Biol Reprod Lalor Postdoctoral Fellowships -- Application Deadline January 15, 2009
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print January 4, 2006.
Biol Reprod 2006, 10.1095/biolreprod.105.044354
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
74/4/714    most recent
biolreprod.105.044354v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yang, L.
Right arrow Articles by Hinton, B. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, L.
Right arrow Articles by Hinton, B. T.
Agricola
Right arrow Articles by Yang, L.
Right arrow Articles by Hinton, B. T.
BIOLOGY OF REPRODUCTION 74, 714–720 (2006)
DOI: 10.1095/biolreprod.105.044354
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Putative Regulation of Expression of Members of the Ets Variant 4 Transcription Factor Family and Their Downstream Targets in the Rat Epididymis1

Ling Yang , Sallie A. Fox , Jennifer L. Kirby , Brigid V. Troan , and Barry T. Hinton 2 

Department of Cell Biology, University of Virginia Health System, Charlottesville, Virginia 22908

ABSTRACT

Several genes expressed in the initial segment of the epididymis depend on factors from the testis that reach the epididymis via the luminal system. These include gamma-glutamyl transpeptidase mRNA IV (Ggt_pr4), steroid 5 alpha reductase (Srd5a1), glutathione peroxidase 5 (Gpx5), and cystatin-related epididymal spermatogenic (Cst8) genes. Promoter analyses indicated that these genes contain several ETS DNA-binding sites. Members of the polyomavirus enhancer activator 3 (ETV4) family bind to ETS sites on the promoter of target genes to regulate transcription. In this study, the role of ETV4 family members (ETV4, ETV5, ETV1) in the transcription of initial segment specific genes was evaluated. All three ETV4 family mRNAs are expressed in the principal cells of the initial segment and depend upon the presence of testicular luminal fluid factors. ETV4 protein was localized to principal cell nuclei and displayed the highest expression in the most proximal region of the initial segment. In addition, ETV4 protein levels were diminished after loss of testicular luminal fluid factors. A dominant-negative construct of ETV5 was in vivo electroporated into the initial segment to determine if ETV4 family members can regulate the transcription of testicular luminal fluid factor-regulated genes. Quantitative PCR indicated that 1 day postelectroporation, all three ETV4 family member mRNAs were significantly decreased. In addition, Ggt_pr4, Srd5a1, and Gpx5 mRNA levels were also significantly decreased. The data suggest that ETV4 family members regulate their own expression, and that they regulate transcription of a subset of genes that are dependent upon testicular luminal fluid factors.

epididymis, male reproductive tract, signal transduction

INTRODUCTION

Many of the genes expressed within the epididymis are dependent on androgens for their expression and/or their regulation. However, several genes expressed within the most proximal region of the epididymis, the initial segment, are regulated by factors that reach the epididymis via the rete testis and efferent ducts, called testicular luminal fluid factors. Evidence suggests that these may be growth factors such as members of the fibroblast growth factor (FGF) family [1, 2]. Genes that are regulated by testicular luminal fluid factors include Cst8 [3], Gpx5 [4], Srd5a1 [5], Etv4 [6], and Ggt_pr4 [7]. Ggt_pr4, which is expressed under the control of GGT promoter IV, is highly expressed in the initial segment under the control of testicular luminal fluid factors [7, 8]. Recent work on GGT promoter IV using an in vivo electroporation method revealed that three polyomavirus enhancer activator 3 (ETV4) DNA-binding sites within the first 530 bp of the promoter are responsible for expression of this gene [9]. Previous work has shown that a protein from initial segment nuclear extracts can bind to one of these three sequences by electromobility shift assay, and binding is eliminated by the addition of an ETV4 monoclonal antibody directed at the DNA-binding motif [10].

The ETV4 subfamily, comprised of ETV4, ETV5, and ETV1, belongs to a larger family of transcription factors known as ETS proteins. Members of this family are characterized by an 85-amino acid region known as the ETS domain that encodes for a helix-turn-helix DNA-binding motif [11]. The founding member of the group, ETV4, was originally described as a DNA-binding activity that protected the sequence 5'-AGGAAG-3' from digestion with DNaseI [12]. Although each of the three ETV4 family members is similar in structure, they have unique characteristics that may be important for the differential regulation of transcriptional activity. All ETV4 family members have an N-terminal transactivation domain characterized by a stretch of conserved acidic residues [13], but ETV5 and ETV1 have an additional transactivation domain in their C-terminus [14, 15]. Furthermore, the transcriptional activity of the ETV4 family is differentially modified by several signaling molecules, including mitogen-activated protein kinases (MAPKs) and cAMP [14, 16–22]. Finally, the ETV4 family genes show distinct patterns of expression. Etv5 expression is the most universal [13], whereas Etv4 and Etv1demonstrate more limited patterns of expression. Etv1 is expressed in several tissues, with its highest levels in heart, lung, brain, and testis [23–25]. Etv4 has the most restricted pattern of expression, with high levels in brain and epididymis and moderate levels in mammary tissue [26].

In the epididymis, Etv4 mRNA is highly expressed in the initial segment of the mouse and rat, with little to no expression in the remainder of the epididymis [6, 27]. Interestingly, the expression of Etv4 itself is controlled by testicular luminal fluid factors. Etv4 mRNA levels decrease by approximately 40% 12 h after efferent duct ligation (EDL) and 90% 24 h after EDL [6]. In addition, ETV4 protein levels are highest in the nuclear extracts from initial segments [6]. However, little is known about the cell types within the initial segment that express Etv4 or how quickly the ETV4 protein is degraded following the loss of Etv4 mRNA. Further, there are no studies evaluating the other members of the ETV4 family in the male reproductive tract. In an attempt to understand the role of the ETV4 family in the regulation of testicular luminal fluid factor-controlled genes, experiments were performed to characterize the expression, localization, and function of the different ETV4 family members in the initial segment of the rat epididymis.

MATERIALS AND METHODS

Animals

Normal adult male Sprague-Dawley rats (Hilltop Laboratories) between the ages of 50 and 100 days were maintained on a 12L:12D cycle with free access to food and water in the University of Virginia vivarium. All experiments complied with the regulations set forth by the Animal Welfare Act (Public Law 91–579), the Guide for the Care and Use of Laboratory Animals (NRC, 1996) published by the Department of Health and Human Services, and the policies and procedures of the University of Virginia Institutional Animal Care and Use Committee.

Efferent Duct Ligation

To prevent luminal testicular factors from reaching the epididymis, unilateral EDL surgeries were performed as described previously [7]. For control, a sham operation was performed on the contralateral side within the same animal. Animals were killed with carbon dioxide gas at the indicated times.

In Vivo Electroporation

The dominant-negative Etv5-En1 plasmid was kindly provided by Dr. Brigid Hogan [28]. Briefly, the pFLAG-CMV-2 vector housed a construct containing the ETV5 DNA binding domain (amino acids 290–489) fused to the Engrailed repressor domain (amino acids 1–298) with an N-terminal FLAG tag. This is a useful construct to evaluate the role of all three ETV4 family members because there is high sequence homology of ETV4 family members between mouse and rat, and there is redundancy between the ETV4 family members in activating downstream genes [28].

Three micrograms per microliter of Etv5-En1 plasmid at a volume of 15 µl for a total of 45 µg was electroporated into the interstitium of the initial segment region 1a using previously published conditions [9]. The contralateral side served as an intact control. Rats were allowed to recover and were then killed 1 day postinjection. The injected tissue and the control side were recovered and frozen in liquid nitrogen. RNA was extracted in Trizol (Invitrogen). A second set of experiments was performed in which the empty pFLAG-CMV-2 vector was electroporated into the interstitium of the initial segment and compared to the contralateral intact side (data not shown).

RT-PCR and Quantitative PCR on Initial Segment Principal Cell Total RNA

The isolation of total RNA from initial segment principal cells by laser capture microdissection and whole tissue from rats and subsequent RT-PCR were performed as described in detail previously [2] and using the Invitrogen Superscript III First Strand Synthesis RT-PCR kit. Oligonucleotides used for PCR were synthesized by Invitrogen and are listed in Table 1. All reactions were performed in a GeneAmp PCR System 2400 (Perkin Elmer). The products were subsequently cloned into pCR-Blunt II TOPO (Invitrogen) and confirmed by sequencing (Biomolecular Research Facility, University of Virginia).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Primer pairs used for PCR amplification.

Quantitative PCR reactions for all EDL experiments were performed on an ABI Prism 7700 Sequence Detection System (Applied Biosystems) in 50-µl reactions containing 1 µl of RT product diluted 1:10, 0.2 µM of each primer described above, and 1x SYBR Green PCR master mix (Applied Biosystems). Oligonucleotides were designed to span across at least one intron, minimizing the likelihood of amplifying genomic DNA under these cycling conditions. In addition, PCR products were checked by agarose gel electrophoresis to be sure that one reaction product was present, in order to eliminate anomalous readings when using SYBR Green as a fluorescent probe. Each sample was also subjected to PCR amplification of 18s ribosomal RNA using TaqMan ribosomal RNA control reagents (Applied Biosystems) with 1x TaqMan Universal PCR master mix (Applied Biosystems) according to the manufacturer's instructions. Standard curves were generated with serial dilutions of normal initial segment RT product. Data were analyzed using Sequence Detector v1.7 or v1.9 software (Applied Biosystems). Target gene expression levels were normalized using relative rRNA levels and graphed as percentage of sham control. As an additional control for EDL samples, primers were used to amplify a portion of the androgen receptor; PCR product levels were quantitated by real time PCR (n = 4 animals each run in triplicate).

Quantitative PCR reactions for all Etv5-En1 electroporation and empty vector experiments were performed on the M.J. Research Chromo4 System (M.J. Research Design) in 20-µl reaction volumes with 1- to 2-µl RT reactions diluted 1:10. All PCR amplifications were performed with 1x SYBR Green Master Mix (Biorad) using oligonucleotides for the gene of interest. Reactions were also performed to amplify the 18S rRNA subunit as a load control using the specific oligonucleotides included in the Taqman kit mentioned above and 1- to 2-µl RT reactions (the same as those used for the gene of interest) diluted 1:10. PCR conditions were designed to take into account primer annealing temperature and product length. All samples were run in triplicate and standard curves were generated. Data were analyzed using Graph Pad Prism Software version 3.0.

Preparation of Nuclear and Cytoplasmic Extracts

Nuclear and cytoplasmic extracts were prepared and pooled from six to eight rats per condition using the Nuclear Extract Kit (Active Motif) according to the manufacturer's instructions. Briefly, the proximal half of initial segments were removed, diced into pieces with a razor blade, and homogenized in 1x hypotonic buffer with 1 mM DTT and detergent in a glass dounce homogenizer at 4°C. Samples were centrifuged at 850 x g for 10 min at 4°C. The pellets were gently resuspended in 1x hypotonic buffer and incubated on ice for 15 min. The cells were lysed with detergent and centrifuged at 14000 x g for 30 sec at 4°C. The supernatants were removed and stored at –80°C in small aliquots for analysis as the second cytoplasmic fraction (C2). The pellets were resuspended in Complete Lysis Buffer and incubated at 4°C with agitation for 30 min. The samples were vortexed and centrifuged at 14000 x g at 4°C for 10 min. The supernatants were removed and stored at –80°C for analysis as the nuclear fraction. Prior to use, protein determinations of each fraction were made using the Bradford method (Bio-Rad Laboratories).

Immunoblotting

Thirty micrograms of nuclear and/or cytoplasmic protein in Laemmli sample buffer were loaded onto SDS-PAGE gels. Prior to blotting, the membranes were stained with Ponceau S (Sigma) to ensure equal protein loading. Western blotting was performed as described previously [6] using ETV4 monoclonal antibody 16 (used at 0.4 µg/ml) from Santa Cruz Biotechnology.

Immunohistochemistry

Initial segments were immersion-fixed in 4% paraformaldehyde for 24–48 h at 4°C. Tissues were sent to the Center for Research in Reproduction Cell Science Core at the University of Virginia for paraffin embedding and sectioning. Immunohistochemistry was performed with 1 µg/ml ETV4 monoclonal antibody 16, using a modification of a previously published procedure [29]. The modification included a step to microwave the slides in antigen unmasking solution (Vector Laboratories) for 10 min on high in a 1300-W microwave, after which the slides were cooled for 1 h at room temperature.

RESULTS

ETV4 Family Member Expression in Principal Cells

To determine whether Etv4, Etv5, and Etv1 mRNAs are expressed within the principal cells in the initial segment, RT-PCR was performed on total RNA collected from principal cells isolated by laser capture microdissection (LCM). A discussion regarding the collection and characterization of principal cells by LCM has previously been published [2]. As shown in Figure 1, the mRNAs of all three members of the ETV4 family were expressed in both initial segment tissue and principal cells. These experiments were repeated on separate microdissection samples taken from four different rats with identical results.


Figure 1
View larger version (55K):
[in this window]
[in a new window]
  		FIG. 1. RT-PCR for ETV4 family members. RT-PCR was performed to screen for Etv4, Etv5, and Etv1 genes in total RNA from rat initial segment tissue (T) and from principal cells taken by laser capture microdissection (C). A no-reverse transcriptase (–RT) and a no-template (–Template) control reaction were also performed for all laser capture and total initial segment tissue cDNA samples and no band was detected in either sample.

ETV4 Protein in Initial Segment Tissue

Immunoblot analysis was performed on the cytoplasmic and nuclear fractions of proteins isolated from initial segment tissue to determine whether ETV4 family proteins were present. As shown in Figure 2A, ETV4 protein was found exclusively in the nuclear portion of cellular extracts, in agreement with previous studies [6]. Commercially available antibodies to ETV5 and ETV1 were found to be unreliable in immunoblot analyses.


Figure 2
View larger version (85K):
[in this window]
[in a new window]
  		FIG. 2. Localization of ETV4 by immunoblotting and immunohistochemistry. A) Both nuclear (N) and cytoplasmic fractions (C2) from rat initial segments were electrophoresed on reducing 10% SDS-PAGE gels. Blots were incubated with ETV4 antibody (left panel) or secondary antibody only (right panel). Immunohistochemistry for ETV4 was performed on sections from rat epididymis (B–G). Slides were incubated with ETV4 antibody (B–F) or secondary antibody only (G). B and C) Zone 1a with an unlabeled efferent duct (ED). D) Zone 1b. E) Zone 1c. F) Proximal caput.

To determine the localization of ETV4 within the initial segment, immunohistochemistry was performed on sections of paraformaldehyde-fixed initial segments. ETV4 protein showed segmental expression with no ETV4 found in the efferent ducts (Fig. 2B) or proximal caput (Fig. 2F). The micrographs in Figure 2, C, D, and E, were taken from the same tissue section and show that the highest ETV4 levels observed were in zone 1a, with decreasing levels through zones 1b and 1c (see [25] for description of zones). No immunoreactivity was observed when the secondary antibody alone was used (Fig. 2G). Immunohistochemistry was repeated on sections from initial segments taken from three individual rats with similar results. Attempts at immunohistochemistry with the commercially available ETV5 and ETV1 antibodies were unsuccessful.

Testicular Luminal Fluid Factor-Dependent Expression of ETV4 Family Member mRNA

Previous studies from our lab showed a decrease in Etv4 mRNA expression following EDL [6]. To determine whether Etv1 and Etv5 mRNA expression are also regulated by luminal testicular factors, quantitative RT-PCR was performed on total RNA isolated from initial segments subjected to 4-, 8-, 12-, 18- and 24-h sham and EDL. Ggt_pr4 and Etv4 expression levels after EDL (Fig. 3, A and B) were consistent with data generated previously by RNase protection assay [6]. In addition, the expression of Etv5 and Etv1 mRNA was also modulated by EDL (Fig. 3, C and D). Steady-state Etv5 mRNA levels decreased the most rapidly after EDL. Expression levels of Etv5 were at 10% of sham control 8 h after EDL with no further significant decrease. In contrast, steady-state Etv1 mRNA levels increased to 120% of sham control after 4 h, but eventually decreased to 20% of sham control by 18 h, with no further decrease observed. As a control, we determined that androgen receptor mRNA levels were not significantly affected 24 h after EDL as compared to sham control (100% vs. 110% ± 7% respectively).


Figure 3
View larger version (18K):
[in this window]
[in a new window]
  		FIG. 3. Quantitative PCR analysis of ETV4 family mRNA levels following EDL. Quantitative PCR was performed with the oligonucleotides listed in Table 1 on RT products from rat initial segments subjected to sham and EDL for 4, 8, 12, 18, and 24 h. Data were taken from at least 4 animals per time point and expressed as the mean percentage of sham control ± SEM. Data were normalized to the levels of 18S ribosomal subunit levels within each sample. Mean ratios with different numbers were significantly different (P < 0.05) as assessed by one-way ANOVA followed by Tukey test. Ggt_pr4 (A), Etv4 (B), Etv5 (C), and Etv1 (D).

Testicular Luminal Fluid Factor-Dependent Expression of ETV4 Protein

To examine the role of testicular luminal fluid factors on ETV4 protein levels, immunoblot analysis was carried out on initial segment nuclear extracts from rats subjected to 6-, 12-, and 24-h sham and EDL. ETV4 protein levels were only slightly decreased 6 h after EDL, but were substantially decreased 12 h after EDL and were undetectable after 24 h (Fig. 4). Ponceau S staining before immunoblotting confirmed loading and transfer of equal levels of protein (data not shown).


Figure 4
View larger version (20K):
[in this window]
[in a new window]
  		FIG. 4. Immunoblot analysis of ETV4 following EDL. Nuclear extracts from rat initial segments subjected to sham (S) or EDL (E) for 6, 12, and 24 h were electrophoresed on a reducing 10% SDS-PAGE gel. After transfer, the blot was incubated with ETV4 antibody.

Although ETV4 protein levels decreased in nuclear extracts, the possibility existed that ETV4 changed cellular localization following EDL. Immunohistochemistry was performed on sections of initial segments from rats subjected to 12-h sham and EDL (Fig. 5). ETV4 immunoreactivity in the 12-h sham sections was consistent with that seen in Figure 2. ETV4 immunoreactivity was undetectable in the principal cell nuclei and cytoplasm in zones 1b and 1c and was severely reduced in region 1a (Fig. 5, D, F, and B, respectively) following 12-h EDL. These results were repeated in sections from three separate sham and EDL tissue pairs with equivalent results.


Figure 5
View larger version (141K):
[in this window]
[in a new window]
  		FIG. 5. Immunohistochemistry for ETV4 following EDL. Immunohistochemistry for ETV4 was performed on sections from the rat initial segment 12 h after sham (A, C, E) or EDL (B, D, F). Zone 1a (A and B). Zone 1b (C and D). Zone 1c (E and F).

Regulation of Testicular Luminal Fluid Factor-Dependent Genes by ETV4 Family Members

To determine whether ETV4 family members can regulate the transcription of testicular luminal fluid factor-dependent genes, a FLAG-tagged dominant-negative construct of the mouse ETV5 protein was in vivo electroporated into the initial segment as previously described [9] and gene expression evaluated after 1 day by quantitative RT-PCR. Expression of the fusion protein was confirmed by RT-PCR (Fig. 6A). Attempts to detect the presence of the fusion protein by immunoblotting and immunohistochemistry with a FLAG antibody were unsuccessful because of antibody cross-reactivity.


Figure 6
View larger version (18K):
[in this window]
[in a new window]
  		FIG. 6. Quantitative PCR of ETV4 family-regulated genes following Etv5-En1 in vivo electroporation. A) PCR for En1 mRNA using specific oligonucleotides listed in Table 1 on RT samples 24 hours postelectroporation of the Etv5-En1 plasmid from 4 injected (I) and control (C) initial segment tissue samples. In lane 9, the reverse transcriptase enzyme was omitted. Lane 10 is PCR using the pFLAG-CMV-2 Etv5-En1 vector as a positive control. B) Quantitative PCR on RT samples from rat initial segment tissue samples 24 hours postelectroporation of the Etv5-En1 plasmid using specific oligonucleotides listed in Table 1. Data were taken from 4 animals (same animals used for RT-PCR in Fig. 6a) per time point and expressed as the mean percentage of uninjected control ± SEM. Data were normalized to the levels of 18S ribosomal subunit levels within each sample. Mean ratios with different numbers were significantly different (*P < 0.05, ** P < 0.01, ***P < 0.001) as assessed by Student t-test.

The expression of all three ETV4 family members was significantly reduced (Fig. 6B) with Etv5 showing the greatest reduction in expression (>50%). Interestingly, Etv5 mRNA levels were also reduced by the largest amount as compared to Etv4 and Etv1 levels upon withdrawal of testicular luminal fluid factors (Fig. 3, C vs. B and D). The expression of the testicular luminal fluid factor-regulated genes Ggt_pr4, Gpx5, and Srd5a1 were significantly reduced. However, the expression of Cst8 and Lcn8, which are regulated in a similar manner, was not affected by the expression of dominant-negative ETV5. These experiments were repeated by in vivo electroporation of an empty vector control in comparison to the intact control, and quantitative RT-PCR was performed for the same gene targets with no significant change in expression detected in any of these genes (data not shown).

DISCUSSION

Previous characterizations have shown that Etv4 mRNA and protein are highly expressed in the epididymis, with highest levels in the initial segment [6, 26, 27]. In addition, Etv4 mRNA levels in the initial segment decrease substantially after EDL, suggesting that their expression is dependent upon testicular luminal fluid factors [6]. The studies presented here confirm these findings (Figs. 1 and 3) and also show the localization of ETV4 protein in principal cell nuclei by immunoblotting of nuclear extracts and immunohistochemistry of initial segment tissue (Fig. 2). Interestingly, ETV4 protein shows a highly segmental pattern of expression, with high levels in zone 1a that decrease through zones 1b and 1c. In addition, the protein is completely absent in the efferent ducts and proximal caput cells. In this study, ETV4 protein levels were also shown to be dependent upon testicular luminal fluid factors. ETV4 protein was slightly reduced following EDL for 6 h as determined by immunoblotting (Fig. 4). ETV4 immunohistochemistry 6 h after EDL revealed that even the most proximal tubules still expressed ETV4, although the luminal contents had cleared from this region first (data not shown). ETV4 protein levels were substantially reduced 12 h after EDL in both the immunoblotting and the immunohistochemistry experiments, suggesting that ETV4 protein expression decreases sometime between 6 and 12 h after EDL (Figs. 4 and 5). From these data, one can also infer that the half-life of the ETV4 protein is likely to be much less than 12 h in the initial segment. Although some transcription factors can go between the cytoplasm and the nucleus depending on their state of activation, ETV4 was found exclusively in the nucleus, and this localization was not altered by EDL (Fig. 5).

Etv5 and Etv1 mRNAs were also found in the principal cell population (Fig. 1) and their expression was dependent upon testicular luminal fluid factors (Fig. 3). Etv5 and Etv1 mRNA steady state levels decreased with different rates following EDL. Whereas Etv5 steady state levels dropped rapidly, reaching a nadir (10% of sham) by 8 h after EDL (Fig. 3C), the decrease in Etv1 mRNA to approximately 20% of sham (lowest level) occurred much later—18 h after EDL (Fig. 3D). The decrease in Etv4 mRNA levels following EDL (Fig. 3B) was consistent with previously published RNase protection assay results [6].

One way to explain the decrease in Ggt_pr4 levels after EDL and expression of the ETV5 construct would be that the concomitant decrease in ETV4 protein levels presumably leads to a loss of ETV4 binding to and transactivation of GGT promoter IV. Ggt_pr4 expression begins to decrease at 8–12 h (Fig. 3A), and the reduction in ETV4 protein levels begins between 6 and 12 h (Figs. 4 and 5). However, because the immunoblot was not quantitative (Fig. 4), it is impossible to determine a relationship between ETV4 protein levels and Ggt_pr4 levels. An alternative hypothesis is that transcriptional activity of Etv4 is regulated by testicular luminal fluid factors. ETV4 family members are targets of the RASA/RAF/MEK/MAPK3/1 pathway, which increases their transcriptional activity [14, 16, 17]. The phosphorylation of MAPK3/1 is dependent upon testicular luminal fluid factors, and the decrease in phosphorylated MAPK3/1 (active MAPK3/1) can be seen as soon as 4 h after EDL [30]. In addition, the expression patterns of active MAPK3/1, Ggt_pr4, and ETV4 are all highly segmented and show similar patterns of expression (Fig. 2; M.A. Palladino and B.T. Hinton, personal communication). The highest levels of expression of all three are found within zone 1a, with less expression in zone 1b and minimal to no expression in zone 1c.

The use of a dominant-negative construct of the ETV5 protein revealed that ETV4 family members can regulate the expression of a particular subset of testicular luminal fluid factor-regulated genes (Fig. 6B). Promoter analysis of Gpx5, Lcn8, and Ggt_pr4 [10, 27, 31] indicates the presence of multiple ETS binding sites within the 5' flanking region of these genes. In addition, we performed an analysis of approximately 5 kb of mouse and rat sequence 5' to the transcription start site for ETV4 family members, as well as for the genes regulated by testicular luminal fluid factors Gpx5, Lcn8, Cst8, Ggt_pr4, and Srd5a1. Multiple ETS binding sites were discovered in the 5' flanking region of these genes as well, and are listed in Table 2. Hence, ETV4 family members are capable of regulating the expression of all of these testicular luminal fluid factor-regulated genes. The reduction in expression of all three ETV4 family members suggests the presence of a positive feedback loop in which ETV4 members can induce their own expression by using some of these ETS-binding domains. Benz et al. [32] showed that mouse ETV4 can bind to elements within its own promoter to activate transcription, implicating a mechanism by which ETV4 can upregulate its own expression. However, it appears that ETV4 family members are unable to affect the expression of Cst8 and Lcn8. It is possible that prolonged expression of the dominant-negative construct may reveal a role for ETV4 family members in the regulation of Cst8 and Lcn8, but the regulation of these two genes does not appear to be primarily through ETV4 family members and may involve other transcription factor families.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Putative ETS sites in ETV4 family members and their potential downstream target genes.a

Expression of Gpx5 and Srd5a1 was significantly reduced after expression of the dominant-negative construct, suggesting that ETV4 family members may regulate their expression. Interestingly, expression of both of these genes was also significantly reduced by the expression of a dominant-negative FGFR1 construct in the initial segment (unpublished results). Recent studies have implicated the FGF family of growth factors as regulators of ETV4 family expression, particularly during development. In Xenopus mesoderm, Etv1 mRNA expression is stimulated by bFGF and blocked by expression of a dominant-negative FGF receptor [33]. In zebrafish, both Etv5 and Etv4 were shown to be controlled by both FGF3 and FGF8 [34, 35]. Etv5 and Etv4 were recently identified as downstream targets of FGF8 in the development of the nasal region in chick embryos [36]. We have previously published the existence of both FGF2 and FGF8 in rat rete testis fluid [2]. This could indicate that Gpx5 and Srd5a1 are regulated by an FGF-specific pathway through activation of MAPK3/1 and a ETV4 family member.

ACKNOWLEDGMENTS

The authors acknowledge helpful insight from Drs. D. Brautigan, R. Ogle, A. Sutherland, and T. Turner. Thanks to Dr. R. John Lye for the in silico promoter analyses.

FOOTNOTES

1 Supported by NIH-NICHD HD32979, Ernst Schering Research Foundation, and CONRAD to B.T.H., and by NICHD through the assistance of the U54 Specialized Cooperative Centers Program for Reproduction Research: Cell Science Core Facility, located at the University of Virginia, Charlottesville, VA (U54 HD28934), and the Lasercapture Microdissection Core Facility located at the University of Maryland School of Medicine, Baltimore, MD (U54 HD36207). J.L.K. was supported by a grant from the Medical Scientist Training Program, NIH, grant 2T32 GM07267. Back

2 Correspondence: Barry T. Hinton, Department of Cell Biology, University of Virginia Health System, PO Box 800732, Charlottesville, VA 22908. FAX: 434 982 3912; bth7c{at}virginia.edu Back

Received: 2 June 2005.

First decision: 5 July 2005.

Accepted: 19 December 2005.

REFERENCES

  1. Lan ZJ, Labus JC, Hinton BT, Regulation of gamma-glutamyl transpeptidase catalytic activity and protein level in the initial segment of the rat epididymis by testicular factors: role of basic fibroblast growth factor. Biol Reprod 1998 58:197-206[Abstract/Free Full Text]
  2. Kirby JL, Yang L, Labus JC, Hinton BT, Characterization of fibroblast growth factor receptors expressed in principal cells in the initial segment of the rat epididymis. Biol Reprod 2003 68:2314-2321[Abstract/Free Full Text]
  3. Cornwall GA, Orgebin-Crist M-C, Hann SR, The CRES gene: a unique testis regulated gene related to the cystatin family is highly restricted in its expression to the proximal region of the mouse epididymis. Mol Endocrinol 1992 6:1653-1664[Abstract]
  4. Rigaudiere N, Ghyselinck NB, Faure J, Dufaure JP, Regulation of the epididymal glutathione peroxidase-like protein in the mouse: dependence upon androgens and testicular factors. Mol Cell Endocrinol 1992 89:67-77[CrossRef][Medline]
  5. Viger RS, Robaire B, Immunocytochemical localization of 4-ene steroid 5{alpha}-reductase type 1 along the rat epididymis during postnatal development. Endocrinology 1994 134:2298-2306[Abstract]
  6. Lan ZJ, Palladino MA, Rudolph DB, Labus JC, Hinton BT, Identification, expression, and regulation of the transcriptional factor polyomavirus enhancer activator 3, and its putative role in regulating the expression of gamma-glutamyl transpeptidase mRNA-IV in the rat epididymis. Biol Reprod 1997 57:186-193[Abstract]
  7. Palladino MA, Hinton BT, Expression of multiple {gamma}-glutamyl transpeptidase messenger ribonucleic acid transcripts in the adult rat epididymis is differentially regulated by androgens and testicular factors in a region-specific manner. Endocrinology 1994 135:1146-1156[Abstract]
  8. Palladino MA, Laperche Y, Hinton BT, Multiple forms of gamma-glutamyl transpeptidase messenger ribonucleic acid are expressed in the adult rat testis and epididymis. Biol Reprod 1994 50:320-328[Abstract]
  9. Kirby JL, Yang L, Labus JC, Lye RJ, Hsia N, Day R, Cornwall GA, Hinton BT, Characterization of epididymal epithelial cell-specific gene promoters by in vivo electroporation. Biol Reprod 2004 71:613-619[Abstract/Free Full Text]
  10. Lan ZJ, Lye RJ, Holic N, Labus JC, Hinton BT, Involvement of polyomavirus enhancer activator 3 in the regulation of expression of gamma-glutamyl transpeptidase messenger ribonucleic acid-IV in the rat epididymis. Biol Reprod 1999 60:664-673[Abstract/Free Full Text]
  11. Karim FD, Urness LD, Thummel CS, Klemsz MJ, McKercher SR, Celada A, Van Beveren C, Maki RA, Gunther CV, Nye JA, The ETS-domain: a new DNA-binding motif that recognizes a purine-rich core DNA sequence. Genes Dev 1990 4:1451-1453[Free Full Text]
  12. Martin ME, Piette J, Yaniv M, Tang WJ, Folk WR, Activation of the polyomavirus enhancer by a murine activator protein 1 (AP1) homolog and two contiguous proteins. Proc Natl Acad Sci U S A 1988 85:5839-5843[Abstract/Free Full Text]
  13. Monté D, Baert JL, Defossez PA, de Launoit Y, Stéhelin D, Molecular cloning and characterization of human ERM, a new member of the Ets family closely related to mouse PEA3 and ER81 transcription factors. Oncogene 1994 9:1397-1406[Medline]
  14. Janknecht R, Analysis of the ERK-stimulated ETS transcription factor ER81. Mol Cell Biol 1996 16:1550-1556[Abstract]
  15. Laget MP, Defossez PA, Albagli O, Baert JL, Dewitte F, Stehelin D, de Launoit Y, Two functionally distinct domains responsible for transactivation by the Ets family member ERM. Oncogene 1996 12:1325-1336[Medline]
  16. Janknecht R, Monté D, Baert JL, de Launoit Y, The ETS-related transcription factor ERM is a nuclear target of signaling cascades involving MAPK and PKA. Oncogene 1996 13:1745-1754[Medline]
  17. O'Hagan RC, Tozer RG, Symons M, McCormick F, Hassell JA, The activity of the Ets transcription factor PEA3 is regulated by two distinct MAPK cascades. Oncogene 1996 13:1323-1333[Medline]
  18. Brown LA, Amores A, Schilling TF, Jowett T, Baert JL, de Launoit Y, Sharrocks AD, Molecular characterization of the zebrafish PEA3 ETS-domain transcription factor. Oncogene 1998 17:93-104[CrossRef][Medline]
  19. Bosc DG, Goueli BS, Janknecht R, HER2/Neu-mediated activation of the ETS transcription factor ER81 and its target gene MMP-1. Oncogene 2001 20:6215-6224[CrossRef][Medline]
  20. Janknecht R, Cell type-specific inhibition of the ETS transcription factor ER81 by mitogen-activated protein kinase-activated protein kinase 2. J Biol Chem 2001 276:41856-41861[Abstract/Free Full Text]
  21. Baert JL, Beaudoin C, Coutte L, de Launoit Y, ERM transactivation is up-regulated by the repression of DNA binding after the PKA phosphorylation of a consensus site at the edge of the ETS domain. J Biol Chem 2002 277:1002-1012[Abstract/Free Full Text]
  22. Wu J, Janknecht R, Regulation of the ETS transcription factor ER81 by the 90 kDa ribosomal S6 kinase 1 and protein kinase A. J Biol Chem 2002 277:27517-27527[Abstract/Free Full Text]
  23. Brown TA, McKnight SL, Specificities of protein-protein and protein-DNA interaction of GABP alpha and two newly defined ets-related proteins. Genes Dev 1992 6:2502-2512[Abstract/Free Full Text]
  24. Jeon IS, Davis JN, Braun BS, Sublett JE, Roussel MF, Denny CT, Shapiro DN, A variant Ewing's sarcoma translocation (7;22) fuses the EWS gene to the ETS gene ETV1. Oncogene 1995 10:1229-1234[Medline]
  25. Monté D, Coutte L, Baert JL, Angeli I, Stéhelin D, de Launoit Y, Molecular characterization of the ets-related human transcription factor ER81. Oncogene 1995 11:771-779[Medline]
  26. Xin JH, Cowie A, Lachance P, Hassell JA, Molecular cloning and characterization of PEA3, a new member of the Ets oncogene family that is differentially expressed in mouse embryonic cells. Genes Dev 1992 6:481-496[Abstract/Free Full Text]
  27. Drevet JR, Lareyre JJ, Schwaab V, Vernet P, Dufaure JP, The PEA3 protein of the Ets oncogene family is a putative transcriptional modulator of the mouse epididymis-specific glutathione peroxidase gene gpx5. Mol Reprod Dev 1998 49:131-140[CrossRef][Medline]
  28. Liu Y, Jiang H, Crawford HC, Hogan BLM, Role for ets domain transcription factors pea3/erm in mouse lung development. Dev Biol 2003 261:10-24[CrossRef][Medline]
  29. Rodriguez CM, Labus JC, Hinton BT, Organic cation/carnitine transporter, OCTN2, is differentially expressed in the adult rat epididymis Biol Reprod 2002; 67:314–319
  30. Kirby JL, Hinton BT, Testicular luminal growth factors as modulators of epididymal function. In: Francavilla F, Francavilla S, Forti G (eds.) Proceedings of the 1st European Congress of Andrology Chieti, Italy: Collana Di Studi Abruzzesi 2000: 411-425
  31. Lareyre J-J, Winfrey VP, Kasper S, Ong DE, Matusik RJ, Olson GE, Orgebin-Crist M-C, Gene duplication gives rise to a new 17-kilodalton lipocalin that shows epididymal region-specific expression and testicular factor(s) regulation. Endocrinology 2000 142:1296-1308
  32. Benz CC, O'Hagan RC, Richter B, Scott GK, Chang CH, Xiong X, Chew K, Ljung BM, Edgerton S, Thor A, Hassell JA, HER2/Neu and the Ets transcription activator PEA3 are coordinately upregulated in human breast cancer. Oncogene 1997 15:1513-1525[CrossRef][Medline]
  33. Münchberg SR, Steinbeisser H, The Xenopus Ets transcription factor XER81 is a target of the FGF signaling pathway. Mech Dev 1999 80:53-65[CrossRef][Medline]
  34. Raible F, Brand M, Tight transcriptional control of the ETS domain factors Erm and Pea3 by Fgf signaling during early zebrafish development. Mech Dev 2001 107:105-117[CrossRef][Medline]
  35. Roehl H, Nüsslein-Volhard C, Zebrafish Pea3 and Erm are general targets of FGF8 signaling. Curr Biol 2001 11:503-507[CrossRef][Medline]
  36. Firnberg N, Neubuser A, FGF signaling regulates expression of Tbx2, Erm, Pea3, and Pax3 in the early nasal region. Dev Biol 2002 247:237-250[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
74/4/714    most recent
biolreprod.105.044354v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yang, L.
Right arrow Articles by Hinton, B. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, L.
Right arrow Articles by Hinton, B. T.
Agricola
Right arrow Articles by Yang, L.
Right arrow Articles by Hinton, B. T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS