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Mechanisms of Hormone Action |
Wadsworth Center,3 David Axelrod Institute for Public Health, New York State Department of Health, Albany, New York 12208
Human Genetics Program,4 Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
Department of Biomedical Sciences,5 State University of New York at Albany, Albany, New York
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) was chosen for further analysis. APPL1 appears to coimmunoprecipitate with FSHR in HEK 293 cells stably expressing FSHR (293/FSHR cells), confirming APPL1 as a potential FSHR-interacting partner. The phosphorylation status of members of the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway was also examined because of the proposed role of APPL1 in the antiapoptotic PI3K/Akt pathway. FOXO1a, also referred to as forkhead homologue in rhabdomyosarcoma, is a downstream effector in the pathway and tightly linked to expression of proapoptotic genes. FOXO1a, but not the upstream kinase Akt, is rapidly phosphorylated, and FOXO1a is thereby inactivated when 293/FSHR cells are treated with FSH. In addition, FSHR coimmunoprecipitates with Akt. The identification of APPL1 as a potential interactor with FSHR and the finding that FOXO1a is phosphorylated in response to FSH provide a possible link between FSH and PI3K/Akt signaling, which may help to delineate a survival mechanism whereby FSH selects the dominant follicle to survive.
apoptosis, follicle-stimulating hormone, follicle-stimulating hormone receptor, mechanisms of hormone action, signal transduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Postnatally, the process continues through atresia, in which granulosa cells, which surround the developing oocyte in the follicle, go through apoptosis. During the menstrual cycle, a cohort of follicles is recruited to begin the process of maturation. Recently, multiple waves of follicular development have been documented in women [3], with an ovulatory follicle developing in the last wave of the menstrual cycle. By a process that is poorly understood, one follicle is selected to become the dominant follicle, which will continue to mature until a fertilizable oocyte is released. The remaining follicles in the cohort go through atresia [4].
In the early stage of maturation, FSH has little impact on preantral follicle growth except in combination with cGMP analogs [5]. Acting through the FSHR in the membrane of granulosa cells, however, FSH is a major factor in the survival of early antral follicles [6, 7]. In vitro, FSH and LH prevent atresia of cultured preovulatory follicles [8]. Analogs of cAMP mimic this effect [9].
A large body of evidence supports the idea that FSH induces cAMP and activates the protein kinase A pathway when it binds to FSHR [10]. Recent evidence, however, suggests that alternative signaling pathways may also be activated by FSH.
It has been shown that FSH activates p38 mitogen-activated protein kinase (MAPK) in granulosa cells [11] and stimulates growth of neoplastic ovarian cells via the MAPK signaling cascade [12]. It also phosphorylates Akt via the phosphatidylinositol-3-kinase (PI3K) signaling pathway in granulosa cells and induces both synthesis and phosphorylation of Sgk (serum- and glucocorticoid-induced kinase) in a mechanism involving protein kinase A, PI3K, and p38 MAPK [13, 14]. In Sertoli cells, FSH further enhances the activation of Akt by insulin-like growth factor (IGF)-I [15].
It seemed to be a reasonable hypothesis that FSHR activates signaling pathways via interaction with cytoplasmic proteins. The present study identifies APPL1 as an interacting partner with FSHR intracellular loops 1 and 2 (iL1 and iL2, respectively). APPL1 (referred to as APPL) interacts with the p110 catalytic subunit of PI3K and with inactive Akt [16]. Recent studies have found that APPL1 (referred to as APPL) associates with androgen receptor and with the p85 regulatory subunit of PI3K [17]. APPL1 also associates with Rab5, a critical regulator of endocytosis [18]. Also referred to as DIP13, APPL1 has been found to interact with the cytoplasmic domain of DCC (deleted in colorectal cancer) [19]. APPL1 shares 54% homology with APPL2 (DIP13ß) [20]. The interaction of APPL1 with FSHR provides a potential link between G protein-coupled receptor (GPCR) activation and the PI3K signaling pathway.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Yeast two-hybrid screening was performed essentially as described previously [21, 22]. A bait plasmid was constructed in which a concatemer of human FSHR iL1 and iL2 was fused to the Escherichia coli-repressor lexA cloned into pJK202 (kindly provided by Steve Hanes, Wadsworth Center, Albany, NY). The yeast expression plasmid pJK202 contains lexA with a multiple cloning site. Both iL1 and iL2 were inserted such that they fused in-frame to the C-terminus of lexA. The selectable marker is HIS3. The concatemer consisted of amino acids (a.a.) L370–M384 (iL1), a glycyl-seryl linker, and a.a. Y441–S471 (iL2). The library plasmid pJG4-5 contains the activation domain of the B42 acid blob under the control of the inducible GAL1 promoter. The selectable marker is TRP1. The reporter plasmid pSH18-34 contains the lacZ gene with the URA3 selectable marker. A human ovarian cDNA library (Origene Technologies, Bethesda, MD) that had been fused to the activation domain of B42 in pJG4-5 was used to generate approximately 105 transformants. Transformants were initially selected on leucine-deficient media to test for expression of the LEU2 reporter gene and then screened for expression of the ß-galactosidase reporter. Colonies were also screened on leucine-deficient plates with galactose or glucose to verify that the positive phenotype was dependent on library plasmid expression. Clones demonstrating specificity were sequenced and compared to the nr database, which includes all GenBank, RefSeq Nucleotides, EMBL, DDBJ, and PDB sequences, by BLAST [23].
ß-Galactosidase Assay
Clones that passed the initial screens were then tested in a ß-galactosidase assay as described previously [24] to verify that potential clones specifically interacted with iL1 and iL2 of the FSHR. Full-length, N-terminal (a.a. 1–460), and C-terminal (a.a. 460–709) APPL1 were inserted into the yeast library plasmid pJG4-5 and transformed into yeast RFY206 (MATa, trp1
::hisG, his3200, ura3–52, lys2201, and leu2–3) containing the pSH18-34 reporter and individual bait plasmids. The bait plasmids consisted of pJK202 in which lexA was fused to a.a. L370–M384 (iL1), a.a. Y441–S471 (iL2), a.a. C531–K555 (iL3), and a.a. I611–N678 (iL4). In addition, iL1iL2 (described above), 4-loop (iL1-linker-iL2-linker-iL3-linker-iL4), and lexA alone were tested. Colonies were isolated on synthetic medium lacking histidine, tryptophan, and uracil; were grown in the same medium with galactose; and were assayed for ß-galactosidase activity to test specificity.
Plasmid Construction
Full-length APPL1 [16] was inserted into the mammalian expression vector p3XFLAG-CMV10 (Sigma, St. Louis, MO) that had been modified to insert a XhoI site to tag APPL1 with a FLAG epitope on the N-terminus. In addition, the N-terminal (a.a. 1–460) and C-terminal (a.a. 461–709) portions of APPL1 were cloned into the modified p3XFLAG-CMV10 vector. HA-tagged Akt2 is described elsewhere [25].
Tissue Culture and Transfections
HEK 293 cells stably expressing human FSHR (293/FSHR cells) [26] were maintained in Eagle medium containing 10% fetal bovine serum and supplemented with 100 units penicillin and 100 µg streptomycin. Subconfluent, 60-mm dishes were transfected (Lipofectamine Plus; Invitrogen, Carlsbad, CA) with FLAG-tagged (Sigma) APPL1 constructs (2 µg/dish) and incubated for an additional 24–48 h. Cells were treated with 1.1 nM human pituitary FSH in serum-free Eagle medium for the indicated times. Where indicated, cells were treated with 6.6 nM IGF-I (National Hormone & Peptide Program, Harbor-UCLA Medical Center, Torrance, CA) for 30 min in serum-free Eagle medium.
Protein Expression
Cells were harvested in Igepal-DOC lysis buffer (10 mM Tris-HCl [pH 7.5], 1% Igepal [Sigma], 0.4% deoxycholate, and 6.6 mM EDTA) with protease inhibitors (10 µg/ml of pepstatin A, 10 µg/ml of aprotinin, 10 µg/ml of leupeptin, 16 µg/ml of benzamide, 10 µg/ml of 1,10-phenanthroline, and 1 mM PMSF). Sodium orthovanadate (1 mM) was included in experiments examining phosphorylated proteins. Cell lysates were sonicated, and protein concentrations were determined in a BCA assay (Pierce, Rockford, IL). Laemmli sample buffer (2x) [27] was added, the samples were boiled for 5 minutes, and 20 µg/lane were loaded on 10% SDS-polyacrylamide gels [27].
For coimmunoprecipitation experiments, cells were harvested in Igepal-DOC lysis buffer with protease inhibitors and immunoprecipitated as described by Nechamen and Dias [26]. In some cases, 140 mM NaCl was included in the lysis buffer.
Monoclonal antibodies (mAbs) specific for FSHR (mAb 106.105 [28]) and for the FLAG epitope (FLAG M2 mAb; Sigma) were used in the present study. The following polyclonal antibodies (Abs) were obtained from Cell Signaling Technology (Beverly, MA): Akt Ab, phospho-Akt (Ser473) Ab, FKHR (forkhead homolog in rhabdomyosarcoma) Ab, phospho-FKHR (Ser256) Ab, and phospho-FKHR (Thr24) Ab. Polyclonal HA-tag Ab was obtained from Upstate Cell Signaling Solutions (Charlottesville, VA). The anti-APPL Ab was raised in rabbits against GST-tagged, C-terminal APPL1 as described previously [16]. Purified mouse immunoglobulin (Ig) G1 was obtained from Pharmingen (San Diego, CA), and IgG2b was kindly provided by Gary Winslow (Wadsworth Center, Albany, NY).
Immunoblot Analysis
After electrophoresis, proteins were electroblotted onto Immobilon-P membranes (Millipore, Bedford, MA) according to the method described by Towbin et al. [29]. The membranes were blocked in TBST (10 mM Tris-HCl [pH 7.2], 150 mM NaCl, and 0.5% Tween 20) with 5% nonfat milk (TBSTM) overnight at 4°C. Blots were washed briefly and probed with anti-HA Ab (1:2000) or mAb 106.105 (5 µg) in TBSTM for 1 h at room temperature. Blots were washed and incubated with goat anti-rabbit (1:5000) or goat anti-mouse (1:10 000) Ab, respectively, conjugated to horseradish peroxidase (HRP) for 1 h at room temperature. In the case of HRP-conjugated M2 FLAG mAb (Sigma), blots were blocked in TBSTM for 1 h at room temperature and then probed with HRP-M2 FLAG mAb (1:1000) in TBSTM overnight at 4°C.
Alternatively, when blots were probed with Abs from Cell Signaling Technology, blots were blocked with TBSTM for 1 h at room temperature, washed briefly in TBST, and incubated in primary Ab diluted 1:1000 in TBST containing 5% bovine serum albumin overnight at 4°C. Blots were washed with TBST and probed with goat anti-rabbit Ab conjugated to HRP (1:2000) for 1 h at room temperature.
Signal was developed using the ECL Western blotting detection kit (Pierce), and blots were immediately exposed to Kodak XAR-2 film (Eastman Kodak, Rochester, NY). Equal protein loading was confirmed on a gel run in parallel and stained with Coomassie blue.
In some cases, blots were stripped before reprobing by incubation in 50 ml of stripping buffer (62.5 mM Tris-HCl [pH 6.7], 2% SDS, and 0.7% ß-mercaptoethanol) for 30 min at 60°C. Blots were washed three times for 5 min each in TBST and reblocked.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In a yeast two-hybrid screen of 105 transformants, eight unique cDNA clones were identified as specifically interacting with iL1 and iL2 of human FSHR. One clone, corresponding to the C-terminal 250 a.a. of APPL1 [16, 19], was chosen for further analysis (Fig. 1).
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Because FSH is a survival factor for ovarian follicles, APPL1 is of particular interest, because it interacts with Akt2 [16], which promotes cell survival [30], and also interacts with the transmembrane protein DCC to enhance DCC-mediated apoptosis [19]. The C-terminal 250 a.a. of APPL1 encompasses a phosphotyrosine-binding (PTB) domain, which is also the domain that interacts with Akt2 and DCC [16, 19].
To determine which portions of FSHR specifically interact with APPL1, a ß-galactosidase assay was performed in yeast. Both full-length and the C-terminal 250 a.a. of APPL1 specifically interact with iL1 (Fig. 2, A and B). Surprisingly, the concatemer of iL1 and iL2, which was used to initially identify APPL1 as an interacting partner, has little activity with full-length and C-terminal APPL1. The N-terminal 460 a.a. of APPL1 do not show a specific interaction with FSHR intracellular loops (Fig. 2C).
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LexA alone exhibits a high level of ß-galactosidase activity with C-terminal APPL1 (Fig. 2B) but not with full-length or N-terminal APPL1. The C-terminal fragment of APPL1 has a PTB domain, the activity of which normally may be dampened by the N-terminus. In addition, the lexA bait plasmid has an exposed C-terminus that is fused to additional amino acids in the other bait plasmids. The combination of an active PTB domain in C-terminal APPL1 and exposed residues in lexA may have contributed to the high background. With the exception of the high background observed with C-terminal APPL1 and lexA, APPL1 shows little nonspecific binding to FSHR iL2 and iL3 and to the C-terminal tail.
Interaction of APPL1 and FSHR in Mammalian Cells
To confirm that the interaction between APPL1 and receptor observed in yeast also occurred in mammalian cells, 293/FSHR cells were transfected with full-length, FLAG-tagged APPL1. Complexes of FSHR and FLAG-tagged APPL1 were immunoprecipitated with mAb 106.105, specific for the extracellular domain of FSHR [28], and FLAG-tagged APPL1 was detected with FLAG mAb. As seen in Figure 3, the interaction between FSHR and APPL1 is detectable in the absence of hormone. However, the interaction is significantly enhanced by treatment of cells with FSH in as little as 30 min of treatment, and APPL1 appeared in stable association with FSHR for up to 8 h.
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In agreement with the observation that the C-terminal fragment of APPL1 was identified as the interactor with receptor in the initial yeast two-hybrid screen, Figure 4 shows that the C-terminal portion of APPL1 appears to be coimmunoprecipitated with FSHR by mAb 106.105 in mammalian cells and that the association is enhanced by FSH. In addition, FSHR was detected in APPL Ab-derived immune complexes (Fig. 5). Both monomer and higher-molecular-weight, FSHR-specific oligomers are visible in Figure 5.
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Although N-terminal APPL1 does not interact with FSHR in yeast, Figure 6 demonstrates that the N-terminal fragment of APPL1 appears to associate with full-length FSHR expressed in mammalian cells in a hormone-dependent manner. This is likely because of the difference in conformation between individual FSHR intracellular loops expressed in the yeast cytoplasm versus FSHR expressed as a full-length protein with the transmembrane segments and intracellular loops oriented in the native conformation in the mammalian cell membrane.
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Activation Status of PI3K/Akt Pathway
A number of recent reports indicate that FSH acts, at least in part, by amplification of the PI3K/Akt signaling pathway [13, 31–33]. The interaction of APPL1 with Akt2 [16] and with FSHR suggests a possible mechanism whereby FSH activates the PI3K/Akt pathway. Activation of the PI3K/Akt pathway triggers a cascade of phosphorylation [30]. Accordingly, the activation/phosphorylation state of various members of the PI3K/Akt pathway was assessed by immunoblot analysis of total cell lysates.
One of the terminal effectors in the PI3K/Akt pathway, FKHR, activates transcription of proteins involved in apoptosis, including FasL [34]. According to the new nomenclature [35], FKHR has been renamed FOXO1a. Phosphorylation of FOXO1a leads to repression of apoptotic gene expression [34]. The ultimate effect of PI3K/Akt activation, therefore, is cell survival.
As seen in Figure 7A, FOXO1a is rapidly phosphorylated on Ser256 and, thereby, is inactivated when 293/ FSHR cells are treated with FSH. A similar finding has been observed in porcine [32] and rat [36] granulosa cells. Synthesis of FOXO1a does not appear to be hormone-dependent, because total levels of FOXO1a protein did not change on FSH treatment (Fig. 7B). Although IGF-I stimulates phosphorylation of FOXO1a on Thr24, FSH has no effect and does not further enhance phosphorylation of Thr24 in conjunction with IGF-I (Fig. 7C).
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Because FOXO1a is phosphorylated in response to FSH, the phosphorylation/activation status of Akt, the upstream kinase in the cascade, was examined. Although FSH induces the phosphorylation of FOXO1a in as little as 5 min, FSH stimulates only a slight increase in phosphorylation of Akt, which is not observed until after 60 min of treatment (Fig. 8A). Although phosphorylation of Akt likely is important in FSH-induced cell signaling and consequent follicular survival and atresia, the time course of Akt phosphorylation is inconsistent with the rapid phosphorylation (5–10 min) of FOXO1a observed in response to FSH treatment in HEK 293 cells.
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Likewise, whereas IGF-I stimulates phosphorylation of Akt on Ser473, FSH does not have a similar effect in HEK 293 cells and does not interact with IGF-I to further enhance phosphorylation of Akt (Fig. 8C), in contrast to the synergy observed in Sertoli cells [15]. It is important to point out, however, that basal phosphorylation of Akt is observed. Total levels of Akt appear to remain stable in response to hormone (Fig. 8B).
Although FSH does not induce a rapid phosphorylation of Akt, it was of interest to determine whether FSHR associates with Akt, particularly because APPL1 and Akt are known to interact and because the present work suggests that APPL1 and FSHR interact. Accordingly, 293/FSHR cells were cotransfected with HA-tagged Akt2 and FLAG-tagged APPL1 and then immunoprecipitated with mAb 106.105 to immunoprecipitate FSHR and associated proteins. Figure 9 shows that FSHR appears to associate with Akt and with APPL1. As expected from previous work [16], APPL1 also interacts with Akt (Fig. 9).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Activation of Akt promotes cell survival by phosphorylating and downregulating the activity of proapoptotic proteins, including FOXO1a [34], BAD [37], and caspase-9 [38]. Amplification of the Akt2 gene is associated with human ovarian and pancreatic tumors [39, 40], and overexpression of Akt is oncogenic [39].
A recent report by Yang et al [17] indicates an interaction between APPL1 and androgen receptor, a nuclear steroid receptor, in prostate tumor cell lines. Their findings suggest that APPL1 expression may regulate Akt activity in some, but not all, prostate cancer cell lines. The observation that APPL1 associates with androgen receptor expands the repertoire of APPL1-interacting proteins to include nuclear receptors.
Additionally, APPL1 has been found to associate with the GTPase Rab5 [18], which mediates fusion of clathrin-coated vesicles with early endosomes [20]. Treatment with epidermal growth factor results in movement of APPL1 to the nucleus, where it interacts with the nucleosome remodeling and histone deacetylase complex, suggesting a novel signaling pathway from the cell membrane to the nucleus.
Initially, DCC was identified because of its deletion in colorectal cancers at high frequency [41], although its role as a tumor suppressor is controversial. Loss of expression of DCC has since been found in a number of tumors, including prostate and pancreatic tumors [42, 43]. Overexpression of DCC has been observed to induce apoptosis [44], and overexpression of APPL1 with DCC further enhances apoptosis in certain cell lines [19].
Apoptosis and cell survival can be viewed as flip sides of the same coin and tumorigenesis as an extreme example of either failed apoptosis or unregulated cell survival. As such, it is interesting to note that APPL1 interacts with at least three proteins involved in apoptosis/cell survival/tumorigenesis and that inhibition of APPL1 activity [18] reduces DNA synthesis.
Apoptosis and cell survival are also key processes in regulating follicular development in the ovary. In a cohort of developing follicles, one follicle is selected to continue maturing, and the remaining follicles go through atresia or apoptosis. The precise mechanism whereby the dominant follicle escapes atresia is poorly understood, but it may involve the action of FSH through FSHR. Interestingly, Castrillon et al. [45] showed that FOXO3a (FKHRL1) knockout mice had accelerated follicular recruitment, resulting in premature ovarian failure [45] and suggesting an involvement of forkhead transcription factors in follicular development.
As a protein that interacts with both membrane receptors and components of the PI3K/Akt signaling pathway, APPL1 may function as an adaptor or docking protein, coordinating inputs from multiple signaling pathways. Adaptors typically have an amino terminal membrane targeting region, such as a PH domain and a PTB domain, that recognizes an NPXpY motif in receptors [46]. APPL1 has such a domain organization [16]. On association with a receptor, adaptors become phosphorylated and interact with the SH2 domains of additional signaling proteins. Although FSHR and DCC lack a potential tyrosine phosphorylation site, the PTB domains of adaptor proteins X11, FE65, and Numb [47, 48] recognize nonphosphorylated peptide motifs. APPL1 may have a similar mode of action.
It has been shown that APPL1 interacts with the p110 catalytic subunit of PI3K and with inactive Akt [16]. The p110
subunit of PI3K is generally thought to be the mediator of PI3K signaling for GPCR. The suggestion that APPL1 interacts with p110 as well as with FSHR enlarges the scope of GPCR activation of this pathway. As such, APPL1 is an adaptor that may tether PI3K and Akt in the cytoplasm.
The lack of correlation between FOXO1a and Akt phosphorylation in response to FSH has been observed by other investigators [13, 32]. This suggests that a redundant pathway is available, and future studies will be aimed at determining which protein kinase is responsible for phosphorylation of FOXO1a. A likely candidate is Sgk, which phosphorylates the forkhead transcription factor family [49, 50] and appears to be phosphorylated in granulosa cells following FSH activation [13, 36].
Interesting parallels can be found between our studies and those of Cottom et al. [51], in which they found that a downstream effector (i.e., ERK) was phosphorylated in granulosa cells in response to FSH but that upstream kinases and regulators were not activated by FSH treatment. The ERK signaling pathway was constitutively active in granulosa cells, and an FSH-regulated, phosphotyrosine phosphatase controlled ERK phosphorylation. A similar possibility will be investigated with regard to FOXO1a.
A model is thus proposed in which APPL1 associates with FSHR on FSH stimulation to stimulate the PI3K/Akt signaling cascade. APPL1 has been shown to associate with Akt [16], and the present study shows that FSHR may interact with APPL1 and with Akt. Although FSH does not stimulate the phosphorylation of Akt, the basal level of phosphorylated Akt may be sufficient to phosphorylate FOXO1a, in which case the localization of Akt would be a critical factor. The potential interaction between FSHR and Akt may serve to sequester inactive Akt in an APPL1-dependent process. Future experiments will determine whether FSHR exists in a trimolecular complex with APPL1 and Akt or as two bimolecular complexes composed of FSHR with APPL1 and FSHR with Akt. The potential placement of FSHR, Akt, and APPL1 together could also allow Akt to be involved in a Rab5 endosome-mediated signaling pathway.
The FSHR has been found to constitutively form oligomers, and this process is augmented by FSH (unpublished results). Oligomerization of FSHR provides a mechanism for drawing multiple signaling pathways (i.e., PI3K/Akt and protein kinase A) together in microdomains with the opportunity for finely controlled interaction and regulation.
The studies reported here also demonstrate that FSH induces the rapid phosphorylation of FOXO1a in HEK 293 cells. Phosphorylated forkhead family members translocate to the cytoplasm [52], thereby preventing transcription of genes encoding proapoptotic proteins, such as FasL. Interestingly, a hormone-dependent interaction between 14-3-3 and FSHR has been identified [53]. The observation that FSH stimulates the association of 14-3-3 with FSHR (data not shown) and increases the phosphorylation of FOXO1a, together with the results of studies showing that phosphorylated FOXO1a binds to 14-3-3 in the cytoplasm [34], encourages the authors to speculate on a mechanism in which FSH causes FOXO1a to be sequestered and thereby inhibits apoptosis. APPL1 may facilitate this sequence of events in its role as adaptor protein.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: James A. Dias, Wadsworth Center, David Axelrod Institute for Public Health, New York State Department of Health, 120 New Scotland Avenue, Albany, NY 12208. FAX: 518 474 5978; james.dias{at}wadsworth.org
Received: 25 November 2003.
First decision: 9 December 2003.
Accepted: 30 March 2004.
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