Báo cáo khoa học: Regulation of the human leukocyte-derived arginine aminopeptidase/endoplasmic reticulum-aminopeptidase 2 gene by interferon-c pot

Regulation of the human leukocyte-derived arginine aminopeptidase/endoplasmic reticulum-aminopeptidase 2 gene by interferon-c Toshihiro Tanioka 1, *, Akira Hattori 1 , Shigehiko Mizutani 2 and Masafumi Tsujimoto 1 1 Laboratory of Cellular Biochemistry, RIKEN, Wako, Saitama, Japan 2 Department of Obstetrics and Gynecology, Nagoya University School of Medicine, Showa, Nagoya, Japan Keywords aminopeptidase; antigen-presentation; interferon regulatory factor; interferon-c; PU.1 Correspondence M. Tsujimoto, Laboratory of Cellular Biochemistry RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako-shi, Saitama 351-0198 Japan Fax: +81 48 462 4670 Tel: +81 48 467 9370 E-mail: tsujimot@riken.jp *Present address Laboratory of Medical Information, Showa University School of Pharmaceutical Sciences, Shinagawa, Tokyo 142-8555, Japan Note The nucleotide sequence of human L-RAP(s)1 and L-RAP(s)2 reported in this paper have been submitted to the GenBank TM ⁄ EMBL ⁄ DDBJ Data Bank with accession numbers AY028805 and AB163917, respectively. (Received 12 October 2004, revised 26 November 2004, accepted 8 December 2004) doi:10.1111/j.1742-4658.2004.04521.x The leukocyte-derived arginine aminopeptidase (L-RAP) is the second ami- nopeptidase localized in the endoplasmic reticulum (ER) processing anti- genic peptides presented to major histocompatibility complex (MHC) class I molecules. In this study, the genomic organization of the gene encoding human L-RAP was determined and the regulatory mechanism of its expres- sion was elucidated. The entire genomic structure of the L-RAP gene is similar to both placental leucine aminopeptidase (P-LAP) and adipocyte- derived leucine aminopeptidase (A-LAP) genes, confirming the close relation- ship of these three enzymes. Interferon (IFN)-c up-regulates the expression of the L-RAP gene. Deletion and site-directed mutagenic analyses of the 5¢-flanking region of the L-RAP gene and electrophoretic mobility shift assay indicated that while interferon regulatory factor (IRF)-2 is important in the basal condition, IRF-1 is the primary regulator of IFN-c-mediated augmentation of the gene expression. In addition, PU.1, a member of the E26 transformation-specific family of transcription factors, also plays a role in the regulation of gene expression. The maximum expression of the gene was achieved by coexpression of IRF-1 and PU.1 in HEK293 cells and IRF-2 suppressed the IRF-1-mediated enhancement of gene expression, suggesting that IFN-c-induced L-RAP gene expression is cooperatively regulated by IRFs and PU.1 transcription factors. Abbreviations A-LAP, adipocyte-derived leucine aminopeptidase; BAC, bacterial artificial chromosome; CHX, cycloheximide; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; ER, endoplasmic reticulum; ERAP, endoplasmic reticulum aminopeptidase; Ets, E26 transformation- specific; IFN, interferon; IL, interleukin; IRF, interferon regulatory factor; L-RAP, leukocyte-derived arginine aminopeptidase; MHC, major histocompatibility complex; P-LAP, placental leucine aminopeptidase; PMSF, phenylmethanesulfonyl fluoride; TAP, transporter associated with antigen processing. 916 FEBS Journal 272 (2005) 916–928 ª 2005 FEBS Aminopeptidases hydrolyze N-terminal amino acids of proteins or peptide substrates. They are distributed widely in animal and plant tissues as well as in bacteria and fungi, suggesting that they play important roles in various biological processes [1]. Among them, M1 zinc-metallopeptidases (gluzincins) share the consensus GAMEN and HEXXH(X) 18 E zinc-binding motifs essential for enzymatic activity [2,3]. So far, nine enzymes belonging to the family were identified and laeverin was recently reported as the tenth member although its enzymatic activity was not reported [4,5]. In our previous work, we cloned a cDNA for the pla- cental leucine aminopeptidase (P-LAP) ⁄ oxytocinase, a type II membrane-spanning protein which belongs to the M1 family of aminopeptidases [6]. Subsequently we cloned cDNAs encoding adipocyte-derived leucine aminopeptidase (A-LAP) [7], which is also designated as puromycin-insensitive leucine-specific aminopeptidase (PILS-AP) or ER-aminopeptidase (ERAP)-1 [8,9], and leukocyte-derived arginine aminopeptidase (L-RAP) ⁄ ERAP2 [4] as highly homologous proteins to P-LAP. Structural and phylogenetic analyses indicated the close relationship between these three enzymes. Therefore we proposed that they should be classified into the oxyto- cinase subfamily of M1 aminopeptidases [10]. Recent evidence facilitates new insights into the biological significance of the oxytocinase subfamily of aminopeptidases. P-LAP ⁄ oxytocinase, which is also designated as insulin-regulated aminopeptidase (IRAP), was shown to translocate from the intracellu- lar compartment to the plasma membrane in a stimu- lus-dependent manner and may regulate concentration of substrate peptide hormones on the cell surface [11–14]. This enzyme was recently shown to be the angiotensin IV receptor and may play a role in memory retention and retrieval [15,16]. On the other hand, we and others reported that A-LAP ⁄ ERAP1 is a final processing enzyme of the precursors of the major histocompatibility complex (MHC) class I-presented antigenic peptides [9,17]. A-LAP ⁄ ERAP1 was also shown to play roles in blood pressure regulation and angiogenesis [18–20]. Moreover, the enzyme was shown to bind to cytokine receptors such as tumour necrosis factor type I receptor, interleukin (IL)-6 a-receptor and IL-1 type II receptor, and facilitate ectodomain shedding of these receptors [21–23]. As for L-RAP ⁄ ERAP2, we have shown that the enzyme is retained in the endoplasmic reticulum (ER) and can trim some precursor peptides to MHC class I ligands [4]. These results indicate that the mammalian amino- peptidases belonging to the oxytocinase subfamily play important roles in the regulation of several biological processes. Precursors of MHC class I-presented peptides with extra N-terminal residues are trimmed to mature epi- topes in the ER [24,25]. The peptides are first cleaved from endogenously synthesized proteins by proteasome or tripeptidyl peptidase II in the cytoplasm, transpor- ted into ER-lumen and then trimmed by certain aminopeptidases. Until now, only two aminopeptid- ases (A-LAP ⁄ ERAP1 and L-RAP ⁄ ERAP2) have been identified in the ER-lumen and have shown their ability to trim antigenic peptides [4,9,17]. Characteris- tically, as in the case of other components included in the presentation of MHC class I ligands such as MHC class I molecules, transporter associated with antigen processing (TAP) and proteasome b-subunits, inter- feron (IFN)-c enhances the expression of these enzymes [24]. Although IFN-c-inducible aminopeptid- ases including lens LAP, A-LAP, and L-RAP play roles in the processing or degradation of antigenic peptides [4,9,17,26], the mechanisms of IFN-c-medi- ated regulation of aminopeptidase gene expression have never been examined. In the current study, we have elucidated the genomic structure of human L-RAP gene for the first time. Characterization of the promoter region of the gene indicates that while interferon regulatory factor (IRF)- 2 is important in the basal condition, IRF-1 is the primary regulator of IFN-c-mediated augmentation of the gene expression. It is also shown that PU.1, a member of the E26 transformation-specific (Ets) family of transcription factors, plays a role in the regulation of gene expression. Our data provide the molecular basis of regulatory mechanisms of enzymatic activity of L-RAP, which may play an important role in the processing of antigenic peptides presented to MHC class I molecules in the ER. Results Genomic organization of human L-RAP gene To elucidate the genomic organization of the L-RAP gene, we screened a human genome database prepared in a bacterial artificial chromosome (BAC). This led to the identification of BAC clone RPCI-11-496M2 (accession number AC009126), which contains all exons of the L-RAP gene. All exons and intron–exon junctions were determined from the database. The sequences of splice junctions obey the GT-AG rule [27]. As shown in Fig. 1, the gene spans 45 kb and contains 19 exons ranging in size from 28 bp (exon 1) to 697 bp (exon 2) and the overall structure of the gene is quite similar to both P-LAP and A-LAP genes. The first exon includes only the 5¢-untranslated region. T. Tanioka et al. Regulation of L-RAP ⁄ ERAP2 gene expression FEBS Journal 272 (2005) 916–928 ª 2005 FEBS 917 Exon 2 includes the remaining 5¢-untranslated region and coding sequence for the first 192 amino acids. The GAMEN and HEXXH motifs are encoded in exon 6 and an essential glutamic acid residue located 19 amino acids downstream from the HEXXH motif is encoded in exon 7. Exon 19 contains the coding sequence for the last 47 amino acids, stop codon (TAA) and the 3¢-untranslated region. In our previous work, we obtained a cDNA enco- ding the truncated form of L-RAP [4]. Exon 10 that is deleted in the P-LAP gene encodes a sequence that may function as a hinge region [28]. As shown in Fig. 1, a transcript encoding this form, termed L- RAP(s)1 (accession number AY028805), is generated by differential usage of exon 10. When an intermediate nucleotide sequence (AACATGgtaag) matching the GT-AG rule in the exon functions as a splicing donor, full length L-RAP transcript is generated. If the sequence does not act as a splicing donor, the stop codon (TGA) in the exon causes the generation of a truncated form. Thereafter, another transcript, L-RAP(s)2 (accession number AB163917), encoding the same truncated form was also cloned. Differential usage of exon 15 causes the generation of two trun- cated forms of the transcripts. These results indicate that at least three mRNAs encoding either the full length or truncated form of L-RAP protein are gener- ated from a single gene. Isolation and characterization of human L-RAP gene promoter Figure 2 shows the nucleotide sequence of the 5¢-flank- ing region of the human L-RAP gene. The transcrip- tional initiation site of the gene was determined by 5¢ RACE and is shown as nucleotide position +1 in the figure. The site (TCAGTC) matches well with the pyrimidine-rich initiator consensus sequence, PyPy(A+1)N(T ⁄ A)PyPy, in which Py represents a pyrimidine residue [29]. Computer analysis of the sequence revealed no canonical TATA- or CCAAT- box, suggesting a housekeeping nature for the gene. On the other hand, several potential transcription factor binding motifs, such as IRF, GATA-1, Sp-1 and AP-1 were identified in the promoter region of the gene [30]. To characterize the regions regulating transcriptional activity of the L-RAP gene, chimeric reporter plasmids encoding the luciferase gene and different lengths of L-RAP gene were constructed. The resultant chimeric constructs were then transfected into HEK293 cells to analyze the promoter activity. As a negative control, the promoter-less pGL3 basic plasmid was transfected into the cells. As shown in Fig. 3A, substantial promo- ter activity was detected when chimeric constructs con- taining the 5¢-flanking sequence upstream from the A at position )10 were transfected, confirming that the 5¢-flanking sequence of the L-RAP gene is indeed able to support transcriptional initiation. The maximum Fig. 1. Genomic structure of the human L-RAP gene. Schematic exon–intron structure of the gene is shown at the center. Exons are numbered and depicted as boxes. Asterisks indicate the sites used for the generation of different mRNAs described in the text. Generation scheme of L-RAP and L-RAP(s) proteins is also shown in the figure. Numbers shown in each protein molecule are number of amino acids derived from the respective exons. Fig. 2. Nucleotide sequence of the 5¢-flanking region of the human L-RAP gene. The 5¢-flanking region was searched for transcription factor binding sites by TF-SEARCH. The exon sequence is shown in uppercase letters, while that of the untranscribed region is given in lowercase letters. The transcriptional initiation site shown as +1 was determined by 5¢-RACE. For the measurement of promoter activity, the start point of each construct is indicated by an arrowhead. Regulation of L-RAP ⁄ ERAP2 gene expression T. Tanioka et al. 918 FEBS Journal 272 (2005) 916–928 ª 2005 FEBS promoter activity was obtained with the constructs containing the sequence from )33 to +5. Deletion of the sequence from )33 to )17 caused a decrease in the promoter activity by nearly 50%. Further deletion to T at position )9 caused almost complete loss of activ- ity. Employing Jurkat-T cells, we obtained nearly the same results. The promoter sequence from )33 to )17 contains a sequence (AGAAAGTGAAAGC) with resemblance to the IRF-E consensus sequence [G(A)AAASYGAA ASY]. We next examined the role of this site on the basal promoter activity by constructing mutant plasmid [phLP5(MuI)] from phLP5 plasmid. We describe this sequence as the IRF-E site hereafter [31]. Fig. 3. Role of the IRF-E site in IFN-c-induced enhancement of L-RAP gene expression in HEK293 cells. (A) Luciferase expression plasmids (1 lg) containing sequentially deleted fragments of the L-RAP chimera were transfected into HEK293 cells. Luciferase activity was measured as described in Experimental procedures and normalized to the b-galactosidase activity of a cotransfected internal control plasmid. The luci- ferase activity obtained in the basal condition from cells transfected with phLP1 was taken as 100%. (B) Enhancing effect of IFN-c is shown by fold increase in the figure. (C) Functional analysis of the IRF-E site of the L-RAP gene promoter in HEK293 cells. The phLP5 plasmid hav- ing either an intact or mutated IRF-E site was transfected into HEK293 cells and the luciferase activity was measured as described above. Open bars indicate luciferase activity in the basal condition and closed bars in the IFN-c-stimulated condition. T. Tanioka et al. Regulation of L-RAP ⁄ ERAP2 gene expression FEBS Journal 272 (2005) 916–928 ª 2005 FEBS 919 Although substantial activity was retained, mutation of the IRF-E site caused a significant decrease in the promoter activity (Fig. 3C). These results suggest that the IRF-E site is crucial for the maximum promoter activity of the L-RAP gene in the basal condition. Mechanism of IFN-c-mediated regulation of L-RAP gene expression Because L-RAP gene expression is enhanced by IFN-c, we next examined its regulatory mechanism. As an ini- tial experiment, decay rates of L-RAP mRNAs pre- pared from Jurkat-T cells treated with or without IFN-c were compared in the presence of actinomycin D. As shown in Fig. 4A, there was little difference between decay rates of L-RAP mRNAs in the cells treated with or without IFN-c, indicating that the cytokine-induced enhancement of mRNA accumula- tion could not be attributed to the change of stability of L-RAP mRNA. In addition, it was found that the increase in mRNA accumulation was not observed in the presence of cycloheximide (CHX), indicating that de novo protein synthesis was required for the action of IFN-c (Fig. 4B). To elucidate the role of IFN- c in the regulation of L-RAP gene expression, the luciferase-reporter assay was conducted to identify cytokine responsive elements in the gene. As shown in Fig. 3A,B, IFN-c induced about a fourfold increase in the expression of luci- ferase activity in HEK293 cells transfected with con- structs containing the sequence from )33 to )17. After deletion of this sequence, IFN-c had no enhancing activity. These results indicate that the sequence is essential for the cytokine-induced increase in L-RAP gene expression. Because the sequence from )33 to )17 contains the IRF-E site, we next examined the role of this site in L-RAP gene regulation. As shown in Fig. 3C, muta- tion in this site caused complete loss of IFN-c-induced increase in the luciferase activity, confirming the role of the IRF-E site in the cytokine-mediated L-RAP gene regulation. These results indicate that the IRF-E site plays an important role both in the basal and IFN-c-mediated L-RAP gene expression. On the other hand, it was found that in addition to the IRF-E site, the Ets site in the promoter also plays a role in gene expression in Jurkat-T cells. As in the case of HEK293 cells, the IRF-E site was required for the maximum expression of the gene. IFN-c induced an eightfold increase in the gene expression in cells transfectied with phLP5 plasmid. When Jurkat-T cells were transfected with plasmids containing the sequence from )67 to )33 that contains the Ets site, a further increase (about 13- to 15-fold) was observed (Fig. 5A,B). Mutation of either the IRF-E [phLP4(MuI)] or Ets site [phLP4(MuE)] caused a substantial decrease in the IFN-c-mediated gene expression. Plasmid having mutations in both sites [phLP4(MuE ⁄ MuI)] had little activity, indicating that in Jurkat-T cells both the IRF and Ets sites are required for maximum enhancement of IFN-c-induced gene expression (Fig. 5C). In contrast, it was found that the Ets site had no effect on the promoter activity in HEK293 (data not shown). We found by RT-PCR A B Fig. 4. Requirement of de novo protein synthesis for IFN-c -medi- ated enhancement of L-RAP gene expression. (A) Effect of IFN-c on the stability of L-RAP mRNA. Jurkat-T cells were treated with or without 30 ngÆmL )1 IFN-c for 12 h at 37 °C and further incubated in the presence of 1 l M actinomycin D (ActD) for indicated times. After incubation, expression levels of L-RAP mRNAs were meas- ured by Northern blot analysis. Densitometric data showing the decay rates of mRNAs are also shown. (B) Effect of CHX on the IFN-c-induced enhancement of L-RAP gene expression. Jurkat-T cells were preincubated for 3 h in the presence or absence of 1 lgÆmL )1 cycloheximide (CHX) and further incubated with 30 ngÆmL )1 IFN-c for 12 h at 37 °C. L-RAP mRNAs were detected by Northern blot analysis. Regulation of L-RAP ⁄ ERAP2 gene expression T. Tanioka et al. 920 FEBS Journal 272 (2005) 916–928 ª 2005 FEBS that expression of PU.1 was detectable in Jurkat-T cells but not in HEK293 cells (data not shown). Although the Oct-1 site is located proximal to the Ets site, mutational analysis indicated that this site had little effect on the gene expression (data not shown). Role of IRFs in the regulation of L-RAP gene expression Transcription factors, IRF-1 and -2 are known to bind to the IRF-E site and regulate IFN-c action [31]. To examine whether IFN-c affects the expression levels of Fig. 5. Role of the IRF-E and Ets sites in IFN-c-induced enhancement of L-RAP gene expression in Jurkat-T cells. (A) Luciferase expression plasmids (10 lg) containing sequentially deleted fragments of the L-RAP chimera were transfected into Jurkat-T cells. Cells were then trea- ted with or without 30 ngÆmL )1 IFN-c for 15 h at 37 °C. Luciferase activity was measured as described in Experimental procedures and nor- malized to the b-galactosidase activity of a cotransfected internal control plasmid. The luciferase activity obtained in the basal condition from cells transfected with phLP1 was taken as 100%. (B) Enhancing effect of IFN-c is shown by fold increase in the figure. (C) Mutational analy- sis of the IRF-E and Ets sites of the L-RAP gene promoter in Jurkat-T cells. The phLP4 plasmid having either intact or mutated sites shown in the left panel of the figure was transfected into Jurkat-T cells and luciferase activity was measured. Open bars indicate luciferase activity in the basal condition and closed bars in the IFN-c-stimulated condition. T. Tanioka et al. Regulation of L-RAP ⁄ ERAP2 gene expression FEBS Journal 272 (2005) 916–928 ª 2005 FEBS 921 IRF-1 and -2 proteins, HEK293 cells were cultured in the presence or absence of the cytokine (Fig. 6A). When compared with untreated cells, an increase in the expression level of IRF-1 was observed in cells treated with IFN-c, indicating that IRF-1 is a candidate for the mediator of IFN-c action. In contrast, no effect was observed on the expression of IRF-2 protein. To determine whether IRF-1 and -2 bind to the IRF-E site in the L-RAP promoter sequence, we per- formed an electrophoretic mobility shift assay (EMSA) using nuclear extracts from HEK293 cells treated with or without IFN-c (Fig. 6B). An oligonucleotide probe corresponding to the IRF-E site bound to nuclear extracts prepared from both IFN-c-treated and untreated cells. In spite of the presence of an unex- pected nonspecific large band, bands shown as IRF-1 and -2 in Fig. 6 were replaced by unlabeled oligo- nucleotide competitor, indicating their specificity. The specificity of these bands was also confirmed by oligo- nucleotide with mutations at the IRF-E site. Although repeated trials were made, we could not remove the nonspecific bands. To identify transcription factors bound to the IRF- E site, supershift assay was performed using antibodies raised against IRF-1 and IRF-2. When anti-(IRF-1) IgG was employed, supershift was observed only in the IFN-c-treated cells. On the other hand, anti-(IRF-2) IgG caused supershift in both treated and untreated cells. Moreover, appearance of two supershift bands and complete disappearance of the oligonucleotide spe- cific bands was observed in the presence of both anti- bodies. These results suggest that while in untreated cells IRF-2 but not IRF-1 was bound to the IRF-E site, IRF-1 and IRF-2 co-occupied the site after treat- ment with IFN-c. We obtained the same results in Jur- kat-T cells. However, it should be noted here that an IFN-c-inducible band other than IRF-1 and -2 was also observed in this experiment, suggesting that tran- scription factors other than IRF-1 and -2 also partici- pate in the regulation of L-RAP gene expression. A B CD Fig. 6. Role of IRF-1 and PU.1 in the expression of the L-RAP gene. (A) IFN-c-induced increase in the expression of IRF-1 protein in HEK293 cells. HEK293 cells were treated with or without 30 ngÆmL )1 IFN-c for 5 h at 37 °C. IRF-1 and IRF-2 in the nuclear extracts were measured by Western blot analysis. (B) Binding of IRF-1 and IRF-2 to the IRF-E site of the L-RAP gene. HEK293 cells were treated with or without 30 ngÆmL )1 IFN-c for 5 h at 37 °C. EMSAs were then performed in nuclear extracts using a probe con- taining the IRF-E site from the L-RAP gene promoter. Supershifts were conducted using antibodies against indicated factors. Arrow- heads indicate the positions of IRF-1 and IRF-2. Arrow indicates the position of unidentified IFN-c-inducible IRF-E binding protein. Lanes shown as probe are loaded only with labeled probe. (C) Enhance- ment of promoter activity of the L-RAP gene by transcription fac- tors. HEK293 cells were cotransfected with phLP4 (0.5 lg) and various expression plasmids (0.5 lg) shown in the figure. After 24 h, luciferase activity was measured as described in Experimental procedures. Enhancing effects of the transcription factors are shown by fold increase using cells transfected only with phLP4 or as a control. (D) Induction of endogenous L-RAP gene expression by transcription factors. HEK293 cells were transfected with expression plasmids (0.5 lg) shown in the figure. For RT-PCR ana- lysis, total RNA was extracted after 24 h-incubation. Relative expression level is shown by fold increase in the figure using mock-transfected cell as a control. Regulation of L-RAP ⁄ ERAP2 gene expression T. Tanioka et al. 922 FEBS Journal 272 (2005) 916–928 ª 2005 FEBS To further elucidate the role of IRF-1 and -2 in the regulation of L-RAP gene expression, phLP4 plasmid was cotransfected either with pTARGET-IRF-1 or pTARGET-IRF-2 plasmid into HEK293 cells (Fig. 6C). IRF-1 overexpressed in HEK293 cells induced 8.6-fold increase in the luciferase activity, indi- cating that IRF-1 is indeed able to augment L-RAP gene expression. IRF-2 and PU.1, a transcription fac- tor which is known to bind to the Ets site [32], also had enhancing effects on the luciferase activity, and a 4.7- and 2.1-fold increase in the gene expression was observed, respectively. Furthermore, maximum gene expression (18.3-fold increase) was achieved in a syner- gistic manner when IRF-1 and PU.1 were coexpressed. On the other hand, coexpression of IRF-1 and IRF-2 caused lower expression of luciferase activity than expected, suggesting that IRF-2 suppressed IRF-1- mediated augmentation of the gene expression. We also examined other Ets site-binding factors such as Ets-1 and Ets-2 and found that they had little effect on the gene expression (data not shown). When the same experiments were performed using either phLP4(MuE), phLP4(MuI) or phLP4(MuE ⁄ MuI), no synergistic effect between IRF-1 and PU.1 was observed (data not shown), confirming that native sites of both IRF-E and PU.1 are required for the synergis- tic action. Taken together these results indicate that while IRF-2 plays a role in the basal condition, IRF-1 can mediate IFN-c-stimulated L-RAP gene expression synergistically with PU.1. To determine whether IRF-1 indeed mediates L-RAP gene expression in vivo, we next examined the effect of transcription factors on the expression of endogenous L-RAP gene. Several combinations of plasmids were transfected into HEK293 cells and their ability to enhance gene expression was examined by RT-PCR (Fig. 6D). In mock-transfected cells, L-RAP mRNA was barely detectable. Among trancription fac- tors tested, the highest expression was achieved when IRF-1 was overexpressed. Further increase in the gene expression was observed when IRF-1 and PU.1 were coexpressed. In contrast, IRF-2 suppressed IRF-1- mediated increase in the gene expression, although IRF-2 alone had some enhancing activity. These results further confirm the roles of transcription factors in the expression of the L-RAP gene. Discussion L-RAP ⁄ ERAP2 is a newly identified ER aminopepti- dase and the third member of the oxytocinase sub- family of M1 family of aminopeptidases [4]. It was suggested by its ability to generate certain antigenic peptides that the enzyme plays an important role in the processing of antigenic peptides presented to MHC class I molecules in the ER. In this study, we deter- mined the genomic structure of the human L-RAP gene and characterized the regulatory mechanisms of the gene expression. It was found that two forms of the L-RAP proteins are generated by alternative spli- cing. While the full length form exerts distinct amino- peptidase activity, the truncated form has no enzymatic activity [4]. It is necessary to elucidate the relationship between these two forms. The entire genomic structure of the L-RAP gene is quite similar to the P-LAP and A-LAP genes [28,33]. The GAMEN and HEXXH(X) 18 E motifs essential for the enzymatic activity are encoded by exons 6 and 7 of the respective genes. Becasue these three genes are located contiguously around human chromosome 5q15, between versican and calpastatin [28], these data further confirm the latest divergence of the genes from a common ancestral gene. Expression of L-RAP is up-regulated by IFN-c [4]. IFN-c also stimulates the induction of components included in the processing of MHC class-I ligands [24]. Considering the evidence that suggest a role of L-RAP as an antigen-trimming enzyme in the ER, it is import- ant to elucidate the mechanism of the IFN-c-induced increase in L-RAP gene expression. The transient expression of the 5¢-flanking region of the L-RAP gene fused to the luciferase gene in either HEK293 or Jurkat-T cells allowed the analysis of basal and IFN-c- mediated regulation of promoter activity. We demon- strated by deletion analysis that the sequence ranging from )16 to )10 carries the minimum promoter activ- ity of the gene. In addition, mutational analysis sug- gested that the IRF-E site is required for the maximum enhancement of gene expression in the basal condition. Because EMSA indicated that IRF-2 is associated with the IRF-E site in unstimulated HEK293 cells, our data suggest that IRF-2 can aug- ment the transcription of the L-RAP gene in the basal condition. In fact, IRF-2 had some enhancing effect on the gene expression when overexpressed in HEK293 cells. Although it is generally considered that IRF-2 is a negative regulator of gene expression [31], it has also been shown to up-regulate the expression of several genes such as histone 4 and VCAM-1 [34,35]. However, we could not completely rule out the possible contribu- tion of other IRF-E site-binding proteins [31], because an IFN-c-inducible IRF-E binding protein other than IRF-1 and IRF-2 was detectable by EMSA, even in the basal condition. Identification and elucidation of the role of this protein in L-RAP gene expression is required. T. Tanioka et al. Regulation of L-RAP ⁄ ERAP2 gene expression FEBS Journal 272 (2005) 916–928 ª 2005 FEBS 923 It is worthy of noting here that both P-LAP and A-LAP promoters also contain IRF-E sites [28,33]. It was reported that IFN-c had no enhancing effect on P-LAP gene expression [33]. In our preliminary results, the IRF-E site in the A-LAP promoter had little effect on IFN-c-induced gene expression, raising the possib- lity that IFN-c affects the expression of the A-LAP gene differently from that of the L-RAP gene. IFN-c-induced increase in L-RAP gene expression required de novo protein synthesis and it was found that IRF-1 was induced by IFN-c. Deletion and muta- tional analyses indicated that the IRF-E site is res- ponsible for the cytokine-mediated increase in gene expression in HEK293 cells. Mutation of the site caused loss of the cytokine action. EMSA indicated that while IRF-2 but not IRF-1 bound to the IRF-E site in the basal condition, binding of both IRF-1 and -2 were detectable after treatment with IFN-c. More- over, transfection of the IRF-1 expression plasmid into HEK293 cells caused an increase in the gene expression. These results strongly suggest that IRF-1 is a primary mediator of IFN-c-mediated enhancement of L-RAP gene expression in HEK293 cells. Because coexpression of IRF-1 and IRF-2 caused a decrease in L-RAP gene expression, it is plausible that IRF-2 acts as a negative regulator of IRF-1-mediated enhance- ment of gene expression, by co-occupation of the IRF-E site with IRF-1 in IFN-c-treated cells. On the other hand, the mechanism of IFN-c-induced L-RAP gene regulation in Jurkat-T cells is rather com- plex. It is obvious that in addition to the IRF-E site, the Ets site located between )63 and )56 also plays a role in the maximum enhancement of IFN-c-induced gene expression. It is unlikely that another Ets site located between )352 and )345 plays a role in the gene expression, because deletion of this sequence had little effect on the enhancement of the gene expression. Among the Ets family transcription factors tested, only PU.1 could mediate the cytokine action. The expres- sion of PU.1 is limited to hematopoietic lineages and is necessary for the differentiation of myeloid cell line- ages such as macrophages and osteoclasts [36,37]. Our analysis by RT-PCR indicated the expression of PU.1 in Jurkat-T cells but not HEK293 cells. Therefore, it is plausible that the lineage-dependent expression of PU.1 may determine the expression level of the L-RAP gene. Consistent with this notion, IRF-1 and PU.1 mediated the maximum expression of both reporter and endogenous genes when overexpressed, even in HEK293 cells. The molecular basis of the cooperative action of transcription factors is considered to be the physical interaction of the transcription factors involved [37]. For instance, it was reported that IRFs and PU.1 synergistically mediate the transcriptional enhancement of human interleukin-1 (IL-1)b gene expression. It was suggested by physical interaction analysis that the fac- tors might function together as an enhanceosome [38]. As shown in other genes [39,40], it is plausible that IRF-1 mediates the IFN-c-stimulated L-RAP gene expression through interaction with PU.1. On the other hand, it is possible that IRF-2 acts independently from PU.1 to enhance the gene expression in the basal con- dition. Because binding of IRF-2 to the IRF-E site was still observed after IFN-c treatment, it is possible to speculate that IRF-2 modulates the gene expression by interacting with IRF-1 in the IRF-E site of the L-RAP gene. As with the L-RAP gene, Gobin et al. reported that binding of IRF-2 to the MHC class I molecule promoter was observed after IFN-c treatment [41]. It was also reported that IRF-2 co-occupies the IRF-E site of the class II transactivator type IV promoter with IRF-1 and synergistically activates the promoter [42]. L-RAP ⁄ ERAP2 is the second ER-lumenal amino- peptidase to be determined, and can trim certain pre- cursors of antigenic peptides presented to MHC class I molecules [4], suggesting the potential significance of this enzyme in antigen processing. In this study, we characterized the L-RAP gene for the first time. We have shown that the IRF-E site located in the proximal region of the transcription initiation site plays a pivotal role in the regulation of human L-RAP gene expres- sion both in the basal and IFN-c -stimulated condi- tions. As shown in several genes [43–45], it was found that while IRF-2 plays a role in the basal condition, IRF-1 is the primary regulator of the gene expression induced by IFN-c. However, transcription factor(s) other than IRF-1 and -2 may also regulate L-RAP gene expression. Further works are required to exam- ine the involvement of other transcription factors that bind to the IRF-E site. Considering the significance of the processing of antigen presented to MHC class I molecules in several pathophysiological conditions such as virus infection, tumor generation and self-antigen generation, it is important to elucidate the total aspects of the antigen presentation process including the regu- latory mechanisms of L-RAP gene expression. Experimental procedures Identification of the human L-RAP gene A genomic sequence of  178 kb from the Gen- Bank TM ⁄ EMBL ⁄ DDBJ Data Bank was obtained. This sequence encompasses a region on human chromosome 5q15 where the known P-LAP gene is located. The loca- Regulation of L-RAP ⁄ ERAP2 gene expression T. Tanioka et al. 924 FEBS Journal 272 (2005) 916–928 ª 2005 FEBS tions of the exons of the L-RAP gene were determined using the blast program. Cell culture Jurkat-T cells were obtained from the RIKEN Cell Bank (Tsukuba, Japan) and maintained in RPMI 1640. Human embryonic kidney (HEK) 293 cells were purchased from ATCC (Manassas, VA, USA) and maintained in RPMI 1640. All media used in this study was from Sigma (St. Louis, MO, USA) and supplemented with 10% fetal bovine serum (JRH Biosciences, Lenexa, KS, USA), peni- cillin G, and streptomycin (Meiji Seika Co., Kanagawa, Japan). The cells were cultured in a humidified 5% CO 2 and 95% air incubator at 37 °C. Analysis of promoter activity The human L-RAP 5¢-flanking region and its fragments, which include the transcriptional initiation site, were amplified by PCR from a BAC clone. PCR products were inserted into the promoter-less plasmid, pGL3 basic (Promega, Madison, WI, USA). The nucleotide sequence of the primers used for PCR amplification were as follows (5¢-3¢): CGGGTACCTGAACCAGCTAGTACT TACTG (sense strand of the sequence from )88 to )68) for phLP3; CGGGTACCTACTCAGGAAGCATGC AAGT (sense strand of the sequence from )67 to )47) for phLP4; CGGGTACCACAGAAAGTGAAAGCA (sense strand of the sequence from )33 to )18) for phLP5; CGACGCGTTGACTGAAGGGGAATTTACTTT (antisense strand of the sequence from )17 to +5) for all constructs. For phLP6 and 7, plasmids were construc- ted by using synthetic oligonucleotides corresponding to the 5¢-flanking region. We also constructed phLP1 and 2 plasmids by sequetial deletion of SmaI and Van91I fragment from phLP0 covering the sequence from )1042 to +5. Mutagenesis of the IRF and Ets sites was performed by using mutated oligonucleotides when constructing the respective plasmids. For making mutations in IRF, 5¢-CACAGA GGGTGAGGGCAAAAGTAAATTCCCCTT CAGTCAA-3¢ (sense sequence of mutant IRF-E site) and 5¢-CGCGTTGACTGAAGGGGAATTTACTTTTGC CCT CAC CCTCTGTGGTAC-3¢ (antisense sequence of mutant IRF-E site) were used. For the Ets mutant, 5¢-GGGGTA CCTACTCA AAGAGCATGCAAAGT-3¢ (sense sequence of mutant Ets site) and 5 ¢-CGACGCGTTGACTGAAGG GGAATTTACTTT-3¢ (antisense sequence of mutant Ets site) were used. For the construction of the double mutant plasmid, 5¢-GGGGTACCTACTCA AAGAGCATGCAAA GT-3¢ and 5¢-CGACGCGTTGACTGAAGGGGAATTTA CTTTTGC CCTCACCCTCTGTTCTAA-3¢ (antisense sequ- ence of mutant IRF-E site) were used. The mutated nucleo- tide sequences are underlined. The PU.1 expression plasmid pcDNA3-PU.1 which was constructed by using pcDNA3 (Invitrogen, Carlsbad, CA, USA) was obtained from M Matsumoto of Saitama Med- ical School (Saitama, Japan) [46]. Transfection and luciferase assay For transfection of the reporter plasmid, HEK293 cells were plated on 24 well plates at a density of 1 · 10 5 cells per well on the day before transfection, while Jurkat-T cells (1 · 10 6 cells) were plated in a 100 mm dish. Plasmid DNA was mixed with LipofectAmine (Invitorogen) and transfected into HEK293 cells following the manufacturer’s protocol. A pCMVb (0.5 lg) plasmid was employed as an internal control of transfection efficiency. To transfect into Jurkat-T cells, the electroporation (225 V, 350 lF) method was employed, using Electro Cell Manipulator (BTX, San Diego, CA, USA). After 24 h of transfection, the cells were washed three times with NaCl ⁄ P i and then lysed in reporter lysis buffer (Promega). The luciferase activity was then measured with a luciferase assay system (Promega) accord- ing to the manufacturer’s instruction. Luciferase activity was measured in triplicate, averaged, and then normalized to b-galactosidase activity to correct for transfection effi- ciency. b-Galactosidase activity was measured using o-nitro- phenyl-b-d-galactopyranoside as a substrate. Electrophoretic mobility shift assay Double-stranded oligonucleotides containing the consensus sequence IRF-E site were radiolabeled with [ 32 P]dCTP[aP] at the 3¢ end with a Klenow fragment and then purified with a MicroSpin column (Amersham Biosciences, Piscata- way, NJ, USA). The sense sequences of the synthesized oligonucleotides used were as follows: for the native IRF-E site (GAACAGAAAGTGAAAG) in the human L-RAP 5¢-flanking region, and for mutation in the IRF-E site (GAACAGA GGGTGAGGG). and the antisense sequences of synthesized oligonucleotides used were as follows: for native IRF-E site (TTTGCTTTCACTTTCT) in the human L-RAP 5¢-flanking region, for mutation in IRF-E site (TTTGC CCTCACCCTCT). The mutated nucleotide sequ- ences are underlined. Nuclear extracts of the cells were prepared as follows: the cells suspended in 500 lL of ice-cold buffer A [10 mm He- pes, 10 mm KCl, 1.5 mm MgCl 2 , 0.5 mm dithiothreitol (DTT), 0.2 mm phenylmethanesulfonyl fluoride (PMSF): pH 7.9] were lysed in a Dounce-homogenizer and then the nuclei were pelleted by centrifugation at 3300 g for 15 min. The pellets were then suspended in an equal volume of low salt buffer (20 mm Hepes, 20 mm KCl, 1.5 mm MgCl 2 , 0.2 mm EDTA, 0.5 mm DTT, 0.2 mm PMSF, 25% glycerol: pH 7.9). Thereafter, half volume of high salt buffer (20 mm Hepes, 1.4 m KCl, 1.5 mm MgCl 2 , 0.2 mm EDTA, 0.5 mm DTT, 0.2 mm PMSF, 25% glycerol: pH 7.9) was added T. Tanioka et al. Regulation of L-RAP ⁄ ERAP2 gene expression FEBS Journal 272 (2005) 916–928 ª 2005 FEBS 925 [...]... 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The maximum expression of the gene was achieved by coexpression of IRF-1 and PU.1 in HEK293 cells and IRF -2 suppressed