Peroxynitrite inhibits the activity of ornithine decarboxylase

Life Sciences 68 (2001) 1477–1483

Peroxynitrite inhibits the activity of ornithine decarboxylase Edward R. Seidel, Vernon Ragan, Lin Liu* Department of Physiology, Brody School of Medicine East Carolina University, Greenville, NC 27858, U.S.A. Received 11 July 2000; accepted 6 October 2000

Abstract Polyamines are required during cell proliferation, whereas NO has anti-proliferative properties. Ornithine decarboxylase (ODC) is a critical enzyme for the synthesis of polyamines. We tested the hypothesis that the modification of ODC by peroxynitrite (OONO2), a short-lived free radical formed from NO and superoxide produces a fall in ODC activity, and therefore polyamine synthesis and cell proliferation. The treatment of a rat recombinant ODC (rODC) with OONO2 resulted in a dose-dependent inhibition of rODC activity with an IC50 of approximately 100 mM. A Western blot employing a specific antibody to nitrotyrosine revealed a dose-dependent nitration of rODC tyrosine residues. When intact IEC-6 cells were treated with ONOO2, ODC activity decreased by 49%. These data suggest a correlation between ODC activity and nitration, and a possible mechanism by which NO synthesis may modulate polyamine synthesis. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Ornithine decarboxylase; Peroxynitrite; Polyamines; Nitrotyrosine

Introduction The enzyme ornithine decarboxylase (ODC) is a tightly regulated, highly inducible enzyme which synthesizes the parent polyamine putrescine by decarboxylating the amino acid ornithine. Arginase converts arginine to ornithine during urea cycle transition. Most growth factors, hormones and other mitogenic stimuli produce an increase in ODC activity that occurs during the G1 phase of the cell cycle. Mitogenic factors control enzyme induction at the levels of ODC gene transcription, mRNA translation, and protein stability although posttranscriptional events appear to be of primary importance [1;2]. ODC has a half-life of only ten to thirty minutes in most systems and the peak in enzyme activity is short-lived. The mechanisms regulating the degradation of ODC are less well documented than those that regulate enzyme induction. However, the protein ornithine decarboxylase antizyme (ODC* Corresponding author: Department of Physiological Sciences, 264 McElroy Hall, Oklahoma State University, Stillwater, OK 74078. Tel.: (405) 744-4526; fax: (405) 744-8263. E-mail address: [email protected] (L. Liu) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 0 9 4 1 -9

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AZ) plays a major role. (for review see [3]). AZ binds ODC and directs translocation of the enzyme to the 26S proteosome whereupon ODC is degraded. While the action of ODC-AZ is of consequence in the inhibition of ODC activity, other posttranslational events may also participate. Like ODC, nitric oxide (NO) is also synthesized from the amino acid arginine by a family of enzymes: nitric oxide syntheases (NOS). NO can be synthesized in the gut by the constutitive NOS isoform in numerous intestinal tissues including epithelium, mast cells and smooth muscle under resting conditions and by the inducible NOS isoform in leukocytes under inflammatory conditions [4;5]. NO has been shown to inhibit cell proliferation in a number of cells [6–8], whereas polyamines are intimately involved in G1 progression and proliferation. In line with these reciprocal effects on cell proliferative status, increases in ODC activity are accompanied by a fall in NO synthesis while during periods of high NO synthesis, polyamine synthesis falls to low levels. The mechanisms underlying these reciprocal events remain to be determined experimentally. Ignarro and coworkers [9] have recently demonstrated that NO inhibits ODC activity by S-nitrosylation of an unidentified but critical cysteine residue. We tested the hypothesis that an alternative regulatory event involved in coordinating polyamine and NO synthesis is the modification of ODC by peroxynitrite (OONO2), a short-lived oxygen radical formed by the reaction of NO and superoxide. In contrast to the S-nitrosylation produced by NO, OONO2 nitrates tyrosine residues. The modification of ODC by ONOO2 should produce a fall in ODC activity and thereby inhibit polyamine synthesis and ultimately cell cycle progression and cell proliferation. Materials and methods Materials [14C]ornithine was purchased from NEN (Boston, MA). Anti-nitrotyrosine antibodies were from Upstate (Lake Placid, NY). Enhanced chemiluminescence (ECL) reagents were from Amersham (Arlington Heights, IL). A pGEX plasmid vector was from Pharmacia Biotech (Boston, MA). Synthesis of ONOO2 ONOO2 was synthesized from acidified nitrite and hydrogen peroxide according to the method of White et al. [10]. Excess hydrogen peroxide was removed by passing the solution through a granular manganese dioxide column. ONOO2 concentration was determined spectrophotometrically using an extinction coefficient («302 nm) of 1670 M21cm21. Purification of recombinant ODC A recombinant ODC (rODC) protein was synthesized in E. coli. A 2.4 Kb rat ODC cDNA containing a coding segment of 1383 nucleotides [11;12] was generously provided by Dr. J. L. Mitchell, Northern Illinois University, and ligated into a pGEX plasmid vector. The resultant glutathione S-transferase/ODC fusion protein was purified from bacterial lysates by affinity chromatography using glutathione sepharose 4B. rODC was cleaved from the fusion protein by overnight incubation with thrombin at room temperature. ODC/GST fusion pro-

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teins and thrombin-purified ODC yielded similar rates of ornithine decarboxylase and most experiments were performed using fusion protein. ONOO2 treatment of rODC 1 ml of concentrated stock ONOO2 was added to 0.7 mg of rODC/GST in 50 ml 0.1 M sodium phosphate buffer, pH 7.0 to make up the final concentrations of ONOO2 indicated in the figures. The mixture was incubated at room temperature for 10 min and ODC activity assay or Western blot analysis was performed. For the decomposition of ONOO2, 1 ml of 50 mM ONOO2 was added to 50 ml of buffer and incubated for 2–3 hours before rODC was added. ODC activity assay ODC activity was measured as previously described [1;2]. Briefly, 0.7 mg rODC/GST fusion protein per assay point or a 30,000 3g (30 min) supernatant of cell homogenate was incubated in the presence of [14C]ornithine (spec act 50 mCi/mmol), pyridoxal 59-phosphate, and Cleland’s reagent at 378 C for 2 hrs and the rate of 14CO2 generation estimated after scintography. Western blot analysis To detect nitrotyrosine formation in ONOO2-treated rODC, the samples were separated on 10 % SDS-PAGE and transferred to a nitrocellulose membrane. The blot was probed using anti-nitrotyrsine antibodies and horseradish peroxide-conjugated goat anti-rabbit IgG and visualized with ECL reagents. Cell culture The rat epithelial cell line IEC-6 was cultured as previously described [1;2]. Subconfluent cultures were serum-deprived for 24 hrs and treated with 5% fetal bovine serum (FBS) for the subsequent 4 hr period to induce ODC enzyme activity. At the end of the incubation, the medium was changed to modified PBS (50 mM Na2HPO4, 90 mM NaCl, 5 mM KCl, 0.8 mM MgCl2, 1 mM CaCl2 and 5 mM glucose, pH 7.4). Cells were treated with 1 mM ONOO2 or decomposed ONOO2 for 10 min. Cells were then collected for ODC activity assay. Results Recombinant ODC was incubated with increasing concentrations of OONO2 for 10 min at room temperature. ONOO2 inhibited rODC activity in a dose-dependent manner (Figure 1). The IC50 for OONO2 induced inhibition of rODC enzyme activity was approximately 100 mM and concentrations of OONO2 above 1 mM produced a near complete inhibition of enzyme activity. In contrast, OONO2 which had been decomposed prior to use was without effect on activity of rODC. To examine whether cofactor or substrate influenced ONOO2 inhibition of rODC activity, rODC was first pre-incubated with 0.1 mM pyridoxal 59-phosphate or 3 mM ornithine or both for 10 min, followed by the addition of 1 mM ONOO2. In all cases, a similar percent in-

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Fig. 1. Effect of peroxynitrite (OONO2) on rODC activity. Data are presented as per cent of control rODC activity which was not incubated with OONO2. DECOMP represents rODC that was incubated with 1 mM decomposed ONOO2. Each bar represents the mean 6 SEM of six separate determinations.

hibition of approximately 90% was observed (data not shown), indicating that neither cofactor nor substrate can provide protection of rODC protein from ONOO2 inhibition. ONOO2 is a strong nitrating agent. Therefore, ONOO2 inhibition of rODC activity could be due to the nitration of tyrosine residues in rODC protein. To test the possibility, we determined the formation of nitrotyrosine in rODC treated with ONOO2 by Western blot. As shown in Figure 2, nitrotyrosine levels in rODC were increased as a function of ONOO2 concentrations, which roughly correspond to the fall in rODC activity. The effect of ONOO2 treatment on ODC activity in intact cells was also evaluated. Under basal conditions provided by a 24 hr serum deprivation, putrescine was decarboxylated at a rate of only 6.8 pmol CO2/mg protein-hr. Four hours of 5% FBS stimulation resulted in a 16fold induction in ODC activity to a level of 111.8 pmol CO2/mg protein-hr (Figure 3). Pretreatment of cells with 1 mM OONO2 inhibited enzyme activity by 49% to a level of 57.0 pmol CO2/mg protein-hr. Decomposed ONOO2 had only a minor effect. Discussion The amino acid arginine serves a common precursor molecule in the synthesis of polyamines and NO. These two products have opposite effects on cell proliferation. The present study shows that ONOO2, a product of excess NO and superoxide, nitrates ODC protein and inhibits ODC activity. Furthermore, this inhibition was observed in the rat duodenal

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Fig. 2. Nitration of rODC. rODC was treated with various concentrations of ONOO2 or 1 mM decomposed ONOO2 (DECOMP) and analyzed by Western blot using anti-nitrotyrosine antibodies.

mucosa-derived cell line, IEC-6 cells treated with ONOO2. The results support our hypothesis that overproduction of NO leads to the formation of ONOO2, the inhibition of ODC activity and a fall in polyamine synthesis. The polyamines are a group of ubiquitously distributed organic cations, which are intimately involved in regulation of growth of the gastrointestinal mucosa. Treatment of cells of gastrointestinal origin with difluoromethylornithine, a suicide substrate inhibitor of ODC which induces depletion of intracellular polyamines, inhibits proliferation [13]. Conversely, intraluminal infusion of the parent polyamine putrescine into the rat ileum induces mucosal proliferation. Sixty-six hrs of putrescine infusion produced an approximate 1.5-fold increase in ileal mucosal cellularity [14]. NO is an intracellular signalling molecule and plays an important role in many physiological functions. However, it can be toxic when generated in excess. NO is considered as an antiproliferative agent. For example, NO donors have been shown to inhibit proliferation of Caco-2 and HT-29 human colon carcinoma cells as well as the transformed kidney proximal tubule cell line [6–8]. However, the mechanisms for this inhibition are still not fully understood. One possibility is that NO attacks the enzymes involved in the synthesis of polyamines. ODC is the first enzyme in polyamine biosynthesis. NO was reported to inhibit ODC activity in a number of cell lines including Caco-2, HT-29 and MCT cells (6–8). Inactivation of ODC by NO was

Fig. 3. Effect of peroxynitrite (OONO2) on serum (FBS) induction of ODC in intact IEC-6 cells. Decomposed OONO2 (DECOMP) is included as negative control. For treatment conditions see Materials and Methods. Each bar represents the mean 6 SEM of six determinations. * P,0.05 vs FBS alone.

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also observed using purified ODC (9). Since dithiothreitol reverses NO-mediated inhibition of ODC activity, it was thought that the inhibition is likely due to the modification of the cysteine residues by S-nitrosylation [9]. The present study provides an additional mechanism for NO inhibition of ODC activity, i.e. nitration of tyrosine residues of ODC by ONOO2. ONOO2 is a potent oxidant that causes sulfhydroyl oxidation, lipid peroxidation and tyrosine nitration. ONOO2 reacts with biological molecules and contributes to the pathophysiology of a large variety of diseases associated with inflammation. Increased levels of nitrotyrosine, an indicator of ONOO2 formation, were observed in the some of these diseases [15]. In addition, the nitration of many important proteins including annexin II, surfactant protein A, glutamine synthetase and superoxide dismutase leads to the inhibition of protein function [16–19]. Treatment of rODC with ONOO2 results in a decrease of rODC activity and nitration of the protein. However, neither the ODC cofactor, pyridoxal 59-phosphate, nor its substrate, the amino acid ornithine provided protection of the rODC enzyme from ONOO2mediated inhibition, suggesting that the tyrosine residues in the binding sites of substrate or cofactor are not involved in enzyme activity inhibition. This modification likely also occurs in intact cells, because ODC activity was inhibited in intact IEC-6 cells when treated with ONOO2. However, it is noteworthy that the ONOO2 independent pathways for the formation of nitrotyrosine also exist [20]. Acknowledgments This work was supported in part by grants from NIH HL52146 and North Carolina Institute of Nutrition. We thank Dr. J. L. Mitchell (Northern Illinois University) for kindly providing ODC cDNA clone.

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