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Biology of Reproduction 60, 664-673 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Involvement of Polyomavirus Enhancer Activator 3 in the Regulation of Expression of Gamma-Glutamyl Transpeptidase Messenger Ribonucleic Acid-IV in the Rat Epididymis1

Zi-Jian Lana, R. John Lyea, Nathalie Holicb, Jacquelyn C. Labusa, and Barry T. Hinton2,a

a Department of Cell Biology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908 b INSERM U.99, Hopital Henri Mondor, F 94010 Creteil, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gamma-glutamyl transpeptidase (GGT) mRNA-IV and polyomavirus enhancer activator 3 (PEA3) mRNA are highly expressed in the initial segment of the rat epididymis, and both are regulated by testicular factors. PEA3 protein in rat initial segment nuclear extracts has been shown to bind to a PEA3/Ets binding motif, which is derived from the partially characterized GGT mRNA-IV promoter region. This suggests that PEA3 may be involved in regulating transcription from the rat GGT mRNA-IV gene promoter in the initial segment. Using DNA oligonucleotide primers and DNA sequencing analysis, an approximately 1500-basepair (bp) DNA sequence at the 5' region of the promoter was obtained. Using transient transfection, PEA3 activated transcription of the rat GGT mRNA-IV promoter only in cultured epididymal cells from the rat initial segment, but not in Cos-1 or NRK-52E cells. Promoter deletion analysis indicated that a PEA3/Ets binding motif between nucleotides -22 and -17 is the functional site for PEA3 to activate transcription of GGT promoter IV and that an adjacent Sp1 binding motif is also required to maintain promoter IV activity in epididymal cells. Transcriptional activation of promoter IV was shown to be epididymal cell-specific and PEA3-specific. In addition, PEA3 may act as a weak repressor for transcription of promoter IV, probably using a PEA3/Ets binding motif(s) distal to the transcription start site. A model of how PEA3 is involved in the regulation of transcription of GGT promoter IV in epididymal cells is proposed.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gamma-glutamyl transpeptidase (GGT; EC 2.3.2.2) is an important enzyme involved in glutathione metabolism [1–3]. Previous studies have shown that high GGT enzyme activity is present in the epididymis [4–10]. Male infertility and hypoplastic epididymal epithelia of GGT "knock out" mice [11] indicate that epididymal GGT may function to protect spermatozoa from oxidative stress in the epididymal duct and/or function to recover extracellular cysteine for the synthesis of epididymal proteins that may be required for maturation of spermatozoa [12]. Molecular studies have shown that multiple forms of mRNA are transcribed from a single copy of the rat GGT gene using different promoters [13–20]. Our previous studies have shown that three forms of GGT mRNAs (II, III, and IV) are expressed in the rat epididymis [19, 21]. Presumably under the influence of promoter IV [18], GGT mRNA-IV is one of the major forms of GGT mRNAs expressed in the initial segment [19, 21] and may play a role in maintaining high GGT enzyme activity in the initial segment [4]. In addition, the expression of GGT mRNA-IV and GGT enzyme activity and protein level in the initial segment are all under the regulation of testicular factors, probably basic fibroblast growth factor (bFGF) [4, 19, 21]. A previous study has also shown that the expression of GGT mRNA-IV in the initial segment could be up-regulated by reactive oxygen species [22]. It appears that GGT mRNA-IV plays an essential role in maintaining high GGT enzyme activity to ensure the maturation of spermatozoa and/or the protection of spermatozoa from potential oxidative stress in the initial segment. Therefore, elucidating the regulation of expression of GGT mRNA-IV in the initial segment could provide new insight into the mechanisms of sperm maturation and/or pathogenesis of male infertility.

PEA3 (Polyomavirus Enhancer Activator 3) is an Ets transcriptional factor [23], as it contains an Ets domain that is conserved among all members of the Ets transcription family including Ets-1, Ets-2, spi-1/PU.1, GABP-{alpha}, ER81, ERM, E74A, and E74B [24–26]. Most of these Ets family members bind to a core DNA motif (5'-GGAA/T-3') and can act either as activators or repressors to regulate transcription of their target gene promoters [24–26]. PEA3 binds to a conserved PEA3/Ets binding motif (5'-AGGAAG-3') in the promoters of a number of genes including mouse haptoglobin [27], human synapsin II [28], human pregnancy specific glycoprotein gene 12 [29], ribosomal protein L32 gene [30], mouse PEA3 [31], and urokinase-type plasminogen activator [32, 33] and activates transcription of these genes. PEA3 and/or other Ets factors may also be required to initiate transcription from TATA-less promoters via interactions with other transcriptional factors (e.g., Sp1) and/or with components of the general transcriptional complex [24, 34–42]. In addition, PEA3 may also act as a repressor of gene promoter activity, for example, gpx5 [43], even though PEA3 is known as an activator of transcription. The transcriptional activity of PEA3 and its highly related Ets members ERM [44] and ER81 [45] has been shown to be regulated by the classical Ras/Raf/MAPK (mitogen-activated protein kinase) signal transduction pathway [46–48].

PEA3 has been shown to be highly expressed in the epididymis [23, 49]. A previous study has shown that PEA3 is highly expressed in the initial segment and that the expression of PEA3 mRNA in the initial segment is under the regulation of testicular factors [49]. This expression pattern of PEA3 mRNA is similar to that of GGT mRNA-IV in the rat epididymis [19, 21]. Multiple PEA3/Ets binding sites are present in the published partial DNA sequence of rat GGT promoter IV [18, 49]. Using an electrophoretic mobility shift assay (EMSA), PEA3 protein in nuclear extracts from the rat initial segment was shown to bind to a 16-basepair (bp) DNA probe whose sequence was derived from rat GGT promoter IV and has a conserved PEA3/Ets binding motif (AGGAAG) [49]. Therefore, PEA3 may bind to PEA3/Ets binding motifs in rat GGT promoter IV and function to regulate transcription of rat GGT mRNA-IV.

In this communication, we characterize a further 1500-bp DNA sequence at the 5' region of rat GGT promoter IV. Using transient transfections, we found that PEA3 could activate transcription of rat GGT mRNA-IV gene promoter only in primary epididymal cells that were isolated from the initial segment. Our data also show that PEA3/Ets and Sp1 binding motifs in the proximal region of promoter IV are both required to maintain GGT promoter IV activity, and that a PEA3/Ets binding motif between nucleotides -22 and -17 is the functional site for PEA3 to activate transcription of GGT promoter IV. In addition, our results suggest that PEA3 may act as a weak repressor of transcription of the GGT mRNA-IV gene, probably using a PEA3/Ets binding motif(s) in the distal region of the promoter. A model of how PEA3 is involved in the regulation of transcription of GGT promoter IV in epididymal cells is presented.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Normal adult male retired breeder Sprague-Dawley rats (450–550 g; Hilltop Laboratories, Philadelphia, PA) were maintained on a 12L:12D cycle, with free access to food and water, in the University of Virginia vivarium. All experiments in this study were approved by the University of Virginia Animal Welfare Committee.

Plasmid Constructs

A mouse PEA3 cDNA plasmid was generously provided by Dr. John Hassell [23]. To obtain sense and antisense PEA3 expression vectors, the PEA3 cDNA (pGEM 3.9) was excised with EcoRI and inserted into pSI vector (Promega, Madison, WI) to form sense pSI-PEA3 and antisense pSI-PEA3 plasmids. A ß-galactosidase plasmid, PSV-ß-Gal, was purchased from Promega and used as a transfection efficiency control in two cell lines (NRK-52E cells and Cos-1 cells). Another ß-galactosidase plasmid, pCMV-SPORT1-ß-Gal, was purchased from Gibco-BRL (Gaithersburg, MD) and used as a transfection efficiency control in primary epididymal cells. pCMV-ETS2 repressor factor (ERF [50]) was generously provided by Dr. Richard Day (Dept. of Cell Biology, University of Virginia). The plasmid for promoter IV of the rat GGT gene (pGEM-G-IV) [18] was kindly provided by Dr. Yannick Laperche (INSERM, Creteil, France). Different lengths of 5' DNA sequences of the promoter IV were obtained by using various restriction enzymes, linked to a HindIII-XmnI adaptor (New England Biolabs, Beverly, MA), and then inserted into the HindIII site of the pGL-3-enhancer plasmid (Promega). The orientation of DNA inserts in all vectors was determined by restriction enzyme digestion and/or by DNA sequencing analysis. The p(-16-36)LUC and p(-8-36)LUC plasmids were obtained by digesting p135LUC plasmid with Bsu36I, and then religating with DNA ligase. Neither plasmid contained PEA3/Ets binding motifs, as confirmed by DNA sequencing analysis.

Cell Culture

All cell lines used in this study were obtained from the American Type Culture Collection (ATCC, Rockville, MD). NRK-52E cells (epithelial cells) are derived from normal rat kidney [51], and Cos-1 cells (fibroblast cells) are derived from the CV-1 cell line (SV40 transformed Africa green monkey kidney) [52, 53]. These cells were grown in ATCC-recommended media. Primary epididymal epithelial cells from rat initial segments were isolated and cultured according to previous reports [54, 55] with minor modifications. Briefly, initial segments from adult rats were collected, immediately washed four times with cold Hanks' balanced salt solution (HBSS) without calcium and magnesium, and then minced with sharp scissors into small pieces. The minced tissues were then washed twice with HBSS and subjected to three pronase digestions (0.2% pronase in Ca2+- and Mg2+-free HBSS). The first two digestions were performed at room temperature for 10 min, while the last digestion was performed in a 35°C water bath with gentle shaking for 25 min. The last digestion solution was then gently passed through a pipette 20 times. Epididymal epithelial cell-enriched preparations were obtained by filtering the last digestion solution through a 215-µm steel mesh (Spectrum, Laguna Hills, CA). These epithelial cell-enriched preparations were washed twice with Dulbecco's Modified Eagle's medium (DMEM) and then seeded in 6-well culture plates. Usually, cells isolated from four initial segments were seeded into 20–24 wells (35-mm diameter; Corning Glass Works, Corning, NY). After incubation at 35–37°C for 5 days, the attached epididymal cells were used for transfection studies.

Transient Transfection

Lipofectamine transfection agent (Gibco-BRL) was used for NRK-52E cell transfections. Briefly, cells were plated in 6-well culture dishes at a density of 1.0 x 105 on the day before transfection. On the day of transfection, the cells were washed twice with serum-free DMEM (Gibco-BRL), then fed with 0.8 ml of serum-free DMEM and kept at 35–37°C for 30–45 min. DNA samples used for transfection (0.5 µg luciferase plasmid, 1.2 µg RSV-ß-Gal, and 1 µg PEA3 plasmid) were mixed with 0.2 ml of serum-free DMEM, and then 10 µl of lipofectamine was added to the DNA solution and allowed to stand at room temperature for 30 min to form DNA-lipofectamine complexes. The DNA-lipofectamine mixture was then added to the cells, and the transfected cells were incubated at 35–37°C for 5 h. After 5 h, 1 ml of 10% fetal calf serum (FCS) in DMEM was added to the culture. Twenty-four hours later, the culture medium was replaced with normal medium (DMEM, single-strength nonessential amino acids, 5% FCS), and incubation was continued at 35–37°C. Two days after transfection, cells were washed twice with PBS, and cell lysates were prepared for measurement of luciferase and ß-galactosidase activity, and protein level.

LipofectAmine PLUS reagent (Gibco-BRL) was used for Cos-1 and primary epididymal cell transfections. Cos-1 cells were plated in 6-well culture dishes at a density of 1.0 x 105 per well on the day before transfection. Epididymal cells were incubated at 35–37°C for 5 days after isolation as described above. On the day of transfection, cells were washed with serum-free DMEM twice, then fed with 0.8 ml of serum-free DMEM and kept at 35–37°C for 30–45 min. DNA samples used for transfection (Cos-1 cells: 0.2 µg luciferase plasmid, 0.2 µg RSV-ß-Gal, and 0.25 µg PEA3 plasmid; epididymal cells: 0.3 µg luciferase plasmid, 0.3 µg pCMV-SPORT-ß-Gal, and 0.4 µg PEA3 plasmid) were mixed with 0.1 ml of serum-free DMEM and 6 µl of PLUS reagent at room temperature for 15 min, then mixed with 0.1 ml of serum-free DMEM containing 4 µl of lipofectamine, and incubated at room temperature for 20 min to form DNA-PLUS-lipofectamine complexes. The DNA-PLUS-lipofectamine mixture was then added to the cells and incubated at 35–37°C for 3 h. Then, the cell media were replaced with normal media, and incubation was continued at 35–37°C. Twenty-four hours after transfection, Cos-1 cells were washed twice with PBS, and cell lysates were prepared using single-strength reporter lysis buffer (Promega) for luciferase, protein, and ß-galactosidase assays. The epididymal cells were harvested at 66–72 h after transfection.

DNA Sequencing

Plasmid DNA was purified using a Qiagen kit (Valencia, CA) according to the manufacturer's protocol. The purified plasmid DNA was sequenced by the Molecular Core Laboratory of the Center for Reproduction Research or the Central Facility for Molecular Studies at the University of Virginia using an automated fluorescent sequencer (Applied Biosystems Inc., Foster City, CA) or a Sequenase Version 2.0 DNA sequencing kit (USB, Cleveland, OH) with GLprimer 2 (Promega). DNA sequences were assembled using either the Genetics Computer Group (Madison, WI) suite of programs or the GeneRunner computer program (Hastings Software).

RNA Isolation and Ribonuclease (RNase)Protection Assay

Total RNA from the rat initial segment and from nontransfected or transfected Cos-1, NRK-52E, and epididymal cells was prepared using TriZol reagent (Gibco-BRL) according to the manufacturer's protocol. The PEA3 template between nucleotides +557 and +867 was obtained by digesting pGEM 3.9 [23] with KpnI. This DNA fragment was purified on a 1.5% agarose gel and ligated into the KpnI site of the pGEM7Zf (+) vector. The orientation of this DNA insert was determined by restriction digestion mapping. The colony containing pGEM-Kpn (5'-3' orientation) plasmid was amplified in Luria broth (LB) containing ampicillin, purified using a Qiagen plasmid purification kit, and then linearized with BamHI. The antisense 382-nt PEA3 probe was obtained after the linearized pGEM-Kpn was subjected to in vitro transcription using T7 polymerase [19, 21]. The antisense cyclophilin probe and the four GGT cRNA probes were prepared as described previously [19, 21]. RNase protection analysis was performed as described previously [21, 56].

Protein, Luciferase, and ß-Galactosidase Assays

Protein concentrations of transfected cell extracts were determined by the Bio-Rad (Richmond, CA) protein assay. Luciferase activity in each sample was determined by a luciferase assay kit (Promega) on a luminometer (LKB, Piscataway, NJ). Beta-galactosidase activity in each sample was biochemically determined using a galactosidase assay kit (Promega) according to the manufacturer's protocol. Both luciferase and ß-galactosidase activity in each sample were normalized per milligram of total protein. Luciferase and ß-galactosidase activities in nontransfected cells were regarded as endogenous luciferase and ß-galactosidase activities, respectively. Relative luciferase activity (RLA) in each sample was obtained using the following equation: RLA = (Luc/mg proteins-endogenous Luc/mg proteins)/(ß-Gal/mg proteins-endogenous ß-Gal/mg proteins).

In every experiment, RLA in samples transfected with p135LUC, ß-gal plasmid, and PSI vector (no PEA3 cDNA insert) was regarded as control RLA in each cell line or in epididymal cells. Within the same experiment, RLAs in all other samples were expressed as a percentage of control RLA in each cell line or in primary epididymal cells. Data represent the mean percentage ± SEM from two to eight individual experiments.

Statistical Analysis

To compare data between promoter constructs and within each promoter construct, a one-way ANOVA followed by Tukey's range test was used. Results were considered significantly different at p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
DNA Sequences of Promoter IV of the Rat GGT Gene

The isolation and characterization of a plasmid for the rat promoter IV gene (pGEM-G-IV) was reported previously [18]. The proximal 395-nt DNA sequence in the 5' direction from the transcriptional initiation site (+1) of this promoter has been published [18]. Using oligonucleotides designed from the cDNA and subsequent genomic DNA sequence, the DNA sequences between nucleotides -1976 and -395 of promoter IV were determined (Fig. 1). As shown in Figure 1, this promoter does not contain a consensus TATA box near the transcriptional start site. However, multiple PEA3/Ets binding motifs (5'-AGGAAN-3') [23, 24, 57] and an Sp1 binding site [58] are present in this promoter (Fig. 1).



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  		FIG. 1. DNA sequence of promoter IV of the rat GGT gene. The promoter region for GGT mRNA-IV is aligned at the major transcriptional initiation site (+1). Potential PEA3/Ets binding motifs in promoter IV are single-underlined while a potential Sp1 binding motif at the proximal region of promoter IV is double-underlined. Numbers at the left represent the location of DNA sequence in the 5' direction from the major transcription start site. A partial DNA sequence (nucleotides-395 to +1) of this promoter has been published by Okamoto et al. [18].

Cell Culture

Cos-1 and NRK-52E cells attached to the plastic surface within 24 h and grew to confluence after 2–3 days in culture. The attached epithelial-like NRK-52E cells had dense granules in their cytoplasm and distinct nuclei with one or more nucleoli as reported [51], while round Cos-1 cells usually formed clusters, a characteristic of their parental CV-1 cell line [53]. Isolated epididymal cells began to attach to the plastic surface on Day 2 of culture. By Day 5 of culture, more than 93% (93.3 ± 4.2, n = 3) of the attached cells were epithelial cells, as demonstrated by the presence of numerous dense granules in the cytoplasm and the presence of round or oval nuclei with one or more nucleoli. About 99% of the attached epididymal cells were in interphase while less than 1% of cultured cells were in anaphase. Under phase contrast microscopy, these epithelial cells had morphological characteristics similar to those described previously [54, 55]. Less than 5% of the total attached cells were fibroblasts, as identified by their long spindle shape and very dark contrast under phase contrast microscopy. Epididymal cells were maintained for 8 days without obvious changes in cell morphology.

As shown in Figure 2, cyclophilin mRNA was expressed in the initial segment and in all cultured cells including primary cultured epididymal cells from the rat initial segment. PEA3 mRNA and GGT mRNA-II, -III, and -IV, but not GGT mRNA-I, were expressed in the rat initial segment. Neither PEA3 mRNA nor GGT mRNAs were expressed in Cos-1 cells. GGT mRNA-I, -II, and -III were not detected in normal NRK-52E cells or primary epididymal cells. However, PEA3 mRNA and GGT mRNA-IV were both expressed in NRK-52E cells and primary epididymal cells, although GGT mRNA IV was expressed at a reduced level compared to the in vivo level.



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  		FIG. 2. RNase protection analysis of total RNAs from cultured NRK-52E and Cos-1 cells, and epididymal cells from the rat initial segment. Fifteen micrograms of total RNA was hybridized with antisense cyclophilin (Cyc., 107 nt), GGT mRNA-I (I, 155 nt), GGT mRNA-II (II, 160 nt), GGT mRNA-III (III, 256 nt), GGT mRNA-IV (IV, 341 nt), and PEA3 (PEA3, 382 nt) cRNA probes. An autoradiogram exposed for 48 h with an intensifying screen is shown. P, undigested probes; t, tRNA control hybridization; S, total RNA from the rat initial segment; N, total RNA from NRK-52 E cells cultured in DMEM for 4 days; Co: total RNA from Cos-1 cells cultured in DMEM for 3 days; Epi: total RNA from initial segment epididymal cells cultured for 8 days. The position of each protected RNA is shown to the right.

Effects of Exogenous PEA3 on GGT Promoter IVActivity in Cos-1, NRK-52 E, and Epididymal Cells

A series of luciferase reporter plasmids containing promoter-IV DNA sequences from nucleotides -1976 to +38 of the rat GGT gene are shown in Figure 3A. The schematic locations of the conserved PEA3/Ets binding motif (5'-AGGAAG-3') and/or the degenerate PEA3/Ets binding motif (5'-AGGAA[A/C]-3') are also included in each reporter plasmid. These plasmids were transiently transfected into cultured cells to determine whether PEA3 can regulate the expression of GGT mRNA-IV.



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  		FIG. 3. Effects of exogenous PEA3 on promoter IV activity of the rat GGT gene in cultured cells. A) Schematic representation of a series of reporter plasmids containing rat GGT promoter IV. Various lengths of promoter IV DNA sequences were inserted into a promoterless luciferase reporter plasmid (pGL-enhancer-3; Promega). Solid boxes represent canonical PEA3/Ets binding motif (5'-AGGAAG-3'), while open boxes represent degenerate PEA3/Ets binding motifs (5'-AGGAAa/c-3'). B) Different GGT promoter IV constructs (200 ng) were transfected into cultured Cos-1 cells with 250 ng of PSI DNA (basal), PSI-PEA3 sense DNA (sense), or PSI-PEA3 antisense DNA (antisense), along with 200 ng of PSI-ß-Gal DNA. Twenty-four hours after transfection, cell extracts were prepared and analyzed for ß-galactosidase and luciferase activity. C) Different GGT promoter IV constructs (500 ng) were transfected into cultured cells with 1000 ng of PSI DNA (basal), PSI-PEA3 sense DNA, or PSI-PEA3 antisense DNA, along with 1200 ng of PSI-ß-Gal DNA. Forty-eight hours after transfection, cell extracts were prepared and analyzed for ß-galactosidase and luciferase activity. D) Different GGT promoter IV constructs (300 ng) were transfected into cultured epididymal cells from rat initial segments with 400 ng of PSI DNA (basal), PSI-PEA3 sense DNA, or PSI-PEA3 antisense DNA, along with 300 ng of pCMV-SPORT-ß-Gal DNA. Sixty-six to seventy-two hours after transfection, cell extracts were prepared and analyzed for ß-galactosidase and luciferase activity. For B–D, RLA in each sample was measured after the luciferase activity was normalized to ß-galactosidase activity (transfection efficiency control). RLA in samples transfected with p135LUC, PSI, and ß-gal plasmids was regarded as control (100%). RLA in other samples within an experiment was expressed as a percentage of the RLA in control. Data shown represent mean percentage ± SEM from four to eight individual experiments for basal, sense, or antisense groups. Histogram bars showing different numbers are significantly different (p < 0.05) in a within-group, between-construct analysis. Histogram bars showing different letters are significantly different (p < 0.05) in a within-construct, between-group analysis.

As shown in Figure 3B, basal RLAs were p135=p250=p530=p903>p1410=p1976LUC in Cos-1 cells. After cotransfection with a sense PEA3 plasmid, the RLA mean values for each reporter plasmid group, except the p135LUC group, decreased compared to their respective basal activity. The RLAs in p903, p1414, and p1976LUC plasmid groups decreased significantly when compared to each basal level. However, the RLA in each plasmid group did not decrease after cotransfection with an antisense PEA3 plasmid.

In NRK-52E cells, basal reporter activity was p135=p250=p530>p903=p1410>p1976LUC in the absence of the PEA3 expression vector (Fig. 3C). After cotransfection with either a sense or an antisense PEA3 plasmid, the RLA in each reporter plasmid group, except p903LUC, did not change significantly compared to its corresponding basal activity. In the p903LUC group, the RLA increased after cotransfection with an antisense PEA3 plasmid (Fig. 3C).

In primary epididymal cells, basal RLA mean values were p135=p530=p903>p1976LUC=p1410LUC (Fig. 3D). After cotransfection with a sense PEA3 plasmid, the RLA in each reporter plasmid group increased significantly compared to its corresponding basal activity. Even though no significant changes in luciferase activity in each plasmid group were observed after cotransfection with an antisense PEA3 plasmid, the RLA mean values in p1410LUC and p1976LUC groups were slightly increased.

As shown in Figure 4, PEA3 mRNA levels in cells cotransfected with a sense PEA3 plasmid increased when compared to their levels in cells cotransfected with a PSI vector plasmid. However, the cyclophilin mRNA level remained relatively constant in cells transfected with either PSI or PSI-PEA3-sense plasmid (Fig. 4).



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  		FIG. 4. RNase protection analysis of total RNA from transfected NRK-52E (NRK) and Cos-1 (Cos) cells, and epididymal cells (Epi) from the rat initial segment. Cultured cells were cotransfected with a p135LUC, a ß-galactosidase plasmid (NRK and Cos-1: PSI-ß-Gal; epididymal cells: pCMV-SPORT-ß-Gal), and a PSI vector (B) or a PSI-PEA3 sense expression plasmid (T) as described under Materials and Methods. Total RNA (15 µg) isolated from transfected cells was hybridized with an antisense Cyclophilin (Cyc., 107 nt), GGT mRNA I (I, 155 nt), GGT mRNA II (II, 160 nt), GGT mRNA III (III, 256 nt), GGT mRNA IV (IV, 341 nt), and PEA3 (PEA3, 382 nt) cRNA probes. An autoradiogram exposed for 8 h with an intensifying screen is shown. P, Undigested probes; C, tRNA control hybridization; S, total RNA from the rat initial segment. The position of each protected RNA is shown to the right.

Requirements of PEA3 and Sp1 Binding Motifs for GGT Promoter IV Activity in Primary Epididymal Cells

In primary epididymal cells, deletion of DNA sequences between nucleotides -36 and -16 (no PEA3/Ets binding motif) and between nucleotides -36 and -8 (no PEA3/Ets and no Sp1 binding motifs) of promoter IV in the p135LUC reporter plasmid caused a 50% and 80% decrease in basal promoter IV activity, respectively (Fig. 5). The basal RLA in p+38LUC was about the same value as the RLA of p-8-36LUC. A promoterless plasmid, pGL-3-enhancer, had the lowest basal RLA (about 10% of the RLA in p135LUC). After the cells were cotransfected with a sense PEA3 plasmid, the RLAs in p135LUC and p+38LUC groups, but not in the other tested plasmid groups, were increased significantly when compared to the corresponding basal activities. Cotransfection of an antisense PEA3 plasmid did not significantly affect promoter activity of any tested plasmid.



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  		FIG. 5. Requirement of both PEA3/Ets and adjacent Sp1 binding sites for GGT promoter IV activity in primary epididymal cells. A) Schematic representation of several reporter plasmid constructs of rat GGT promoter IV (nucleotides-135 to +38). The plasmids were constructed such that the sequence was either left intact, was altered by deletion of nucleotides, or was inverted. Putative PEA3/Ets binding motifs (5'-AGGAAc/t-3') and an Sp1 binding motif within this promoter region are shown. p+38LUC plasmid was obtained by inserting a 173-bp DNA in the 3' to 5' orientation into a luciferase reporter plasmid. Coincidentally, a cryptic PEA3/Ets binding motif in the antisense direction was also present and shown in p+38LUC. p(-16-36)LUC and p(-8-36)LUC were obtained by deleting DNA sequences from nucleotides -36 and -16 and from nucleotides -36 to -8 of promoter IV in p135LUC, respectively. B) The above plasmid constructs (300 ng) were transfected into cultured epididymal cells with 400 ng of PSI DNA (basal), PSI-PEA3 sense DNA, or PSI-PEA3 antisense DNA, and with 300 ng of pCMV-SPORT-ß-Gal DNA. Sixty-six to 72 h after transfection, cell extracts were prepared and analyzed for ß-galactosidase and luciferase activity. RLA in each sample was measured after luciferase activity was normalized to ß-galactosidase activity (transfection efficiency control). RLA in samples transfected with p135LUC, PSI, and ß-gal plasmids was regarded as control (100%). RLA in other samples within an experiment was expressed as a percentage of the RLA in control. Data shown represent mean percentage ± SEM from two to five individual experiments for basal, sense, or antisense groups. Statistical analysis of the data is similar to that described in Figure 3.

Effects of ETS2 Repressor Factor (ERF) on Promoter IV Activity in Primary Epididymal Cells

To determine whether the activation effect on promoter activity in p135LUC is specific for PEA3 and whether promoter activities in p1410LUC and p1976LUC can be regulated, ETS2 repressor factor (ERF), a transcription repressor of the Ets transcription family [50], was transfected into epididymal cells. As shown in Figure 6, the basal RLAs in p1410LUC and p1976LUC groups decreased significantly when compared to the basal RLA in p135LUC group. After cotransfection with an ERF expression plasmid, luciferase activity in p135LUC group did not differ from its basal control activity. However, the RLAs in the p1410LUC and p1976LUC groups increased to their control values after cotransfection with an ERF expression plasmid.



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  		FIG. 6. Effects of exogenous ERF on promoter IV activity in primary epididymal cells from the rat initial segment. Different GGT promoter IV constructs (300 ng) were transfected into cultured epididymal cells with 400 ng of PSI DNA (basal) or pCMV-ERF (ERF) along with 300 ng of pCMV-SPORT-ß-Gal DNA. Sixty-six to 72 h after transfection, cell extracts were prepared and analyzed for ß-galactosidase and luciferase activity. RLA in each sample was measured after the luciferase activity was normalized to ß-galactosidase activity (transfection efficiency control). RLA in samples transfected with p135LUC, PSI, and ß-gal plasmids was regarded as control (100%). RLAs in other samples within an experiment were expressed as a percentage of the RLA in control. Data shown represent mean percentage ± SEM from three to eight experiments. Mean percentages for RLA within basal or ERF groups, or within each promoter IV-luciferase plasmid, with different letters are significantly different (p < 0.05).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Primary epididymal cells from the rat initial segment had morphological characteristics similar to those described previously [54, 55] and maintained the GGT mRNA-IV and PEA3 mRNA expression levels of their parental tissue (Fig. 2). It is unclear why GGT mRNA-II and mRNA-III were not expressed in primary epididymal cell cultures (Fig. 2). A previous study had shown that FCS in culture media caused a decrease in GGT activity in primary liver cells [59]. FCS has also been shown to decrease GGT enzyme activity in incubated initial segment pieces (unpublished data). Thus, FCS could be a factor in culture media that may be involved in the suppression of GGT mRNA-II and GGT mRNA-III expression. The presence of PEA3 mRNA in NRK-52E cells (Fig. 2) was consistent with our observations that PEA3 protein was present in rat kidney tissue as measured by Western blot analysis (unpublished data) and immunocytochemistry (unpublished data). The lack of expression of PEA3 mRNA in Cos-1 cells (Fig. 2) confirms a previous report that PEA3 protein is absent in those cells [48].

Using transient transfection assays, we found that p135LUC had high promoter activity (basal activity) and that extension of the promoter sequence to nucleotide -1976 caused a decrease of basal promoter activity in Cos-1, NRK-52E, and primary epididymal cell cultures (Fig. 3). These results are consistent with a previous observation that a reporter plasmid containing a promoter IV sequence between nucleotides -164 and +38 has the highest chloramphenicol acetyltransferase (CAT) reporter activity and that extension of the promoter sequences up to nucleotide -903 resulted in no change of reporter activity in Fao cells or a decrease of promoter IV activity in HTC hepatoma cells [18]. Using GCG computer software, no responsive elements for known transcriptional factors were observed to be present between nucleotides -164 and -135 of promoter IV. These results suggest that DNA sequences between nucleotides -135 and +38 of the rat GGT promoter IV are important to drive the expression of GGT mRNA-IV and that possible repressors or silencers are present in the 5' region upstream of promoter IV.

As multiple PEA3/Ets binding motifs are present in GGT promoter IV (Fig. 1), it is possible that PEA3 may be a factor that regulates the transcription of rat GGT mRNA-IV in the initial segment. Our previous studies have shown that PEA3 mRNA and GGT mRNA-IV are expressed in the initial segment and that the expression of both PEA3 mRNA and GGT mRNA are under the regulation of testicular factors in this epididymal region [21, 49]. Data from electrophoretic mobility shift assays (EMSAs) have shown that PEA3 protein in the nuclei of the initial segment can bind to a PEA3/Ets binding motif in promoter IV [49]. In this study, we also found that both PEA3 mRNA and GGT mRNA-IV are expressed in primary epididymal cells isolated from the initial segment (Fig. 2). All of these results indicate that the expression of PEA3 mRNA and the expression of GGT mRNA-IV in the initial segment are correlated and that PEA3 may be involved in the regulation of promoter IV activity of the rat GGT gene. To determine whether PEA3 is involved in the regulation of GGT promoter IV activity, transient transfection experiments using a PEA3 expression plasmid were performed in two cell lines, Cos-1 (which has no endogenous PEA3 expression) and NRK-52E (which has endogenous PEA3 expression), and in primary epididymal cells. Our results show that exogenous PEA3 enhanced p135LUC reporter activity only in epididymal cells but not in NRK-52E or Cos-1 cells (Fig. 3) even though PEA3 mRNA levels were abundant in all these transfected cells (Fig. 4). These results suggest that PEA3 is involved in the activation of promoter IV in a cell-specific manner. In other words, an epididymal cell-specific factor(s) could also be required to enhance promoter activity by PEA3. Since deletion of the only PEA3/Ets binding motif in p135LUC plasmid resulted in a significant decrease of basal reporter activities in epididymal cells (basal p-16-36LUC or p-8-36LUC vs. basal p135LUC, Fig. 5) and exogenous PEA3 did not affect promoter activities in p-16-36LUC and p-8-36LUC (Fig. 5), it suggests that the PEA3/Ets binding motif between nucleotides -22 and -17 is the functional site for PEA3 to activate transcription of GGT promoter IV. Transfection experiments were performed in epididymal cells using an ERF expression plasmid to examine the specificity of PEA3 promoter activation. Like PEA3, ERF is also a member of the Ets transcriptional family [50]. ERF binds DNA sequences containing a PEA3/Ets binding motif and acts as a transcriptional repressor [50]. In the presence of endogenous PEA3, ERF neither inhibited nor activated the basal promoter activity of p135LUC (Fig. 6) in transfected epididymal cells, indicating specific activation effects of PEA3 on the proximal PEA3/Ets binding motif in promoter IV. Deletion of the PEA3/Ets binding motif between nucleotides -36 and -16 of the rat promoter IV only resulted in a 50% decrease in basal promoter activity (Fig. 5). This result suggested that in addition to PEA3, one or more other cis element(s) between nucleotides -135 and +38 is also required for maintaining its high promoter activity. A previous study has shown that transcription from a TATA-less promoter requires a multisubunit TFIID complex that includes TATA-binding protein (TBP), TBP-associated factors (TAFs), coactivators, and Sp1 [60]. Sp1 is a transcription factor that can bind to the consensus DNA sequence 5' (G/T)(G/A) GGC(G/T)(G/A)(G/A)(G/T) 3' and is involved in transcription from a TATA-less promoter [58, 60–62]. Rat GGT promoter IV is such a TATA-less promoter (Fig. 1) and contains an Sp1 consensus binding sequence adjacent to the PEA3/Ets binding site (Fig. 1). Sp1 protein has been shown to be present in the nuclei of mouse epididymal cells [62]. Using EMSA, nuclear extract isolated from the rat initial segments can bind to a radiolabeled GC-rich Sp1 consensus DNA(unpublished data). In addition, a number of studies have shown that both Sp1 and Ets transcriptional factors are involved in the regulation of transcription of other promoters [34–41, 63]. Thus, Sp1 could be one of the factors, along with PEA3, that regulate GGT promoter IV activity in the initial segment. To test this possibility, transfection studies using a reporter plasmid that has no PEA3 and no Sp1 binding motifs (p-8-36LUC) were performed. Our results show that deletion of the Sp1 binding site (nucleotides -16 to -8) along with the adjacent PEA3/Ets site significantly reduced GGT promoter IV activity (Fig. 5). We conclude that both the Sp1 and the PEA3/Ets binding motifs in promoter IV are required for maintaining high promoter activity in primary epididymal cells.

Our data (Fig. 2) would also suggest that additional factors may be important for the maintenance of promoter activity. Figure 2 shows that PEA3 mRNA expression in 8-day primary epididymal cell cultures remained unchanged relative to its expression in a normal intact initial segment. However, GGT mRNA IV expression, although present in these cell cultures, was reduced. Hence, although it appears that there are sufficient amounts of PEA3 mRNA in these cultures, the level of GGT mRNA IV expression was lower than expected. It is tempting to speculate that additional factors are needed in primary epididymal cell cultures to bring the amount of GGT mRNA IV back to its control levels. Candidate factors include growth factors such as bFGF [4] and androgens. In addition, there are a number of important cis elements, for example SF-1 and GATA, in the promoter region of GGT mRNA IV which may play a critical role in the maintenance of promoter activity.

Our results also indicate that PEA3 could act as a weak repressor to inhibit transcription of GGT mRNA-IV in epididymal cells and that the responsive elements for this inhibiting activity could be PEA3/Ets binding motifs in the distal region (nucleotides -1976 to -1414) of promoter IV. Our reasoning is that 1) basal promoter activity in p1976LUC and p1410LUC, but not in p903LUC and p530LUC, decreased significantly when compared to the activity in p135LUC (Fig. 3, Fig. 6); 2) cotransfection of an antisense PEA3 plasmid resulted in slightly increased reporter activities only in p1976LUC and p1410LUC in epididymal cells (Fig. 3); 3) ERF selectively recovered the decreased basal promoter activities in p1410luc and p1976LUC to basal promoter activity in p135LUC (Fig. 6); 4) the potency of activation in epididymal cells by exogenous PEA3 in p1976LUC was considerably less than that in other tested promoter-reporters (Fig. 3); 5) cotransfection with a sense PEA3 expression plasmid, but not with an antisense PEA3 expression plasmid, also caused a significant decrease in promoter activity in the p1976 and p1410LUC groups in Cos-1 cells (a nonendogenous PEA3 cell line) (Fig. 3) ; 6) basal promoter activity of p1976LUC was also lower than that of other tested promoter-reporters in NRK-52E cells (a cell line that has endogenous PEA3) (Fig. 3); and 7) a cluster of PEA3/Ets binding sites are present in the region between nucleotides -1579 and -1043 of promoter IV (Fig. 1). A recent report has also shown that PEA3 can inhibit the reporter activity of an epididymal-specific glutathione peroxidase gene promoter [43].

On the basis of our results and previous studies with respect to Sp1 and other Ets transcriptional factors [34, 36, 37, 39–42, 64–69], an initial model of how PEA3 may be involved in the regulation of transcription of GGT promoter IV in epididymal cells is presented in Figure 7. By using different PEA3/Ets binding sites, PEA3 can regulate transcription of GGT by acting as a strong activator and/or as a weak repressor in epididymal cells from the initial segment. Besides PEA3, Sp1 and ERF may also be involved in the regulation of transcription of GGT promoter IV. It is anticipated that further studies such as protein-protein interaction will define the mechanisms of regulating the expression of GGT mRNA-IV in the initial segment of the rat epididymis by PEA3, other transcriptional factors, and an unknown epididymal cell-specific factor(s).



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  		FIG. 7. A simplified model describing the functions of PEA3 in regulating transcription of rat GGT promoter IV. PEA3 could bind to the proximal PEA3/Ets binding motif and interact with an unknown epididymal cell-specific factor(s), Sp1, and/or some components of general transcriptional factors to activate transcription of the rat GGT mRNA-IV gene in the initial segment. The strong activation effects could be PEA3-specific and may be unaffected by ERF. PEA3 may also bind to distal PEA3/Ets binding motifs to function as a weak repressor of transcription of promoter IV. The inhibiting effects of PEA3 could be modulated by ERF. PEA3/Ets binding motifs in the middle region of promoter IV do not appear to be functional sites for PEA3 to activate or inhibit transcription of promoter IV. Both testicular factors such as bFGF [4] and circulating androgens are probably very important for the overall regulation of this pathway.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. John Hassell (McMaster University, Hamilton, ONT, Canada) for PEA3 cDNA; to Dr. James Douglass (Oregon Health Sciences University, Portland, OR) for cyclophilin cDNA; and to Dr. Yannick Laperche (Unite INSERM 99, France) for rat GGT cDNA plasmids. We thank Michelle A. Christgen and Nena S. Fox (Tissue Culture Core, the Diabetes Center, University of Virginia) for preparing Cos-1 and NRK-52E cells. DNA sequencing was performed by the Molecular Core Laboratory of the Specialized Cooperative Centers Program in Reproduction Research, and the Facility Center in Molecular Biology of the University of Virginia. We also thank Drs. Marie Hanigan, Margaret Shupnik, Terry Turner, and an unknown reviewer for their critical evaluation and helpful comments.


   FOOTNOTES
 
1 This study was supported by NIH grants HD 32979 (B.T.H.) and U54-HD28934, Specialized Cooperative Centers Program in Reproduction Research, The Rockefeller Foundation, and The Ernst Schering Research Foundation. Back

2 Correspondence: Barry T. Hinton, Dept. of Cell Biology, University of Virginia, Box 439, 1300 Jefferson Park Avenue, Charlottesville, VA 22908. FAX: 804 982 3912; bth7c{at}virginia.edu Back

Accepted: October 23, 1998.

Received: July 29, 1998.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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J. L. Kirby, L. Yang, J. C. Labus, and B. T. Hinton
Characterization of Fibroblast Growth Factor Receptors Expressed in Principal Cells in the Initial Segment of the Rat Epididymis
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M. K. Rao, C. M. Wayne, and M. F. Wilkinson
Pem Homeobox Gene Regulatory Sequences That Direct Androgen-dependent Developmentally Regulated Gene Expression in Different Subregions of the Epididymis
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R. Telgmann, J.J. Brosens, K. Kappler-Hanno, R. Ivell, and C. Kirchhoff
Epididymal epithelium immortalized by simian virus 40 large T antigen: a model to study epididymal gene expression
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S. D. Zabludoff, M. Charron, J. N. DeCerbo, N. Simukova, and W. W. Wright
Male Germ Cells Regulate Transcription of the Cathepsin L Gene by Rat Sertoli Cells
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O. Fröhlich, C. Po, and L. G. Young
Organization of the Human Gene Encoding the Epididymis-Specific EP2 Protein Variants and Its Relationship to Defensin Genes
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