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Endocrinology Vol. 139, No. 12 4839-4848
Copyright © 1998 by The Endocrine Society

ARTICLES

DEFT, a Novel Death Effector Domain-Containing Molecule Predominantly Expressed in Testicular Germ Cells1

Chandra P. Leo2, Sheau Yu Hsu, Elizabeth A. McGee3, Michele Salanova and Aaron J. W. Hsueh

Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305-5317

Address all correspondence and requests for reprints to: Aaron J. W. Hsueh, Department of Gynecology and Obstetrics, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, California 94305-5317. E-mail: aaron.hsueh{at}forsythe.stanford.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Apoptosis is a physiological process by which multicellular organisms eliminate unwanted cells. Death factors such as Fas ligand induce apoptosis by triggering a series of intracellular protein-protein interactions mediated by defined motifs found in the signaling molecules. One of these motifs is the death effector domain (DED), a stretch of about 80 amino acids that is shared by adaptors, regulators, and executors of the death factor pathway. We have identified the human and rat complementary DNAs encoding a novel protein termed DEFT (Death EFfector domain-containing Testicular molecule). The N-terminus of DEFT shows a high degree of homology to the DEDs found in FADD (an adaptor molecule) as well as procaspase-8/FLICE and procaspase-10/Mch4 (executors of the death program). Northern blot hybridization experiments have shown that the DEFT messenger RNA (mRNA) is expressed in a variety of human and rat tissues, with particularly abundant expression in the testis. In situ hybridization analysis further indicated the expression of DEFT mRNA in meiotic male germ cells. In a model of germ cell apoptosis induction, an increase in testis DEFT mRNA was found in immature rats after 2 days of treatment with a GnRH antagonist. Unlike FADD and procaspase-8/FLICE, overexpression of DEFT did not induce apoptosis in Chinese hamster ovary cells. Although cotransfection studies indicated that DEFT is incapable of modulating apoptosis effected by FADD and procaspase-8/FLICE, interactions between DEFT and uncharacterized DED-containing molecules in the testis remain to be studied in the future. In conclusion, we have identified a novel DED-containing protein with high expression in testis germ cells. This protein may be important in the regulation of death factor-induced apoptosis in the testis and other tissues.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
APOPTOSIS is a fundamental biological process through which multicellular organisms eliminate unwanted cells. This form of programmed cell death plays a major role in normal development and tissue homeostasis as well as in the pathogenesis of a variety of diseases (1, 2, 3). Apoptosis can be induced by different stimuli including death factors, genetic damage, growth factor deprivation, or viral infection. The separate pathways triggered by each of these stimuli ultimately converge in the activation of a group of enzymes called caspases (4). These so-called killer proteases then cleave key cellular components, disabling critical homeostatic and repair processes essential for cell survival as well as causing disassembly of the cytoskeleton and nuclear DNA fragmentation (5).

The mechanisms by which death factors induce apoptosis have been studied extensively because of their importance in the immune system (reviewed in Ref. 6). Death factors are members of the tumor necrosis factor (TNF) family of cytokines and include Fas ligand (also known as Apo-1/CD95 ligand), TNF-{alpha} and TRAIL/Apo-2 ligand. These membrane-bound or soluble proteins induce apoptosis in target cells by binding to specific members of the TNF receptor superfamily called death receptors (see Ref. 7 and references therein). The stimulation of death receptors by their respective ligands induces the formation of a death-inducing signaling complex (DISC) at the cell membrane, a process best studied in apoptosis induced through Fas/Apo-1/CD95 (8). The intracellular domain of the death receptor binds to the adaptor molecule Fas-associating death domain-containing protein (FADD), which in turn recruits catalytically inactive procaspase-8/FADD-like interleukin-1ß-converting enzyme (FLICE) molecules. This critical step of DISC formation is mediated by the death effector domain (DED), a structural motif of about 80 amino acids in length found in both FADD and procaspase-8 (6, 9). The DED-dependent recruitment of procaspase-8 molecules into the DISC induces their intermolecular autocatalytic cleavage (10), thus releasing active caspase-8 molecules to activate other downstream procaspases in a cascade-like manner. Jointly, these caspases act as executors of apoptosis by effecting a systematic and orderly disassembly of the dying cell.

The development of both the male and female gonad is characterized by massive apoptosis, and it has been estimated that up to 75% of all male germ cells in the testis undergo programmed cell death (11, 12, 13). Recently, the death factor Fas ligand has been implicated as a physiological stimulus involved in germ cell apoptosis in both testis and ovary (14, 15, 16). Immunohistochemical studies of the testis in rat and mouse have localized Fas ligand to Sertoli cells and Fas to germ cells in seminiferous tubules (14, 17). Apart from the regulation of germ cell survival, the expression of Fas ligand in Sertoli cells has also been demonstrated as the mechanism responsible for the unique immune privileged nature of the testis (18).

To further elucidate the regulation of gonadal cell apoptosis by death factors, we searched for new proteins possessing the consensus DED motif. Here, we report the molecular cloning, sequence analysis, and expression pattern of a novel mammalian gene product designated DEFT (for Death EFfector domain-containing Testicular molecule) based on its unique sequence homology to the DED. Because the DEFT message is predominantly expressed in testis germ cells and increases following treatment with a GnRH antagonist that induces testicular apoptosis, this protein may be important in the regulation of male germ cell apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation/cloning of human and rat DEFT complementary DNAs (cDNAs)
The National Center for Biotechnology Information database of expressed sequence tags (ESTs) was searched using the BLAST program (19) with amino acid sequences of different DED-containing proteins as queries. We identified a human EST (GenBank accession no. aa127788) as having extensive homology to the first DED of procaspase-8. Further searches yielded a number of overlapping human (GenBank accession nos. h12060 and aa382778) and murine ESTs (GenBank accession nos. aa270899, aa124451, aa611579, and aa163060). Two EST clones (I.M.A.G.E. Consortium Clone IDs 47690 and 490307) were sequenced and found to contain the partial and full-length open reading frame (ORF) of a novel gene. The human ORF sequence was independently confirmed by performing PCR with EST-derived primers from a human ovary cDNA library (CLONTECH Laboratories, Inc., Palo Alto, CA), followed by cloning and sequencing of the amplicons. The full-length rat ORF sequence was established by performing a 3' rapid amplification of cDNA ends using primers based on the murine EST information to amplify downstream sequences from rat testis and ovary cDNA libraries (Marathon-ready cDNA; CLONTECH Laboratories, Inc.). A comparison of the predicted amino acid sequence against the SWISS-PROT protein sequence database (19) showed homologies between the N-terminal part of DEFT and DEDs in FADD and procaspases-8 and -10. In addition, a search against PROSITE (20), a database of biologically significant patterns and profiles, confirmed the presence of a DED motif in residues 25–103 of DEFT. The phylogenetic analysis was carried out using the GCG software package (Genetics Computer Group, Madison, WI).

Isolation of Sertoli and germ cells
Sertoli cell-enriched primary cultures were prepared from testes of 20-day-old Sprague-Dawley rats (Simonsen Laboratories, Inc., Gilroy, CA). Explants of seminiferous epithelium were isolated by dissection and cultured according to established procedures (21). Following dispersion using collagenase (Worthington Biochemical Corp., Lakewood, NJ), the cell suspension was centrifuged at 480 x g for 10 min at room temperature. After several washings, the cells thus obtained were cultured in DMEM for 3 days; contaminating germ cells were removed following a short hypotonic treatment (22). Enriched Sertoli cells were then harvested and used to extract messenger RNA (mRNA).

Enriched germ cells were isolated from adult rat testes (60 days of age) by two sequential collagenase digestions (0.33% collagenase type I, 220 U/mg; Ref. 22). After the first digestion, the cell preparation was diluted with PBS, and the supernatant containing interstitial cells was discarded. Following an additional digestion of the sediment, collagenase was removed by several washings with PBS, and the tubules were disrupted mechanically. The obtained sediment containing Sertoli cells and peritubular cells was discarded, and the supernatant centrifuged at 480 x g for 10 min. After several washings of the pellet in PBS, the enriched germ cell suspension was ready for RNA extraction.

Preparation of mRNA
For analysis of mRNA expression during development, male Sprague-Dawley rats at 10, 20, 30 and 60 days of age were killed and the testes collected. Poly(A)+ RNA from whole rat testis as well as Sertoli and germ cell preparations was extracted using the Quick prep micro mRNA purification kit (Pharmacia, Piscataway, NJ) following the manufacturer’s protocol. The isolated mRNA (5 µg per lane) was fractionated by agarose-formaldehyde gel electrophoresis and transferred to nitrocellulose membranes.

Northern and Southern blot analysis
For mRNA expression analysis, human and rat DEFT cDNA probes (nucleotides 1–580 of the ORFs) were 32P-radiolabeled by random priming using a commercial kit (Gibco BRL, Gaithersburg, MD). Blots containing poly(A)+ RNA from various adult human and rat tissues (CLONTECH Laboratories, Inc.) or from rat testis preparations (see above) were hybridized with the probe for the respective species and processed according to the manufacturer’s instructions. To estimate RNA loading, multiple tissue Northern blots were subsequently hybridized with a radiolabeled ß-actin cDNA probe. Similarly, blots containing poly(A)+ RNA from rat testes were hybridized with a cDNA probe for the Sertoli cell-specific androgen-binding protein (23). In addition, rat testis RNA blots were reprobed with a cDNA probe for cyclophilin A, an abundant transcript in different testicular cell types, the expression of which does not change during apoptosis (24, 25).

For studies of cross-species conservation of the deft gene, a Zoo blot (CLONTECH Laboratories, Inc.) containing EcoRI-digested genomic DNA from different species was probed with a 32P-labeled rat DEFT cDNA probe.

In situ hybridization studies
For in situ hybridization analysis of DEFT mRNA expression, testes from 21-day-old Sprague-Dawley rats were isolated and fixed at 4 C for 8 h in 4% paraformaldehyde in PBS (pH 7.4), followed by overnight dehydration in 0.5 M sucrose. Tissue blocks were embedded in Tissue-Tek solution (Sakuraus Finetek U.S.A., Inc., Torrence, CA) and snap frozen in liquid nitrogen. Cryosections (10–12 µm thick) were mounted on charged microscopic slides (Fisher Scientific International, Inc., Pittsburgh, PA), postfixed in 4% paraformaldehyde, and stored at -70 C. Hybridization and washes of cryosections were performed as described previously (26). After 3 weeks of exposure under NTB2 emulsion (Eastman Kodak Co., Rochester, NY), the slides were developed, counterstained, and mounted with Permount (Fisher Scientific International, Inc.) for photography using an Axioplan 2 microscope (Carl Zeiss, Oberkochen, Germany).

Regulation of DEFT message following induction of germ cell apoptosis
Immature, male Sprague-Dawley rats were treated with a GnRH antagonist (Org 30850, Organon, Oss, The Netherlands; Ref. 27) at a dose of 1000 µg/kg body weight once daily. Subcutaneous injections were administered in a volume of 100 µl/injection [aqueous solution of gelatin (5 mg/ml) and mannitol (50 mg/ml)]. Animals were killed after 1, 2, and 4 days of treatment, and total body weight was recorded. The testes were dissected and weighed individually before being snap frozen in a dry ice-ethanol bath and storage at -70 C. RNA extraction was performed using the RNeasy Midi kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Twenty micrograms total RNA per lane were run on agarose-formaldehyde gels before transfer to nitrocellulose membranes. Northern blots were hybridized with radiolabeled rat DEFT and cyclophilin A cDNA probes as described above. Densitometric quantification of the major transcript bands on autoradiograms was carried out using the Quantity One software (PDI, Huntington Station, NY).

Expression vectors, mammalian cell transfection, and apoptosis assay
Expression vectors for DEFT, reverse DEFT, FADD, procaspase-8, and p35 were generated by PCR amplification of ORFs, followed by subcloning into the pcDNA3 vector (Invitrogen, Carlsbad, CA). All constructs were tested for their ability to direct the synthesis of appropriate [35S]Met-labeled proteins in an in vitro coupled transcription/translation system (Promega Corp., Madison, WI). Five microliters of the radiolabeled translation products were separated on a 10% SDS-polyacrylamide gel. Following electrophoresis, the gel was fixed, treated with a fluorography-enhancing solution (Amplify; Amersham Pharmacia Biotech, Arlington Heights, IL), dried, and subjected to autoradiography.

For transfection studies, Chinese hamster ovary (CHO) cells (2x105/well) were cultured in DMEM/F12 supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine. One day later, cells were transfected using the lipofectamine procedure (Life Technologies, Gaithersburg, MD) with the empty pcDNA3 expression vector and/or the same vector containing different cDNAs (total of 2.0 µg/well). All transfections included 1/20 fraction of the reporter plasmid pCMV-ß-galactosidase (ß-gal) to allow the identification of transfected cells. Inclusion of 20-fold excess of expression vectors as compared with the reporter plasmid ensured that most of the ß-gal-expressing cells also expressed the protein(s) under investigation. Cells were incubated with plasmids in serum-free medium for 4 h, followed by addition of FBS to a final concentration of 10% and further incubation for 20 h. Cells were fixed in 0.25% glutaraldehyde and stained with 5-bromo-4-chloro-3-indolyl-D-galactoside (0.4 mg/ml) to detect ß-gal expression. Cell survival was determined by counting the number of viable blue cells from eight random fields of view per well (26). Each data point shown represents the mean (± SEM) of cell numbers from three cultures of a representative experiment.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification and cloning of human and rat DEFT
To identify novel apoptosis-regulatory gene products, we screened public EST databases for cDNAs encoding proteins with homology to known DED sequences. Several overlapping human and murine EST sequences homologous to the first DED of procaspase-8 were identified. Based on this information, primers were designed and used to perform PCR and 3' rapid amplification of cDNA ends reactions from human and rat cDNA libraries. The cloned products and human EST cDNAs were sequenced and found to contain the ORF sequences for the human and rat forms of a novel gene. We designated the protein encoded by these cDNAs as DEFT.

Sequence analysis of DEFT and homology to related proteins
The human and rat DEFT cDNA sequences contain ORFs encoding a protein of 318 amino acids with a predicted relative mol wt of about 36,800, preceded by an upstream in-frame terminator codon (Fig. 1Go). The nucleotide coding sequence is 93.3% identical between human and rat DEFT, whereas the predicted amino acid sequences share 98.4% identity with variations in only five residues. Structurally, the DEFT protein can be subdivided into two parts: The N-terminal third contains a stretch of amino acid sequence completely conserved between human and rat that is homologous to the DEDs of other known molecules (Fig. 2AGo). By contrast, the amino acid sequence corresponding to the C-terminal two-thirds of DEFT shows no substantial homology to any previously identified proteins.



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  		Figure 1. Nucleotide and deduced amino acid sequence of human and rat DEFT. Numbers to right of sequences indicate amino acids (in italics) and nucleotides. Initiation codon as well as in-frame terminator codons (marked by asterisks) up- and downstream of ORF are depicted in boldface. Dots in rat nucleotide sequence represent identity with human sequence. Human amino acid sequence shown is identical to rat sequence except for substitutions in five underlined residues (16 H->R, 17 G->V, 105 R->K, 159 P->S, 171 A->T). Shaded residues (25–103) correspond to death effector domain of DEFT, whereas underlined tetrapeptides indicate putative caspase cleavage sites (see text).

 


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  		Figure 2. Alignment of DEFT with DEDs of related human proteins and phylogenetic analysis. A, Amino acid sequence corresponding to DED of human/rat DEFT is aligned with N-terminal DEDs of human FADD, procaspase-8 and -10, and c-FLIP. For each block of aligned sequences, black boxes indicate >50% amino-acid sequence identity, whereas gray shading indicates >50% sequence similarity through conservative amino acid substitutions. Positions of residues contributing to a hydrophobic region important for protein-protein interactions (asterisks) in DED of FADD are marked. B, Phylogenetic analysis of DED motifs of DEFT, procaspases-8 and -10, c-FLIP, PEA-15, and Bap31. Numbers in parentheses refer to N-terminal (1 ) and C-terminal (2 ) DEDs of specific proteins.

 
We further investigated the exact relationship between residues 25–103 of DEFT and the DED sequences of all other known mammalian DED proteins, i.e. FADD (an adaptor in the death factor pathway), procaspases-8 and -10 (executors of apoptotic cell death), c-FLIP (cellular FLICE-like inhibitory protein) and PEA-15 (a phosphoprotein of unknown function; see Refs. 28, 29, 30, 31, 32). Also included in the analysis was Bap31, a molecule with a weak DED homology that forms a complex with procaspase-8 and Bcl-2 proteins in the endoplasmatic reticulum (33). The DED of DEFT was found to share a particularly high degree of identity (22–27%) and homology (37–41%) with the N-terminal DEDs of FADD, procaspases-8 and -10, and c-FLIP. Intriguingly, the residues found to be important for mediating protein-protein interactions in FADD (9) are well conserved in DEFT (Fig. 2AGo). The C-terminal DEDs of procaspases-8 and -10, c-FLIP, and PEA-15 are less closely related to DEFT (15–22% identity, 33–39% homology), whereas the sequence of Bap31 is most distant from DEFT (10% identity, 22% homology) and the other DED proteins. The putative phylogenetic relationship among the DEDs of these proteins is shown in Fig. 2BGo.

Expression of DEFT mRNA in human and rat tissues
Northern blot analysis revealed that the DEFT mRNA is predominantly expressed in the human and rat testis (Fig. 3Go, A and B). In addition, low level expression was also detected in a variety of other adult human and rat tissues, including the ovary. In human tissues, one main transcript with a size of approximately 2.1 kb was found; in rat tissues, a major transcript of about 2.0 kb together with two less prominent transcripts of approximately 2.7 kb and 4.1 kb were detected. These different mRNA species could result from the use of alternative polyadenylation sites and/or alternative splicing of DEFT.



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  		Figure 3. Distribution of DEFT transcripts in human and rat tissues. Northern blots containing poly(A)+ RNA of various human (A) and rat tissues (B) were probed with DEFT cDNA probes for the according species. Subsequent hybridization using a ß-actin probe is shown as a control for mRNA loading. Numbers on left indicate kilobases and arrowheads DEFT transcripts of different sizes. (Small int., small intestine; PBL, peripheral blood leukocyte; sk. muscle, skeletal muscle.) C, DEFT mRNA expression in testis of rats at different ages (10, 20, 30, and 60 days) was investigated by performing Northern blot hybridizations of poly(A)+ RNA (5 µg/lane). mRNA expression was additionally assessed in preparations of poly(A)+ RNA from enriched Sertoli and germ cells (5 µg/lane). Results of hybridizations using rat androgen-binding protein (ABP) and cyclophilin A (Cyp) probes are shown for comparison.

 
To characterize the testicular expression of DEFT in more detail, we isolated poly(A)+ RNA from rat testes at different stages of postnatal development and probed the blots with a radiolabeled rat DEFT cDNA probe. Being undetectable at 10 days of age, the DEFT message first becomes apparent at 20 days of age, and reaches its highest expression level in the testis of 30-day-old rats (Fig. 3CGo). Northern blot analysis of poly(A)+ RNA extracted from preparations of enriched Sertoli and germ cells indicated that the DEFT message is highly expressed in germ cells but not Sertoli cells. We further performed in situ hybridization analysis to study the localization of the DEFT message in the testis of immature 21-day-old rats. As shown in Fig. 4Go, the most intense signal was observed in meiotic germ cells (spermatocytes) present in the central portions of the seminiferous tubules. No hybridization signal was detected using the sense DEFT probe.



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  		Figure 4. Localization of DEFT message in immature rat testis. Expression of DEFT mRNA was studied by in situ hybridization of sections of 21-day-old rat testis counterstained with hematoxyline. Brightfield (A) and darkfield (B) views of same section hybridized with an antisense DEFT cRNA probe (x20). At a higher magnification, brightfield views of sections hybridized with an antisense DEFT probe (C) showed gene expression in meiotic germ cells in center of seminiferous tubules, whereas hybridization with a sense DEFT probe (D) resulted in no signal (x40).

 
Regulation of DEFT message during germ cell apoptosis
Because DEFT contains a DED, and its message is expressed in male germ cells, we studied the regulation of the DEFT mRNA in an established model of testicular germ cell apoptosis. Gonadotropins are known survival factors in the testis of immature rats (34), whereas treatment with GnRH antagonists induces massive germ cell apoptosis (34, 35). Immature male rats at 28 days of age were injected with the GnRH antagonist Org 30850 for 1–4 days, resulting in a marked decrease of testis weight after 4 days of treatment (Fig. 5AGo). As shown in Fig. 5Go, B and C, treatment with the GnRH antagonist led to a 170% increase in DEFT message levels after 2 days of antagonist treatment. This was followed by a decrease of DEFT mRNA after 4 days of treatment when overall testis weight had decreased to 64% as compared with control animals. These results suggest that the induction of germ cell apoptosis is correlated with an increase in DEFT mRNA expression.



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  		Figure 5. Regulation of DEFT message following induction of germ cell apoptosis in rat. A, Testis weight of immature rats after 1, 2, and 4 days of GnRH antagonist treatment (closed bars) is compared with testis weight of control animals (open bars). Mean ± SEM, n = 6 per group. B, Northern blot hybridization of total testis RNA (20 µg/lane) from three individual animals per group was performed with a radiolabeled DEFT cDNA probe. Hybridization results of same blot using a cyclophilin A (Cyp) cDNA probe are shown for estimation of sample loading. C, Densitometric quantification of DEFT message normalized to cyclophilin message. Mean ± SEM are given for each group.

 
Conservation of deft among different vertebrate species
Conservation of the deft gene was investigated by Southern blot hybridization of genomic DNA from diverse species. The rat DEFT cDNA probe hybridized strongly with DNA from various mammalian species, implying a substantial degree of conservation, but not with genomic DNA from chicken or yeast (Fig. 6Go).



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  		Figure 6. Genomic conservation of DEFT in different vertebrate species. Southern blot analysis of genomic DNA from different vertebrate species was performed. DNA (4 µg/lane) was digested with EcoRI restriction enzyme and blotted onto nitrocellulose membranes before hybridization with a rat DEFT cDNA probe. Numbers on left indicate migration of DNA marker fragments (size in kilobases).

 
Effects of overexpression of DEFT and other DED-containing proteins on apoptosis induction in mammalian cells
Because the DED-containing molecules FADD and procaspases-8 and -10 have been shown to potently induce apoptosis when overexpressed in mammalian cells (28, 29, 30), we investigated the effects of DEFT overexpression in CHO cells using a ß-gal cotransfection assay (26). DEFT overexpression at concentrations of up to 1 µg DNA per well did not significantly affect survival of CHO cells (Fig. 7AGo), whereas overexpression of FADD and procaspase-8 induced extensive cell death at 0.1 µg DNA per well (Fig. 7Go, B and C).



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  		Figure 7. Effects of overexpression of DEFT and coexpression with FADD and procaspase-8 on apoptosis induction. A-C, CHO cells were transiently transfected with indicated expression vectors together with a ß-gal reporter construct. Unless marked otherwise, DEFT, reverse DEFT, and p35 vectors were used at 1.0 µg/well and FADD and procaspase-8 vectors at 0.1 µg/well. Where appropriate, empty pcDNA3 vector was added to keep total amount of transfected DNA constant. Data shown are percentages of viable ß-gal-expressing cells as compared with control (empty pcDNA3 vector). Overexpression of DEFT in CHO cells did not induce apoptosis (A). Coexpression of a 10-fold excess of p35 but not DEFT inhibits cell death induced by overexpression of FADD (B) and procaspase-8 (C). D, Autoradiogram of [35S]Met-labeled translation products generated from pcDNA3 expression vectors in an in vitro transcription/translation system. Numbers on left denote migration of protein molecular weight markers (in kilodaltons). Arrowheads indicate expected bands for DEFT (36.8 kDa), FADD (23.3 kDa), procaspase-8 (55.4 kDa), and p35 (34.8 kDa).

 
The DED protein c-FLIP and its viral homologs have been demonstrated to inhibit death factor-induced apoptosis by interacting with FADD and/or procaspase-8 (31, 36) and to block FADD-induced, but not procaspase-8-induced, cell death (37). To test whether DEFT interferes with these components of the death factor pathway, we performed coexpression experiments in CHO cells (Fig. 7Go, B and C). However, DEFT did not suppress apoptosis when coexpressed with either FADD or procaspase-8, whereas the baculoviral caspase inhibitor p35 (38) effectively counteracted cell death induced by either molecule. Because no DEFT antibody is available at the present time, it was difficult to verify the expression of recombinant DEFT in transfected CHO cells. Instead, we analyzed the expression of recombinant DEFT using an in vitro transcription/translation assay. As shown in Fig. 7DGo, [35S]Met-labeled translation products of the expected molecular size could be generated from the expression vector for DEFT as well as those for FADD, procaspase-8, and p35.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We identified the novel DED-containing gene product DEFT with a predominant expression in testis germ cells. Following induction of germ cell apoptosis using a GnRH antagonist, an increase in testis DEFT message level was detected. Sequence analysis indicates that DEFT contains a well-conserved N-terminal DED (amino acids 25–103) fused to a unique C-terminal domain. DEFT therefore represents the sixth member of an emerging group of cellular DED molecules that also includes FADD, procaspases-8 and -10, c-FLIP, and PEA-15. Four of the five previously identified DED proteins have been shown to function as integral components of the death factor pathway: FADD serves as an adaptor molecule, linking activated death receptors to procaspases-8 and -10 (6), whereas c-FLIP (also known as Casper/FLAME/CASH/I-FLICE, reviewed in Ref. 39) appears to regulate death factor-induced apoptosis. The function of PEA-15 (32), a phosphoprotein shown to be a substrate for protein kinase C and predominantly expressed in the central nervous system, is currently unknown. In addition, two other proteins involved in the regulation of apoptosis, Bap31 and Ced-4, have recently been suggested to possess regions with weak homology to the DED (33, 40). Ced-4 serves as a molecular bridge between the antiapoptotic Bcl-2 homolog Ced-9 and the caspase Ced-3 in the nematode Caenorhabditis elegans (41), whereas the mammalian Bap31 was found to associate with the Bcl-2 family member Bcl-xL and procaspase-8 in the endoplasmatic reticulum (42).

The C terminus of DEFT (amino acids 104–318) is remarkable for the absence of any previously identified motifs. Unlike FADD, DEFT lacks the death domain sequence responsible for binding to the intracellular region of death receptors (6). Unlike procaspases-8 and -10, DEFT shows no sequence homology to catalytic domains of other caspases and is missing the universally conserved QACxG catalytic motif (5). Furthermore, overexpression studies did not reveal a potential of DEFT to induce apoptosis in CHO cells, in contrast to the known apoptosis-inducing effects of FADD and procaspase-8 (28, 29). These findings argue against a possible function of DEFT as either a FADD-like adaptor molecule or a caspase. Interestingly, however, DEFT contains three tetrapeptide regions (amino acids 51–54, 112–115, and 297–300, see Fig. 1Go) that closely resemble recognition sequences found in caspase substrates (5, 43), raising the possibility that it may itself be cleaved by active caspases.

Another mechanism of action for DED proteins is exemplified by c-FLIP and its viral homologs (31, 36, 39). These molecules have been shown to bind to FADD and/or procaspase-8 and appear to function, at least in part, by disrupting the interaction between these two molecules. However, in coexpression studies, DEFT was unable to inhibit cell death induced by overexpression of FADD or procaspase-8. Furthermore, yeast two-hybrid system experiments, which have successfully been used in the past to assay interactions between DED proteins (44), indicate that DEFT does not interact with FADD or procaspase-8 (data not shown). It is, however, possible that cell-specific posttranslational modifications are required for such binding to occur. Based on the observed increases in DEFT mRNA during induction of germ cell apoptosis, DEFT may interact with other (known or still unidentified) DED-containing molecules in the testis to regulate apoptosis, a question that remains unanswered. Similarly, the functional properties of the unique C-terminal domain of DEFT remain to be explored.

The 3' untranslated region of the human DEFT cDNA contains the sequence-tagged site SHGC-14821. Preliminary mapping data suggest the localization of this sequence-tagged site and, consequently, the deft gene on human chromosome 1 (The Genome Database, see Ref. 45). Because three of the other five known genes for DED-containing proteins (procaspases-8 and -10 and c-FLIP) are clustered in region 33–34 on the long arm of human chromosome 2 (39), these genes presumably arose by a relatively recent gene duplication event. In contrast, the deft gene may have diverged from other DED-containing genes at an earlier point during evolution (Fig. 2BGo).

Although most of the aforementioned DED-containing proteins are expressed at comparable levels in multiple tissues (28, 29, 30, 31), DEFT is unique in showing a particularly abundant expression in the testis. Cell death by apoptosis is an important physiological feature of spermatogenesis and can be suppressed by gonadotropins and androgens (12, 34). Significantly increased rates of testicular germ cell apoptosis have been found under pathological conditions including certain forms of male infertility (46) and experimentally induced cryptorchidism (12, 47). Developmental studies showed that the expression of DEFT mRNA rises from undetectable levels in the testis of 10-day-old rats and reaches its maximum at 30 days of age. This pattern matches the developmental increase of apoptotic DNA fragmentation observed in the rat testis (35), consistent with a role for DEFT in the regulation of apoptosis. In situ hybridization analysis of immature rat testis further indicated the strongest DEFT message levels in meiotic spermatocytes, which represent the main cell type affected by apoptosis in the male gonad (35). In contrast, Northern blot hybridizations did not detect the expression of DEFT mRNA in Sertoli cells. Of interest, induction of germ cell apoptosis by a GnRH antagonist was associated with a significant increase in testicular DEFT mRNA levels, suggesting that DEFT may have an important role during the hormonal regulation of apoptosis in the male gonad.

Recent studies showed that expression of the death factor Fas ligand in the seminiferous tubule is limited to Sertoli cells, whereas the death receptor Fas, like DEFT, is found exclusively on germ cells (14). In vitro, mouse germ cells were susceptible to apoptosis induced by Fas activation, whereas antisense oligonucleotides to Fas ligand increased the survival of germ cells in coculture with Sertoli cells (14). These findings suggest that death factor-induced apoptosis, a process mediated by DED-containing proteins, represents an important mechanism for the regulation of germ cell numbers in the testis. In addition, the death factor system has been demonstrated as the basis for the immune privileged nature of the testis (18) and has been implicated in experimental models of testicular tumor formation (48). Although the exact cellular function of DEFT remains unclear, it is possible that this novel DED-containing molecule may act as a component of the death factor pathway in testis germ cells. Future studies on DEFT-interacting proteins in the testis may allow elucidation of germ cell-specific pathways in death factor-induced apoptosis.


   Acknowledgments
 
We thank Ms. Caren Spencer for editorial assistance and Dr. John Whitin for help with the densitometric analysis. We also thank Dr. V. M. Dixit (University of Michigan, Ann Arbor, MI) for providing the FADD and procaspase-8 cDNAs, Dr. L. K. Miller (University of Georgia, Athens, GA) for the p35 cDNA, and Dr. Lenus Kloosterboer (Organon, The Netherlands) for the gift of Org 30850.


   Footnotes
 
1 This work was supported by NIH Grant HD-31566 and by NIH Training Grant HD7493 (to S.Y.H.). Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession number AF043733 and AF053362 for human and rat DEFT, respectively). Back

2 Recipient of a postdoctoral fellowship from the German Academic Exchange Service. Back

3 Fellow of the Reproductive Scientist Development Program supported by Grant K12-HD-0084908. Back

Received March 26, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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