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Endocrinology, doi:10.1210/en.2007-0018
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Endocrinology Vol. 148, No. 7 3196-3204
Copyright © 2007 by The Endocrine Society

Bfk, a Novel Member of the Bcl2 Gene Family, Is Highly Expressed in Principal Cells of the Mouse Epididymis and Demonstrates a Predominant Nuclear Localization

Dwi Ari Pujianto, Anastasios E. Damdimopoulos, Petra Sipilä, Jenni Jalkanen, Ilpo Huhtaniemi and Matti Poutanen

Department of Physiology (D.A.P., A.E.D., P.S., J.J., I.H., M.P.), Institute of Biomedicine, University of Turku, 20520 Turku, Finland; and Department of Reproductive Biology (I.H.), Imperial College London, Hammersmith Campus, London W12 0NN, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
B-cell lymphoma 2 (BCL2) family kin (BFK) is a recently identified novel protein that is similar to proteins of the BCL2 family. In the present study, we discovered that the mouse Bfk transcript is expressed at the highest level in the epididymis. Two transcripts of 0.9 and 2.6 kb in size were identified, with alternative exon 4 structures, resulting in a difference in the last three to five amino acids of the variants. However, the 0.9-kb transcript was found to be the predominant form in the epididymis and mammary gland, another tissue with strong Bfk expression. Epididymal Bfk expression was regulated both by androgens and other testicular factors. It is thus one of the few initial-segment enriched genes under androgen control, the majority of them being regulated by other testicular factors. BFK protein was expressed specifically in the principal cells of the epididymis. Its nuclear localization was evident in the initial segment and caput epididymis and in the epithelium of pregnant female mammary gland. The expression of BFK-enhanced green fluorescent protein recombinant protein in epididymal cells further confirmed the predominant nuclear localization of BFK with nucleo-cytoplasmic shuttling. Overexpressing BFK in epididymal cells did not induce apoptosis. However, enhanced caspase 3 activation was observed in the presence of BFK upon staurosporine-induced apoptosis. This suggests that BFK may have a proapoptotic role only after the process has been initiated by other mechanisms. Being exceptionally highly expressed in the initial segment, Bfk is suggested to have a role in the differentiation of this segment of the epididymis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE EPIDIDYMIS IS characterized by region-specific expression of genes that are involved in creating a unique luminal microenvironment suitable for sperm maturation. Several such genes have been identified (1, 2, 3, 4, 5, 6, 7), and it is likely that these genes have specific roles in the epididymis rather than having general housekeeping functions. In addition to the region-specific expression in the functionally mature epididymis, the function of several genes is associated with the developmental changes of the epididymis during sexual maturation. It is known that cell proliferation rate does not differ significantly among epididymal regions during development of the organ, but the outcome of the proliferating activity (the number of principal cells) shows remarkable differences between the regions (8). This suggests that the difference in cell populations among the various epididymal regions could be the result of different rates of apoptosis.

The B-cell lymphoma 2 (Bcl2) gene family is involved in the regulation of cell death and survival without affecting cell proliferation. Bcl2 plays an important role in the survival of various epithelial cells by inhibiting apoptosis during embryogenesis, morphogenesis, development of the immune system, cell maturation, and differentiation (9, 10, 11, 12). In the reproductive organs, Bcl2 has been reported to be expressed at least in the ovary (13), testis (14, 15), and epididymis (16). Bcl2 has been shown to be involved in protecting cells in the cauda epididymis from apoptosis induced by the abdominal temperature (17), and also to regulate epithelial cell proliferation during the postnatal development (8).

A new member of the BCL2 protein family, BCL2 family kin (BFK), has recently been identified (18). It was found that overexpression of Bfk promoted weak apoptosis and antagonized the prosurvival function of Bcl2. In the mouse, Bfk expression was detected in the stomach, ovary, bone marrow, and spleen (18). In the human, four alternative Bfk splice variants were found to be expressed at least in the gastrointestinal tract. Two of them (variants 1 and 2) were able to induce weak apoptosis when overexpressed, and they contained the BCL2 homolog region 3 (BH3) that is required for initiation of apoptosis (19, 20). Variant 1 is the longest form of the human variants and constitutes the human homolog for mouse Bfk (18). Variants 3 and 4 did not induce apoptosis due to the lack of the functional domain or being incomplete for the BCL2 homolog region (21). Furthermore, it was reported that the expression of the human Bfk transcript variants 1 and 2 was reduced in gastrointestinal malignomas (21). In the present study, we discovered that the mouse Bfk transcript is specifically expressed in the epididymis in males and in mammary gland of pregnant females. We characterized the expression, regulation, and cellular localization of BFK and provide evidence for its role in epididymal differentiation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Identification of the gene model 566 (GM566) as Bfk and analyses in silico
GM566 was discovered in our previous study (22), whereas in the present study, the GM566 was identified as mouse Bfk by the identity of their amino acid sequences. The cDNA sequences of the mouse Bfk transcripts were deduced from the expressed sequence tag (EST) sequences available at Unigene (http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Mm&CID=297245). The Unigene database was used to analyze the distribution of the ESTs in various tissues. Ensembl Mouse GeneView (http://www.ensembl.org/Mus_musculus/index.html) and UCSC Genome Bioinformatics (http://genome.ucsc.edu/) were used to predict gene structure and exon-intron boundaries. InterProScan Sequence Search (http://www.ebi.ac.uk/InterProScan/) was used to predict functional domains. MultAlin (http://prodes.toulouse.inra.fr/multalin/multalin.html) was used to align the analyzed transcripts.

Experimental animals and RNA extraction
FVB/N male and female mice were used throughout the study. All mice were handled in accordance with the institutional animal care policies of the University of Turku (Turku, Finland). To analyze tissue distribution of the gene expression, 7- to 8-wk-old mice were used. To analyze androgen dependency of the gene expression, 28 sexually mature male mice (divided into seven groups of four mice) were gonadectomized under anesthesia. Testosterone (T) treatment was performed by inserting sc a silicon tube (SF Medical, Hudson, MA; inner diameter = 1.5 mm; outer diameter = 2.0 mm) filled with T powder (Sigma-Aldrich Corp., St. Louis, MO). The epididymides were collected 1, 3, 7, and 14 d after gonadectomy from the untreated mice and after 7- and 14-d-long T treatment. The epididymides were cut into four different regions (initial segment, caput, corpus, and cauda). For analyzing postnatal gene expression, five to 15 mice at different ages (1, 4, 8, 13, 17, 20, 30, 40, and 60 d old) were killed, and the whole epididymides were dissected out. To analyze expression in the female mammary gland, the tissue was isolated from virgin, pregnant, and lactating mice. Total RNA was isolated using the single-step method (23). Poly-A mRNA was isolated using the Oligotex mRNA kit (QIAGEN, Valencia, CA). The epididymides from untreated mice and the mammary glands from various stages were also prepared for histological sections.

Northern hybridization
Twenty micrograms total RNA or 4 µg poly-A mRNA were denatured, resolved on a 1% denaturing agarose gel, and transferred onto nylon membrane (Hybond-XL, Amersham Pharmacia, Buckinghamshire, UK). The membranes were hybridized with the [32P]{alpha}CTP-labeled cDNA probe for Bfk using standard techniques. To generate the probe, total RNA from the initial segment of epididymis was reverse transcribed and amplified (RT-PCR) using specific oligos Bfk-F and Bfk-R (supplemental Table S1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). The 502-bp product was used for labeling. Hybridization signals were detected by autoradiography using x-ray film (Fuji Photo Film Co. Ltd., Tokyo, Japan) or a phosphor imager (Fuji).

RT-PCR and quantitative real-time RT-PCR
RT-PCR using 1 µg total RNA from the initial segment was performed to amplify and confirm the predicted transcripts of Bfk available at Unigene and Ensembl Mouse Geneview. Quantitative real-time RT-PCR was carried out for analyzing androgen dependency, tissue distribution, and postnatal expression of Bfk in the developing mammary gland and comparison of transcript levels between the two mRNA variants. DNase-treated total RNA (100 ng) was reverse transcribed and amplified in the same reaction tube using QuantiTect SYBR-Green RT-PCR kit (QIAGEN), according to the manufacturer’s instructions. The primers and annealing temperatures used are described in supplemental Table S1. The samples and standard curves were run in triplicate. The relative standard curve method was used to calculate relative gene expression. Expression value from the ß-actin (Actb) gene was used for normalization.

In situ hybridization
cRNA probes (sense and antisense) were generated from a 400-bp fragment of the Bfk cDNA including exon 1 and exon 2. The cDNA was cloned into pBluescript I KS+ vector. The recombinant plasmid was used as a template for the [{alpha}-35S]UTP-labeled RNA probes produced by in vitro transcription by applying the T3 and T7 RNA polymerases. The probes were denatured by heating at 70 C for 5 min and dissolved in hybridization buffer [50% formamide, 0.3 M NaCl, 20 mM Tris-HCl, 5 M EDTA, 50 mM dithiothreitol (DTT), 0.5 g/liter tRNA, 1x Denhardt’s solution, and 10% dextran sulfate] to obtain a specific activity of about 100,000 cpm/µl. The probes were then applied to the tissue sections on glass slides, followed by overnight incubation in a moisturized chamber at 55 C. After hybridization, the slides were washed with 2x standard saline citrate, 50% formamide, and 10 mM DTT for 30 min at 55 C. Subsequently, the slides were treated with RNase A solution (10 µg/ml in 0.5 M NaCl, 10 mM Tris-HCl, and 5 mM EDTA, pH 8.0) for 30 min at 37 C and washed again with 2x standard saline citrate, 50% formamide, and 10 mM DTT for 15 min at 55 C, followed by dehydration with ascending concentrations of ethanol. The slides were then dried at room temperature, dipped into NTB2 emulsion (Eastman Kodak Co., Rochester, NY), and exposed in the dark at 4 C for 3–14 d. The slides were developed with Dextol developer (Eastman Kodak) for 4 min, rinsed in distilled water, fixed for 5 min in Kodak fixer (Eastman Kodak), and rinsed in distilled water. The slides were counterstained with hematoxylin and Hoechst 33258 (Sigma-Aldrich). Hybridization with a sense probe was used as a control.

Immunohistochemistry
Histological sections (5 µm thick) were used in immunohistochemical analysis. After deparaffinization and rehydration, the sections were exposed to 3% hydrogen peroxide in distilled water for 10 min. Sections were then boiled in 0.01 M sodium citrate, pH 6.0, for 15 min and cooled slowly to room temperature. For immunohistological staining, the PowerVision Poly-HRP IHC Kit (ImmunoVision Technologies, Brisbane, CA) was used as suggested by the manufacturer. The rabbit anti-BFK antiserum (kindly provided by Dr. A. Strasser, The Walter and Elisa Hall Institute of Medical Research, Melbourne, Australia) was diluted 1/15,000 in blocking solution for staining the epididymis and at 1/500 for staining the mammary gland. The slides were counterstained with hematoxylin and dehydrated in ethanol and xylene before mounting with pertex mounting solution (Histolab, Göteborg, Sweden).

Western blot analysis
Proteins from four different regions of the epididymis (initial segment, caput, corpus, and cauda) were separated in 15% SDS-PAGE, followed by transfer to polyvinylidene fluoride membrane (Amersham Biosciences). The membrane was subsequently blocked in PBS with 5% nonfat milk overnight at 4 C and incubated with the BFK antibody (dilution 1/2,000 in blocking solution) for 2 h at room temperature. The membrane was then rinsed with PBS and incubated with horseradish peroxidase (HRP)-conjugated goat antirabbit IgG (dilution 1/4,000 in blocking solution) for 1 h at room temperature, followed by three rinses with PBS. The HRP was visualized by using a chemiluminescence substrate (ECL plus Western blot detection system; Amersham Biosciences). To obtain the protein-loading control, the same membrane was stripped by incubating in buffer containing 65 mM Tris-HCl (pH 6.7), 100 mM ß-mercaptoethanol, and 2% SDS at 50 C for 1 h, followed by blocking with 5% nonfat milk and incubated with glyceraldehyde-3-phosphate dehydrogenase antibody (dilution 1/4000 in blocking solution).

Intracellular localization of BFK
Bfk cDNA was amplified using primers Bfk-F and Bfk-R (supplemental Table S1). The product was purified, digested with XhoI and PstI, and ligated into the pEGFPN3 (Clontech, Palo Alto, CA) vector upstream of EGFP. This resulted in a fusion protein with the BFK at the N terminus of EGFP. The Bfk-pEGFPN3 and pEGFPN3 (as a negative control) plasmids were transfected into mECap18 epididymal cells (24) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The EGFP fluorescence was monitored 24–48 h after transfection. For the localization studies, cells were fixed with 4% paraformaldehyde for 30 min at room temperature. After fixation, the cell nuclei were counterstained using 4',6-diamidino-2-phenylindole (Molecular Probes, Eugene, OR) for 5 min and washed three times in PBS before mounting using fluorescent mounting media (DakoCytomation, Glostrup, Denmark). Cells were imaged using a CellR fluorescence microscope (Olympus, Tokyo, Japan). The fluorescence loss in photobleaching (FLIP) experiment was performed on a Leica SP2 confocal microscope. Live cells transfected with either Bfk-EGFP or EGFP alone were imaged using the 488-nm laser line. Using the same laser line, cells were then spot bleached at two locations in the cytoplasm for 10 sec at each spot, and then a new image was captured; after 1 min, the procedure was repeated, and the cells were imaged, then bleached, and imaged again followed by a 1-min interval for the next round.

Generating a stable cell line overexpressing BFK
To obtain stable transfected cells overexpressing Bfk, we amplified the Bfk cDNA using primer pair Bfk-F2 and Bfk-R2 (supplemental Table S1) and cloned the fragment into the pEGFPN3 vector (Clontech). mECap18 cells (24) were transfected with either empty vector or pEGFPN3-Bfk vector, and cells with stable integration of the plasmid were obtained by selection in media (DMEM, 10% fetal calf serum) containing 1 mg/ml G418 (Sigma). The overexpression of BFK was confirmed through Western blot analysis.

Apoptotic studies
Cells were treated with 200 nM staurosporine for the indicated times and then trypsinized, collected through centrifugation, and lysed through repeated freeze/thaw cycles. The protein concentration was measured using the BCA Protein Assay Kit (Pierce, Rockford, IL). Twenty micrograms of the extracts were loaded onto a 15% SDS-PAGE gel and after separation transferred to Hybond-P (Amersham Biosciences) by Western blotting. The membranes were blocked by 10% nonfat milk powder in PBS with 0.1% Tween 20 and then analyzed using the antibodies Asp175 against cleaved caspase 3 (Cell Signaling Technology, Danvers, MA) at 1/2500 and monoclonal mouse anti-actin clone C4 (ICN, Irvine, CA) at 1/100,000. The Aps175 antibody was detected using an antirabbit IgG HRP-conjugated antibody and the anti-actin antibody with an antimouse IgG HRP antibody (Amersham Biosciences). To visualize the antibodies, we used the ECL plus Western blotting detection kit (Amersham Biosciences).

Statistical analysis
SigmaStat for Windows was used to perform Kruskal-Wallis one-way ANOVA for the androgen dependency of mRNA expression analyzed by real-time RT-PCR. Significant differences among the groups were subsequently assessed by the Student-Newman-Keuls test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
On searching for segment-specific and androgen-regulated genes in the initial segment and caput of the mouse epididymis, we recently identified an initial segment-enriched gene (Gm566) that was also expressed in the distal caput at a lower level (22). In the present study, we verified the structure of the mouse Gm566 gene and characterized the mRNAs expressed in the epididymis. Based on the sequence identity, Gm566 was found to be identical to mouse BFK (18), a newly identified proapoptotic member of the BCL2 protein family.

Bfk exists as two splice variants with different levels of expression
The Bfk gene is located on chromosome 3 (3F2.2), and it consists of four exons (Fig. 1AGo). Two alternative spliced transcripts were predicted by in silico analysis and confirmed by Northern blot analysis (Fig. 1BGo) showing a strong signal with a size of 0.9 kb (variant 1) and an additional faint signal with a size of 2.6 kb (variant 2). Both transcripts were also identified by RT-PCR (Fig. 1AGo) with primers that covered the full coding regions of the two transcripts (Fig. 1CGo). The two splice variants share the first three exons, whereas two alternative exon 4 structures with different transcript sequences were identified. Accordingly, the amino acid structures of the two forms vary in the last three to five amino acids (Fig. 1DGo). The existence of two transcripts was also proven by EST mapping, with several ESTs present for both transcripts (supplemental Fig. S1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Northern blot analysis indicated that variant 1 is the major form in the epididymis and mammary gland. This was further confirmed by quantitative real-time RT-PCR, which also showed that both variants were expressed at a much higher level in the epididymis as compared with the mammary gland (data not shown).


Figure 1
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  		FIG. 1. Analysis of gene structure and confirmation of the two splice variants of the mouse Bfk. A, Structures of Bfk variants 1 and 2. Shaded areas represent the translated region. Both variants were predicted in silico and confirmed by RT-PCR using overlapping primers covering the whole transcript. Positions of the corresponding primer pairs are indicated by horizontal arrows. F, Forward; R, reverse. B, Northern blot analysis using poly-A mRNA and total RNA from the mouse epididymis (Epid) and 1-d lactating mammary gland (Mg). C, RT-PCR products using primers pairs indicated in A. D, Amino acid sequences of the BFK variants with a difference in the C-terminal end of exon 4.

 
Bfk is an epididymis-specific gene in males with region-specific expression
Predominant expression of Bfk in the epididymis was also indicated by a tissue distribution analysis using real-time RT-PCR (Fig. 2AGo). Within the epididymis, Bfk was enriched in the initial segment. Moderate expression was detected in the caput and cauda regions, whereas in the corpus, Bfk expression was low. In addition to the epididymis, moderate expression was also detected in the mammary gland, whereas the expression in other tissues was close to the detection limit of the method used. The predominant expression of Bfk in the initial segment was further confirmed by in situ hybridization (Fig. 2BGo), which demonstrated that Bfk expression was clearly defined to the initial segment.


Figure 2
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  		FIG. 2. A, Tissue distribution analysis of mouse Bfk expression measured by quantitative real-time RT-PCR using RNA samples from four different regions of the epididymis, initial segment (Is), caput (cap), corpus (cor), cauda (cau), and several other tissues as shown on the x-axis. intest, Intestine; semves, seminal vesicles; vasdef, vas deferens. Expression of ß-actin (Actb) was used for normalization. Error bars indicate SEM; n = 3. B, In situ hybridization analysis of Bfk expression in the initial segment and caput. Hybridization with sense probe was included as a negative control. Scale bar, 200 µm.

 
Bfk is regulated by androgens and other testicular factors
We observed that Bfk expression in the epididymis was significantly reduced after gonadectomy at all time points studied (1–14 d; P < 0.05). On d 1 after gonadectomy, Bfk expression in the proximal epididymis (initial segment plus caput) was reduced to 16% of that in intact males, and the expression reached its lowest level (5% of intact mice) on d 3 after gonadectomy. T replacement significantly restored the expression (P < 0.05), to a level of 37% of intact mice (Fig. 3Go). Androgen dependency of Bfk expression was also observed in the cauda region (P < 0.05) but not in the corpus. The study also showed a similar regulation for Bfk in initial segment and caput: a 3- to 4-fold reduction 3 d after gonadectomy (P < 0.05), with a significantly restored expression after T replacement (data not shown). However, the expression did not recover to the level detected in intact nongonadectomized mice.


Figure 3
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  		FIG. 3. Androgen dependency of Bfk in three different regions of the epididymis. Black bars represent the proximal part of the epididymis (initial segment plus caput), white bars the corpus, and diagonally striped bars the cauda. Quantitative real-time RT-PCR was performed using epididymal RNA from intact mice (WT) 1–14 d after gonadectomy and 7–14 d after gonadectomy with T replacement therapy. Significant changes (P < 0.05) of Bfk expression in the proximal region of the epididymis were observed at all time points after gonadectomy (WT vs. 1–14 d) and T replacement (7–14 d vs. 14 d plus T). Expression of ß-actin (Actb) was used for normalization. Error bars indicate SEM; n = 3.

 
By analyzing postnatal expression, it became evident that Bfk expression was detectable from d 20 onward, with a gradual increase along pubertal maturation (Fig. 4AGo). Because Bfk was also significantly expressed in the mammary glands, we examined by real-time RT-PCR the expression pattern during mammary gland development. Bfk expression was not detected in virgin female mammary glands; it was low on d 5–10 of pregnancy, increased sharply on d 15–18, and reached the highest level on d 1 of the lactating period (Fig. 4BGo). The expression was thereafter sharply down-regulated on d 5 of lactation, reaching the level observed on d 10 of pregnancy. The data suggested that Bfk might also be regulated by female steroid hormones. However, treating gonadectomized male mice with progesterone and/or estradiol for 3 d did not significantly increase Bfk expression in the initial segment or caput (data not shown).


Figure 4
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  		FIG. 4. Quantitative real-time RT-PCR analyses of postnatal expression of Bfk in the epididymis using RNA samples from 1- to 60-d-old mice (A) and Bfk expression during mammary gland development using RNA obtained from virgin (V), 5- to 18-day pregnant (5P–18P) and 1- to 15-d lactating (1L–15L) mice (B). Expression of ß-actin (Actb) was used for normalization. Error bars indicate SEM; n = 3.

 
BFK protein is localized in the nucleus of epididymal principal cells
The specificity of the antibody used for BFK detection was confirmed by Western blot analysis in four different regions of the epididymis, and in all of them we identified an expected 18-kDa protein (Fig. 5AGo). Interestingly, immunohistochemical analysis of epididymal tissue showed an intense nuclear staining only in principal cells, whereas no expression was detected in basal, narrow, or halo cells (Fig. 5BGo). Staining of the principal cells diminished toward the more distal regions of the organ. Also, in the mammary gland, staining was detected exclusively in the nucleus of epithelial cells lining the alveolar ducts (Fig. 5CGo).


Figure 5
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  		FIG. 5. A, Western blot analysis of the BFK protein in four different epididymal regions: initial segment (IS), caput (Cap), corpus (Cor), and cauda (Cau). Antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the control for equal loading. B, BFK immunohistochemistry with counterstaining showed positive nuclear staining in principal cells (P), whereas the finding was negative in basal (B), apical (A), and narrow (N) cells. Scale bars, 25 µm. C, BFK immunohistochemistry in the late pregnant female mammary gland (Mg) with nuclear staining in epithelial cells lining the alveolar ducts. Scale bar, 20 µm.

 
In agreement with the immunohistochemistry on tissue sections, the BFK-EGFP fusion proteins were predominantly localized in the nucleus (Fig. 6AGo). However, a weak cytosolic staining was present as well. The FLIP studies revealed that bleaching of the cytosolic portion also leads to bleaching of the protein in the nucleus, suggesting that although the BFK protein concentrates in the nucleus, it is shuttling between the nucleus and cytoplasm (Fig. 6BGo, lower panel). As shown in Fig. 6BGo (upper panel), there was a considerable difference in the speed at which the EGFP and BFK-EGFP proteins shuttle, whereas the BFK-EGFP fusion protein was retained in the nucleus considerably longer than the EGFP alone. Control cells imaged in the same frame that were not subjected to spot bleaching showed that the bleaching observed was specific (Fig. 6BGo, lower panel). In the Western blot, a protein corresponding to the size of the BFK-EGFP fusion was identified, indicating that the antibody detected the fusion protein, and the protein was intact in the transfected cells (data not shown).


Figure 6
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  		FIG. 6. Immunofluorescence analysis using epididymal mECap18 cells transfected with the Bfk-EGFP construct. A, Bfk-EGFP is indicated by green color in the fluorescent image, and 4',6-diamidino-2-phenylindole staining is shown in blue. B, A representative FLIP experiment in the mouse epididymis caput cells expressing Bfk-EGFP or EGFP alone. The graph shows nuclear fluorescence at different time points, and the confocal fluorescent images of the same cells from the indicated time points are below. The white circles in the prebleached images indicate the area of the spot for bleaching. To control that bleaching was not caused by the imaging procedure, control cells are shown that were imaged in the same frame but not subjected to spot bleaching.

 
BFK increases apoptosis initiated by other means
Although it has been previously suggested that BFK is apoptotic, mECap18 cells overexpressing the protein were viable and could be maintained in culture. As shown in Fig. 7AGo, the cells do indeed overexpress Bfk (pEGFPN3-Bfk), whereas intact cells (pEGFPN3) have apparently lost its expression, although they originate from the mouse caput epididymis. Furthermore, a higher degree of apoptosis did not occur in the overexpressing cells, and Western analysis for activated caspase 3 did not reveal any differences between the wild-type and overexpressing cells (data not shown). However, when the cells were challenged with staurosporine, a protein kinase inhibitor known to induce apoptosis, the cells expressing BFK underwent apoptosis faster than the control cells (transfected with empty vector) as detected by elevated levels of caspase 3 cleavage (Fig. 7Go, B and C). After 8 h, the caspase 3 activation appeared to decline, most likely due to death of apoptotic cells, leading to degradation of the proteins. In addition, by Western blot analysis for actin expression, it was found that in the BFK-overexpressing cells actin degradation was faster than in intact cells.


Figure 7
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  		FIG. 7. BFK enhances caspase 3 activation upon stimulation by staurosporine. A, mEcap18 cells were transfected either with empty expression vector or one containing the Bfk cDNA. Stably transfected cells were obtained under the selective pressure of G418 resistance. Extracts of the cells were analyzed by Western blot for the expression of BFK. B, Control cells (Co) and BFK-overexpressing cells (BFK) were treated with 200 nM staurosporine for the indicated times. Cells were harvested, and 20 µg of the lysates was analyzed by Western blot for the cleaved form of caspase 3 (Casp3) as well as actin. C, Films from the Western blot analysis were scanned and quantified using ImageJ, and a representative quantification is shown. Bars and the left axis represent the amount of actin, whereas the lines and the right axis represent the amount of activated caspase 3. For actin, the untreated control level was set to a value of 100, whereas for the activated caspase 3, because none could be detected in untreated cells (0 h), the 2-h time point of treated control cells was set as 100.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In our recent study, we performed global gene expression profiling of the mouse epididymis to reveal the segment-specific expression and differential regulation of the genes in the initial segment and caput (22). With the strict criteria used, we identified one gene (GM566) that was enriched in the initial segment and was responsive to dihydrotestosterone treatment given after gonadectomy. In the present study, we discovered that GM566 was identical to BFK, a novel member of the BCL2 family (18).

The human homolog of the mouse BFK has been reported to be predominantly expressed in the gastrointestinal tract with four alternative spliced variants (21). However, we could conclude that in the mouse, among all tissues analyzed, the epididymis is the organ with the highest level of Bfk expression. In the mouse epididymis, we identified two splice variants, whereas a 0.9-kb variant was the predominantly expressed form. We did not detect any expression of a 2-kb transcript previously reported for mouse Bfk (18). However, the cDNA structure and amino acid sequence deduced from the 0.9-kb transcript identified in the present study were in full agreement with that reported by Coultas et al. (18).

In a previous study, using NIH3T3 cells infected with a retroviral vector expressing Bfk, a cytosolic localization for BFK was identified (18). To our surprise, we observed a predominant nuclear localization for the protein in the tissue sections of epididymis and mammary gland and by expressing a BFK-EGFP fusion protein in an epididymal cell line (24). The reason for the discrepancy between our results and those of Coultas et al. (18) is not known, but it might be due to differences in properties between the epididymal and NIH3T3 cells. BFK may undergo cell-specific posttranslational modifications that could change its subcellular localization. For example in the rat epididymis, several groups of proteins such as nuclear proteins, transport proteins, chaperones, and enzymes have been reported to undergo androgen-induced posttranslational modification during development (25).

Predominant nuclear localization with nucleocytoplasmic shuttling is of interest for a BCL2 family member. Furthermore, the reported inability of BFK to interact with prosurvival or proapoptotic BCL2 protein family members (18) suggests an exceptional character for BFK. Although overexpression of BFK in epididymal cells did not enhance apoptosis by itself, we could reveal that such cells showed a higher level of caspase 3 cleavage after 2 and 5 h exposure to staurosporine as compared with the control cells. However, at later stages, although the control cells showed continuously increased levels of cleaved caspase 3, the levels in the BFK-overexpressing cells had started to decrease. The decrease in cleaved caspase 3 could be due to the ubiquitination and degradation of the protein by X-linked inhibitor of apoptosis protein as has been shown previously (26).

It has previously been described that in the presence of staurosporine, and at the onset of apoptosis, the actin cytoskeleton is disrupted as one of the early phases of the induced apoptosis (27). Furthermore, actin has been reported to be degraded under the treatment of staurosporine and to contain sites for caspase as well as IL-1ß-converting enzyme-mediated cleavage (28, 29, 30). Data obtained increased actin degradation in the presence of BFK, and to our knowledge, such a drastic degradation of actin has not been reported thus far. Whether BFK could specifically promote actin degradation is currently under investigation.

The shorter time needed for activation and reaching the maximum of caspase 3 cleavage after staurosporine induction, together with the more rapid degradation of actin, suggests that the progress of the apoptotic response in the BFK-overexpressing cells is faster than in control cells. In line with this, it has previously been speculated that BFK could be in a latent form in resting cells and that upon stimulation it transforms into a potent apoptotic BCL2 homolog region 3 (BH3)-only protein (18). We thus assume that BFK can promote apoptosis only when overexpressed or in a condition where apoptosis has been induced by other means and that the protein under normal physiological conditions might have other functions. Similarly, it has been shown that Bcl-2-associated death promoter, a proapoptotic BCL2 family member that counteracts the antiapoptotic properties of BCL-XL, can also promote the growth of cells when it is not challenged by apoptosis-inducing conditions (31).

The BFK expression in epididymis and mammary gland occurs specifically at the stage when the tissues undergo a series of changes, including an increase in cell number and the appearance of different cell types. Thus, it is likely that BFK promotes epithelial differentiation in both organs. In accordance with this finding, it has been reported that a BCL2 family member, the BCL2, appears to be restricted to cells at specific stages of differentiation (32). The role of BFK in epithelial proliferation and differentiation, rather than in apoptosis, is also supported by the data showing that Bfk expression is dependent on both androgens and other testicular factors. These factors are also known to be the key determinants in epididymal cells differentiation. In contrast, the expression of apoptotic-related genes are typically increased in the lack of testicular factors (33), in line with the decrease in tissue volume after gonadectomy. We assume that in the epididymis under physiological conditions, BFK does have a prosurvival activity involved in proliferation and differentiation of epithelial cells in the initial segment. However, when the apoptotic cascade has been initiated by external stimulations, BFK may have a weak proapoptotic activity. Additional studies are needed to reveal the exact biological function of BFK in the epididymis.


   Acknowledgments
 
We thank Nina Messner, Heli Niittymäki, and Erja Mäntysalo for animal handling and Jonna Palmu for histological sections. We also thank Dr. A. Strasser for providing BFK antibody.


   Footnotes
 
Corresponding author and reprint request: Matti Poutanen, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland. E-mail: matti.poutanen{at}utu.fi.

This work was supported by The Academy of Finland (project number 211480) and Sigrid Juselius Foundation.

Disclosure Statement: The authors have nothing to disclose.

First Published Online April 5, 2007

Abbreviations: BFK, BCL2 family kin; BCL2, B-cell lymphoma 2; DTT, dithiothreitol; EST, expressed sequence tag; FLIP, fluorescence loss in photobleaching; GM566, gene model 566; HRP, horseradish peroxidase; T, testosterone.

Received January 10, 2007.

Accepted for publication March 23, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lareyre JJ, Winfrey VP, Kasper S, Ong DE, Matusik RJ, Olson GE, Orgebin-Crist MC 2001 Gene duplication gives rise to a new 17-kilodalton lipocalin that shows epididymal region-specific expression and testicular factor(s) regulation. Endocrinology 142:1296–1308[Abstract/Free Full Text]
  2. Cornwall GA, Orgebin-Crist MC, Hann SR 1992 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 6:1653–1664[Abstract]
  3. Xin JH, Cowie A, Lachance P, Hassell JA 1992 Molecular cloning and characterization of PEA3, a new member of the Ets oncogene family that is differentially expressed in mouse embryonic cells. Genes Dev 6:481–496[Abstract/Free Full Text]
  4. Ghyselinck NB, Jimenez C, Lefrancois AM, Dufaure JP 1990 Molecular cloning of a cDNA for androgen-regulated proteins secreted by the mouse epididymis. J Mol Endocrinol 4:5–12[Medline]
  5. Lareyre JJ, Zheng WL, Zhao GQ, Kasper S, Newcomer ME, Matusik RJ, Ong DE, Orgebin-Crist MC 1998 Molecular cloning and hormonal regulation of a murine epididymal retinoic acid-binding protein messenger ribonucleic acid. Endocrinology 139:2971–2981[Abstract/Free Full Text]
  6. Sonnenberg-Riethmacher E, Walter B, Riethmacher D, Godecke S, Birchmeier C 1996 The c-ros tyrosine kinase receptor controls regionalization and differentiation of epithelial cells in the epididymis. Genes Dev 10:1184–1193[Abstract/Free Full Text]
  7. Eberspaecher U, Roosterman D, Kratzschmar J, Haendler B, Habenicht UF, Becker A, Quensel C, Petri T, Schleuning WD, Donner P 1995 Mouse androgen-dependent epididymal glycoprotein CRISP-1 (DE/AEG): isolation, biochemical characterization, and expression in recombinant form. Mol Reprod Dev 42:157–172[CrossRef][Medline]
  8. Ramirez R, Martin R, Martin JJ, Ramirez JR, Paniagua R, Santamaria L 1999 Changes in the number, proliferation rates, and bcl-2 protein immunoexpression of epithelial and periductal cells from rat epididymis during postnatal development. J Androl 20:702–712[Abstract/Free Full Text]
  9. Alnemri ES, Litwack G 1990 Activation of internucleosomal DNA cleavage in human CEM lymphocytes by glucocorticoid and novobiocin. Evidence for a non-Ca2(+)-requiring mechanism(s). J Biol Chem 265:17323–17333[Abstract/Free Full Text]
  10. Compton MM 1992 A biochemical hallmark of apoptosis: internucleosomal degradation of the genome. Cancer Metastasis Rev 11:105–119[CrossRef][Medline]
  11. Hawkins CJ, Vaux DL 1997 The role of the Bcl-2 family of apoptosis regulatory proteins in the immune system. Semin Immunol 9:25–33[CrossRef][Medline]
  12. Wyllie AH 1980 Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284:555–556[CrossRef][Medline]
  13. Kaipia A, Hsu SY, Hsueh AJ 1997 Expression and function of a proapoptotic Bcl-2 family member Bcl-XL/Bcl-2-associated death promoter (BAD) in rat ovary. Endocrinology 138:5497–5504[Abstract/Free Full Text]
  14. Ross AJ, Amy SP, Mahar PL, Lindsten T, Knudson CM, Thompson CB, Korsmeyer SJ, MacGregor GR 2001 BCLW mediates survival of postmitotic Sertoli cells by regulating BAX activity. Dev Biol 239:295–308[CrossRef][Medline]
  15. Rodriguez I, Ody C, Araki K, Garcia I, Vassalli P 1997 An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis. EMBO J 16:2262–2270[CrossRef][Medline]
  16. Suzuki A, Matsuzawa A, Iguchi T 1996 Down regulation of Bcl-2 is the first step on Fas-mediated apoptosis of male reproductive tract. Oncogene 13:31–37[Medline]
  17. Jara M, Esponda P, Carballada R 2002 Abdominal temperature induces region-specific p53-independent apoptosis in the cauda epididymidis of the mouse. Biol Reprod 67:1189–1196[Abstract/Free Full Text]
  18. Coultas L, Pellegrini M, Visvader JE, Lindeman GJ, Chen L, Adams JM, Huang DC, Strasser A 2003 Bfk: a novel weakly proapoptotic member of the Bcl-2 protein family with a BH3 and a BH2 region. Cell Death Differ 10:185–192[CrossRef][Medline]
  19. Zong WX, Lindsten T, Ross AJ, MacGregor GR, Thompson CB 2001 BH3-only proteins that bind pro-survival Bcl-2 family members fail to induce apoptosis in the absence of Bax and Bak. Genes Dev 15:1481–1486[Abstract/Free Full Text]
  20. Cheng EH, Wei MC, Weiler S, Flavell RA, Mak TW, Lindsten T, Korsmeyer SJ 2001 BCL-2, BCL-XL sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis. Mol Cell 8:705–711[CrossRef][Medline]
  21. Dempsey CE, Dive C, Fletcher DJ, Barnes FA, Lobo A, Bingle CD, Whyte MK, Renshaw SA 2005 Expression of pro-apoptotic Bfk isoforms reduces during malignant transformation in the human gastrointestinal tract. FEBS Lett 579:3646–3650[CrossRef][Medline]
  22. Sipila P, Pujianto DA, Shariatmadari R, Nikkila J, Lehtoranta M, Huhtaniemi IT, Poutanen M 2006 Differential endocrine regulation of genes enriched in initial segment and distal caput of the mouse epididymis as revealed by genome-wide expression profiling. Biol Reprod 75:240–251[Abstract/Free Full Text]
  23. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K 1995 Current protocols in molecular biology. New York: Wiley, Sons
  24. Sipila P, Shariatmadari R, Huhtaniemi IT, Poutanen M 2004 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. Endocrinology 145:437–446[Abstract/Free Full Text]
  25. Umar A, Ooms MP, Luider TM, Grootegoed JA, Brinkmann AO 2003 Proteomic profiling of epididymis and vas deferens: identification of proteins regulated during rat genital tract development. Endocrinology 144:4637–4647[Abstract/Free Full Text]
  26. Suzuki Y, Nakabayashi Y, Takahashi R 2001 Ubiquitin-protein ligase activity of X-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death. Proc Natl Acad Sci USA 98:8662–8667[Abstract/Free Full Text]
  27. Paul C, Manero F, Gonin S, Kretz-Remy C, Virot S, Arrigo AP 2002 Hsp27 as a negative regulator of cytochrome C release. Mol Cell Biol 22:816–834[Abstract/Free Full Text]
  28. Utsumi T, Sakurai N, Nakano K, Ishisaka R 2003 C-terminal 15 kDa fragment of cytoskeletal actin is posttranslationally N-myristoylated upon caspase-mediated cleavage and targeted to mitochondria. FEBS Lett 539:37–44[CrossRef][Medline]
  29. Kayalar C, Ord T, Testa MP, Zhong LT, Bredesen DE 1996 Cleavage of actin by interleukin 1ß-converting enzyme to reverse DNase I inhibition. Proc Natl Acad Sci USA 93:2234–2238[Abstract/Free Full Text]
  30. Mashima T, Naito M, Tsuruo T 1999 Caspase-mediated cleavage of cytoskeletal actin plays a positive role in the process of morphological apoptosis. Oncogene 18:2423–2430[CrossRef][Medline]
  31. Maslyar DJ, Aoki M, Vogt PK 2001 The growth-promoting activity of the Bad protein in chicken embryo fibroblasts requires binding to protein 14-3-3. Oncogene 20:5087–5092[CrossRef][Medline]
  32. Lu QL, Abel P, Foster CS, Lalani EN 1996 bcl-2: role in epithelial differentiation and oncogenesis. Hum Pathol 27:102–110[CrossRef][Medline]
  33. Ezer N, Robaire B 2003 Gene expression is differentially regulated in the epididymis after orchidectomy. Endocrinology 144:975–988[Abstract/Free Full Text]




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