cx26 cDNA insert (nucleotides +501 to +860, relative to the ATG site) that was subcloned in pGEM4Z (Promega, Madison, WI). The labeling of the cRNA probe was done according to the manufacturer’s instructions (Promega). Hybridization was followed by ribonuclease (RNase) treatment (20 µg/ml, 30 min, 37 C) and subsequent washing (2xsaline sodium citrate, 50% formamide, 50 C, 30 min). After blocking in Tris-buffer-saline (TBS), pH 7.5, containing 2% normal sheep serum and 0.05% Triton X-100, the slides were incubated with antidigoxigenin-AP-antibody (Boehringer Mannheim), 1:500 in TBS, pH 7.5, 1% normal sheep serum. After being washed in TBS, pH 7.5 and pH 9.5, the slides were incubated in color-developing solution (nitroblue tetrazolium and x-phosphate in TBS, pH 9.5) in dark jars for at least 3 h. The color reaction were stopped by placing the slides in Tris-EDTA, pH 8.0, and the slides were then counterstained with 0.01% neutral red and mounted in aquamount.

Immunocytochemistry
Six-micron cryosections from frozen tissues were fixed in absolute ethanol for 10 min. After PBS washing and blocking with 1.5% horse serum (Vector Laboratory, Burlingame, CA), they were incubated with a monoclonal antibody against Cx26 (Zymed, San Francisco, CA) at 1:500 dilution and 4 C overnight. Biotinylated horse antimouse IgG antibody, diluted at 1:200 in 3% horse serum/PBS was then added and incubated at room temperature for 2 h. After washing with PBS, streptavidin-fluorescein (1:1000 in PBS)(Amersham, Arlington Heights, IL) was added. The degree of fluorescence was examined under a Zeiss fluorescent microscope (Carl Zeiss, Thornwood, NY).

Nuclear Extract Preparation and Electrophoretic Mobility Shift Assay (EMSA)
Nuclear extracts from rat mammary glands and uteri were prepared according to the method described (33). The nuclear extract was aliquoted and quick frozen in liquid nitrogen before being stored at -70 C. The protein concentration was measured using a protein assay kit (Bio-Rad Laboratories, Hercules, CA) according to the directions provided by the manufacturer. The stored nuclear extracts were used only once after thawing.

For the EMSA, the oligonucleotides (see Table 1Go for sequences) were labeled with [{alpha}-32P]dCTP by filling 3'-recessed termini using Klenow DNA polymerase I. The labeled probes (50,000 cpm, ~0.5 ng) were incubated with 2 µg of the nuclear extract proteins and 1.0 µg of poly[d(I-C)] in a total volume of 20 µl of buffer containing 10% glycerol, 15 mM HEPES-NaOH, pH 7.8, 5 mM MgCl2, 5 mM dithiothreitol, and 50 mM KCl. The reaction mixtures were incubated at room temperature for 30 min and then were resolved on a 4% nondenaturing polyacrylamide gel in 0.25x TBE buffer (1xTBE, 89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0) at room temperature for 2 h at 200 V, and the products were detected by autoradiography.

For competition studies, the specific oligonucleotides were synthesized and their sequences are listed in Table 1Go. The consensus Sp1 oligonucleotide and the mutated form of Sp1 oligonucleotide were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). These excess unlabeled double-stranded DNA competitors were added at the same time as the probes in the EMSA. For the antibody supershift assay, specific rabbit polyclonal antibodies against Sp1, Sp2, or Sp3 (all from Santa Cruz Biotechnology, Inc.) were added and incubated with the reaction mixture for an additional 15 min at room temperature.


    ACKNOWLEDGMENTS
 
We thank Dr. David Paul for providing the rat Cx26 cDNA probe and Dr. B. J. Kennedy for his invaluable comments.


    FOOTNOTES
 
Address requests for reprints to: David T. Kiang, M.D., Ph.D., Box 286, University of Minnesota Medical School, 420 Delaware Street SE, Minneapolis, Minnesota 55455.

Supported by NIH Grant R01-CA72044 and Minnesota Medical Foundation, Inc.

Received for publication June 24, 1998. Revision received September 2, 1998. Accepted for publication September 4, 1998.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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