Cloning and characterization of the highly expressed ETEA gene from blood cells of atopic dermatitis patients

BBRC Biochemical and Biophysical Research Communications 297 (2002) 1282–1290 www.academicpress.com

Cloning and characterization of the highly expressed ETEA gene from blood cells of atopic dermatitis patients Yukiho Imai,a Akiko Nakada,a Ryoichi Hashida,a Yuji Sugita,a Toshio Tanaka,b Gozoh Tsujimoto,c Kenji Matsumoto,c Akira Akasawa,c Hirohisa Saito,c and Tadahilo Oshidaa,* a

b

Genox Research, Inc., Teikyo University Biotech Center, 907 Nogawa, Miyamae, Kawasaki, Kanagawa 216-0001, Japan Department of Molecular and Cellular Pharmacology, Mie University School of Medicine, 2-174 Edobashi, Tsu, Mie 514-0001, Japan c National Research Institute for Child Health and Development, Tokyo 154-8567, Japan Received 9 September 2002

Abstract Analysis of patients with atopic dermatitis (AD) for differential expression of genes, as compared to normal individuals, will be useful for understanding the molecular pathogenesis of AD. We found that the expression of the gene ETEA in human peripheral blood CD3-positive cells from patients with atopic dermatitis was significantly higher than in normal individuals. Eosinophils from AD patients expressed ETEA at a significantly higher level than the healthy controls. The overall sequence of the 445 aa deduced polypeptide from the cloned ETEA cDNA showed homology to human Fas-associated factor 1 (FAF1), which is involved in Fasmediated apoptosis. However, the interaction of ETEA with the Fas death domain was weaker than that of FAF1, as studied in yeast two-hybrid experiments. The ETEA-EGFP fusion protein was expressed in cytoplasm. During the course of activation-induced cell death of primary T cells, transcription levels of ETEA and FAF1 were upregulated with similar kinetics. The enhanced expression of ETEA may play a role in the regulating the resistance to apoptosis that is observed in T cells and eosinophils of AD patients. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: T cells; ETEA; Eosinophils; Transcription; Atopic dermatitis; Expression; Fas; FAF1

Atopic dermatitis (AD), a chronic and relapsing inflammatory skin disease that often begins in infancy, is characterized by pruritus, elevated serum IgE levels, and peripheral blood eosinophilia. A complex interplay of both genes and environmental factors is likely to be involved in AD pathogenesis. A two phase model of the pathogenesis of AD has been proposed, in which T cells play an immunoregulatory role in the physiological and pathological immune responses. Helper T cells can be divided into two subsets based on their cytokine production profiles. Th1 cells produce IL-2 and IFN-c and Th2 cells produce IL-4, IL-5, and IL-13 [1,2]. The AD episode, without clinically apparent skin lesions, is initiated by an inflammatory response predominated by the Th2 cells. The second, an eczematous phase is *

Corresponding author. Fax: +81-44-797-2622. E-mail address: [email protected] (T. Oshida).

dominated by Th1 cells [3,4]. Eosinophils are believed to be involved in mediating the late-phase allergic inflammation. The effector functions of eosinophils are derived primarily from release of lipid mediators and proteins, such as cytokines and cytotoxic cationic granule proteins [5–7]. Analysis of genes that are differentially expressed in AD patients and normal individuals will provide information about the molecular pathogenesis of AD. During differential gene expression analysis in human peripheral blood CD3-positive cells from patients with atopic dermatitis, we observed that the expression levels of the gene, ETEA, were higher in the AD patients than in normal individuals. Interestingly, the expression levels of ETEA were also higher in eosinophils from the AD patients than those from the normal controls. Therefore, we cloned and expressed the ETEA for additional molecular studies. Because the aa sequence of ETEA was

0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 2 3 8 0 - X

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found to be similar to that of Fas-associated factor 1 (FAF1) [8], we compared the kinetics and expression of FAF1 with that of ETEA in T cells during activationinduced cell death (AICD).

Materials and methods Cloning of ETEA gene. The differential display analysis in human peripheral blood T cells from patients with atopic diseases will be described in detail elsewhere (Matsumoto et al., in press). A 230 bp DNA fragment, amplified by differential display PCR with an anchor primer 50 -GT15 A-30 and an arbitrary primer 50 -GCCGAATAAC-30 , was targeted for full length cDNA cloning. The public EST sequences homologous to the sequence were clustered and DNA fragments were cloned by rapid amplification of cDNA ends method [3,4] using Marathon cDNA amplification kit and Molt4 Marathon ready cDNA as template (Clontech, Palo Alto, USA) to obtain the 3.2 kb cDNA containing the original sequence. The correct sequence was determined by sequencing double strands of at least two fragments amplified from independent PCRs. The cDNA sequence exactly matched KIAA0887, a cDNA sequence identified in the Kazusa cDNA Project [9]; however, the 50 end of KIAA0887 was truncated. To determine 50 end of ETEA cDNA sequence, a human T cell (Jurkat) lambda cDNA library (Stratagene, La Jolla, USA) was screened by plaque hybridization using a probe amplified by PCR with primers 50 -GGATCAGTGTCGCCATACCTTG-30 and 50 -GCCGTCCCACCACAGTCAT-30 . We obtained a clone which had extended 50 end sequence by 11 bp length. Resulted full length cDNA sequence was 4503 bp encoding a deduced protein of 445 aa residues (Fig. 1A). The cDNA sequence was submitted to DDBJ with Accession Number of AB088120. This gene was designated as ETEA, based on its characteristic high transcriptional expression levels in T cells and eosinophils of AD patients as described below. After determining the full length sequence of ETEA, a DNA fragment coding the full ORF region was obtained by PCR using primer 419sSac carrying a EcoRI restriction site (50 -GAGCTCAAAATGGCGGCGCCTGAGGA-30 ) and primer 419asHind carrying a HindIII site (50 -AAGCTTATGTCAT CGTCAGTTAGGTCCTG-30 ). A first strand cDNA was synthesized using total RNA of human peripheral blood T cells and used as a PCR template. The amplified fragment was cloned into pGEM-T Easy vector (Promega, Madison, USA) to produce pGEM-ETEA. Northern blot hybridization. For Northern blot analysis of a variety of human tissues for ETEA mRNA, commercially available human MTN blot kits (Clontech) were used. A 1.6 kb fragment containing the complete coding region of ETEA was amplified by PCR with primers of 419sSac and 419asHind from a human T cell k cDNA library. The 1.6 kb fragment was labeled with 32 dCTP using a random primer labeling kit (Takara, Kyoto, Japan) and used as probe (Fig. 2). Hybridization was carried out using ExpressHyb Hybridization Solution (Clontech) as described in manufacturerÕs protocol. Preparation of T cell and eosinophil cDNA samples from AD patients. Transcription levels were measured in our existing stocks of T cell and eosinophil cDNAs. Patient profiles and clinical parameters for the samples in the T cell 2nd set (Matsumoto et al. in press) and in the eosinophil set (Hashida et al. in preparation) will be described in detail elsewhere. Briefly, blood samples were collected from AD patients in different phases and of differing severity, as judged by a modified version of LeicesterÕs scoring system for AD [10]. Samples collected from healthy volunteers were used as controls. Written informed consent to participate in the study was obtained from all participants. The T cell 2nd set consisted of 40 samples (10 from healthy volunteers and 30 from AD patients) and the eosinophil set consisted of 59 samples (13 from healthy volunteers and 46 from AD patients).

Fig. 1. Amino-acid sequence of ETEA and its region of homology with FAF1. (A) The amino-acid sequence was deduced from the cDNA sequence. The UAS domain (UAS) and the domain present in ubiquitin-regulatory proteins (UBX) are indicated by a solid and dotted underline, respectively. (B) Two homologous regions between ETEA and FAF1 are indicated by dotted lines. UAX, UBX, and NLS (nuclear localization signal) are shown in boxes. (C) Amino-acid sequence arrangement of the two homologous regions between ETEA and FAF1. Pluses (+) indicate conserved amino-acid substitutions. Hyphens represent breaks introduced to maximize homology.

Peripheral blood mononuclear cells (PBMC) were prepared from 10 to 20 ml of venous blood. After dextran sedimentation, white cells were separated by Ficoll–Hypaque gradient centrifugation, using standard methods. CD3 antigen-positive cells were isolated using a magnetic cell sorter (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The percentage of CD3-positive cells (T cells) was >97%. CD16-negative cells (eosinophils), obtained from the sedimentary granulocyte fraction of the Ficoll–Hypaque gradients, were isolated by negative selection using a magnetic cell sorter. The percentage of eosinophils was >97%. CD14 antigen-positive cells (monocytes) were prepared from cells remaining after separation of CD3-positive cells. The cells remaining after separation of CD14-positive cells were used as a source of B cells. CD16-positive granulocytes remaining after separation of CD16-negative cells were the source for neutrophils. Total RNA was prepared using an RNA extraction kit (RNeasy Mini, Qiagen GmbH, Hilden, Germany). DNase-treated total RNAs were

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Fig. 2. Expression of ETEA RNA in various human tissues. Human multiple tissue Northern blot (Clontech) was probed with a 1.6 kb fragment with the ETEA coding region. RNA molecular size marker are in kbp are shown on the left. b-Actin probe was hybridized to the same blot for comparison. mixed with oligo ðdTÞ12–18 primer or random primers and first strand cDNAs were synthesized using SuperScript II reverse transcriptase. Real-time quantitative RT-PCR. Real-time RT-PCR for quantitation of gene expression was performed using an ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems, USA). Based on the target gene sequence, primers and a dual-labeled fluorogenic probe (TaqMan Probe) were designed using the computer program Primer Express 1.0 (PE Applied Biosystems, Foster City, USA). Primer and probe sequences are available from the author on request. The quantitative PCR was performed using a TaqMan PCR Reagent Kit according to manufacturerÕs protocol (PE Applied Biosystems). Sample cDNAs, equivalent to 5 ng of starting RNA, were used for each reaction in a 96-well PCR plate. Plasmid DNA of known target sequence was used to prepare absolute standards. Levels of b-actin mRNA were used as an internal standard for each sample. The average copy number of b-actin, based on values from all samples, and a ratio relative to this was determined for each individual sample. The copy number of the target sequence in each sample was normalized by dividing this value by the value obtained for the relative b-actin ratio. For in vitro experiments using primary T cells, ribosomal RNA was used for an internal standard instead of b-actin. Quantity of ribosomal RNA was measured using commercially available control reagents (VIC probe, PE Applied Biosystems). After the full length cDNA of ETEA was determined, the primers and probe for ETEA were redesigned using the sequence coding for the C-terminal region. Plasmid construction. For expression of ETEA-EGFP fusion protein, a fragment containing the C-terminus of the ETEA sequence, modified to allow fusion with the EGFP, was amplified by PCR using pGEM-ETEA as a template and primer 419sSacI and primer 419asSalIGFP containing a SalI site (50 -TACCGTCGACTGCAG AATTTCGTCAGTTAGGTCCTGAACA-30 ). The PCR product was digested with SacI and SalI, and cloned into pEGFP-N1 (Clontech), also digested with SacI and SalI, to generate pETEA-EGFP. A DNA fragment coding FAF1 was used for cloning. The DNA was amplified by PCR using the human T cell cDNA as a template, and primer FAF1sSac containing a SacI site (50 -GAGCTCAAAATGGCGTCC AACATGGACC-30 ) and primer FAF1asHind containing a HindIII

site (50 -AAGCTTCGCTGGGCCGTGTTTACTCT-30 ). The amplified fragment was cloned into pGEM-T Easy vector to produce pGEMFAF1. Yeast two-hybrid assay. Yeast two-hybrid assays were performed using the Matchmaker LexA two-hybrid system (Clontech). FasDD was cloned into bate vector pGilda for expression of binding domainFasDD fusion protein. ETEA and FAF1 were cloned into pray vector pB42AD for expression of activation domain fusion proteins, respectively. For expression of the ETEA-activation domain fusion protein, PCR amplification was done using pGEM-ETEA as a template, with primer 419sEco with an EcoRI site (50 -AGTGAATTCAAAATGGCGGCG CCTGAGGA-30 ) and primer 419asSalI with a SalI site (50 -CTC GTCGACATGTCATCGTCAGTTAGGTCCTG-30 ). The product was cloned into pGEMR -T Easy(Promega) to generate pGEM-ETEAB. A EcoRI digested fragment carrying ETEA was obtained by digestion of pGEM-ETEAB and cloned into a EcoRI site of pB42AD (Clontech) to generate pB42AD-ETEA. For expression of FAF1-activation domain fusion protein, PCR amplification was done using pGEM-FAF1 as a template and primer FAF1sEco, with an EcoRI site (50 -AGTGAA TTCAAAATGGCGTCCAACATGGACC-30 ) and primer FAF1asSacI with a SacI site (50 -CTCGAGCTCCGCTGGGCCGTGTTT ACTCT-30 ). The PCR product was digested with EcoRI and SacI and cloned into pGADT7, digested with EcoRI and SacI, to generate phFAFAD. A fragment carrying FAF1 was obtained by digestion of phFAFAD by EcoRI and XhoI and cloned into pB42AD, digested with EcoRI and XhoI, to generate pB42AD-FAF1. For expression of FasDDbinding domain fusion protein, PCR amplification was done using pRXhFas-ires-hCD80 (obtained from RIKEN GenBank, Tsukuba, Japan) as a template and primer FasDDEcoRI with an EcoRI site (50 -GGC CGAATTCGGTTCTCATGAATCTCCAAC-30 ) and primer FasSalI with a SalI site (50 -CGACGTCGACCTAGACCAAGCTTTGGAT TTC-30 ). The PCR product was digested with EcoRI and SalI and cloned into pGilda (Clontech), digested with the same enzymes, to generate pGilda-FasDD. Plasmids were cotransformed with various combinations into yeast EGY48 cells bearing plasmid p8op-lacZ. The pLexA-53 and pB42AD-

Y. Imai et al. / Biochemical and Biophysical Research Communications 297 (2002) 1282–1290 T were positive controls for strong interaction. Colonies containing both hybrid plasmids were selected and replica-plated to a series of selection plates containing X-gal. The b-galactosidase activity was evaluated after incubation for 4 days at 30 °C by development of blue color in transformants. Activation-induced cell death of T cells. T cells were isolated from PBMC by negative selection using a Pan T cell isolation kit and a magnetic cell sorter (Miltenyi Biotec). Isolated T cells were maintained in RPMI1640 supplemented with 5% FCS, 2 mM L -glutamine, 1 mM sodium pyruvate, penicillin (100 U/ml), and streptomycin (100 lg/ml). For the first activation, T cells (5  105 cells/ml) were placed into 6-well plates, pre-coated with 10 lg/ml anti-CD3 monoclonal antibody (mAb) as antigen for activation (Orthoclone OKT3, Ortho Biotech, South Raritan, USA), using media both with and without IL-2 (100 U/ ml, Imunace, Shionogi Pharmaceutical, Osaka, Japan). The plates were centrifuged at 1000 rpm for 2 min and the cells were cultured for various periods before analysis for gene expression. Cells cultured in plates without anti-CD3 were negative controls. To prepare cells for the second activation, the initial activation was done with a cell density of 1  105 cells/ml. After a 5-day expansion, the cells were harvested and incubated in medium containing IL-2 (10 U/ml) for an additional 3 days. For the second activation, cells were harvested and placed into anti-CD3 coated 6-well plates in medium in the presence or absence of IL-2. For stimulation with PMA (25 ng/ml) and ionomycin (1 lg/ml) cells were incubated in plates with no anti-CD3. Cell viability was determined by trypan blue exclusion. Statistical analysis: The Wilcoxon rank-sum test was used for comparisons between the normal controls and patient groups. When multiple comparisons were made between groups, significant betweengroup variability was first established using the ANOVA. FisherÕs PLSD was then used for intergroup comparisons. Probability values of p < 0:05 were accepted as significant. StatView 5 software (SAS Institute, Cary, USA) was used throughout the analyses.

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Expression of ETEA in tissues In a survey of human tissues, a 4.6 kb mRNA was detected in various tissues when the mRNA blots were hybridized with the ETEA probe (Fig. 2). The ETEA mRNA was identical in size to the cloned ETEA cDNA. The abundant expression was observed in the brain. Particularly high expression was detected in the cerebellum and caudate nucleus. In immune tissues, ETEA mRNA expression was higher in spleen and lymph node than in the thymus and peripheral blood leukocytes. Transcript levels of ETEA and FAF1 in different subsets in peripheral blood leukocytes were examined by quantitative RT-PCR. ETEA transcription was detected at similar levels in all subsets of leukocytes. The levels of FAF1 were higher in T cells than in other cell types (Fig. 3A). Transcription levels of ETEA in T cells and eosinophils from AD patients ETEA mRNA was found, by quantitative RT-PCR, to be expressed at a significantly higher level in T cells from patients with moderate or severe AD than from either normal individuals or patients with mild AD (Fig. 3B). Furthermore, ETEA was expressed at a higher level in eosinophils from the AD patients than in eosinophils from the healthy controls. Expression of FAF1 mRNA, in contrast to ETEA, was similar between the AD patients and the healthy controls both in T cells and eosinophils.

Results

Subcellular localization

Cloning and primary structure analysis of ETEA

Following transfection of HEK293 cells with an ETEA-EGFP fusion plasmid, the fusion protein was expressed in cytoplasm (Fig. 4).

During differential display analysis in human peripheral blood CD3-positive cells from patients with atopic diseases, quantitative RT-PCR using primers and a probe designed for a 230 bp DNA fragment showed that the fragment was expressed at a higher level in AD patients than in normal individuals (data not shown). The ETEA aa sequence, deduced after cloning and sequencing of the full length cDNA, encoded a protein of 445 aa residues (Fig. 1A). This 445 aa sequence was used for domain and homology searches (CD-Search and BLAST, National Center for Biotechnology Information). ETEA was found to contain a UAS domain (a conservative sequence with unknown function) at the center region and a UBX domain, similar to the one present in ubiquitin-regulatory proteins, at the C-terminus. The sequence showed homology to FAF1, which also contains UAS and UBX domains (Fig. 1B). As shown in the aa alignment of homologous region of ETEA and FAF1 (Fig. 1C), the degree of identity is 39% for ETEA Nterminal region (from 17 to 57 aa) and 23% for ETEA C-terminal region (from 112 to 308 aa).

Interaction of ETEA with Fas death domain Interaction of ETEA with the Fas death domain was determined by a yeast two-hybrid assay (Table 1). After cotransformation with the Fas death domain and FAF1, the cells were blue, confirming the reported interaction of the two proteins. In contrast, cells cotransformed with the Fas death domain and ETEA were pale blue, indicating that the level of binding affinity of ETEA to Fas death domain was weaker than that of FAF1. Expression profiles of transcripts during AICD To examine possible involvement of ETEA in T cell activation and AICD, transcription levels of ETEA together with other apoptosis-related genes were examined in the course of activation of freshly isolated T cells. In the first activation, transient upregulation of transcription levels of ETEA as well as the pro-apoptotic genes (Fas, Fas ligand, Bax, caspase 3, caspase 8, and FAF1)

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Fig. 3. Transcription of ETEA in T cells and epsinophils in AD patients. (A) Transcription levels of ETEA and FAF1 were examined in subsets of PBMC cells from five healthy individuals. The subsets are abbreviated as T (T cells), B (B cells), E (eosinophils), M (monocytes), and N (neutrophils). (B) ETEA transcription levels were examined in T cells and eosinophils from AD patients and healthy controls (H). T cells and eosinophils were prepared from AD patients at different severity levels as indicated by mild (ML), moderate (MD), and severe (S) as described in Materials and methods. Transcription levels of ETEA and FAF1 in T cells and eosinophils were compared between the normal controls and patient groups. *p < 0:05, **p < 0:01, and ***p < 0:001.

and the anti-apoptotic genes (Bcl2 and BclxL) was observed for the cells treated with anti-CD3 mAb in the presence of IL-2 (Fig. 5A). In the second activation the AICD was induced for the cells treated with anti-CD3 mAb and IL-2 (64% death) as well as cells stimulated with PMA/ionomycin (79% death). Addition of the caspase inhibitor, zVAD, to the cells activated by antiCD3 and IL-2 completely inhibited cell death, suggesting that observed cell death resulted from apoptosis

(Fig. 5C). In clear contrast to the first activation, the transcription levels of ETEA as well as the pro-apoptotic genes (Fas, Bax, and FAF1) were once upregulated, and then the elevated levels were maintained until 48 h for the cells treated with anti-CD3 mAb and IL-2. The prolongation of elevated levels of these genes was accompanied by the induction of apoptosis. Fas ligand (FasL), caspase 8, and the anti-apoptotic genes (Bcl2 and BclxL) showed transient upregulation similar to

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Table 1 Comparison of interaction of ETEA or FAF1 with Fas death domain (FasDD)

Fig. 4. Subcellular localization of ETEA-EGFP fusion protein. The ETEA-EGFP fusion protein expression vector was used for transformation of HEK293 cells. The fluorescent (A) and bright field (B)images were taken 48 h after transformation.

Binding domain

Activation domain

Interaction

pGilda-FasDD pGilda pGilda-FasDD pGilda pGilda-FasDD pLexA-pos pLexA-53

pB42AD pB42AD-ETEA pB42AD-ETEA pB42AD-FAF1 pB42AD-FAF1 ) pB42AD-T

) ) +) ) + ) ++

Interaction of ETEA with FasDD was determined by a yeast twohybrid method. Strength of protein interaction was determined by the color of yeast colonies 4 days after transformation and designated as deep blue, ++; blue, +; pale blue, +); and white, ). pLexA-53 and pB42AD-T were positive controls for strong interaction.

Fig. 5. Upregulation of ETEA transcription levels during activation-induced cell death of primary T cell blasts. (A) First activation. Freshly prepared CD3+ cells were stimulated with anti-CD3 and/or IL-2 (100 U/ml). Closed circles, anti-CD3 + IL-2; open circles, IL-2; and crosses, control. (B) Second activation. T cell blasts made by anti-CD3 stimulation were re-stimulated with anti-CD3 and/or IL-2 (100 U/ml). Closed circles, antiCD3 + IL-2; open circles, IL-2; and crosses, control. Total RNA was extracted from each sample and the transcription levels of ETEA and apoptosisrelated genes were measured by RT-PCR. Transcription levels were normalized to ribosomal RNA. (C) Induction of apoptosis. In the second activation of T cell blasts, the number of viable cells was counted by a trypan blue exclusion method. Closed circles, anti-CD3 + IL-2; open circles, IL-2; closed triangles, anti-CD3 + IL-2 + zVAD (12.5 lM); open squares, PMA + ionomycin; and crosses, control.

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Fig. 5. (continued)

that in the first activation (Fig. 5B). The expression kinetics of ETEA were similar to those of the pro-apoptotic genes (Fas, Bax, and FAF1), suggesting the possible involvement of ETEA in modulation of AICD of T cells. Treatment with PMA/ionomycin showed the rapid increase in cell death rate from 6 to 24 h. FasL was

transiently upregulated at 2 and 6 h by PMA/ionomycin. Transient upregulation of transcription levels of ETEA as well as FAF1 and BclxL was observed at 24 h. The levels of these genes were higher than those observed for the cells treated with anti-CD3 mAb and IL-2. The marked upregulation at 24 h in cells stimulated with PMA and ionomycin was accompanied by the highest

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death rate. The transcription levels of Fas and Bax were once upregulated at 24 h and then the elevated levels were maintained until 48 h (data not shown).

Discussion Our data point to the similarities between ETEA and FAF. There is sequence homology. ETEA and FAF1 were expressed in various tissues. The ETEA-EGFP fusion protein was located in the cytoplasm of HEK293 cells. FAF1 is reported to localize in the peri-nuclear cytoplasm and in the nucleoli [11]. We did not observe translocation of ETEA into nuclei during expression analysis of the ETEA-EGFP fusion protein. This lack of translocation into nuclei is consistent with the absence of a nuclear location signal in ETEA. FAF1 binds to the intracellular portion of the apoptosis signal transducing receptor Fas/Apo-1. The function of the N-terminal region of FAF1 is binding to FasDD. The observed truncation of the corresponding 50 region in ETEA predicts the observation in the yeast two-hybrid assay that binding affinity of ETEA to FasDD would be weaker than that of FAF1. The functions of the UAS and UBX domains, common to both ETEA and FAF1, are unknown. Based on sequence homology to hypothetical ubiquitin conjugating enzyme from C. elegans, it is speculated that FAF1 may modulate degradation of FasL/Fas complex by a proteosome and potentiate intracellular apoptosis signaling. However, the role of FAF1 in the ubiquitination pathway has not been studied. The similarity of ETEA to FAF1 strongly implicates it as a factor, possibly related to apoptosis. There is ample evidence to implicate FasL–Fas interaction as a mediator of programmed cell death in the lymphocytes and eosinophils. AICD in T cells, mediated mainly by FasL–Fas apoptosis, acts as a feedback mechanism for terminating an ongoing immune response [12] and induces peripheral tolerance [13,14]. Although the mechanism of modulating apoptosis is not known, mouse FAF1 is reported to enhance, but not initiate, Fasmediated apoptosis when overexpressed in L-cells [15]. Transient overexpression of human FAF1 (hFAF1) in BOSC23 cells caused apoptosis [16]. Recently, it was reported that protein kinase CK2 interacts with hFAF1 and phosphorylates its two serine residues at positions 289 and 291 [17]. Subcellular localization experiments showed that both CK2 subuntis and hFAF1 were colocalized in the perinuclear cytoplasm. The majority of the CK2 subunits were in the nucleus and the hFAF1 was in the nucleoli. The CK2/FAF1 complex formation was significantly increased after induction of apoptosis with various DNA damaging agents, suggesting an important role of the complex in certain steps of apoptosis [11].

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Our observations suggest the possible involvement of ETEA in modulation of AICD of immunoreactive cells. AICD of mature T cells is a major physiologic pathway that regulates T cell homeostasis and peripheral tolerance. Prolonged stimulation renders T cells susceptible to AICD. AICD in T cells is mediated mainly through upregulation of FasL expression and its subsequent interaction with Fas. Upregulation of FasL and Fas was observed in our experiments. Similar kinetics of expression were observed for ETEA and FAF1 in the cells activated with both anti-CD3 and IL-2, as well as PMA and ionomycin. The marked upregulation of these genes was accompanied by the AICD. The high transcription levels of ETEA in T cells and eosinophils, which are probably more resistant to apoptosis in AD subjects, suggest that ETEA may be involved in modulating their sensitivity to apoptosis in the AD patient. It is shown that mitogen-stimulated peripheral blood T cells of asthmatic subjects were resistant to Fas-mediated apoptosis [18]. In addition, some data point to the differences in the Th1 and Th2 subsets in their susceptibility to apoptosis. Fas-mediated AICD was observed only in Th1 clones, whereas Th2 clones resisted AICD [19,20]. Our recent experiments of transcript analysis using the same set of T cell cDNA samples demonstrated that a number of genes, including CC-chemokine receptor 4 as well as T cell-specific tyrosine kinase (Emt/Itk), were more highly expressed in patients with moderate and/or severe AD than in controls or patients with light AD. This suggests that the T cells in moderate and severe AD are polarized towards the Th2 phenotype. Eosinophil apoptosis is delayed in patients with atopic diseases. Prolonged peripheral blood eosinophil survival, probably the result of delayed programmed cell death, is a feature of inhalant allergy and both extrinsic and intrinsic atopic dermatitis [21]. In mild asthmatic patients demonstrating a late response, the survival of peripheral blood eosinophils is prolonged after allergen challenge [22]. Eosinophils from asthmatic patients not taking steroid medication survived longer than those of healthy control subjects [23]. The delay of eosinophil apoptosis may be due, at least in part, to overproduction of T cell-derived cytokines. Besides cytokines, it has also be suggested that FasL–Fas receptor interactions are involved in the regulation of eosinophil apoptosis [24,25]. It is difficult to speculate whether ETEA is a pro- or anti-apoptotic factor in this stage of study. Considering the high transcription levels of ETEA in T cells and eosinophils that are likely to be resistant to apoptosis in AD patients, one can speculate that ETEA works as a decoy for FAF1 and prevents it from potentiating apoptosis. To examine the direct involvement of ETEA in Fasmediated apoptosis, ETEA was transiently expressed in cell lines of HEK293Fas1 (a stable transfectant expressing Fas), HeLa, and CEM-C7. Cells were

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cotransfected with GFP expression vector DNA and either the ETEA plasmid expression vector or a mock vector DNA control. Apoptosis was induced by anti-Fas antibody and the number of live GFP positive cells in both the ETEA+GFP and vector control+ GFP expressing cells was quantified by fluorescence activated cell sorting analysis. The GFP positive cells were considered to be expressing ETEA. A comparison of the numbers of cells in each of the two sets of transfectants was made to evaluate the effect of ETEA on apoptosis. The expression of ETEA did not significantly change the sensitivity of each cell line to Fas-induced apoptosis under the experimental conditions used in this study (data not shown). Further studies are needed to determine whether ETEA has a role in modulating apoptosis and in pathophysiology of T cells and eosinophils in AD. In conclusion, we have identified the ETEA gene that is abundantly expressed both in T cells and eosinophils from AD patients. ETEA has homology with FAF1, which is involved in Fas-mediated apoptosis. The T cells and eosinophils from AD patients are likely to be resistant to apoptosis, pointing to a modulating role for the ETEA gene.

Acknowledgments We are grateful to Ms Kaori Takeuchi for the excellent assistance and to Dr. Yukio Oya and Dr. Masaji Ohno for their continuous encouragement for this work.

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