Expression of the Angiogenic Factor Thymidine Phosphorylase in THP-1 Monocytes: Induction by Autocrine Tumor Necrosis Factor- and Inhibition by Aspirin

The angiogenic factor thymidine phosphorylase (TP) is highly expressed in human monocytes and macrophages, and its expression has been linked to the pathology and progression of solid tumors, rheumatoid arthritis, and gastric ulcers. In this study, TP mRNA and enzyme activity were found to be upregulated upon the induction of differentiation of the human monocyte cell line THP-1 by phorbol 12-myristate 13-acetate (PMA). TP expression in THP-1 cells was similarly increased by tumor necrosis factor(TNF ). Because monocytes and macrophages are a predominant source of TNF , the up-regulation of TP upon THP-1 differentiation could have been caused by the autocrine production of TNF . In support of this hypothesis, PMA increased TNF mRNA levels; furthermore, the increase in TP expression with PMA treatment was partially blocked by a neutralizing antibody to TNF , particularly at the earlier time points. This data also suggested there may be additional mechanisms regulating TP expression upon PMA treatment of the cells. The induction of TP by TNF was mimicked by an antibody to the TNF receptor R2 (TNF-R2; p75), but not by an antibody to TNF-R1 (p55), suggesting that the TNF-R2 plays a role in the regulation of TP expression. The PMA-induced increase in TP expression was blocked by aspirin but not by the related agent indomethacin, suggesting that aspirin’s effect was not caused by the inhibition of cellular cyclooxygenases. An alternative mechanism by which aspirin inhibits gene expression is the modulation of the transcription factor NF B, and the TNF -induced increase in TP mRNA was blocked by a cell-permeable NF B inhibitory peptide. Furthermore, TNF increased and aspirin (but not indomethacin) decreased NF B DNA-binding activity in THP-1 cells. In conclusion, the modulation of TP expression in monocytes by proand anti-inflammatory agents suggests that its angiogenic-related actions could contribute to the inflammatory response associated with a number of pathophysiological conditions. Thymidine phosphorylase (TP; also known as platelet-derived endothelial cell growth factor) is an angiogenic factor that has been found to be chemotactic for endothelial cells and to induce neovascularization in vivo (Miyazono et al., 1987; Ishikawa et al., 1989; Finnis et al., 1993; Sumizawa et al., 1993). These actions are not mediated by TP directly but rather by 2-deoxyribose, a metabolite of the 2-deoxyribose-1phoshophate formed from thymidine via the catalytic activities of TP and cellular phosphatases (Haraguchi et al., 1994; Hotchkiss et al., 2003a,b). In normal human tissues, TP is strongly expressed in macrophages, including Kupffer cells and alveolar macrophages, other stromal cells, glial cells, and more weakly in some epithelia (Fox et al., 1995). TP was found to be frequently overexpressed in human solid tumors compared with adjacent uninvolved tissue, and its expression has been correlated with higher tumor microvessel density, increased tumor invasion and metastasis, and shorter patient survival time (Takebayashi et al., 1996). In human colon and other gastrointestinal tumors, TP overexpression occurred more often in tumor-associated macrophages and other stromal cells compared with expression in the colon cancer epithelial cells. High levels of expression of TP in tumor-associated macrophages have also been observed in human breast, prostate, lung, and brain tumors (Engels et al., 1997; Koukourakis et al., 1998; Lee et al., 1999; Toi et al., 1999; Okada et al., 2001; Yao et al., 2001; Sivridis et al., 2002). These findings suggested that TP in tumor-associated macrophages may play a more direct role in tumor angiogenesis, and it has been hypothesized that tumor cells can amplify their own angiogenic activity by recruiting or activating macrophages, which then express high level of angiogenic factors (Polverini and Leibovich, 1984). This work was supported by grants R01-CA54422, RO1-CA89352, and P01CA13330 from the National Cancer Institute. ABBREVIATIONS: TP, thymidine phosphorylase; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor; PMA, phorbol 12myristate 13-acetate; NF B, nuclear factor B; RT-PCR, reverse transcription-polymerase chain reaction; PBS, phosphate-buffered saline; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; NSAID, nonsteroidal anti-inflammatory drug; COX, cyclooxygenase; GAPDH, glyceraldehyde3-phosphate dehydrogenase. 0026-895X/03/6405-1251–1258$7.00 MOLECULAR PHARMACOLOGY Vol. 64, No. 5 Copyright © 2003 The American Society for Pharmacology and Experimental Therapeutics 2646/1106809 Mol Pharmacol 64:1251–1258, 2003 Printed in U.S.A. 1251 at A PE T Jornals on O cber 3, 2017 m oharm .aspeurnals.org D ow nladed from Elevated levels of TP expression have also been associated with the pathophysiology of other inflammatory diseases, including: 1) rheumatoid arthritis, where TP was found to be highly elevated in synovial fluid and where there was an increase in TP mRNA in cultured rheumatoid arthritis fibroblast-like synoviocytes; 2) psoriasis, where there was an increase in TP expression in psoriatic lesions, including increased TP mRNA in lesional epidermis and increased TP expression in basal keratinocytes and suprabasal layers; and 3) gastric ulcers, in which TP was elevated near gastric ulcer margins compared with uninvolved fundic and pyloric stomach (Takeuchi et al., 1994; Creamer et al., 1997; Kusugai et al., 1997; Muro et al., 1999). Plasma TP was found to be higher in intractable gastric ulcer patients compared with either healthy persons, patients with duodenal ulcers, or patients with gastric ulcer with significant resolution (Kusugai et al., 1997). An increase in TP expression was also noted in interstitial mononuclear infiltrates in scarred kidneys occurring secondary to urinary tract diseases, suggesting that TP plays a role in the inflammatory and/or neovascularization response to renal interstitial fibrosis (Konda et al., 1999). Tumor necrosis factor (TNF ) is an important mediator of inflammatory responses, and it regulates multifunctional cytokines and several cellular functions in a wide variety of cells. A number of genes that can mediate angiogenesis have also been shown to be induced by TNF , including basic fibroblast growth factor, plasminogen activator, platelet-activating factor, angiopoietin, ephrin A1, and VEGF and its receptors (Bussolino et al., 1988; Okamura et al., 1991; Ryuto et al., 1996; Giraudo et al., 1998; Kim et al., 2000; Cheng and Chen, 2001). In our previous study, we found that TNF induced TP expression in human colon cancer WiDr cells by transactivation of the TP gene (Zhu et al., 2002). Activated macrophages are a major source of TNF , and it has been proposed that the angiogenic activity of macrophages was mediated primarily by TNF (Beutler and Cerami, 1986; Leibovich et al., 1987). It was reasonable to hypothesize, therefore, that the angiogenic activity associated with monocyte/macrophages was caused, in part, by the autocrine regulation of angiogenic factor expression by TNF . The THP-1 cell line is often used as a model of human monocytes, and it represents a relatively immature stage of monocyte differentiation. Treatment of THP-1 cells with agents such as lipopolysaccharide or phorbol-12-myristate 13-acetate (PMA) has been shown to both induce further monocytic differentiation of the cells (characterized by increased adhesion, loss of proliferation, and higher CD14 and CD54 expression) and increase the expression and release of TNF by the cells (Sugimoto et al., 1984; Hmama et al., 1999; Rutault et al., 2001). In the present study, we used PMAdifferentiated THP-1 cells as a model to test the hypothesis that an autocrine circuit mediated by TNF regulates TP gene expression in macrophages. TNF mediates its biological effects by interacting with two distinct receptors, p55 (TNF-R1) and p75 (TNF-R2) (reviewed in Vandenabeele et al., 1995). Although these receptors have been shown to share partially overlapping signaling pathways, they can also mediate distinct cellular functions. The role of TNF-R1, independent of TNF-R2, in the TNF -mediated induction of apoptosis and other cellular actions has been well described; recent studies have suggested that TNF-R2 can also mediate cell proliferation and/or apoptosis, both independent of and in conjunction with TNF-R1 (Tartaglia et al., 1991; Vandenabeele et al., 1995; Murray et al., 1997; Grell et al., 1998; Baxter et al., 1999; Chan and Lenardo, 2000; Amrani et al., 2001). A second objective of these studies, therefore, was to begin to discern the signal transduction pathways responsible for the regulation of TP by TNF . The capacity of TNF to induce its pleiotropic effects is attributable partly to its ability to activate the NF B family of transcription factors. Thus, we also investigated whether NF B activation was involved in the regulation of TP expression. These studies included an examination of the effect on TP expression of aspirin, which at higher therapeutic concentrations has been shown to inhibit the activation of NF B through the stabilization of its inhibitory protein, I B (Kopp and Ghosh, 1994; Yin et al., 1998). Materials and Methods Cell Lines and Reagents. Human monocyte THP-1 cells (American Type Culture Collection, Manassas, VA) were maintained in RPMI 1640 medium with 10% fetal bovine serum and gentamicin in a humidified CO2 incubator at 37°C. Anti-TP antibody was from Oncogene Research Products (San Diego, CA); anti-TNF , anti-TNFR1, and anti-TNF-R2 antibodies, and recombinant hTNF were purchased from R&D Systems (Minneapolis, MN); anti-p65 NF B (Rel A) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). PMA, aspirin, and indomethacin were from Sigma (St. Louis, MO); the NF B cell-permeable inhibitor peptide SN50 and the inactive control peptide SN50M were from Calbiochem (San Diego, CA). Measurement of TP and TNF mRNA Levels. RNA was isolated from THP-1 cells using TRIzol reagent (Invitrogen, Carlsbad, CA). TP, TNF , and GAPDH mRNA levels were determined by RT-PCR, as described previously (Zhu et al., 2002). Briefly, 2 g of total RNA was reverse transcribed into cDNA with 200 units of Maloney leukemia transcriptase (Invitrogen) in 20 l of reaction buffer containing 10 units of RNasin, 0.2 g of random primers, and 0.8 mM dNTPs at 42°C for 1 h. Reactions were terminated by heating at 95°C for 10 min. The mixture was diluted 2.5 times with RNasefree water. An aliquot (2.5 l) was used for PCR amplification with primer for TP: sense, 5 -GCTTCGTGGCCGCTGTGGTG-3 ; antisense, 5 -TCTGCTCTGGGCTCTGGATGA-3 ; TNF : sense, 5 -GTCTACTTTGGGATCATTG-3 ; antisense, 5 -TCAGGGATCAAAGCTGTA-3 GAPDH: sense, 5 -CATCTCTGCCCTCTGCTG-3 ; antisense, 5 -CCCTCCGACGCCTGCTTCAC-3 . The TP primer corresponds to sequences in exons 2 and 4 of the human genomic TP sequence. Reactions contained 25 l of 10 mM Tris-HCl, pH 8.3, containing 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.4 M of each primer, and 1.25 units of Taq DNA polymerase. The TP reaction proceeded for 25 cycles of denaturation at 94°C for 1 min, annealing at 56°C for 1 min, and extension at 72°C for 1 min. Amplified cDNAs were electrophoresed on 2% agarose gels containing ethidium bromide, gels were photographed, and bands were scanned and quantitated by densitometry using ImageQuant (Amersham Biosciences, Piscataway,