Interferon γ-dependent Induction of Thymidine Phosphorylase/Platelet-derived Endothelial Growth Factor through γ-Activated Sequence-like Element in Human Macrophages¹

TP³is a unique enzyme involved not only in the reversible conversion of thymidine to thymine but also in endothelial cell chemotaxis, and it is identical to the angiogenic factor, PD-ECGF (1, 2). TP/PD-ECGF is a M_r-55,000 intracellular protein consisting of a M_r-110,000 homodimer (3),and the TP/PD-ECGF gene consists of 10 exons that code for a 1.8-kb mRNA with a translation start codon at exon 2 (4). Haraguchi et al. (5) first showed that 2-deoxy-d-ribose, a product from thymidine through the catalytic action of TP, showed angiogenic activity in vitro and in vivo. Administration of a TP-inhibitor inhibits angiogenic activity of human cancer cells overexpressing TP in a mouse dorsal air sac assay (6). These studies suggest that TP might be somehow involved in angiogenesis.

The expression level of TP is low in normal tissues, but is very high in various tumor tissues (7). Many studies have consistently reported that the expression of TP in cancer cells is closely associated with malignancy and/or angiogenesis in various types of cancer (8, 9, 10). On the other hand, TP expression is up-regulated by TNF-α, IL-1α, and IFN-γ in various human cancer cell lines (11). Moreover,the exposure of human breast cancer cells to hypoxia or low pH results in up-regulation of the TP gene (12). In contrast, the expression of TNF-α and its receptor is closely associated with TP expression in monocyte/macrophages infiltrated in invasive breast cancer (13). Toi et al. (14) have examined further the clinical implications of TP expression in monocytes/macrophages infiltrating in breast carcinoma tissues, and they showed that infiltration of TP-positive monocyte/macrophages was closely associated with poor prognosis. Torisu et al.(15) have also reported that infiltration of TP-positive monocytes/macrophages is associated with the malignancy of human melanomas. These studies suggest that inflammatory cytokines including TNF-α, IL-1α, and IFN-γ are important for TP expression, and that the expression of TP in monocyte/macrophages might play a key role in malignancy and angiogenesis in human cancers. In this study, we examined how the expression of the TP gene is up-regulated in human monocytic cells in response to inflammatory cytokines.

Human monocytic U937 cells (JCRB9021) were obtained from the Health Science Research Resources Bank (Osaka, Japan). Cells were cultured in RPMI 1640 with 10% fetal bovine serum in a humidified CO₂ incubator at 37°C. hrTNF-α, hrIL-1α,and hrIFN-γ were purchased from R & D Systems (Minneapolis,MN). The concentrations of cytokines used in this study were as follows: IFN-γ, 50 units/ml; IL-1α, 100 units/ml; and TNF-α, 2 ng/ml. Anti-STAT1αp91 antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.).[γ-³²P]dATP and[α-³²P]dCTP were obtained from Amersham(Buckinghamshire, United Kingdom). Actinomycin D was purchased from NACALAI TESQUE, Inc. (Kyoto, Japan).

Protein fractions were electrophoresed by SDS-PAGE on 10%polyacrylamide gels and blotted onto an Immobilon-P membrane. The membrane was then incubated with monoclonal antibody against TP overnight at 4°C and then with a horseradish peroxidase-linked second antibody for 45 min at room temperature. The membrane was developed by chemiluminescence according to the enhanced chemiluminescence protocol (Amersham, Buckinghamshire, United Kingdom).

The DNA probe (360 bp) of the TP gene was amplified using RT-PCR. The oligonucleotides for PCR were 5′-CAGCAGCTTGTGGACAAGCA-3′and 5′-AACTTAACGTCCACCACCAG-3′. The harvested U937 cells were suspended in 4 m guanidinium isothiocyanate, 25 mm sodium citrate (pH 7.0), 0.5% Sarkosyl, and 0.1 m β-mercaptoethanol. Total RNA was extracted and Northern blot analysis was performed as described previously (16, 17).

The concentration of TP in U937 cells was measured by ELISA using the protocol of Nippon Roche Research Center (Kamakura, Japan). U937 cells were treated with TNF-α, IL-1α, or IFN-γ in the medium containing 10% fetal bovine serum for 24 h. After incubation, the cellular protein fraction was collected, and the cell lysates (45 μg of protein) were plated onto antihuman TP monoclonal antibody-coated wells and incubated for 2 h at 37°C. After incubation, the plates were incubated with a second TP monoclonal antibody at room temperature for 2 h and then with a third antibody (antimouse IgG antiserum conjugated with peroxidase) for 1 h at room temperature. The substrate reaction was done by using the tetramethyl benzidine (TMB) substrate system (Kirkegaard &Perry Laboratories Inc., Gaithersburg, MD). The absorbance was read at 450 nm using a precision microplate reader (Wako, Ltd., Osaka,Japan).

The TP-promoter fragment was initially isolated by PCR using following primers: TP-5, 5′-AGGTCAGAACGGCCCATCCC-3′; and TP-3,5′-GTACAAGCTT-AGGGCGCTGCCCTCGCCCG-3′. The primer sequence of TP-3 contained a HindIII site at the 5′ end. Amplified fragments were then digested with XhoI and HindIII. The resulting 1243-bp fragment was then introduced in front of a luciferase gene Basic Vector 2 (Nippon Gene,Tokyo, Japan) and digested with XhoI and HindIII. This reporter construct was designated pTP-Luc1. To construct other deletion constructs, pTP-Luc1 was digested with StuI, SacI, and BstEII, and then digested with SmaI, to remove the promoter region. The digested products were blunt-ended with the Klenow fragment of DNA polymerase1 and self-ligated. To construct pTP-Luc1ΔBS, pTP-Luc1 was digested with BstEII and StuI, blunt-ended, and self-ligated. Site-directed mutagenesis of GAS in pTP-Luc1 (named “pTP-Luc1/mut”)was also performed by a PCR-based method. Using pTP-Luc1 as a template,two fragments were first amplified with following primers: TP-5m,5′-CTAGCTCGAGACCGGGGACCGCC-3′; TP-GAS3,5′-CTCGCAGACTCTAATCGAACACGTGTG-3′; TP-3m,5′-GTACAAGCTTAGGGCGCTGCCCTCGCCCG-3′; and TP-GAS5,5′-CACACGTGTTCGATTAGAGTCTGCGAG-3′. A second PCR was then performed with the first PCR products using TP-5m and TP-3m as a primer pair. Amplified fragments were then digested with XhoI and HindIII, and introduced in front of a luciferasegene Basic Vector 2. The mutations introduced into GAS were confirmed by DNA sequencing.

U937 cells were plated at a density of 4 × 10⁶ cells/well. Cells were cotransfected with 5μg of luciferase plasmid DNA, 100 ng of pRL-CMV vector (Promega) by lipofection using Lipofectamine (Life Technologies, Inc.). The luciferase activity of the transient transfectant was measured using the Dual-Luciferase assay protocol (Promega) as described previously(18).

Nuclear extracts (6 μg of protein) were prepared as described previously (18) and incubated for 30 min at room temperature in a final volume of 20 μl of reaction mixture containing 20 mm HEPES (pH 7.9), 40 mm KCl, 1 mm MgCl₂, 0.1 mm EGTA,10% (v/v) glycerol, 0.5 mm DTT, 0.1 μg of poly(dI-dC), and 1 × 10⁴cpm of a ³²P-labeled oligonucleotide probe in the absence or presence of competitors (19). When using an anti-STAT1αp91 antibody, 2 μg of antibody was incubated with nuclear extract for 30 min at room temperature before adding the ³²P-labeled oligonucleotide probe. Then the samples were electrophoresed on a 6% polyacrylamide gel(polyacrylamide/bis-acrylamide ratio, 79:1) in a Tris-borate buffer. The gel was directly analyzed using a Fujix BAS 2000 Bioimage Analyzer (Fuji Photo Film Co., Japan). The sequence of TP/GAS oligonucleotide was 5′-ACACGTGTTTGCTTAAAGTCTGCGA-3′ (nt −407 to −431 of the TP gene). The consensus/GAS oligonucleotide was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.). The sequence of the consensus/GAS oligonucleotide was 5′-AAGTACTTTCAGTTTCATATTACTCTA-3′.

Statistical comparisons were performed using Student’s ttest.

We first examined whether three inflammatory cytokines, TNF-α,IL-1α, and IFN-γ, could enhance the expression of TP in human monocytic cells. Western blot analysis with anti-TPantibody showed about a 3-fold increase of TP production over the control when treated with IFN-γ for 24 h (Fig. 1,A). By contrast, TP protein levels increased only slightly when treated with TNF-α or IL-1α. Next, Northern blot analysis was done to determine whether mRNA levels of TP are increased by inflammatory cytokines (Fig. 1,B). TP mRNA levels increased ∼4- to 5-fold over the control when treated with IFN-γ for 6 h and 1.5-fold when treated with TNF-α or IL-1α for 6 h. By contrast, mRNA levels of TP showed no apparent increase when treated for 24 h with any cytokine. Time kinetic analysis showed a maximal increase in mRNA levels of TP at 6 to 12 h after treatment with IFN-γ, and a marked decrease in the mRNA level of TP was observed at 24 h (Fig. 1,C). ELISA also showed the effect of IFN-γ on TP production (Table 1). The TP protein level increased ∼3-fold over the control when treated with IFN-γ. By contrast, TP protein levels only slightly increased when treated with TNF-α or IL-1α.

To determine whether IFN-γ might increase the stabilization of TP mRNA, we examined the turnover rates of TPmRNA in monocytic cells incubated with or without IFN-γ. TP mRNA in both untreated and treated monocytic cells was degraded with similar half-lives of about 7 h in the presence of actinomycin D (data not shown). Exogenous IFN-γ did not appear to alter the stability of TP mRNA, suggesting the involvement of other mechanisms besides mRNA stability in the enhanced expression of the TP gene by IFN-γ.

To determine whether IFN-γ might affect the transcription of the TP gene, we isolated the 5′-flanking region up to −1121 from the transcription initiation site. Consistent with the previous study by Hagiwara et al. (4), seven copies of the SP-1 binding sites were located on the promoter region. We made various deletion constructs of the TP gene promoter and fused them to the reporter luciferase plasmid (Fig. 2,A). Using these reporter plasmids, we examined whether inflammatory cytokines increased the transcriptional activity of the TP gene promoter. Treatment with IFN-γ alone caused a 2.5-fold increase over the basal level of the luciferase activity in cells transfected with pTP-Luc1, but did not cause an increase with pTP-Luc2 and pTP-Luc3 (Fig. 2,B). By contrast, the luciferase activity of pTP-Luc1 did not increase when treated with TNF-α or IL-1α. These results strongly suggest that any element between −1121 and −355 could be responsible for the IFN-γ-dependent promoter activation. IFN-γ phosphorylates the dormant cytoplasmic protein STAT1, which then translocates to the nucleus and binds to GAS(20). Because the consensus GAS sequence was assumed to be TTNCNNNAA, we observed that the sequence TTGCTTAAA was located at −423 bp from the transcription start site, and we named it “TP/GAS”(Fig. 2 A). Moreover, we also observed one ISRE, consistent with a previous report (21).

To determine whether these sequences are necessary for transcriptional activation of the TP gene by IFN-γ, two other deletion constructs were made: pTP-Luc4 containing TP/GAS and pTP-Luc1ΔBS containing TP/ISRE but no TP/GAS (Fig. 2,A). We then examined whether IFN-γ specifically modulates the four TP promoter constructs pTP-Luc1, pTP-Luc2, pTP-Luc4, and pTP-Luc1ΔBS (Fig. 2 C). Treatment with IFN-γ increased by ∼2- to 3-fold the luciferase activity by pTP-Luc1 and pTP-Luc4. However, IFN-γ could not increase the luciferase activity by pTP-Luc2 and pTP-Luc1ΔBS. These data suggested the plausible involvement of the sequences between −474 and−355 in IFN-γ-dependent activation of the TP gene, and that this sequence contains TP/GAS.

Furthermore, the role of TP/GAS in IFN-γ-dependent induction of TP gene promoter activity was investigated in monocytic cells transiently transfected with luciferase reporter plasmids containing promoter sequences with specific mutations in TP/GAS;TTGCTTAAA was converted to TCGATTAGA (pTP-Luc1/mut; Fig. 3,A). As compared with pTP-Luc1, the pTP-Luc1/mut construct showed no decrease in basal transcriptional activity (Fig. 3,B). The luciferase activity of pTP-Luc1 was increased∼3-fold when treated with IFN-γ, whereas only a 1.5-fold increase was observed in pTP-Luc1/mut (Fig. 3 B). The mutations in TP/GAS thus caused more than 70% inhibition on the IFN-γ-dependent promoter activity of the TP gene.

We performed EMSA to investigate whether STAT1 can bind TP/GAS in human monocytic cells. An oligonucleotide corresponding to the sequence from the TP/GAS region of the TP gene was used as a probe in EMSA with nuclear extracts from untreated or IFN-γ-treated cells. No apparent protein-DNA complex appeared in the untreated cells (Fig. 4). By contrast, the binding activity increased when treated with IFN-γ. The binding activity to TP/GAS was almost totally competed by using a 40-fold excess of unlabeled TP/GAS oligonucleotide and unlabeled consensus/GAS oligonucleotide. The antibody against STAT1αp91 blocked the DNA-binding activity of the slower migrating complex, but not that of the faster migrating complex(Fig. 4), suggesting that the upper band corresponds to the specific band of the STAT1αp91-TP/GAS complex. A supershifted complex,however, was not observed when this antibody was used.

Infiltrating activated macrophages are a major component of the stroma responsible for tumor development and neovascularization(22, 23, 24). TNF-α and IL-1α, representative cytokines of activated macrophages, induce angiogenesis through the enhanced expression of various angiogenic factors such as vascular endothelial growth factor, interleukin-8, and basic fibroblast growth factor(17, 25). However, a critical question is: What is the biochemical characteristic marker in activated macrophages in close association with angiogenesis or malignancy in human tumors? Clinical studies have hypothesized that TP expression in monocyte/macrophages could be a diagnostic marker for malignancy,angiogenesis, and prognosis in human breast cancers (13, 14) and melanomas (15, 25).

The promoter region of the TP gene has no TATA box or CCAAT box, but has a high G-C content and seven copies of the SP-1 binding site upstream from the transcription start site. Our previous study showed that TNF-α enhances the promoter activity of the vascular endothelial growth factor gene, which, like TP,contains five SP-1 binding sites, and that deletion of the SP-1 binding site showed that four clustered SP-1 binding sites in the proximal promoter are essential for TNFα-dependent activation(16). We also showed previously that TNF-α-induced up-regulation of the low-density lipoprotein receptor gene is caused by activating SP-1 in vascular endothelial cells (26). It was reported previously that TNF-α strongly induced NF-κB-dependent DNA binding in these monocytic cells (27). We first thought that TNF-α could activate the TP gene expression by SP-1,but we did not observe any apparent up-regulation of the TPgene in monocytic cells by TNF-α. In this study, we observed that TP production was enhanced by exposing monocytic cells to IFN-γ. Schwartz et al. (21) reported that in human colon cancer cells, the IFN-α-induced up-regulation of TP is attributable to both transcriptional activation and increased mRNA stability, and that the transcriptional activation of the TPgene by IFN-α is attributable to increased nuclear factors binding to a putative IFN-α response element. Our study strongly indicated that IFN-γ-induced up-regulation of TP is attributable to only transcriptional activation. And IFN-γ did not affect the TP mRNA stability in monocytic cells.

IFN-γ is a key cytokine in macrophage activation, and it uses the JAK-STAT pathway for its signal transduction. Binding of IFN-γ to its receptor results in tyrosine phosphorylation of the dormant cytoplasmic protein STAT1, which then translocates to the nucleus and binds to GAS(20). Moreover, other DNA-responsive elements mediated by IFN-γ have been reported, such as ISRE, with the consensus sequence GTTTCNNTTTCNC, and γ-IRE, with the consensus sequence CWKKANNY(28, 29). In this study, reporter gene assays showed that the sequence between −474 and −355 has a leading role in the transcriptional activation of the TP gene by IFN-γ. In this region, only TP/GAS was detected, but no other response element was located. Introduction of mutation in this TP/GAS region also abolished the IFN-γ-induced TP promoter activity. This GAS-like element thus appears to have an important role in induction of TP by IFN-γ in monocytic cells. Using EMSA, two DNA-protein complexes appeared, a fast migrating complex and a slow migrating complex. As described in a previous study (24), the antibody against STAT1αp91 disrupted the slow migrating complex instead of supershifting. The disruption cannot be explained clearly, but this antibody may recognize the DNA-binding domain of STAT1αp91. The fast migrating complex may include STAT1αp84, which does not activate IFN-γ-induced transcription (20), but this remains to be studied further.

In conclusion, expression of TP was up-regulated in human monocytic cells when activated in the presence of IFN-γ. One mechanism for this up-regulation of the TP gene seemed to be attributable to transcriptional activation through the TP/GAS element in the TP gene promoter region. The up-regulation of the TP gene in monocyte/macrophages might provide a favorable condition for angiogenesis and malignant state in human tumors. However, the activated macrophages that are infiltrated in tumors have an ability not only to induce the tumor growth and malignancy, but also to block the proliferation of cancer cells (30). These different functions of macrophages might depend on their state of activation and differentiation and also on the condition of the surrounding environment. Additional study is needed to understand the function of tumor-associated macrophages expressing TPin vivo.

We thank the Nippon Roche Research Center for the gift of anti-TP antibody for ELISA. We also thank T. Uchiumi and J. Fukushi(Kyushu University, Fukuoka, Japan) for fruitful discussion.