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Endocrinology, doi:10.1210/en.2003-0831
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Endocrinology Vol. 145, No. 1 437-446
Copyright © 2004 by The Endocrine Society

Immortalization of Epididymal Epithelium in Transgenic Mice Expressing Simian Virus 40 T Antigen: Characterization of Cell Lines and Regulation of the Polyoma Enhancer Activator 3

Petra Sipilä, Ramin Shariatmadari, Ilpo T. Huhtaniemi and Matti Poutanen

Department of Physiology (P.S., R.S., I.T.H., M.P.), Institute of Biomedicine, and Turku Graduate School of Biomedical Sciences (P.S.), University of Turku, FIN-20520 Turku, Finland

Address all correspondence and requests for reprints to: Dr. Matti Poutanen, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland. E-mail: matti.poutanen{at}utu.fi.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the present study epididymal epithelium was immortalized in transgenic mice by expressing simian virus 40 T antigen under a 5.0-kb mouse glutathione peroxidase 5 promoter (GPX5-Tag1). Epididymal tumorigenesis was associated with an increase in c-Myc expression, and a marked decrease in B-Myc expression, with a 500-fold lower level in the GPX5-Tag1 caput epididymis compared with wild-type caput. Furthermore, B-Myc was undetectable in the immortalized corpus and cauda epididymis. Hence, it is possible that the normally high B-Myc expression in the epididymis is one of the factors contributing to the highly resistant nature of epididymis toward immortalization. Morphologically different epithelial cell lines were generated from the immortalized epididymides, and the cells expressed several genes typical for epididymal epithelium, such as mouse epididymal 1, mouse epididymal protein 9, androgen and estrogen receptors, anion exchangers 2 and 4, retinoic acid receptor {alpha}, and polyoma enhancer activator 3 (PEA3). This indicated the differentiated status of the cells and their usefulness for analyzing epididymal gene expression in vitro. As PEA3 is considered to be one of the transcription factors responsible for epididymal gene expression, we further studied its regulation in epididymal cells in vitro. The data showed that PEA3 mRNA expression is regulated in the epididymis via protein kinase A and ERK signaling cascades. Inhibiting protein kinase A resulted in up-regulation and inhibiting ERK resulted in down-regulation of PEA3 mRNA, whereas no significant effect on PEA3 expression was found by modulating the protein kinase C, stress-activated p38, phosphoinositol 3-kinase and p70 S6 kinase cascades.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEVERAL ONCOGENES have been used in studies aimed at developing mouse models of tumor development and generating various immortalized cell lines. Some of the results indicate that the combination of activated oncogene and the tissue context are the major determinants of malignant transformation (1). In contrast to tumor development in the prostate (2), testis (3), and adrenal gland (4) in transgenic (TG) mouse models, the epididymis seems to be more resistant to tumorigenesis induced by oncogenes such as HER2/Neu and H-ras, and until recently only hyperplasia has been reported (1, 5, 6). Interestingly, B-Myc, which has been shown to inhibit cellular proliferation (7), has been found to be predominantly expressed in the epididymis (8). Further, it has been shown that B-Myc also inhibits the function of its family member c-Myc (9), which is powerful stimulator of cell proliferation. c-Myc also inhibits the differentiation of several cell types, and this can lead to transformation and tumorigenesis (10). Polyoma enhancer activator 3 (PEA3), also called ets variant gene 4, is a family member of the Ets transcription factors and is expressed in the adult brain and epididymis (11). High PEA3 levels have also been found in 76% of all human breast tumors (12), suggesting the possibility that it could have a role in carcinogenic transformation. However, conflicting results have been reported (13, 14, 15), with data showing that PEA3 can act as a tumor suppressor rather than a tumor inducer both in vitro and in vivo. Hence, it is possible that high B-Myc and PEA3 expression in the normal epididymis contributes to the rarity of epididymal tumors in humans (16) and to the highly resistant nature of this organ to immortalization brought about by various oncogenes (1, 5, 6) in TG mice.

The epididymis is a tubular organ that can be divided into four histologically different regions: initial segment, caput, corpus, and cauda. In each of these regions, the type and function of the epithelium differ, the main functions being sperm concentration, maturation, transport, and storage. The epididymis can be further subdivided into several segments by way of the septa of connective tissue (17, 18). Epididymal gene expression is characterized by strong tissue, segment, and cell specificity, leading to checkerboard-like expression patterns for many genes (19, 20). To date, very little is known about the transcriptional mechanisms controlling this regionalized gene expression. However, several epididymal genes have been reported to be under the control of androgens (21, 22) or unknown testicular factors (23). Furthermore, male estrogen receptor {alpha} (ER{alpha}) knockout mice are infertile (24), and epididymal dysfunction in these mice (25) has been shown to be caused by a decrease in Na+/H+ exhanger-3 gene expression (26). In addition, retinoids, acting through retinoic acid receptors (RAR) or retinoid X receptors, are thought to be required for the normal development and maturation of most epithelia (for a review, see Ref. 27). In particular, RAR{alpha} is expressed in the adult epididymis (28), and TG mice expressing a dominant negative mutant of RAR{alpha} have reduced fertility due to an abnormally dense ductal fluid blocking the epididymal lumen (29). To date, only two transcriptional regulators have been shown to regulate epididymal gene expression. Of these, PEA3 has been shown to regulate glutathione peroxidase 5 (GPX5) (30) and {gamma}-glutamyl transpeptidase mRNA IV expression (31), and CCAAT/enhancer-binding protein ß has been found to regulate cystatin-related epididymal spermatogenic gene expression (32). Hence, in addition to possessing tumorigenic properties, PEA3 serves as a transcription factor for epididymal gene regulation.

A variety of cell types from different organs have been immortalized by introducing the gene encoding simian virus 40 large T antigen (SV40 Tag), a powerful oncogene, into the cell type of interest. The oncogene can be introduced into primary cells by transfection in vitro (33, 34) or by using transgenic techniques in vivo (3, 4, 35). We have previously described the establishment of two transgenic mouse lines (GPX5-Tag1 and -2), expressing SV40 Tag under a 5-kb murine GPX5 promoter (36). In GPX5-Tag1 mice, severe dysplasia of the epididymal epithelium was found in all epididymal regions at the age of 4 months. In the present study we characterized further the immortalization process in these mice, and the cell lines established from the immortalized caput epididymides were used to study the epididymal regulation of PEA3 expression in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
All of the inhibitors of signal transduction pathways were obtained from Calbiochem (Merck & Co., Darmstadt, Germany), and stock solutions included dimethylsulfoxide (Sigma-Aldrich Corp., St. Louis, MO). The final concentration of dimethylsulfoxide in the medium was adjusted to 0.1% of the total volume in all samples.

Experimental animals
In GPX5-Tag1 mice, tumorigenesis in the epididymal epithelium was found in all regions, and caput epididymides of these mice were used to generate epididymal cell lines. All mice were handled in accordance with the institutional animal care policies of University of Turku (Turku, Finland); they were specific pathogen-free, fed complete pelleted chow and tap water ad libitum, and kept in a room with controlled light (12 h of light, 12 h of darkness) and temperature (21 ± 1 C).

Establishment of cell lines
Epididymides from 4- to 5-month-old GPX5-Tag1 mice were dissected out, and the caput region was isolated. The capsule surrounding the epididymal tubule was removed, and the tubule was cut into small pieces and dispersed with collagenase II (3 x 105 U/liter; Sigma-Aldrich Corp.) for 20 min at 37 C, followed by two incubations (30 min each) in serum-free medium containing collagenase II (3 x 105 U/liter) and deoxyribonuclease I (50 mg/liter; Sigma-Aldrich Corp.). The isolated cells were plated on culture dishes and cultured at 35 C in a humidified atmosphere containing 5% CO2. After 8 h of incubation, most of the fibroblasts and smooth muscle cells were attached to the bottom of the culture dishes, and the culture media with all unattached cells were placed in new culture dishes. These cells were cultured in D-valine-modified MEME (Modified Eagle’s Medium, Sigma-Aldrich Corp.), containing heat-inactivated fetal calf serum (5%; Bioclear, Berks, UK), 0.1 g/liter gentamicin (Biological Industries, Bet-HaEmek, Israel), fungizone (1.25 mg/liter; Invitrogen, Life Technologies, Inc., Gaithersburg, MD), and 50 nM testosterone (T; Fluka BioChemika, Buchs, Switzerland). To minimize fibroblast growth, the cultures were briefly trypsinized to detach most of the fibroblasts, which were then discarded. For further selection, cells were plated in 96-well plates, and clones originating from 1–10 epithelial cells were selected for more detailed characterization.

Immunohistochemistry
To characterize the activation of Tag expression in the GPX5-Tag1 mouse epididymides, frozen sections of epididymides from 10-, 15-, 20-, 30-, and 40-d-old and 2-month-old GPX5-Tag1 and wild-type (WT) mice were immunostained using a rabbit polyclonal anti-SV40 Tag antibody (1:3,000 to 1:10,000 in PBS, supplemented with 1% normal goat serum; Vector Laboratories, Inc., Burlingame, CA). The antigen-antibody complexes were visualized using biotinylated antirabbit antibody (Vector Laboratories, Inc.) combined with streptavidin-fluorescein isothiocyanate (Dako, Glostrup, Denmark) complex.

To characterize the cell lines generated, immunohistochemistry with anti-pan-cytokeratin antibody (Sigma-Aldrich Corp.) was carried out on cell lines mE-Cap13, -18, -19, -20, -27, and -28. For staining, 2 x 104 cells were plated on eight-well chamber slides and incubated overnight. The cells were then fixed for 10 min in 4% paraformaldehyde, and permeabilized for 4 min in methanol at -20 C and for 2 min in acetone at -20 C. After fixing and permeabilizing, the primary antibody was used at dilutions of 1:50 to 1:200 in PBS supplemented with 1% goat serum. The antigen-antibody complexes were visualized using biotinylated antimouse antibody (Vector Laboratories, Inc.) combined with streptavidin-fluorescein isothiocyanate complexes. For ER{alpha}, a rabbit polyclonal ER{alpha} antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used at dilutions of 1:50 to 1:200 in PBS with 1% goat serum. ER{alpha} antigen-antibody complexes were visualized using biotinylated antirabbit antibody (Vector Laboratories, Inc.) combined with avidin-biotin complex (Vectastain Elite ABC kit, Vector Laboratories, Inc.) and 3,3'-diaminobenzidine tetrahydrochloride (DAB-Plus kit, Zymed Laboratories, Inc., San Francisco, CA). Finally, the sections were slightly counterstained with Mayer’s hematoxylin.

RNA analysis
Total RNA from the epididymal cell lines and epididymal tissues (caput, containing initial segment; corpus; cauda) of WT and GPX5-Tag1 mice was isolated using the single step method. The expression of several epididymal genes and certain ion transporters was studied using RT-PCR. One microgram of deoxyribonuclease I (amplification grade, Invitrogen, Life Technologies, Inc.)-treated total RNA was reverse transcribed (10 min, 50 C) using avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI) and amplified using Dynazyme II-polymerase (Finnzymes, Espoo, Finland) in the same reaction tube. After amplification reaction, the products were resolved on 1% agarose gel and stained with ethidium bromide. The primers and annealing temperatures used are presented in Table 1Go. To confirm that all of the RT-PCR products were correct, Southern blot analysis with an independent oligonucleotide was performed using previously described protocols (37). Real-time RT-PCR measurements were performed by using the DNA Engine Opticon system (MJ Research, Inc., Waltham, MA) with continuous fluorescence detection. One hundred nanograms of deoxyribonuclease I (Invitrogen, Life Technologies, Inc.)-treated total RNA were used, and the reactions were performed using the QuantiTect SYRB-Green RT-PCR Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The samples and standard curves were run in triplicate. The relative standard curve method (38) was used to calculate relative gene expression. ß-Actin was included as the endogenous normalization control to adjust for unequal amounts of RNA.


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  		TABLE 1. Genes analyzed in the epididymis of GPX5-Tag1 mice and the mE-Cap cell lines: primer pairs used in RT-PCR and real-time RT-PCR, and specific RT-PCR conditions

 
Cell doubling time assay
Cells (2 x 104) were plated on 24-well cell culture plates and incubated for 4 d in DMEM without phenol red in the presence or absence of 50 nM T or 20 nM 5{alpha}-dihydrotestosterone (DHT). The cell doubling time was assessed in quadruplicate using a colorimetric Thiazol Blue [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich Corp.] assay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (500 µg/liter) in serum-free DMEM was added into the wells. After 6-h incubation, 0.5 ml 2-propanol was added, and the plates were shaken for 10 min. Absorbance at 570 nm was measured using a Victor 1420 Multilabel Counter (PerkinElmer, Turku, Finland), using 630 nm as the reference wavelength.

Analyzing the regulation of PEA3 and ME1 by androgens
The effects of 50 nM T and 20 nM DHT on the expression of PEA3 and mouse epididymal 1 (ME1) were studied by extracting total RNA from epididymal cell lines grown with or without the androgens, and performing quantitative real-time RT-PCR as described above. To increase androgen receptor expression in the cells, 2 x 105 mE-Cap18 cells were plated on six-well culture plates and incubated for 24 h. The next day, triplicate aliquots of cells were transfected with 0.5 µg pSG5-rAR (an expression vector for rat androgen receptor) (39) using 5 µl Lipofectamine (Invitrogen, Life Technologies, Inc.) according to the instructions of the manufacturer. After 4 d, RNA was isolated from the cells as described previously, and the expression of androgen receptor (AR), PEA3, and ME1 was studied by quantitative real-time RT-PCR as described above.

Analyzing the regulation of PEA3 by signaling pathway inhibitors
Aliquots of 3 x 105 mE-Cap18 and -28 cells were plated on six-well culture plates and incubated at 37 C for 24 h. Thereafter, different signaling pathway inhibitors at various concentrations (Table 2Go) were added in 2 ml MEME in the absence of androgens. After 18-h incubation, RNA was isolated from the cells as described previously, and the expression of ME1, AR, and PEA3 mRNA was studied using quantitative real-time RT-PCR as described above. Based on the results obtained, time-course studies with the protein kinase A (PKA) pathway inhibitor, H-89, were carried out. mE-Cap18 cells (4 x 105) were plated on six-well culture plates and incubated at 37 C for 24 h. Thereafter, 10 µM H-89 was added to the cells in 2 ml MEME. After various time periods (0, 0.5, 1, 2, 4, 8, or 18 h), RNA was isolated from the cells as described previously, and the expression of PEA3 was studied using quantitative real-time RT-PCR.


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  		TABLE 2. Signal transduction pathway inhibitors used

 
Statistical analysis
Statistical analyses were performed using the SigmaStat-program (version 2.0 for Windows 95, SPSS, Inc., Chicago, IL). For quantitative real-time RT-PCR and cell doubling time results, one-way ANOVA or Kruskal-Wallis one-way analysis was performed. In the case of statistically significant results (P < 0.05), Dunn’s test was performed in pairwise multiple comparisons after Kruskal-Wallis one-way analysis, and Bonferroni’s t test was performed after one-way ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of GPX5-Tag1 epididymides
Using immunohistochemistry, we identified SV40 Tag expression in the GPX5-Tag1 epididymides from the age of 10 d on, and the intensity of staining reached a maximum at the age of 40 d (Fig. 1Go). The expression levels of two other endogenously expressed oncogenes, mouse telomerase reverse transcriptase (mTERT) and PEA3 were measured using real-time RT-PCR. The data revealed that compared with WT mice, c-Myc expression was higher in GPX5-Tag1 caput and corpus regions, where it was increased 2.6-fold (P < 0.05). However, in the cauda epididymis, no significant increase was detected (Fig. 2AGo). Furthermore, a dramatic difference in B-Myc expression was detected between WT and GPX5-Tag1 mice, where it was 500-fold (P < 0.05) lower in GPX5-Tag1 caput epididymides compared with WT caput, and it was undetectable in GPX5-Tag1 corpus and cauda epididymis (Fig. 2BGo). In addition, mTERT expression levels were significantly lower in GPX5-Tag1 caput (8.3-fold; P < 0.05) compared with WT mice (Fig. 2CGo), but not in the corpus and cauda regions. Further, endogenous PEA3 expression in GPX5-Tag1 caput was over 180-fold (P < 0.001) lower than that in WT caput. PEA3 expression in the corpus region was unchanged, and in the cauda it was 7-fold (P < 0.001) lower in GPX5-Tag1 vs. WT mice (Fig. 2DGo).



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  		FIG. 1. Immunohistochemical staining of SV40 T-antigen in the GPX5-Tag1 epididymides at different ages: B and C, 10 d; E and F, 20 d; H and I, 40 d; K and L, 2 months of age. A, D, G, and J, Light microscopic image from the adjacent fluorescence image, shown at two magnifications.

 


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  		FIG. 2. Real-time RT-PCR results of expression of c-myc (A), B-myc (B), mTERT (C), and PEA3 (D) mRNA in WT and GPX5-Tag1 caput (Cap), corpus (Cor), and cauda (Cau) epididymides. The data represent the mean ± SD. *, P < 0.05; ***, P < 0.001.

 
Generation and basic characterization of epididymal cell lines
Eighteen epithelial cell lines, named mE-Cap11–28, were generated from the caput region of 4-month-old GPX5-Tag1 mice. Morphologically, several different cell types were present in the cell lines: cobblestone-shaped, spindle-shaped, and irregularly shaped cells (Fig. 3Go, A and B). All of the cell lines grew in a monolayer, and one of them, mE-Cap28, formed tubule-like structures even when cultured on a plastic surface (Fig. 3BGo).



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  		FIG. 3. Morphology and immunohistochemical staining for cytokeratin of two immortalized epididymal epithelial cell lines. Phase contrast microscopic image of mE-Cap18 (A) and mE-Cap28 cells (B). Tubule-like structures (indicated by an arrow) were formed by epithelial cells from the cell line mE-Cap28. Cytokeratin staining in cell lines mE-Cap18 and mE-Cap28 (C and D, respectively).

 
To further characterize the cells, the expression patterns of various epididymal genes were analyzed using RT-PCR. The data revealed that most of the cell lines maintained the mRNAs of several epididymal genes (Table 3Go), such as ME1, MEP9, PEA3, AR, ER{alpha}, and anion exchanger 2 (AE2) and 4 (AE4). Some cell lines also expressed ERß and Na+/H+ exchanger 2. However, some epididymal genes, such as GPX5, cysteine-rich secretory protein-1 (CRISP-1), c-ros, murine epididymal retinoic acid binding protein (mE-RABP), mouse epididymal protein 17 (MEP17), human epididymal 5 (HE5), and human epididymal 6 (HE6), were not expressed in any of the cell lines (Table 3Go). All of the cell lines tested also expressed RAR{alpha} and cyclooxygenase-2 (COX-2), and three cell lines, mE-Cap16, -18, and -28, also expressed cyclooxygenase-1 (COX-1).


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  		TABLE 3. Gene expression in different mE-Cap cell lines

 
Immunohistochemistry of the cell lines
Based on cell morphology and the gene expression pattern identified in RT-PCR studies, six cell lines were selected for further studies, namely mE-Cap13, -18, -19, -20, -27, and -28. In these lines, all cells showed positive staining for cytokeratins, confirming their epithelial origin and indicating that the cell lines were free of fibroblast contamination (Fig. 3Go, C and D). The cell lines tested also showed positive nuclear staining for ER{alpha} (data not shown).

Androgen dependency of cell doubling times and gene expression
As the structure and function of the epididymis are highly regulated by androgens, we tested the doubling times of the six cell lines in the presence and absence of androgens. Without androgens, cell doubling times ranged from 30 h for the mE-Cap27 cell line to 67 h for mE-Cap13. The presence of androgens did not affect the growth rate of the cell lines, except for mE-Cap28 cells, in which the doubling time was significantly (P < 0.05) shorter in the absence of androgens (43 h) vs. that in the presence of DHT (49 h).

The androgen dependency of PEA3 and ME1 mRNA expression was next analyzed in the presence of 50 nM T or 20 nM DHT in the culture medium. The presence of androgens did not significantly affect the expression levels of PEA3 (Fig. 4AGo) or ME1 (data not shown) in any of the cell lines. This prompted us to study the expression of AR in the cell lines using real-time RT-PCR. The data revealed that AR mRNA in the GPX5-Tag1 epididymis was 3.5-fold lower in the caput (P < 0.001) and 2.2-fold lower in the corpus and cauda regions with WT epididymis (Fig. 4CGo). Furthermore, the expression levels in the cell lines tested were 20-fold (mE-Cap27) to 69-fold (mE-Cap28) lower than those in WT caput epididymis, and in cell lines mE-Cap18 and mE-Cap20 (P < 0.05; Fig. 4CGo), the expression was at the detection limit. Increasing AR expression in mE-Cap18 cells by transfecting them with the expression plasmid for rat AR resulted in a mild decrease in PEA3 expression (1.6-fold) when the cells were grown in the presence of DHT, and a 2-fold decrease was found (P < 0.05) when they were grown with T (Fig. 4BGo). A similar tendency toward reduced, rather than increased, expression of PEA3 was detected in cells treated with androgens without transfecting the AR (Fig. 4AGo). Furthermore, high AR expression after transient transfection of the receptor into cells did not restore the expression of the endogenously androgen-dependent GPX5 gene (data not shown).



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  		FIG. 4. A, Androgen regulation of PEA3 expression in the mE-Cap13, -18, -19, -20, -27, and -28 cell lines. As analyzed by real-time RT-PCR, there were no significant differences in mRNA expression in cells grown in the absence (-A) or presence of 20 nM DHT (+ DHT) or 50 nM T (+ T). B, Relative PEA3 mRNA expression in the mE-Cap18 cells transfected with AR expression plasmid and grown in the absence (-A) or presence of 20 nM DHT (+ DHT) or 50 nM T (+ T). C, AR mRNA expression in WT and GPX5-Tag1 caput (Cap), corpus (Cor), and cauda (Cau) epididymis in different mouse epididymal epithelial cell lines (mE-Cap13, -18, -19, -20, -27, and -28) and in mE-Cap18 cells after transfecting expression plasmid for AR in to the cells (C). The data represent the mean ± SD. GPX5-Tag1 Cap differed significantly from WT Cap, mE-Cap13, and mE-Cap18 differed significantly from WT Cap (*, P < 0.05; ***, P < 0.001).

 
Effects of inhibitors of signal transduction pathways
As androgens did not regulate PEA3 or ME1 expression, we next studied other signaling pathways putatively involved in the regulation of PEA3 and ME1. Two cell lines were chosen: mE-Cap18, resembling most of the cell lines, and mE-Cap28, which differed morphologically from the other cell lines. The signal transduction pathway inhibitors used are shown in Table 2Go. The data from cell line mE-Cap18 revealed that of the two genes, PEA3 was more sensitive to the regulatory pathways tested. Incubating the cells for 18 h in the presence of suramin (inhibits the binding of G proteins to the membrane receptors) or PD 98059 (an inhibitor of the ERK pathway) decreased PEA3 expression levels 4-fold (P < 0.01; Fig. 5AGo). In contrast, inhibiting the cAMP-dependent protein kinase (PKA) pathway with H-89 resulted in a 3.5-fold (P < 0.05) increase in PEA3 mRNA (Fig. 5AGo). A similar phenomenon was seen when using the cell line mE-Cap28, but the changes were more moderate (data not shown). Inhibiting any of the signaling pathways did not change ME1 expression more than 2.0-fold, and the changes observed were not statistically significant (Fig. 5BGo). In addition, AR mRNA levels did not change significantly with any of the inhibitors tested (data not shown). As the initial studies showed strong up-regulation of PEA3 expression by inhibiting the PKA pathway for 18 h, the time course for PKA inhibition was assessed. However, a highly significant increase in PEA3 mRNA expression was found only after 18 h of inhibition (10.5-fold change; P < 0.001; Fig. 5CGo).



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  		FIG. 5. Real-time RT-PCR results after inhibiting signal transduction pathways in the mE-Cap18 cell line with various kinase inhibitors. A, Relative PEA3 mRNA expression. B, Relative ME1 mRNA expression compared with nontreated cells (controls). Cells were incubated in the presence of bisindolylmaleimide I (Bis), PD 98059 (PD), rapamycin (Rap), SB230580 (SB), H-89, suramin (Sur), or wortmannin (Wort) for 18 h. C, Time course for the cAMP-dependent protein kinase inhibitor H-89. The data represent the mean ± SD. *, P < 0.01; **, P < 0.005; ***, P < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The epididymis is a nonproliferating and highly differentiated organ, which might explain why epididymal tumors in humans are rare (16). In addition, immortalization of epididymal epithelium had not been described until very recently. In these studies canine epididymal cells were transformed in vitro by a powerful oncogene SV40 Tag (33), and mouse epididymal cells were transformed using a temperature-sensitive mutation of the SV40 Tag gene (tsA58 mutant) (35). We have generated transgenic mice expressing the SV40 Tag under a 5-kb murine GPX5 promoter (GPX5-Tag1 and -Tag2) (36). In one of the mouse lines generated (GPX5-Tag1 mice) severe dysplasia of the epididymal epithelium was found in all epididymal regions at the age of 4 months. When the initiation of SV40 Tag expression was characterized in the GPX5-Tag1 epididymides, it was found that temporal expression of the GPX5-Tag transgene mimicked that of endogenous GPX5, which has been reported to appear at the age of 10 d, reaching a maximum level at the age of 40 d (40). Transformation of the cells with SV40 Tag enables their proliferation beyond replicative senescence, as SV40 Tag inactivates the cellular growth suppressor proteins p53 and pRB (41). One of the functions of p53 is to repress transcription of c-Myc (42), which is a powerful stimulator of cell proliferation and inhibitor of differentiation (10). Nevertheless, it has been suggested that for full transformation, cells have to bypass a second obstacle, the shortening of telomeres upon DNA replication, by synthesizing telomeric repeat (TTAGGG) using telomerase RNA (mTR) as a template for mTERT (43). However, several immortal cell lines have no detectable telomerase activity, and a recent report suggests a novel mechanism of telomerase maintenance where telomere length was controlled independently of telomerase activity by retinoblastoma 1 family members (44). The SV40 Tag-c-Myc hypothetical transformation pathway is in line with the observations in this study. In the GPX5-Tag1 epididymides, SV40 Tag is likely to inhibit the function of p53 and pRB, leading to a moderate increase in c-myc mRNA levels. Although, mTERT expression levels were lower in all epididymal regions of the GPX5-Tag1 males compared with WT males, the inhibition of pRB protein could be the basis of telomere maintenance in GPX5-Tag1 epididymides.

A Myc family member, B-Myc, inhibits cellular proliferation (7) and has also been shown to inhibit c-Myc function (9). Interestingly, B-Myc has been found to be predominantly expressed in the epididymis (8), and in the GPX5-Tag1 epididymis B-Myc levels were markedly reduced. Hence, it is possible that the normally high B-Myc expression in the epididymis is one of the factors contributing to the rarity of epididymal tumors in humans (16) and to the highly resistant nature of this organ to immortalization via various oncogenes in experimental animals (1, 5, 6). In GPX5-Tag1 mice, elevated levels of c-Myc together with strongly suppressed B-Myc expression are likely to contribute to the severe dysplastic changes observed.

Immortalization of cells by oncogene expression often results in cell lines that have lost some of their differentiated functions (4). Accordingly, results from canine epididymal cell lines immortalized by SV40 Tag showed that of eight epididymal genes tested (CE1, -4, -5, -7/GPX5, -8, -9, -10, and -12), only three were expressed after 4 d in culture, and only two (CE1 and CE4), normally expressed in all regions of the epididymis, showed persistent expression in the immortalized cell lines (33). Similarly, all of the cell lines generated from the tumorigenic GPX5-Tag1 epididymides had lost the expression of some epididymal genes tested that show spatially restricted epithelial expression in the epididymis. Nevertheless, all of the mE-Cap cell lines maintained several specific features of epididymal epithelium, such as the expression of ME1, MEP9, AR (although at low level), and PEA3. Their regulation among other genes can now be studied in the cell lines in vitro. We also analyzed the expression of COX-1 and -2 in the cell lines, as the constitutive expression in rodent epididymis with cell-specific expression pattern suggests a functional importance for their presence. It has been shown that COX-1 is expressed in the basal cells, while COX-2 is expressed especially in the principal cells (45). Accordingly, COX-2 was expressed in all cell lines generated. However, detectable expression of COX-1 was also expressed in some of the cell lines. Interestingly, all of the tested cell lines also expressed ER{alpha}, and five of them expressed ERß, making it possible to study the putative estrogenic regulation of epididymal functions in these cells. The cells furthermore expressed RAR{alpha}. Hence, the mE-Cap cells generated are novel tools to study the downstream targets of retinoic acids in the epididymis. Retinoids are thought to be required for normal maturation and function of a number of epithelial cells. Interestingly, TG mice expressing a dominant negative mutation of RAR{alpha} under the murine mammary tumor virus promoter present squamous metaplasia in the epididymal epithelium (29), resulting in reduced fertility. The data thus suggest a key role for RAR{alpha}-mediated signaling in normal function of the epididymis.

Similarly to the cells presented in the present study some of the mouse epididymal cell lines immortalized with a temperature-sensitive mutation of the SV40 Tag gene have retained the expression of AR and also certain region-specific genes, such as mE-RABP and MEP17 (35), that were not present in the mE-Cap cells. This suggests differences in the cell lines produced by the two different immortalization protocols. Whether the expression of mE-RABP indicates a more differentiated nature of the cell lines produced by the expression of the temperature-sensitive SV40 Tag remains to be studied.

AR was present in the mE-Cap cells, but with markedly reduced expression compared with WT caput. Down-regulation of AR was also noted in SV40 Tag immortalized canine epididymal cells (33). One of the tested mE-Cap cells showed a reduced growth rate in the presence of androgens, which is in line with the differentiating potency of androgens in the epididymis (46). However, no significant change in the expression of PEA3 and ME1 was noted in the cell lines tested. This is in contrast with the data obtained in vivo suggesting androgen-dependent expression of PEA3 in the epididymis (30). The effective transfectability of our cell lines allowed us to restore AR expression to mE-Cap18 cells, but despite the subsequent high AR levels, PEA3 expression did not increase as expected. Instead, it decreased when the cells were grown in the presence of androgens. Furthermore, restored AR expression did not allow recovery of the expression of endogenous GPX5 in the cell lines. Even though GPX5 has been suggested to be an androgen-regulated gene in the epididymis (47, 48), high AR expression alone does not seem to be sufficient for GPX5 expression, at least in epididymal cells in vitro.

PEA3, a member of the family of Ets transcription factors, is particularly expressed in the adult brain and epididymis (11). Low levels of PEA3 expression have also been found in the mammary gland during development and postnatally at the onset of puberty and early pregnancy (49). Furthermore, high levels of PEA3 expression have been found in 76% of all human breast tumors (12), and 93% of HER2/Neu protooncogene-positive breast tumors (50) express PEA3. In breast tumors, PEA3 has been suggested to have a role in stimulating HER2/Neu expression (13). However, conflicting results have also been reported, with data showing that PEA3 inhibits HER2/Neu gene expression in ovarian and breast cancer cell lines in vitro. This was associated with inhibition of tumor growth in vivo in nude mice transplanted with HER2/Neu-overexpressing ovarian cancer cell lines (14). Inhibition of HER2/Neu expression by PEA3 was also supported by the results of a study involving 89 Japanese breast cancer patients, whose overall survival rate was significantly higher in the presence of PEA3 (15). The theory of PEA3 as a tumor suppressor is in line with our observations in GPX5-Tag1 TG mice. In the tumorigenic GPX5-Tag1 caput epididymis (containing the initial segment), PEA3 expression was more than 180-fold lower than that in WT caput (containing the initial segment). In the epididymis, PEA3 is mainly expressed in the initial segment (51), and it has been shown to regulate the expression of two genes, namely GPX5 (30) and {gamma}-glutamyl transpeptidase mRNA-IV (31). In addition, several other genes expressed in the initial segment contain PEA3-binding motifs in their promoter regions, suggesting that PEA3 might be one of the regulators responsible for initial segment-specific gene expression. Hence, in addition to acting as a tumor suppressor, PEA3 is considered to be one of the transcription factors responsible for epididymal gene expression. Using transfections in COS cells, PEA3 has been shown to be regulated through two Ras-dependent MAPK pathways, the ERK cascade and the stress-activated protein kinase/c-Jun N-terminal kinase cascade (52, 53). However, to date, no data have been available on PEA3 regulation in epididymal epithelium. The present results indicate that, as in COS cells, the ERK cascade also up-regulates PEA3 expression in the epididymis. In addition, the data demonstrated that the PKA pathway inhibits PEA3 expression in epididymal cells. It has been previously shown that the PKA pathway, jointly with the ERK cascade, increases the expression of another PEA3 family member, ets-related protein erm, in COS-1 cells (54). However, this might reflect a difference in the signal transduction pathways used to regulate these two family members. PKA has an inhibitory effect on the MAPKK kinase c-Raf, but, in contrast, an activating effect on the MAPKK kinase B-Raf. Further, our results with several cell-permeable kinase inhibitors indicate, that the cascades involving protein kinase C, stress-activated p38, phosphoinositol 3-kinase, and p70 S6 kinase do not significantly participate in the regulation of PEA3 in epididymal epithelial cells in vitro. Compared with PEA3, the expression of ME1 was much less altered. This probably reflects the fact that ME1 is an abundantly expressed protein in all epididymal regions, and it may therefore also show more constant basal expression independent of the cellular signaling cascades.

In conclusion, immortalization of epididymal epithelium is associated with down-regulation of B-Myc and PEA3 expression and up-regulation of c-Myc expression. From the immortalized epididymal epithelia we have generated mouse epididymal epithelial cell lines that retain several features specific to epididymal epithelium. These cell lines will be useful for promoter studies as well as studies on characterization of the regulation of epididymal functions by, for example, androgens, retinoic acids, and estrogens. In the present study the cell lines were also shown to be valuable tools for characterizing the regulation of epididymal genes, and the results indicated PKA-, ERK1-, and ERK2-dependent expression of PEA3 in epididymal epithelium.


   Acknowledgments
 
We thank Dr. D. Hanahan (University of California, San Francisco, CA) for providing the SV40 Tag antibody, A. Rumpunen-Virtanen for animal husbandry, T. Prins and R. Kytömaa for help with cell cultures, and J. Vesa for technical assistance with the tissue sections.


   Footnotes
 
This work was supported by grants from the Rockefeller and Ernst-Schering Research Foundations and the Academy of Finland (Projects 42145 and 43745).

Abbreviations: AR, Androgen receptor; COX, cyclooxygenase; DHT, 5{alpha}-dihydrotestosterone; ER{alpha}, estrogen receptor {alpha}; GPX5, glutathione peroxidase 5; GPX5-Tag1, glutathione peroxidase 5 promoter; ME1, mouse epididymal 1; mTERT, mouse telomerase reverse transcriptase; PEA3, polyoma enhancer activator 3; PKA, protein kinase A; RAR, retinoic acid receptor; SV40 Tag, simian virus 40 large T antigen; T, testosterone; TG, transgenic; WT, wild-type.

Received July 3, 2003.

Accepted for publication September 22, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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