Identification of a Phorbol Ester-responsive Element in the Interferon-γ Receptor 1 Chain Gene

Human monocytic leukemia THP-1 cells differentiate into macrophage-like cells when treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). During this process, interferon-γ (IFN-γ)-inducible expression of human leukocyte antigen-DRα is markedly enhanced. The enhancement of human leukocyte antigen-DRα expression is at least due to the TPA-dependent induction of the IFN-γ receptor 1 chain and IFN-γ receptor 2 chain genes. Here we have studied the mechanism of TPA-induced up-regulation of the IFN-γ receptor 1 chain gene. Reporter gene analyses of 5′-deletion constructs of the IFN-γ receptor 1 gene (IFNGR1) promoter indicated that the critical region for control of transcription and the TPA-responsive element (TRE) were present in the −128 to −109 base pair (bp) region. We confirmed that this region of the IFNGR1 promoter was responsive to TPA-induced signals by using a reporter construct whose promoter consisted of the −128 to −109 bp fragment and the minimal herpes simplex virus thymidine kinase promoter. Moreover, a supershift assay indicated that Sp1 bound to this TRE in TPA-treated THP-1 cells. These results suggest that in TPA-treated cells the binding of Sp1 to the TRE of the IFNGR1 promoter causes the up-regulation of this gene.

Macrophages can recognize and ingest many types of extracellular bacteria, thereby destroying the bacteria, and at the same time present bacterial peptides to CD4 T cells. This can lead to the generation of armed effector CD4 T cells specific for the ingested microorganism. An important function of these armed effector T cells is to enhance the ability of the macrophages to kill the ingested bacteria, many of which have evolved strategies for surviving and proliferating inside phagocytic cells. The induction of antimicrobial activity in macrophages is known as macrophage activation (1)(2)(3), and many of the intracellular events leading to this have been determined. The macrophage-activating factor interferon-␥ (IFN-␥) 1 binds to IFN-␥R and activates a Janus kinase (Jak)/Stat signaling pathway consisting of Jak1 and Jak2 as well as the transcription factor signaling transducer and activator of transcription 1 (STAT1). STAT1 binds to the cytoplasmic portion of the ligandactivated IFN-␥R via the Src homology 2 domain and is phosphorylated by Jak on a single tyrosine residue (Tyr-701). This phosphorylation results in the Src homology 2 domain-mediated formation of a dimer of STAT1, which is then translocated to the nucleus to bind to the IFN-␥-responsive element leading to transcriptional activation of IFN-␥-responsive genes (4 -7). Major histocompatibility complex class II molecules play a key role in macrophage activation by presenting peptides derived from bacteria to CD4 T cells (8). Expression of major histocompatibility complex class II genes is induced by IFN-␥ in macrophages (9,10) and is mediated via the induction of the class II transactivator (CIITA) by the Jak/STAT pathway (11)(12)(13).
We have previously observed that the induction of expression of the HLA-DR␣ gene by IFN-␥ was significantly enhanced in TPA-activated THP-1 cells (14) and showed that this was due to an increase in levels of IFN-␥R1 and IFN-␥R2 following the TPA treatment (15). Here we have investigated the mechanism of the TPA-induced increase in IFN-␥R1 expression and have identified an essential TPA-responsive cis-element in the promoter region of IFNGR1, which is activated by Sp1 in response to TPA treatment.

EXPERIMENTAL PROCEDURES
Cell Culture and Cytokine Treatment-THP-1 cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum. Cells were plated at 1 ϫ 10 6 cells/ml and treated with or without 10 ng/ml TPA for 24 h and then with 100 units/ml IFN-␥ for 15, 30, and 60 min. The human embryonic kidney cell line HEK293T was maintained in Eagle's minimal essential medium supplemented with 10% fetal calf serum. Cells were plated at 1 ϫ 10 5 cells/ml and treated with or without 10 ng/ml TPA for 24 h.
Plasmid Construction-First we constructed a luciferase reporter gene containing the IFNGR1 promoter Ϫ840 to ϩ28 bp sequence. The DNA fragment consisting of the Ϫ840 to ϩ28 bp sequence was synthesized by * This work was supported in part by grants-in-aid for scientific and cancer research from the Ministry of Education, Science and Culture, Japan and by the president research fund of Kochi Medical School. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "adver- polymerase chain reaction using genomic DNA from THP-1 cells as the template. The polymerase chain reaction products were digested with BglII and HindIII, and the digested fragments were ligated into the BglII/HindIII sites of the promoterless luciferase reporter gene vector pGV-B (Toyo Ink Co. Ltd., Tokyo, Japan). The forward primer containing the BglII site and reverse primer containing the HindIII site were as follows: sense, 5Ј-GGAAGATCTACAGTAGGGCGGGGTAA-3Ј; antisense, 5Ј-CCCAAGCTTAAGGGGTAGGAGAAAGAGGA-3Ј. The design of these primers is based on sequence information of the IFNGR1 gene (Gen-Bank TM accession number U19241). To construct the IFNGR1 promoter Ϫ109 to ϩ1 bp sequence luciferase reporter gene, the Ϫ840 to ϩ1 bp sequence reporter gene was digested with NaeI and HindIII, and the DNA fragment consisting of the Ϫ109 to ϩ1 bp sequence was subcloned into the SmaI/HindIII sites of pGV-B. To construct the other luciferase reporter genes, the IFNGR1 promoter Ϫ840 to ϩ28 bp sequence luciferase reporter gene was used as the template in polymerase chain reaction with the following forward primers, each containing a BglII site: Ϫ840 to ϩ1 bp, 5Ј-GGAAGATCTACAGTAGGGCGGGGTAA-3Ј; Ϫ540 to ϩ1 bp, 5Ј-GGA-AGATCTTCTTGGTCAAGCCGATT-3Ј; Ϫ240 to ϩ1 bp, 5Ј-GGAAGATCT-CCTCCCACACCCAGAAG-3Ј; Ϫ160 to ϩ1 bp, 5Ј-GGAAGATCTGCGGC-TTCCCGGACTTG-3Ј; Ϫ128 to ϩ1 bp, 5Ј-GGAAGATCTGGTCCCGCCT-CCTGCCGA-3Ј; and a reverse primer (nucleotide ϩ1) containing a Hind-III site, 5Ј-CCCAAGCTTGCTGCTACCGACGGTCGCTG-3Ј. Polymerase chain reaction products were digested and subcloned into the BglII/ HindIII sites of pGV-B.
The reporter plasmid pGV-TK77 contains the minimal promoter sequence of the HSV thymidine kinase (TK) gene from Ϫ46 to ϩ31 bp fused to the BglII/HindIII sites of pGV-B. pGV-TK77-TRE was constructed by inserting the Ϫ128 to Ϫ109 bp sequence of the IFNGR1 gene upstream of the TK minimal promoter of pGV-TK77.
Transient Transfection Experiment-THP-1 cells were transfected using the DEAE-dextran method (17) with some modifications. The cells were centrifuged and washed once with TBS (25 mM Tris-HCl, pH 7.4, 137 mM NaCl, 5 mM KCl, 0.6 mM Na 2 HPO 4 , 7 mM CaCl 2 , 5 mM MgCl 2 ). The cell pellets were resuspended in TBS, and 1 ϫ 10 7 cells were placed in 15-ml tubes. The tubes were centrifuged, and the cells were resuspended in 1 ml of TBS containing 10 g of the reporter plasmid DNA, 2.5 g of internal control plasmid, HSV-TK promoterdriven Renilla reniformis luciferase (pRL-TK), and 500 g/ml DEAEdextran. The cells were incubated for 30 min at room temperature, and the transfection was stopped by the addition of 10 ml of culture medium. Cells were centrifuged, and the pellets were resuspended in 10 ml of fresh medium and cultured for 42 h. Following this, the transfected cells were harvested for luciferase assays. When required, cells were treated with 10 ng/ml TPA for 24 h before the cell extraction. To normalize the transfection efficiencies, except for the reporter assays using pGV-TK77 or pGV-TK77-TRE, the IFNGR1 promoter-driven luciferase activity was divided by the control thymidine kinase promoterdriven R. reniformis luciferase activity. 293T cells were transfected using LipofectAMINE PLUS TM Reagent (Life Technologies, Inc.) according to the manufacturer's instructions. Cells were plated at 2 ϫ 10 5 cells/3-cm plate, and 100 ng of pGV-TK77-TRE or pGV-TK77 were transfected. After 12 h, transfected 293 cells were treated with 10 ng/ml TPA for 24 h. The cells were then harvested and disrupted, and 20 g of the cell lysates were used to measure luciferase activity according to the manufacturer's protocol (Toyo Ink Co. Ltd.) with a Lumat LB9501 luminometer (EG&G Berthold, Bad Wildbad, Germany). All transfection experiments were repeated three to five times.
Electrophoretic Mobility Shift Assay-Electrophoretic mobility shift assay was performed as described previously (18) with some modifications. A synthetic double-stranded oligonucleotide containing the sequence of the TPA-responsive element (TRE), 5Ј-GGTCCCGCCTCCT-GCCGA-3Ј, was used as a probe. It was end-labeled with [␥-32 P]ATP using T4 polynucleotide kinase. Each 10-l reaction mixture contained 20,000 cpm (6 ng) of the double-stranded, end-labeled TRE fragment, 1.5 g of nuclear proteins, and 0.25 g of poly(dI-dC) in binding buffer (10 mM Tris-HCl, pH 7.5, 5 mM NaCl, 10 mM MgCl 2 , 5 mM CaCl 2 , 5% glycerol, 1 mM EDTA). The mixture was incubated at 25°C for 30 min and then electrophoresed in a 6% polyacrylamide gel in 0.5ϫ Tris borate-EDTA at 100 V for 1 h. The DNA-protein complexes were visualized by autoradiography. For supershift assays, nuclear extracts (1.5 g) were incubated with 1 or 2 g of anti-Sp1 IgG for 30 min at 25°C followed by the addition of the 32 P-labeled probe DNA.

Effect of TPA and IFN-␥ Treatment on Phosphorylation of
Tyr-701 and Ser-727 of STAT1 and Production of STAT1 and IFN-␥R1-In this study, we carried out immunoblotting experiments with phospho-specific antibodies that recognize either Tyr-701 phospho-STAT1 or Ser-727 phospho-STAT1 to examine whether TPA and IFN-␥ signals affect phosphorylation at these residues. Cells were incubated for 24 h with or without TPA and then stimulated with IFN-␥ for 0, 15, 30, and 60 min. Cells were lysed, and equal amounts of lysates (30 g of proteins) were immunoblotted with the phospho-specific antibodies. Under these conditions, phosphorylation on Tyr-701 was markedly increased in TPA-pretreated THP-1 cells stimulated with IFN-␥ for 30 and 60 min compared with cells that had not undergone pretreatment (Fig. 1A, lanes 7 and 8). There was only minimal phosphorylation on Ser-727 in TPA-pretreated cells, and this was not affected by treatment with IFN-␥ (Fig.  1B, lanes 5-8). Lysates were also immunoblotted with an anti-STAT1 antibody that recognizes both phosphorylated and nonphosphorylated forms of STAT1. An increased amount of STAT1 was detected when TPA-pretreated cells were stimulated with IFN-␥ for 30 and 60 min (Fig. 1C, lanes 7 and 8). However, in THP-1 cells that were treated with IFN-␥ but not with TPA, a small amount of STAT1 production and Tyr-701 phosphorylation could only be detected when 300 g of the lysates were used for immunoblotting (data not shown). These observations seem to reflect not only the IFN-␥-inducible phosphorylation of STAT1 but also the TPA-facilitated production of STAT1 molecules.
Since activation of IFN-␥Rs by IFN-␥ increases the production of STAT1 and its phosphorylation on Tyr-701 (12,19), we examined the expression of IFN-␥R1 in THP-1 cells under various conditions (15). The expression of IFN-␥R1 was significantly increased by treatment with TPA but not with IFN-␥ (15). These results possibly explain why pretreatment of THP-1 cells with TPA results in enhanced STAT1 production and phosphorylation on Tyr-701 following treatment with IFN-␥.
Identification of the TRE in the Promoter of IFNGR1-We next investigated the mechanism of transcriptional control of IFNGR1 by TPA. To identify a putative cis-acting element responsive to TPA, we constructed luciferase reporter genes that were regulated by various regions of the promoter of IFNGR1. These constructs were transfected into THP-1 cells, and luciferase activity was measured. Fig. 2 shows the structure of the various deletion derivatives and the corresponding A relative value of 1.0 was equivalent to a normalized relative value of 1.28 (Ϫ840 to ϩ1/ϪTPA), 7.5 was equivalent to 9.7 (Ϫ840 to ϩ1/ϩTPA), 0.2 was equivalent to 0.32 (Ϫ128 to ϩ1/ϪTPA), 8.9 was equivalent to 11.4 (Ϫ128 to ϩ1/ϩTPA), 0.0078 was equivalent to 0.01(Ϫ109 to ϩ1/ϪTPA), and 0.085 was equivalent to 0.11 (Ϫ109 to ϩ1/ϩTPA). These values were the averages of triplicate determinations. The assays with these constructs were performed independently three times with similar results. B, the nucleotide sequence of the IFNGR1 promoter. The DNA sequence shown in bold is a candidate cis-element responsive to TPA in the IFNGR1. promoter activities. The reporter gene containing the promoter fragment Ϫ840 to ϩ1 bp showed a high level of luciferase activity. The ratio of the luciferase activity of the reporter construct containing the Ϫ840 to ϩ1 bp fragment over the internal control (pRL-TK) was arbitrarily set as 1.0. Removal of the 5Ј-region from Ϫ840 to Ϫ160 bp resulted in 0.53 retention of promoter activity. When the 5Ј-region from Ϫ840 to Ϫ128 bp was removed, only 0.28 of the promoter activity remained. The promoter activity was almost completely lost after removal of the 5Ј-region from Ϫ840 to Ϫ109 bp. These results suggest that the element located between positions Ϫ128 and Ϫ109 plays a role in the basal promoter activity of IFNGR1. We further examined the deletion constructs in either untreated or TPAtreated THP-1 cells to identify a TPA-responsive element in the promoter region of IFNGR1. Fig. 3A shows that constructs containing promoter segments from Ϫ840 to ϩ1 bp and Ϫ128 to ϩ1 bp exhibit high levels of induction of luciferase activity following treatment with TPA. However, constructs containing the promoter segment from Ϫ109 to ϩ1 bp produced very little luciferase activity in TPA-treated cells. These results suggest that the promoter segment from Ϫ128 to Ϫ109 bp is responsible for the activation of transcription by TPA.
To confirm that the segment from Ϫ128 to Ϫ109 bp responded to treatment with TPA, we constructed luciferase reporter genes that contained a minimal TK promoter (nucleotides Ϫ46 to ϩ31) (pGV-TK77) and inserted the Ϫ128 to Ϫ109 bp fragment into the upstream region of this TK promoter (pGV-TK77-TRE). We failed to measure any activity for pGV-TK77 or pGV-TK77-TRE using a transient transfection system in THP-1 cells probably because the transcriptional apparatus in THP-1 cells is insufficient to transcribe the minimal TK promoter (data not shown). Therefore, we used 293T cells in which the minimal TK promoter could be transcribed (Fig. 4). In transient transfection assays in 293T cells, constructs containing the Ϫ128 to Ϫ109 bp fragment exhibited high levels of induction of luciferase activity following TPA treatment (Fig.  4). These results indicate that the fragment from Ϫ128 to Ϫ109 bp is essential for TPA responsiveness in IFNGR1.
Interaction of a Nuclear Protein with the TRE of the IFNGR1 Gene in TPA-treated THP-1 Cells-To investigate interactions of proteins with the TRE of IFNGR1, we performed an electrophoretic mobility shift assay using the TRE (Ϫ128 to Ϫ109 bp) oligonucleotide as a probe. The TRE oligonucleotide produced one major DNA-protein complex with the nuclear extract from TPAtreated THP-1 cells (Fig. 5, lane 1). Since the TRE of IFNGR1 contains a nucleotide sequence homologous to the Sp1 motif (Fig.  3B), we used an anti-Sp1 antibody for the electrophoretic mobility shift assay. The nuclear extracts from TPA-treated THP-1 cells were incubated with the anti-Sp1 antibody and then mixed with the TRE. This resulted in the disappearance of the major DNA-protein complex (Fig. 5, lanes 2 and 3). These results suggest that treatment of cells with TPA results in the binding of Sp1 to the TRE of IFNGR1 to activate its expression. DISCUSSION We have been studying the regulatory mechanisms of the IFN-␥-inducible HLA-DR␣ gene for several years. We have previously shown that treatment of THP-1 cells with TPA results in increased levels of IFN-␥Rs that lead to an increase in STAT1 and CIITA, resulting in increased HLA-DR␣ gene expression (15). Thus, elucidation of the mechanism resulting in the TPA-induced increase in IFN-␥R may provide an important clue to help solve the pathogenesis of autoimmune disease, such as Grave's disease in which the number of HLA-DR molecules is anomalously increased (20). In the present study, we first examined the effect of IFN-␥ on phosphorylation of Tyr-701 and Ser-727 of STAT1 in TPA-treated THP-1 cells. Steimle et al. (21) have reported that staurosporine, a specific inhibitor of Tyr-701 phosphorylation (22,23), blocks induction of CIITA by IFN-␥. Van Wagner et al. (24) have demonstrated that H7, an inhibitor of Ser-727 phosphorylation (25), abrogates IFN-␥induced expression of CIITA and major histocompatibility complex class II genes. These observations suggest that phosphorylation of Tyr-701 and Ser-727 of STAT1 leads to CIITA expression, which is a key factor in the induction of HLA-DR␣ by IFN-␥. Consequently, results shown in Fig. 1, A and B, together with these observations suggest that, in TPA-treated THP-1 cells that contain increased levels of IFN-␥R, enhanced expression of HLA-DR␣ (14) is due to the increase in the phosphorylation of Tyr-701 and Ser-727 of STAT1 following treatment with IFN-␥. Recently Kovarik et al. (26) have shown that Ser-727 of STAT1 is phosphorylated through the p38 mitogen-activated protein kinase pathway by using SB 203580, a specific p38 mitogen-activated protein kinase inhibitor. Furthermore, it has been known that TPA activates p38 mitogenactivated protein kinase (27,28), and this activation is inhibited by SB 203580 (28). These observations taken together with our results shown in Fig. 1B suggest that STAT1 is phosphorylated on Ser-727 by p38 mitogen-activated protein kinase following activation by TPA.
IFN-␥ binds to a heterodimeric receptor composed of IFN-␥R1, which is able to bind to the ligand with high affinity (29), and IFN-␥R2, which is required for signal transduction (30). Our previous observations (15) indicate that the expression of IFN-␥R1 is increased by TPA treatment alone, which is in good agreement with the observations of Mao et al. (31). Binding of IFN-␥ to the IFN-␥R complex results in phosphorylation of Tyr-701 of STAT1 (19). Thus, the present results (Fig. 1A) together with our previous results (15) suggest that in the TPA-pretreated cells, the enhanced phosphorylation of Tyr-701 of STAT1 following treatment with IFN-␥ may be due to the increase in the levels of IFN-␥Rs.
We also investigated the transcriptional control mechanisms associated with the TPA-induced up-regulation of IFNGR1. To identify the transcriptional regulatory element that responds to TPA, we performed deletion analysis of the promoter region of IFNGR1 reporter constructs in THP-1 cells. As shown in Fig.  2, the DNA region located between positions Ϫ128 and Ϫ109 plays an important role in the regulation of IFNGR1 expression. Furthermore, we used this cis-element (Ϫ128 to Ϫ109 bp) and the minimal HSV TK promoter to construct a reporter gene and confirmed that this region was a TPA-responsive element (Fig. 4). TPA-responsive elements have been well characterized in some promoters (32)(33)(34) such as the 7-bp sequence 5Ј-TGAGTCA-3Ј and the 8-bp sequence 5Ј-CCCCAGGC-3Ј (35). Although the Ϫ109 to ϩ1 bp fragment of IFNGR1 contains one 5Ј-TGAGTCA-3Ј motif (Ϫ83 to Ϫ76 bp) and one 5Ј-CCCCAGGC-3Ј motif (Ϫ46 to Ϫ35 bp) (Fig. 3B), it did not respond to TPA (Fig. 3A). Therefore, the Ϫ128 to Ϫ109 bp fragment alone is the sole region responsible for TPA activation of the IFNGR1 gene, although this cis-element is different from other previously characterized TPA-responsive elements (35).
The sequence of this TRE in the promoter of IFNGR1 was G ϩ C-rich and correlated well with the known binding sequence for Sp1 (36). The IFNGR1 promoter also lacked a TATA sequence motif (37). Sp1 has been shown to be an important constituent for correct transcription initiation from "TATAless" promoters (38 -40). In addition, recent studies have shown that Sp1 is implicated in the TPA-induced expression of the WAF/CIP1 gene in U937 cells (41) and expression of the thromboxane receptor gene in K562 cells (42). As shown in Fig.  5, Sp1 was identified as the DNA binding factor that bound to the TRE of IFNGR1 in TPA-treated THP-1 cells. Furthermore, deletion of the TRE-containing Sp1 binding motif resulted in almost complete elimination of both basal and TPA-induced promoter activity (Fig. 4A). Therefore, the binding of Sp1 to the TRE of IFNGR1 must be important for the control of both basal IFNGR1 expression and TPA-enhanced expression. Recently, Langmann et al. (43) demonstrated that the amount of Sp1 protein was markedly increased in TPA-treated THP-1 cells. Thus, we suggest that the TPA-induced synthesis of Sp1 is involved in the up-regulation of IFNGR1 expression. However, Sp1 is known to interact with several transcription factors such as Oct-1 (44), TATA-binding protein (45), nuclear factor-Y (46), SREBP-2, Elf-1, and transforming growth factor-␤-inducible early gene (47). Furthermore, other groups have demonstrated that phosphorylation and glycosylation of Sp1 also regulate its activity (48,49). Therefore, identification of how Sp1 activity is controlled and its role in the regulation of IFNGR1 expression will require extensive work in the future.