Transcriptional Regulation of the Mouse IL-7 Receptor Promoter by Glucocorticoid Receptor1
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免疫学杂志 2005年第12期
Abstract
Expression of the IL-7R -chain (IL-7R) is strictly regulated during the development and maturation of lymphocytes. Glucocorticoids (GC) have pleiotypic effects on the growth and function of lymphocytes. Although GC have been reported to induce the transcription of IL-7R gene in human T cells, its molecular mechanism is largely unknown. In this study, we show that GC up-regulate the levels of IL-7R mRNA and protein in mouse T cells. This effect does not require protein synthesis de novo, because protein synthesis inhibitors do not block the process. Mouse IL-7R promoter has striking homology with human and rat, containing consensus motifs of Ikaros, PU.1, and Runx1 transcription factors. In addition, a conserved noncoding sequence (CNS) of 270 bp was found 3.6-kb upstream of the promoter, which was designated as CNS-1. A GC receptor (GR) motif is present in the CNS-1 region. Importantly, we show by reporter assay that the IL-7R promoter has specific transcription activity in T cells. This activity highly depends on the PU.1 motif. Furthermore, GC treatment augments the transcriptional activity through the GR motif in the CNS-1 region. We also demonstrate that GR binds to the GR motif by EMSA. In addition, by chromatin immunoprecipitation assay, we show that GR is rapidly recruited to endogenous CNS-1 chromatin after GC stimulation. These results demonstrate that GR binds to the GR motif in the CNS-1 region after GC stimulation and then activates the transcription of the IL-7R promoter. Thus, this study identifies the IL-7R CNS-1 region as a GC-responsive element.
Introduction
Interleukin-7 is an essential cytokine for development of early lymphocytes and maintenance of mature lymphocytes. At early stages, it controls proliferation of T and B cell precursors, positive selection of CD8+ thymocyte (1), and the recombination accessibility of the TCR and IgH loci (2, 3, 4). At later stages, IL-7 supports survival and homeostatic proliferation of naive and memory T cells (5). IL-7 exerts its effect through interaction with the IL-7R, consisting of a unique -chain (IL-7R -chain (IL-7R)3) (6) and common cytokine receptor -chain (7, 8). The IL-7R also heterodimerizes with the unique thymic stromal-derived lymphopoietin receptor chain (9, 10). IL-7 binding to the IL-7R activates the JAK-1 and -3, which then activate STAT5 (11), phosphoinoside-3 kinase, Ras, and MAPK/ERK.
Expression of the IL-7R is strictly regulated during the development and maturation of lymphocytes. It is induced at the stage of common lymphoid progenitors and maintained during whole T cell life except at two stages: CD4+8+ thymocytes and activated T cells (12, 13, 14). In B cell lineage, in contrast, the IL-7R is down-regulated at the pre-B cell stage and kept off throughout the later stages (15). It is supposed that the IL-7R is actively down-regulated not to transmit unnecessary survival signals, when T cells have to decide their fate solely by the affinity between their TCR and the peptide-MHC complex. Therefore, the strict control of IL-7R expression probably plays an important role in the regulation of selection and immune response. However, the molecular mechanism of IL-7R expression is largely unknown except for reports that an Ets family transcription factor, PU.1, can bind to a motif in the IL-7R promoter in mouse pro-B cells (16) and that GA binding protein binds to the same motif and is essential in the transcription of IL-7R promoter in T cells (17).
Glucocorticoids (GC), immunosuppressive and anti-inflammatory agents, have pleiotypic effects on the growth, differentiation, and function of lymphocytes (18, 19). GC bind to the GC receptor (GR) in cytoplasm, and GR subsequently undergoes nuclear translocation. GR exercises its function either through association with GR binding motif in the promoters or through interaction with other signal molecules and transcription factors. A typical GR motif consists of two six-base palindrome sequences interrupted by a three-base spacer (TGTACANNNTGTTCT). However, GR can also bind to half-palindrome motifs (20, 21). GR regulates the function of T cells either positively or negatively. For example, GC inhibit transcriptional up-regulation of T cell-derived cytokines, such as IL-2, IL-4, and IFN- by inhibiting AP-1 (22) and NF-B (23). Administration of GC results in apoptosis of immature thymocytes and T cell hybridomas (18). In contrast, GC block activation-induced apoptosis by TCR stimulation, through inducing GC-induced leucine zipper gene (24) and GC-induced TNFR family-related gene (25). In addition, it has also been reported that GC induce the expression of the IL-7R in human peripheral T cells (14), which may block activation-induced apoptosis. Based on these findings of mutual inhibition between TCR and GC, the role of GC has received much attention in T cell development.
To identify the molecular mechanism of the induction of IL-7R by GC, we dissected the mouse IL-7R promoter and characterized the molecular mechanism of transcription of the IL-7R gene. In this study, we demonstrated that GC positively induce the expression of IL-7R in mouse T cells and that ligand-activated GR binds to a GR motif in a conserved noncoding sequence (CNS) upstream of the IL-7R promoter, which was designated as CNS-1, and then activates the transcription of the IL-7R promoter. Thus, this study explores the molecular mechanism of IL-7R induction by GC and demonstrates that the CNS-1 region is a GC-responsive element of the IL-7R locus.
Materials and Methods
Transfection was done by electroporation as described previously (28). KKF cells (1 x 107) were transiently transfected by electroporation with 30 μg of luciferase reporter constructs and 100 ng of phRL-TK plasmid (Promega) driven by HSV thymidine kinase promoter by electroporation at 950 μF and 350 V. Reporter gene analysis was performed 24 h after transfection. In cotransfection studies, mixtures also contained 10 μg of mouse GR expression vector (pcDNA3.1-mGR). Dex (10–7 M) was added at 18 h after transfection, and the cells were harvested at 36 h after transfection. The total amount of DNA was kept constant with pGL3-basic or pcDNA3.1 vector. Cell lysates were then subjected to Dual-Luciferase Reporter Assay System (Promega), and luciferase activities were measured with a luminometer (Lumat LB9507; Berthold). Firefly luciferase activity was normalized by Renilla luciferase activity. In each experiment, samples were analyzed in triplicate, and each experiment was repeated at least twice.
Results
GC induce IL-7R expression on mouse T cells
The induction of IL-7R expression by GC was previously reported in human T cells (14). Therefore, we first checked whether GC up-regulate the level of IL-7R mRNA and protein in mouse spleen T cells. Freshly isolated total mouse spleen cells were cultured with 10–7 M Dex, and cell surface level of IL-7R on T cells was analyzed by flow cytometry. IL-7R expression was not changed at 2 and 6 h but slightly increased at 12 h by Dex treatment (1.5-fold at the peak fluorescence intensity) (Fig. 1A and data not shown). To confirm whether GC induce IL-7R mRNA in mouse T cells, we analyzed the levels of IL-7R mRNA after Dex treatment by real-time quantitative PCR (Fig. 1B). The IL-7R mRNA level started to increase within 1 h after Dex treatment, and soared up to 6-fold at 2 h. The level gradually decreased afterward. These results indicate that GC rapidly and transiently increase IL-7R mRNA in mouse peripheral T cells.
Next, we checked whether IL-7R expression is augmented by GC treatment in mouse T cell lines. An IL-7R+ immature thymocyte line, KKF, was treated with 10–7 M Dex for 16 h, and the levels of IL-7R mRNA and protein were analyzed. Dex treatment induced a 4-fold increase of cell surface IL-7R expression (Fig. 1C). IL-7R mRNA was also induced within 1 h after the addition of Dex, peaked at 2 h by 5-fold, and gradually decreased thereafter (Fig. 1D). These results demonstrate that IL-7R mRNA is rapidly induced by GC not only in human but also in mouse T cells.
Induction of mouse IL-7R mRNA is a direct effect of GC
To test whether there is need for new protein synthesis during mouse IL-7R mRNA induction by GC, we used cycloheximide and puromycin, which inhibit protein synthesis by blocking the peptidyl synthesis activity of eukaryotic ribosome (Fig. 1D). When cycloheximide and Dex were simultaneously added, Dex-mediated induction of IL-7R mRNA was not blocked. The treatment with cycloheximide alone did not change the IL-7R mRNA level. Similar results were obtained with puromycin. These observations are comparable with the previous report (14). These results indicate that GC directly induce the IL-7R mRNA, not through de novo protein synthesis.
Comparison of the mouse, rat, and human IL-7R loci
Because IL-7R expression is regulated at the level of transcript, we next characterized cis-control elements of the IL-7R locus (Fig. 2). Another group previously reported a transcription initiation site 945 bp upstream of the translation initiation site (31). However, we could not detect any IL-7R transcripts in this region by RT-PCR analysis (data not shown). Therefore, we determined transcription initiation sites by isolating mouse full-length IL-7R cDNA from 5'-capped mRNA. IL-7R mRNA started from several sites within the region between 46 and 130 bp upstream of the translation initiation site (Fig. 2, A and B, arrows). One of these transcription start sites is very close to the major site reported previously (17). Search between mouse, human, and rat sequences revealed striking homology in the region spanning 320 bp from the translation initiation site. Percent homology was 75% for 197 bp between mouse and human. Consensus motifs of PU.1 and Runx1, indispensable transcription factors for development of hemopoietic stem cells and lymphocyte progenitors (32, 33), were conserved in this region. In addition, Ikaros motif was conserved between mouse and human.
Because a CNS identified by cross-species sequence comparisons has much possibility to be a control element (34), we compared the DNA sequence upstream of the IL-7R promoter between mouse, rat, and human. In addition to the promoter, a CNS of 270 bp was identified at 3.6 kb upstream of the translation initiation site (Fig. 2, A and C). We designate this region as CNS-1. Percent homology was 86% for 300 bp between mouse and human. Consensus motifs of GATA, NF-B, GR, and Evi-1 transcription factors were conserved in CNS-1.
GR and PU.1 motifs are important in activation of the IL-7R promoter
To elucidate the molecular mechanism of transcriptional activation of the mouse IL-7R promoter, we first conducted reporter assay with the KKF cell line. A 320-bp fragment of IL-7R promoter region was cloned into a luciferase reporter vector. This plasmid DNA was transfected into KKF cells by electroporation. As shown in Fig. 3A, the promoter showed specific transcription activity. In contrast, previously reported promoter (31) did not reveal any activity (data not shown).
We next tested whether the conserved motifs are important for this transactivation. A series of mutations was introduced in the motifs of Ikaros, PU.1, and Runx1, and these reporter constructs were transfected into KKF cells. As shown in Fig. 3A, mutations in the Runx1 motif slightly decreased the transcriptional activity of the IL-7R promoter. In contrast, the activity was significantly decreased with mutation of the PU.1 motif, suggesting that the PU.1 motif plays the major role in activation of the IL-7R promoter. We also obtained similar results in a pro-B cell line, 38B9, and a pre-B cell line, NSF 5.3 (data not shown). These results suggest that the PU.1 motif is important for the activity of the IL-7R promoter, and that the Runx1 motif may have positive effects.
Next, we checked whether the GR motif in the CNS-1 region plays an important role in transcriptional activation of the IL-7R promoter by GC stimulation. The reporter constructs of the mouse IL-7R promoter with or without the CNS-1 region were transfected with GR expression vector into KKF cells. After 18 h, the cells were cultured with Dex for 18 h. As shown in Fig. 3B, Dex treatment only slightly increased the transcriptional activity without CNS-1, but greatly increased it by 3.6-fold with CNS-1. This induction by Dex was diminished in the construct with mutation in the GR motif in CNS-1. In addition, similar results were obtained with human counterpart promoter and CNS-1 region. These results indicate that GR, activated by GC, transactivates the IL-7R promoter through the GR motif in CNS-1. It is also suggested that the CNS-1 serves as a GC-responsive element in the IL-7R locus.
GR binds to the GR motif in the IL-7R CNS-1 region
To test whether GR binds directly to the GR motif in the IL-7R CNS-1 region, we performed EMSA with IL-7R CNS-1 GR motif oligonucleotide (Fig. 4A). Nuclear extract of Dex-treated KKF cells showed two kinds of DNA-protein complex with CNS-1 GR motif oligonucleotide probe, which probably represent monomer and dimer of GR as previously reported (35) (Fig. 4B, lane 1, arrows). GC treatment did not change these binding activities (Fig. 4B, lane 2). These activities were reduced by addition of the same GR motif oligonucleotide competitor, but not mutated oligonucleotide competitor (Fig. 4, B, lanes 3 and 4; C and D). Similar results were obtained with cold competitor oligonucleotide of human CNS-1 GR motif and GR consensus motif (Fig. 4, B, lanes 5–8; C and D). The binding activity was further confirmed by inhibition assay using an anti-GR Ab (Fig. 4, E–G). Incubation with the anti-GR Ab resulted in reduced levels of binding activities, whereas a control anti-Stat5 Ab did not affect the activities. The anti-GR Ab probably inhibited the DNA binding of GR. These results, taken together, indicate that GR protein binds to the GR motif in the IL-7R CNS-1 region in vitro.
Recruitment of GR to endogenous CNS-1 chromatin by GC stimulation
Next, we examined whether GR is recruited to endogenous IL-7R CNS-1 chromatin after GC treatment by ChIP assay. KKF cells were cultured with Dex, harvested at various time points, fixed with formalin, lysed, and sonicated to prepare soluble chromatin. The chromatin fraction was immunoprecipitated with anti-GR or control Ab, and purified genomic DNA was measured with the primers for the IL-7R CNS-1 or control region by real-time quantitative PCR. As shown in Fig. 5A, Dex treatment induced accumulation of GR at the IL-7R CNS-1 region. The recruitment peaked at 2 h, and diminished by 4 h. This time course of GR binding to the IL-7R promoter correlated well with that of IL-7R mRNA levels (Fig. 1, B and D). This recruitment of GR was not observed with control primers, which amplify a region 1.7 kb upstream of the translation initiation site (Fig. 5B). These results demonstrate that GR is rapidly recruited to endogenous IL-7R promoter after GC stimulation.
Discussion
In this study, we first showed that GC up-regulate the levels of IL-7R mRNA and protein in mouse T cells. The induction of IL-7R mRNA does not require de novo protein synthesis, because protein synthesis inhibitors do not block the process. The mouse IL-7R promoter contains Ikaros, PU.1, and Runx1 consensus motifs, some of which are also conserved in rat and human. We also identified a highly conserved upstream region designated as CNS-1. The CNS-1 region contains a GR consensus motif. Importantly, we showed by reporter assay that the activity of the IL-7R promoter highly depends on the PU.1 motif and that GC treatment augments the transcriptional activity through the GR motif in the CNS-1 region. We further showed that GR binds to its motifs in the CNS-1 region and that GR is rapidly recruited to endogenous CNS-1 chromatin after GC stimulation. Thus, this study defines the IL-7R CNS-1 as a GC-responsive element, and provides further insights into the molecular mechanism of IL-7R induction by GC.
GC antagonize activation-induced cell death of T cells by TCR stimulation (18). Activation-induced apoptosis is caused by the up-regulation of Fas ligand expression (36). GC induce GC-induced leucine zipper gene, which then blocks the up-regulation of Fas ligand (24). GC-induced TNFR family-related gene may also participate in the antagonism (25). In contrast, TCR signaling transiently down-regulates expression of the IL-7R (13, 14), which probably ensures precise Ag-driven clonal expansion and subsequent activation-induced cell death. It is possible that GC may interfere with this regulation by directly inducing the transcription of IL-7R, as demonstrated by this study. Therefore, this can be one of the mechanisms how GC antagonize the activation-induced cell death of T cells.
GR knockout mice show normal intrathymic T cell development (37, 38). Therefore, it is probable that IL-7R expression in steady state does not require GR signaling. Because the PU.1 motif plays a significant role in the activation of the IL-7R promoter, Ets family transcription factors such as GA binding protein are probably involved in the GR-independent mechanism (17). Therefore, GR-mediated mechanism may be overestimated in our reporter assay. Or, in another way, it can be that the significance of the GR-mediated mechanism of IL-7R induction may be different between early and late stages of T cell development.
The PU.1 motif plays a significant role on the activation of the IL-7R promoter. PU.1 is critical for hemopoietic stem cell maintenance, myeloid and B cell lineage development, and may also support pro-T cell generation (39, 40, 41). PU.1–/– fetal hemopoietic progenitors fail to express IL-7R transcripts. In addition, it has been reported that PU.1 is bound to the same motif in the IL-7R promoter and that a multimerized binding site representing this sequence can stimulate transcription of a reporter gene in pro-B cells (16). Our observation that the IL-7R promoter activity highly depends on the PU.1 motif is comparable with these results. In the T cell lineage, however, PU.1 expression is severely reduced after the pro-T cell stage, and constitutive expression of PU.1 prevents fetal precursors from T cell development (42). Very recently, it has been reported that GA binding protein, a member of Ets transcription factor family, binds to this PU.1 motif and is essential in the transcription of IL-7R promoter in T cells (17).
Ikaros and Runx1 are involved in early hemopoietic and lymphoid development. Our result suggests that Runx1 may positively regulate the IL-7R promoter. Runx1 is expressed not only in hemopoietic progenitors and myeloid cells, but also in T cells in the thymus and spleen (33). Indeed, Runx1 is known to play a role in single-positive thymocytes and naive T cells (43), where IL-7R is highly expressed. Thus, it is possible that Runx family transcription factors may positively control the IL-7R promoter.
In this study, we characterized the IL-7R promoter and identified the molecular mechanism of induction of IL-7R gene by GC. Based on our data, GR is recruited to the GR motif of the CNS-1 region by GC treatment, and induces the transcription of the IL-7R promoter. This study indicates that the CNS-1 region is a GC-responsive element of the IL-7R locus.
Acknowledgments
We thank Dr. K. Igarashi for the 38B9 cell line, and Drs. T. Saito and A. Takeuchi for the KKF cell line; S. Hayashi and S. Kamioka for excellent technical assistance; members of K. Ikuta’s lab for discussion; and Drs. J. Bodor and N. Begum for critically reading the manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This study was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by a grant provided by the Mochida Memorial Foundation for Medical and Pharmaceutical Research. H.-C.L. was supported by a Japan Society for the Promotion of Science postdoctoral fellowship for foreign researchers.
2 Address correspondence and reprint requests to Dr. Koichi Ikuta, Laboratory of Biological Protection, Department of Biological Responses, Institute for Virus Research, Kyoto University, Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail address: ikuta{at}virus.kyoto-u.ac.jp
3 Abbreviations used in this paper: IL-7R, IL-7R -chain; GC, glucocorticoid; GR, GC receptor; CNS, conserved noncoding sequence; Dex, dexamethasone; ChIP, chromatin immunoprecipitation.
Received for publication July 13, 2004. Accepted for publication April 12, 2005.
References
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Expression of the IL-7R -chain (IL-7R) is strictly regulated during the development and maturation of lymphocytes. Glucocorticoids (GC) have pleiotypic effects on the growth and function of lymphocytes. Although GC have been reported to induce the transcription of IL-7R gene in human T cells, its molecular mechanism is largely unknown. In this study, we show that GC up-regulate the levels of IL-7R mRNA and protein in mouse T cells. This effect does not require protein synthesis de novo, because protein synthesis inhibitors do not block the process. Mouse IL-7R promoter has striking homology with human and rat, containing consensus motifs of Ikaros, PU.1, and Runx1 transcription factors. In addition, a conserved noncoding sequence (CNS) of 270 bp was found 3.6-kb upstream of the promoter, which was designated as CNS-1. A GC receptor (GR) motif is present in the CNS-1 region. Importantly, we show by reporter assay that the IL-7R promoter has specific transcription activity in T cells. This activity highly depends on the PU.1 motif. Furthermore, GC treatment augments the transcriptional activity through the GR motif in the CNS-1 region. We also demonstrate that GR binds to the GR motif by EMSA. In addition, by chromatin immunoprecipitation assay, we show that GR is rapidly recruited to endogenous CNS-1 chromatin after GC stimulation. These results demonstrate that GR binds to the GR motif in the CNS-1 region after GC stimulation and then activates the transcription of the IL-7R promoter. Thus, this study identifies the IL-7R CNS-1 region as a GC-responsive element.
Introduction
Interleukin-7 is an essential cytokine for development of early lymphocytes and maintenance of mature lymphocytes. At early stages, it controls proliferation of T and B cell precursors, positive selection of CD8+ thymocyte (1), and the recombination accessibility of the TCR and IgH loci (2, 3, 4). At later stages, IL-7 supports survival and homeostatic proliferation of naive and memory T cells (5). IL-7 exerts its effect through interaction with the IL-7R, consisting of a unique -chain (IL-7R -chain (IL-7R)3) (6) and common cytokine receptor -chain (7, 8). The IL-7R also heterodimerizes with the unique thymic stromal-derived lymphopoietin receptor chain (9, 10). IL-7 binding to the IL-7R activates the JAK-1 and -3, which then activate STAT5 (11), phosphoinoside-3 kinase, Ras, and MAPK/ERK.
Expression of the IL-7R is strictly regulated during the development and maturation of lymphocytes. It is induced at the stage of common lymphoid progenitors and maintained during whole T cell life except at two stages: CD4+8+ thymocytes and activated T cells (12, 13, 14). In B cell lineage, in contrast, the IL-7R is down-regulated at the pre-B cell stage and kept off throughout the later stages (15). It is supposed that the IL-7R is actively down-regulated not to transmit unnecessary survival signals, when T cells have to decide their fate solely by the affinity between their TCR and the peptide-MHC complex. Therefore, the strict control of IL-7R expression probably plays an important role in the regulation of selection and immune response. However, the molecular mechanism of IL-7R expression is largely unknown except for reports that an Ets family transcription factor, PU.1, can bind to a motif in the IL-7R promoter in mouse pro-B cells (16) and that GA binding protein binds to the same motif and is essential in the transcription of IL-7R promoter in T cells (17).
Glucocorticoids (GC), immunosuppressive and anti-inflammatory agents, have pleiotypic effects on the growth, differentiation, and function of lymphocytes (18, 19). GC bind to the GC receptor (GR) in cytoplasm, and GR subsequently undergoes nuclear translocation. GR exercises its function either through association with GR binding motif in the promoters or through interaction with other signal molecules and transcription factors. A typical GR motif consists of two six-base palindrome sequences interrupted by a three-base spacer (TGTACANNNTGTTCT). However, GR can also bind to half-palindrome motifs (20, 21). GR regulates the function of T cells either positively or negatively. For example, GC inhibit transcriptional up-regulation of T cell-derived cytokines, such as IL-2, IL-4, and IFN- by inhibiting AP-1 (22) and NF-B (23). Administration of GC results in apoptosis of immature thymocytes and T cell hybridomas (18). In contrast, GC block activation-induced apoptosis by TCR stimulation, through inducing GC-induced leucine zipper gene (24) and GC-induced TNFR family-related gene (25). In addition, it has also been reported that GC induce the expression of the IL-7R in human peripheral T cells (14), which may block activation-induced apoptosis. Based on these findings of mutual inhibition between TCR and GC, the role of GC has received much attention in T cell development.
To identify the molecular mechanism of the induction of IL-7R by GC, we dissected the mouse IL-7R promoter and characterized the molecular mechanism of transcription of the IL-7R gene. In this study, we demonstrated that GC positively induce the expression of IL-7R in mouse T cells and that ligand-activated GR binds to a GR motif in a conserved noncoding sequence (CNS) upstream of the IL-7R promoter, which was designated as CNS-1, and then activates the transcription of the IL-7R promoter. Thus, this study explores the molecular mechanism of IL-7R induction by GC and demonstrates that the CNS-1 region is a GC-responsive element of the IL-7R locus.
Materials and Methods
Transfection was done by electroporation as described previously (28). KKF cells (1 x 107) were transiently transfected by electroporation with 30 μg of luciferase reporter constructs and 100 ng of phRL-TK plasmid (Promega) driven by HSV thymidine kinase promoter by electroporation at 950 μF and 350 V. Reporter gene analysis was performed 24 h after transfection. In cotransfection studies, mixtures also contained 10 μg of mouse GR expression vector (pcDNA3.1-mGR). Dex (10–7 M) was added at 18 h after transfection, and the cells were harvested at 36 h after transfection. The total amount of DNA was kept constant with pGL3-basic or pcDNA3.1 vector. Cell lysates were then subjected to Dual-Luciferase Reporter Assay System (Promega), and luciferase activities were measured with a luminometer (Lumat LB9507; Berthold). Firefly luciferase activity was normalized by Renilla luciferase activity. In each experiment, samples were analyzed in triplicate, and each experiment was repeated at least twice.
Results
GC induce IL-7R expression on mouse T cells
The induction of IL-7R expression by GC was previously reported in human T cells (14). Therefore, we first checked whether GC up-regulate the level of IL-7R mRNA and protein in mouse spleen T cells. Freshly isolated total mouse spleen cells were cultured with 10–7 M Dex, and cell surface level of IL-7R on T cells was analyzed by flow cytometry. IL-7R expression was not changed at 2 and 6 h but slightly increased at 12 h by Dex treatment (1.5-fold at the peak fluorescence intensity) (Fig. 1A and data not shown). To confirm whether GC induce IL-7R mRNA in mouse T cells, we analyzed the levels of IL-7R mRNA after Dex treatment by real-time quantitative PCR (Fig. 1B). The IL-7R mRNA level started to increase within 1 h after Dex treatment, and soared up to 6-fold at 2 h. The level gradually decreased afterward. These results indicate that GC rapidly and transiently increase IL-7R mRNA in mouse peripheral T cells.
Next, we checked whether IL-7R expression is augmented by GC treatment in mouse T cell lines. An IL-7R+ immature thymocyte line, KKF, was treated with 10–7 M Dex for 16 h, and the levels of IL-7R mRNA and protein were analyzed. Dex treatment induced a 4-fold increase of cell surface IL-7R expression (Fig. 1C). IL-7R mRNA was also induced within 1 h after the addition of Dex, peaked at 2 h by 5-fold, and gradually decreased thereafter (Fig. 1D). These results demonstrate that IL-7R mRNA is rapidly induced by GC not only in human but also in mouse T cells.
Induction of mouse IL-7R mRNA is a direct effect of GC
To test whether there is need for new protein synthesis during mouse IL-7R mRNA induction by GC, we used cycloheximide and puromycin, which inhibit protein synthesis by blocking the peptidyl synthesis activity of eukaryotic ribosome (Fig. 1D). When cycloheximide and Dex were simultaneously added, Dex-mediated induction of IL-7R mRNA was not blocked. The treatment with cycloheximide alone did not change the IL-7R mRNA level. Similar results were obtained with puromycin. These observations are comparable with the previous report (14). These results indicate that GC directly induce the IL-7R mRNA, not through de novo protein synthesis.
Comparison of the mouse, rat, and human IL-7R loci
Because IL-7R expression is regulated at the level of transcript, we next characterized cis-control elements of the IL-7R locus (Fig. 2). Another group previously reported a transcription initiation site 945 bp upstream of the translation initiation site (31). However, we could not detect any IL-7R transcripts in this region by RT-PCR analysis (data not shown). Therefore, we determined transcription initiation sites by isolating mouse full-length IL-7R cDNA from 5'-capped mRNA. IL-7R mRNA started from several sites within the region between 46 and 130 bp upstream of the translation initiation site (Fig. 2, A and B, arrows). One of these transcription start sites is very close to the major site reported previously (17). Search between mouse, human, and rat sequences revealed striking homology in the region spanning 320 bp from the translation initiation site. Percent homology was 75% for 197 bp between mouse and human. Consensus motifs of PU.1 and Runx1, indispensable transcription factors for development of hemopoietic stem cells and lymphocyte progenitors (32, 33), were conserved in this region. In addition, Ikaros motif was conserved between mouse and human.
Because a CNS identified by cross-species sequence comparisons has much possibility to be a control element (34), we compared the DNA sequence upstream of the IL-7R promoter between mouse, rat, and human. In addition to the promoter, a CNS of 270 bp was identified at 3.6 kb upstream of the translation initiation site (Fig. 2, A and C). We designate this region as CNS-1. Percent homology was 86% for 300 bp between mouse and human. Consensus motifs of GATA, NF-B, GR, and Evi-1 transcription factors were conserved in CNS-1.
GR and PU.1 motifs are important in activation of the IL-7R promoter
To elucidate the molecular mechanism of transcriptional activation of the mouse IL-7R promoter, we first conducted reporter assay with the KKF cell line. A 320-bp fragment of IL-7R promoter region was cloned into a luciferase reporter vector. This plasmid DNA was transfected into KKF cells by electroporation. As shown in Fig. 3A, the promoter showed specific transcription activity. In contrast, previously reported promoter (31) did not reveal any activity (data not shown).
We next tested whether the conserved motifs are important for this transactivation. A series of mutations was introduced in the motifs of Ikaros, PU.1, and Runx1, and these reporter constructs were transfected into KKF cells. As shown in Fig. 3A, mutations in the Runx1 motif slightly decreased the transcriptional activity of the IL-7R promoter. In contrast, the activity was significantly decreased with mutation of the PU.1 motif, suggesting that the PU.1 motif plays the major role in activation of the IL-7R promoter. We also obtained similar results in a pro-B cell line, 38B9, and a pre-B cell line, NSF 5.3 (data not shown). These results suggest that the PU.1 motif is important for the activity of the IL-7R promoter, and that the Runx1 motif may have positive effects.
Next, we checked whether the GR motif in the CNS-1 region plays an important role in transcriptional activation of the IL-7R promoter by GC stimulation. The reporter constructs of the mouse IL-7R promoter with or without the CNS-1 region were transfected with GR expression vector into KKF cells. After 18 h, the cells were cultured with Dex for 18 h. As shown in Fig. 3B, Dex treatment only slightly increased the transcriptional activity without CNS-1, but greatly increased it by 3.6-fold with CNS-1. This induction by Dex was diminished in the construct with mutation in the GR motif in CNS-1. In addition, similar results were obtained with human counterpart promoter and CNS-1 region. These results indicate that GR, activated by GC, transactivates the IL-7R promoter through the GR motif in CNS-1. It is also suggested that the CNS-1 serves as a GC-responsive element in the IL-7R locus.
GR binds to the GR motif in the IL-7R CNS-1 region
To test whether GR binds directly to the GR motif in the IL-7R CNS-1 region, we performed EMSA with IL-7R CNS-1 GR motif oligonucleotide (Fig. 4A). Nuclear extract of Dex-treated KKF cells showed two kinds of DNA-protein complex with CNS-1 GR motif oligonucleotide probe, which probably represent monomer and dimer of GR as previously reported (35) (Fig. 4B, lane 1, arrows). GC treatment did not change these binding activities (Fig. 4B, lane 2). These activities were reduced by addition of the same GR motif oligonucleotide competitor, but not mutated oligonucleotide competitor (Fig. 4, B, lanes 3 and 4; C and D). Similar results were obtained with cold competitor oligonucleotide of human CNS-1 GR motif and GR consensus motif (Fig. 4, B, lanes 5–8; C and D). The binding activity was further confirmed by inhibition assay using an anti-GR Ab (Fig. 4, E–G). Incubation with the anti-GR Ab resulted in reduced levels of binding activities, whereas a control anti-Stat5 Ab did not affect the activities. The anti-GR Ab probably inhibited the DNA binding of GR. These results, taken together, indicate that GR protein binds to the GR motif in the IL-7R CNS-1 region in vitro.
Recruitment of GR to endogenous CNS-1 chromatin by GC stimulation
Next, we examined whether GR is recruited to endogenous IL-7R CNS-1 chromatin after GC treatment by ChIP assay. KKF cells were cultured with Dex, harvested at various time points, fixed with formalin, lysed, and sonicated to prepare soluble chromatin. The chromatin fraction was immunoprecipitated with anti-GR or control Ab, and purified genomic DNA was measured with the primers for the IL-7R CNS-1 or control region by real-time quantitative PCR. As shown in Fig. 5A, Dex treatment induced accumulation of GR at the IL-7R CNS-1 region. The recruitment peaked at 2 h, and diminished by 4 h. This time course of GR binding to the IL-7R promoter correlated well with that of IL-7R mRNA levels (Fig. 1, B and D). This recruitment of GR was not observed with control primers, which amplify a region 1.7 kb upstream of the translation initiation site (Fig. 5B). These results demonstrate that GR is rapidly recruited to endogenous IL-7R promoter after GC stimulation.
Discussion
In this study, we first showed that GC up-regulate the levels of IL-7R mRNA and protein in mouse T cells. The induction of IL-7R mRNA does not require de novo protein synthesis, because protein synthesis inhibitors do not block the process. The mouse IL-7R promoter contains Ikaros, PU.1, and Runx1 consensus motifs, some of which are also conserved in rat and human. We also identified a highly conserved upstream region designated as CNS-1. The CNS-1 region contains a GR consensus motif. Importantly, we showed by reporter assay that the activity of the IL-7R promoter highly depends on the PU.1 motif and that GC treatment augments the transcriptional activity through the GR motif in the CNS-1 region. We further showed that GR binds to its motifs in the CNS-1 region and that GR is rapidly recruited to endogenous CNS-1 chromatin after GC stimulation. Thus, this study defines the IL-7R CNS-1 as a GC-responsive element, and provides further insights into the molecular mechanism of IL-7R induction by GC.
GC antagonize activation-induced cell death of T cells by TCR stimulation (18). Activation-induced apoptosis is caused by the up-regulation of Fas ligand expression (36). GC induce GC-induced leucine zipper gene, which then blocks the up-regulation of Fas ligand (24). GC-induced TNFR family-related gene may also participate in the antagonism (25). In contrast, TCR signaling transiently down-regulates expression of the IL-7R (13, 14), which probably ensures precise Ag-driven clonal expansion and subsequent activation-induced cell death. It is possible that GC may interfere with this regulation by directly inducing the transcription of IL-7R, as demonstrated by this study. Therefore, this can be one of the mechanisms how GC antagonize the activation-induced cell death of T cells.
GR knockout mice show normal intrathymic T cell development (37, 38). Therefore, it is probable that IL-7R expression in steady state does not require GR signaling. Because the PU.1 motif plays a significant role in the activation of the IL-7R promoter, Ets family transcription factors such as GA binding protein are probably involved in the GR-independent mechanism (17). Therefore, GR-mediated mechanism may be overestimated in our reporter assay. Or, in another way, it can be that the significance of the GR-mediated mechanism of IL-7R induction may be different between early and late stages of T cell development.
The PU.1 motif plays a significant role on the activation of the IL-7R promoter. PU.1 is critical for hemopoietic stem cell maintenance, myeloid and B cell lineage development, and may also support pro-T cell generation (39, 40, 41). PU.1–/– fetal hemopoietic progenitors fail to express IL-7R transcripts. In addition, it has been reported that PU.1 is bound to the same motif in the IL-7R promoter and that a multimerized binding site representing this sequence can stimulate transcription of a reporter gene in pro-B cells (16). Our observation that the IL-7R promoter activity highly depends on the PU.1 motif is comparable with these results. In the T cell lineage, however, PU.1 expression is severely reduced after the pro-T cell stage, and constitutive expression of PU.1 prevents fetal precursors from T cell development (42). Very recently, it has been reported that GA binding protein, a member of Ets transcription factor family, binds to this PU.1 motif and is essential in the transcription of IL-7R promoter in T cells (17).
Ikaros and Runx1 are involved in early hemopoietic and lymphoid development. Our result suggests that Runx1 may positively regulate the IL-7R promoter. Runx1 is expressed not only in hemopoietic progenitors and myeloid cells, but also in T cells in the thymus and spleen (33). Indeed, Runx1 is known to play a role in single-positive thymocytes and naive T cells (43), where IL-7R is highly expressed. Thus, it is possible that Runx family transcription factors may positively control the IL-7R promoter.
In this study, we characterized the IL-7R promoter and identified the molecular mechanism of induction of IL-7R gene by GC. Based on our data, GR is recruited to the GR motif of the CNS-1 region by GC treatment, and induces the transcription of the IL-7R promoter. This study indicates that the CNS-1 region is a GC-responsive element of the IL-7R locus.
Acknowledgments
We thank Dr. K. Igarashi for the 38B9 cell line, and Drs. T. Saito and A. Takeuchi for the KKF cell line; S. Hayashi and S. Kamioka for excellent technical assistance; members of K. Ikuta’s lab for discussion; and Drs. J. Bodor and N. Begum for critically reading the manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This study was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by a grant provided by the Mochida Memorial Foundation for Medical and Pharmaceutical Research. H.-C.L. was supported by a Japan Society for the Promotion of Science postdoctoral fellowship for foreign researchers.
2 Address correspondence and reprint requests to Dr. Koichi Ikuta, Laboratory of Biological Protection, Department of Biological Responses, Institute for Virus Research, Kyoto University, Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail address: ikuta{at}virus.kyoto-u.ac.jp
3 Abbreviations used in this paper: IL-7R, IL-7R -chain; GC, glucocorticoid; GR, GC receptor; CNS, conserved noncoding sequence; Dex, dexamethasone; ChIP, chromatin immunoprecipitation.
Received for publication July 13, 2004. Accepted for publication April 12, 2005.
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