ANA-1 Murine Macrophages Ribosomal Peptidyl Transferase Activity in Cleavage Is Associated with Inhibition of Nitric Oxide-Depen

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Nitric Oxide-Dependent Ribosomal RNA Cleavage Is Associated with Inhibition of Ribosomal Peptidyl Transferase Activity in ANA-1 Murine Macrophages This information is current as of October 2, 2021. Charles Q. Cai, Hongtao Guo, Rebecca A. Schroeder, Cecile Punzalan and Paul C. Kuo J Immunol 2000; 165:3978-3984; ; doi: 10.4049/jimmunol.165.7.3978

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Nitric Oxide-Dependent Ribosomal RNA Cleavage Is Associated with Inhibition of Ribosomal Peptidyl Transferase Activity in ANA-1 Murine Macrophages1

Charles Q. Cai,* Hongtao Guo,* Rebecca A. Schroeder,† Cecile Punzalan,* Paul C. Kuo2*

NO can regulate specific cellular functions by altering transcriptional programs and protein reactivity. With respect to global cellular processes, NO has also been demonstrated to inhibit total protein synthesis and cell proliferation. The underlying mech- anisms are unknown. In a system of ANA-1 murine macrophages, iNOS expression and NO production were induced by exposure to endotoxin (LPS). In selected instances, cells were exposed to an exogenous NO donor, S-nitroso-N-acetylpenicillamine or a substrate inhibitor of NO synthesis. Cellular exposure to NO, from both endogenous and exogenous sources, was associated with a significant time-dependent decrease in total protein synthesis and cell proliferation. Gene transcription was unaltered. In parallel with decreased protein synthesis, cells exhibited a distinctive cleavage pattern of 28S and 18S rRNA that were the result of two Downloaded from distinct cuts in both 28S and 18S rRNA. Total levels of intact 28S rRNA, 18S rRNA, and the composite 60S ribosome were significantly decreased in the setting of cell exposure to NO. Finally, 60S ribosome-associated peptidyl transferase activity, a key enzyme for peptide chain elongation, was also significantly decreased. Our data suggest that NO-mediated cleavage of 28S and 18S rRNA results in decreased 60S ribosome associated peptidyl transferase activity and inhibition of total protein synthesis. The Journal of Immunology, 2000, 165: 3978–3984. http://www.jimmunol.org/ xpression of inducible NO synthase (iNOS)3 protein in ation in global gene transcription. However, induction of iNOS settings rich in pro-inflammatory cytokines or endotoxin and inhibition of total protein synthesis in this cell line are asso- E is associated with multiple alterations in cellular function. ciated with a specific and reproducible cleavage pattern in 28S and These include inhibition of protein synthesis, induction of cytosta- 18S rRNA that is both NO- and time-dependent. We examined sis, modification of transcriptional programs, inhibition of mito- enzymatic function of the 60S ribosome, which is largely com- chondrial respiration, and altered cellular redox state (1). In some posed of intact 28S and 18S rRNA. Levels of 60S ribosome and instances, the underlying mechanism is related to NO-mediated 60S ribosome-associated peptidyl transferase activity, an essential S-nitrosation of key protein thiol groups with subsequent alteration part of peptide chain elongation are both significantly depressed in in enzymatic function or protein binding properties (2, 3). Alter- the settings of both LPS-mediated endogenous NO synthesis and by guest on October 2, 2021 natively, NO alters the cellular redox balance by virtue of its ox- exposure to an exogenous NO donor, S-nitroso-N-acetylpenicilla- idative properties, and induces downstream counterregulatory mine (SNAP). We conclude that NO-mediated inhibition of total events (4, 5). However, the association between high concentra- protein synthesis is the result of decreased 60S ribosome-associ- tions of NO, as expressed by iNOS, and inhibition of total protein ated peptidyl transferase activity. synthesis has not been extensively examined. The effect of NO upon global cellular translation, gene transcription, or cellular ar- Materials and Methods chitecture critical to the protein synthetic process is unknown. In Cell culture particular, the effect of NO upon the ribosomal scaffolding, which is critical to initiation and potentiation of protein translation, has ANA-1 murine macrophages, a gift from Dr. George Cox (Uniformed Ser- vices University of the Health Sciences, Bethesda, MD), were maintained not been examined. in DMEM with 5% heat-inactivated FCS. LPS (Escherichia coli serotype In this study, using a model of endotoxin-mediated iNOS ex- 0111:B4; 0–10,000 ng/ml) was added to the medium to induce NO syn- pression in ANA-1 murine macrophages, we demonstrate that NO thesis. In selected instances, the competitive substrate inhibitor of NOS, G inhibits total protein synthesis and cell proliferation without alter- N -nitro-L-arginine methyl ester (L-NAME; 4 mM), alone or together with LPS and the NO donors, SNAP or S-nitroso-N-acetylcysteine were added.

Cells were harvested after incubation for 2–20 h at 37°C in 95% O2/5% CO2. To determine cell viability, cells were washed with ice-cold PBS *Departments of Surgery and †Anesthesiology, Georgetown University Medical Cen- three times, stained with 0.2% trypan blue solution (w/v) for 10 min, and ter, Washington, D.C. 20007 viable cells were counted under the light microscope. Cell viability was Ͼ Received for publication February 8, 2000. Accepted for publication July 14, 2000. routinely 95% under all treatment conditions. 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 Determination of NO synthesis with 18 U.S.C. Section 1734 solely to indicate this fact. NO release from cells in culture was quantiﬁed by measurement of the NO 1 This work was supported by the Clowes Faculty Development Award from the metabolite, nitrite, using the technique of Snell and Snell (6). To reduce American College of Surgeons (to P.C.K.) and National Institutes of Health Grant nitrate to nitrite, 200 ␮l conditioned media was incubated in the presence AI44629 (to P.C.K.). of 1.0 U nitrate reductase, 50 ␮M NADPH, and 5 ␮M ﬂavin adenine 2 Address correspondence and reprint requests to Dr. Paul C. Kuo, Georgetown Uni- dinucleotide. Sulfanilamide (1%) in 0.5N hydrochloric acid (50% v/v) was versity Medical Center, Department of Surgery, 4 PHC, 3800 Reservoir Rd, N.W., then added. After a 5-min incubation at room temperature, an equal volume Washington, D.C. 20007. E-mail address: [email protected] of 0.02% N-(1-(naphthyl))ethylenediamine was added; following incuba- 3 Abbreviations used in this paper: iNOS, inducible NO synthase; SNAP, S-nitroso- tion at room temperature for 10 min, absorbance at 570 nm was compared G N-acetylpenicillamine; L-NAME, N -nitro-L-arginine methyl ester. with that of a NaNO2 standard.

RNA and DNA extraction and Northern blot analysis crocentrifuge. The supernatant formed the total ribosomal fraction and a portion was used for peptidyl transferase activity. The remaining superna- Total RNA was purified from cultured ANA-1 macrophages with the use tant was layered onto a linear 10–45% sucrose gradient containing 25 mM of TRIzol Reagent (Life Technologies, Rockville, MD). The purified total Tris-HCl, 80 mM KCl, 4 mM MgCl , and 20 mM sodium fluoride. Fol- ␮ 2 RNA (5 g) was subjected to electrophoresis on a 1% (w/v) agarose gel for lowing centrifugation for4hinaBeckman (Fullerton, CA) SW-41 rotor, 3 h at 80 v. The RNA was blotted onto Nytran nylon transfer membrane ribosomal profiles were monitored by continuously measuring A280. (Schleicher & Schuell, Keene, NH) with 10ϫ SSC (1.5 M NaCl/0.15 M sodium citrate) prepared with diethyl pyrocarbonate-treated water, then Ribosome-associated peptidyl transferase activity cross-linked to the membrane by UV radiation. cDNA probes complementary to murine 28S and 18S rRNA were prepared by PCR amplification Peptidyl transferase activity was measured in ANA-1 macrophage total with primers based on their cDNA sequence and randomly labeled with ribosomal fractions using a peptidyl transferase reaction with full-length [32P]dCTP. Hybridization of the labeled probe was performed in hybrid- formyl-[3H]Met RNA as a donor substrate (7). Typically, cell fractions ization solution (50% formatted, 5ϫ SSC, 5 mM EDTA, 20 mM sodium were incubated with 2.5 pmol formyl-[3H]Met RNA in 40 ␮l buffer con- phosphate buffer (pH 7), 1% SDS, 200 ␮g/ml salmon sperm DNA, and 5ϫ taining 20 mM Tris-HCl (pH 8.0), 400 mM KCl, 20 mM MgCl2,and1mM Denhardt’s solution) at 42°C overnight with3hofprehybridization at puromycin. Reactions were initiated by adding 20 ␮l cold methanol and 42°C. The blots were washed briefly with 2ϫ SSC containing 0.1% SDS incubated for 20 min at 0°C. To terminate the reaction, 10 ␮l4MKOH at room temperature, once for 15 min with 0.5ϫ SSC containing 0.1% SDS was added and incubated for an additional 20 min at 37°C. After the ad- ␮ at 42°C, and once for 15 min with 0.2ϫ SSC containing 0.1% SDS at 50°C dition of 200 l1MKH2PO4, the reaction mixture was extracted with 1 and 65°C. Autoradiographs were exposed at room temperature. Genomic ml ethyl acetate. A portion of the extract was mixed with scintillation DNA was purified from cultured ANA-1 macrophages with the DNAzol mixture and counted. Peptidyl transferase activity is expressed as fmol/mg Reagent (Life Technologies). The purified genomic DNA were subjected to protein/min. electrophoresis on a 0.6% (w/v) agarose gel for3hat100V. Statistical analysis

Determination of protein synthesis Downloaded from Data are presented as mean Ϯ SEM of three or four experiments each Cells were washed with methionine-free DMEM three times and incubated performed in triplicate. Statistical analysis was performed using the Stu- for 20 min in short-term medium (methionine-free DMEM containing 5% dent t test or rank sum test, as appropriate. dialyzed FCS) in a humidiﬁed 37°C, 5% CO2 incubator to deplete intra- cellular pools of methionine. Medium was then replaced with fresh short- Results term labeling medium containing 10 ␮Ci/ml [35S]methionine. After 15-, 30-, 60-, 120-, and 240-min labeling, cells were washed with PBS twice LPS and NO synthesis in ANA-1 macrophages

and lysed in TE buffer supplied with 1% SDS. The cell lysis was then Following stimulation with 10–10,000 ng/ml LPS for a period of http://www.jimmunol.org/ passed through a 21 gauge needle several times. A total of 50 ␮l labeled cell lysis was added to 0.5 ml BSA (0.1 mg/ml) on ice and an equal amount 20 h, ANA-1 murine macrophage production of NO was deter- (0.5 ml) of ice-cold 20% trichloroacetic acid was added. The solution was mined by measuring levels of nitrite and nitrate in the culture me- vortexed vigorously and incubated for 30 min on ice. The precipitate was dia (Fig. 1). The dose response was plotted in a semilogarithmic collected on filter papers and was washed once with ice-cold 10% TCA and fashion. In the absence of LPS, the nitrite level in the media was twice with 95–100% ethanol. When dry, the filter papers were transferred 9.9 Ϯ 1.0 nmol/mg protein. NO production increased in a signif- to a scintillation vial containing 4 ml scintillation fluid. Radioactivity was then measured. icant dose-related fashion at LPS concentrations of 10, 100, 1,000, and 10,000 ng/ml ( p Ͻ 0.01 vs control for LPS ϭ 100, 1,000, and Cell proliferation assays 10,000 ng/ml). The competitive substrate inhibitor L-NAME (4 mM) by guest on October 2, 2021 ANA-1 macrophages were labeled with 5 ␮Ci/ml [3H]thymidine for 20 h was added. In the presence of L-NAME alone, nitrite production was in the presence or absence of LPS. The plates were treated with 0.5 ml/well not significantly altered from that of control cells (10.1 Ϯ 1.1 nmol/ ice-cold 10% trichloroacetic acid and kept at 4°C overnight. The plates were washed three times with distilled water and dried at room temperature. Cells were collected with 0.5 ml/well 0.33 mM NaOH and 300 ␮1 solution was transferred into a scintillation vial containing scintillation fluid. [3H]Thymidine incorporation into DNA was measured using a scintillation counter. Nuclear run-on analysis A total of 100 ␮l ANA-1 macrophage nuclei was incubated for 5 min at 30°C with 150 ␮Ci of [32P]rUTP (800 Ci/mmol) in 100 ␮l 10 mM Tris HCl (pH 8.0), 5 mM MgCl2, 300 mM KCl, and 5.0 mM (each) ATP, CTP, and GTP. Labeled RNA was isolated by the acid-guanidinium thiocyanate method. Before ethanol precipitation, labeled RNA was treated with 0.2 M NaOH for 10 min on ice. The solution was neutralized by the addition of HEPES (acid free) to a final concentration of 0.24 M. After ethanol precipitation, the RNA pellet was re-suspended in 10 mM N-tris(hydroxy- methyl)methyl-2 minoethanesulfonic acid (pH 7.4), 0.2% SDS, and 10 mM EDTA. Target DNA for cyclophillin, ␤-actin, and GAPDH was spotted onto nylon membranes with a slot blot apparatus. ␭-Bacteriophage DNA served as negative controls. Hybridization was performed at 42°C for 48 h with 5 ϫ 106 cpm of labeled RNA in hybridization buffer (50% formamide, 4ϫ SSC, 0.1% SDS, 5ϫ Denhardt’s solution, 0.1 M sodium phosphate (pH 7.2), and 10 ␮g/ml salmon sperm DNA). After hybridization, the membranes were washed twice at room temperature in 2ϫ SSC and 0.1% SDS, and three times at 56°C in 0.1ϫ SSC and 0.1% SDS. The membranes were then exposed to x-ray film and scanned. Cellular ribosome profile FIGURE 1. NO dose-response relationship in LPS-treated ANA-1 mu- ANA-1 murine macrophages were isolated in ice-cold PBS. The cells were pelleted at maximum speed in a microcentrifuge and resuspended in hy- rine macrophages. Murine macrophages were exposed to 1–10,000 ng/ml LPS for a period of 20 h. In selected instances, 4 mM L-NAME was also potonic buffer (40 mM Tris-HCl (pH 7.4), 20 mM KCl, 3 mM MgCl2,20 mM sodium fluoride, and 150 mM sucrose) and kept on ice for an addi- added as a competitive substrate inhibitor of NO synthesis. Results repre- p Ͻ 0.01; LPS vs LPS ϩ ,ء .tional 10 min. Following cell lysis in 1% Triton X-100 and 1% deoxy- sent data from four separate experiments cholate, the preparation was vortexed and centrifuged for 1 min in a mi- L-NAME. ANOVA for LPS curve, p ϭ 0.0005. 3980 NO AND RIBOSOMAL RNA DEGRADATION

mg). In cells treated with LPS ϩ L-NAME, NO production was ab- lated at all concentrations of LPS (10–10,000 ng/ml) and did not signiﬁcantly differ from that of control cells. In addition, trypan blue exclusion and media levels of lactate dehydrogenase were determined as measures of cell viability. Under all treatment conditions, cell viability was routinely Ͼ95%, and LDH levels did not differ among the various treatment groups. These data indicate that LPS-mediated NO production is dose-dependent and does not alter cell viability. Subse- quent studies use 100 ng/ml LPS and 4 mM L-NAME, unless stated otherwise.

Cell proliferation and total protein synthesis Following a 20-h incubation, total protein synthesis was determined by [35S]methionine incorporation in control, LPS-, LPS ϩ L-NAME-, and SNAP 400 ␮M-treated macrophages (Fig. 2A). In all instances, there was a significant linear, time-dependent increase in protein synthesis. In LPS-treated cells, total protein synthesis was consistently decreased by 40–70% at all time points when compared with those of control and LPS ϩ L-NAME ( p Ͻ Downloaded from 0.05 vs control and LPS ϩ L-NAME). The greatest incremental difference in protein synthesis was present following a 15-min pulse. Protein synthesis in control and LPS ϩ L-NAME cells was not significantly different. In SNAP-treated cells, total protein synthesis was significantly decreased at all time points, in comparison to control and LPS ϩ L-NAME and also, when compared with LPS http://www.jimmunol.org/ alone ( p Ͻ 0.05 vs control, LPS ϩ L-NAME, and LPS). When cells were incubated in the presence of LPS ϩ L-NAME ϩ SNAP, the protein synthesis curve was not statistically different from that of SNAP alone (data not shown). These data suggest that NO, whether from an exogenous or endogenous source, is associated with significant depression of total protein synthesis in ANA-1 murine macrophages. The assay was then repeated by determination of [35S]methi-

onine incorporation following a 240-min pulse while varying the by guest on October 2, 2021 duration of LPS stimulation (100 ng/ml), 0, 8, 12, 16, and 20 h (Fig. 2B). There was no difference in protein synthesis following 8 h of LPS stimulation. However, at LPS incubation times greater than 12 h, protein synthesis progressively decreases in a signiﬁcant fashion ( p Ͻ 0.05 vs control and LPS ϩ L-NAME for 16 and 20 h). The time dependence of total NO synthesis was also determined and plotted in Fig. 2B. NO production increases dramati- cally following 12 h of LPS stimulation and reaches a near maxium after 20 h of LPS stimulation. The time course of this increase in NO is associated with a parallel decline in total protein synthesis. When [35S]methionine incorporation is plotted vs nitrite from LPS stimulated cells, an inverse linear relationship (r 2 ϭ 0.70) is present (Fig. 2C). This suggests that NO production is inversely associated with decreased protein synthesis in LPS stimulated macrophages.

rected for total cell protein. In selected instances, 4 mM L-NAME or 400 ␮M SNAP were added. Results represent data from four separate experi- ;p Ͻ 0.05; LPS vs control and LPS ϩ L-NAME. #, p Ͻ 0.05 ,ء .ments SNAP vs LPS, control, and LPS ϩ L-NAME. B, Total cellular protein synthesis and NO production in ANA-1 murine macrophages. ANA-1 murine macrophages were exposed to 100 ng/ml LPS for a designated period of incubation and pulsed with [35S]methionine. Radioactivity was determined following 2 h and corrected for total cell protein. Nitrite production was simultaneously determined in LPS-treated cells. Results represent data FIGURE 2. A, Total cellular protein synthesis in ANA-1 murine mac- from three separate experiments (p Ͻ 0.05, LPS vs control and LPS ϩ rophages. ANA-1 murine macrophages were exposed to 100 ng/ml LPS for L-NAME). C, Total cellular protein synthesis and NO production in 100 a period of 20 h and pulsed with [35S]methionine. Radioactivity was de- ng/ml LPS-treated ANA-1 murine macrophages. First order linear regres- termined following the designated time period; measured values were cor- sion analysis was performed (r 2 ϭ 0.70). The Journal of Immunology 3981

ANA-1 cell proliferation in the setting of LPS-induced NO synthesis was determined by the incorporation of [3H]thymidine (Fig. 3). LPS-mediated NO synthesis was associated with a signiﬁcant 50% decrease in tritiated thymidine incorporation following a 20-h incubation in the presence of LPS ( p Ͻ 0.05 vs control and LPS ϩ L-NAME). These data suggest that LPS-mediated NO synthesis inhibits total protein synthesis and cell proliferation in this model of ANA-1 murine macrophages. Total protein synthesis initially declines following 12 h of exposure to NO.

LPS-mediated NO production and gene transcription To determine the role of NO production in ANA-1 macrophage FIGURE 4. Nuclear run-on analysis in ANA-1 murine macrophages. gene transcription, nuclear run-on analysis was performed using Nuclear run-on studies were performed in ANA-1 murine macrophages exposed to 100 ng/ml LPS and/or 4 mM L-NAME for 20 h. Housekeeping ␤-actin, GAPDH, and cyclophillin as target “housekeeping” genes genes for ␤-actin, GAPDH, and cyclophillin were used to sample global (Fig. 4). Following 20 h of LPS incubation, there was no difference gene transcription. The blot is representative of five experiments. in gene transcription for any of the target genes tested. Trans- cription was not significantly different among control, LPS-, L-NAME-, and LPS ϩ L-NAME-treated cells. These results indi- Downloaded from cate that inhibition of total protein synthesis is not the result of of rRNA was exhibited in all cells treated with LPS. In contrast, decreased global gene transcription in LPS-treated ANA-1 murine the typical rRNA pattern was found in control, L-NAME-, and LPS macrophages. ϩ L-NAME-treated cells. A transition in the intensity of RNA degradation was noted between LPS concentrations of 50–200 ng/ rRNA expression patterns ml. The experiments were then repeated using LPS concentrations The pattern of total RNA expression was then examined in this of 50, 100, 150, 200, and 250 ng/ml to better characterize the experimental model (Fig. 5A). In control cells, the typical electro- transition (Fig. 6B). No distinct transition corresponding to a spe- http://www.jimmunol.org/ phoretic pattern of 28S and 18S rRNA expression was found. In cific LPS concentration was evident from these experiments. ␮ ␮ contrast, in the presence of 50 and 100 ng/ml LPS, a distinctive When an exogenous source of NO, 400 M SNAP, or 400 M ϩ cleavage pattern of 18S and 28S rRNA was found. This pattern is S-nitroso-N-acetylcysteine was added with LPS L-NAME to associated with decreased levels of 18S and 28S rRNA, as deter- ANA-1 macrophages, the distinctive NO-mediated cleavage pat- mined by intensity of the bands on gel electrophoresis. Inhibition tern for rRNA seen in LPS-treated cells was noted (data not of NO production by the addition of L-NAME resulted in restora- shown). The time dependence of this rRNA cleavage pattern was ϩ tion of the normal rRNA pattern. The electrophoretic pattern of examined. Control, LPS-, and LPS L-NAME-treated cells were ϩ incubated for a period of 0, 4, 8, 12, 16, and 20 h before RNA genomic DNA expression in LPS- and LPS L-NAME-treated by guest on October 2, 2021 cells did not differ and specifically did not exhibit an apoptotic extraction was performed. The RNA cleavage pattern appeared in pattern (Fig. 5B). LPS-treated cells at 12, 16, and 20 h of incubation. The cells in- An LPS dose-response experiment was performed using LPS cubated for a period of 20 h exhibited a more intense pattern with concentrations varying from 50 ng/ml to 10 ␮g/ml and an incu- diminished bands corresponding to both 18S and 28S rRNA when bation period of 20 h (Fig. 6A). Again, a specific cleavage pattern compared with those found in cells treated for only 12 or 16 h. Of interest is the finding that the time course of rRNA cleavage parallels that found for inhibition of total protein synthesis and max- imal NO production (Fig. 2, B and C). To determine the identity of these various RNA bands, Northern blot analysis was performed (Fig. 7). A full-length oligonucleotide probe was constructed that was complementary to murine 18S rRNA (1869 bp). Due to the size of 28S rRNA, three separate cDNA probes were made as follows: 28S-1 (nt 0–1500), 28S-2 (nt 1559–3268), and 28S-3 (nt 3275–4712). Based upon the results of the Northern blot analysis, 18S rRNA is cleaved into three fragments of ϳ1.0, 0.8, and 0.6 kb. In contrast, 28S rRNA is cleaved into four fragments. One binds 28S-1 (size ϳ3.3 kb); another (size ϳ2.6 kb) binds 28S-2, and a final two are complementary to 28S-3 (sizes ϳ1.5 and ϳ1.0 kb). These data indicate that NO, from both endogenous and exogenous sources, induces a specific time-dependent cleavage pattern in rRNA resulting in decreased levels of 18S and 28S rRNA expression.

ANA-1 ribosomal proﬁles ANA-1 macrophages were exposed to 100 ng/ml LPS for 20 h. Unstimulated control and LPS ϩ L-NAME-treated cells served as FIGURE 3. ANA-1 cellular proliferation. [3H]Thymidine incorporation comparison groups. In cells exposed to LPS, there was a marked assays were performed in ANA-1 murine macrophages exposed to 100 4-fold decrease in 60S ribosomal A280 (0.025 Ϯ 0.012) compared ng/ml LPS or LPS ϩ L-NAME for 20 h. Results represent data from three with that of control (0.102 Ϯ 0.14; p Ͻ 0.05 vs LPS) and LPS ϩ p Ͻ; 0.05 LPS vs LPS ϩ L-NAME, or control. L-NAME (0.115 Ϯ 0.18; p Ͻ 0.05 vs LPS) cells, indicating a ,ء .separate experiments 3982 NO AND RIBOSOMAL RNA DEGRADATION

FIGURE 5. A, RNA gel of ANA-1 murine macrophages. Total RNA was isolated from ANA-1 murine macrophages exposed to 50 and 100 ng/ml LPS and LPS ϩ L-NAME for 20 h. The bands of the RNA ladder correspond to 0.24, 1.35, 2.37, 4.4, 7.46, and 9.49 kb. Gel is representative of ﬁve experiments. B, DNA gel of ANA-1 murine macrophages. Genomic DNA from ANA-1 murine macrophages exposed to 100 ng/ml LPS and LPS ϩ L- NAME for 20 h. A 500-bp DNA ladder is presented for reference. The gel is representative of four experiments. Downloaded from

decrease in quantity of 60S ribosomes. In addition, predictably, the were associated with a progressive, concentration- and time-de- http://www.jimmunol.org/ A280 of the 80S ribosomal particle composed of 60S and 40S pendent decrease in peptidyl transferase activity beginning at 12 h ribosomes was also decreased in LPS-treated cells (0.012 Ϯ 0.009) of incubation. In contrast, LPS treatment at concentrations of 50, compared with that of control (0.265 Ϯ 0.019; p Ͻ 0.05 vs LPS) 100, and 1000 ng/ml was associated with maximally depressed and LPS ϩ L-NAME-treated (0.281 Ϯ 0.023; p Ͻ 0.05 vs LPS) enzyme activity beginning at 12 h with a subsequent decline there- cells. These data suggest that LPS-mediated NO synthesis is as- after at 16 and 20 h of incubation. At LPS concentrations of 50, sociated with a decrease in relative quantities of 60S and 80S ri- 100, and 1000 ng/ml, peptidyl transferase activity was 2-fold less bosomal particles, which are requisite for protein synthesis. than that of controls at 20 h of incubation. Ablation of NO syn- Ribosome-associated peptidyl transferase activity thesis by addition of 4 mM L-NAME restored peptidyl transferase by guest on October 2, 2021 activity to control levels at all time points studied. The addition of Cleavage of 28S and 18S rRNA may alter 60S ribosomal peptidyl an exogenous source of NO in the form of SNAP signiﬁcantly transferase activity and inhibit total protein synthesis. Peptidyl depressed enzyme activity at all time points compared with both transferase activity from ANA-1 total ribosomal fractions was de- control and LPS cells. L-NAME alone did not alter peptidyl trans- termined using LPS concentrations of 0, 1, 5, 10, 25, 50, 100, and ferase activity in comparison to those of control cells (data not 1000 ng/ml at time points varying from 0 to 20 h (Fig. 8A). In shown). To further examine the threshold characteristic of these certain instances, 4 mM L-NAME was also added. In unstimulated data, the IC of peptidyl transferase activity was plotted as a func- control cells, peptidyl transferase activity was maintained through- 50 out the study period. LPS concentrations of 1, 5, 10, and 25 ng/ml tion of LPS concentration (Fig. 8B). These data suggest that NO,

FIGURE 6. A, Characterization of LPS dose response of rRNA cleavage in ANA-1 murine macrophages. Total RNA was isolated from ANA-1 murine macrophages exposed to 50, 100, 200, 400, 800, 1,200, 2,000, 5,000, and 10,000 ng/ml LPS and LPS ϩ L-NAME for 20 h. The bands of the RNA ladder correspond to 0.24, 1.35, 2.37, 4.4, 7.46, and 9.49 kb. Gel is representative of three experiments. B, Characterization of LPS dose response of rRNA cleavage in ANA-1 murine macrophages. Total RNA was isolated from ANA-1 murine macrophages exposed to 50, 100, 150, 200, and 250 ng/ml LPS for 20 h. The bands of the RNA ladder correspond to 0.24, 1.35, 2.37, 4.4, 7.46, and 9.49 kb. The gel is representative of two experiments. The Journal of Immunology 3983

FIGURE 7. Northern blot analysis of rRNA cleavage pattern in ANA-1 murine macrophages. Northern blot analysis was performed in ANA-1 murine macrophages exposed to 100 ng/ml LPS and/or 4 mM L-NAME for Downloaded from 20 h. Probes were constructed from full-length 18S rRNA, and the following portions of 28S rRNA: 28S-1 (nt 0–1500), 28S-2 (nt 1559–3268), and 28S-3 (nt 3275–4712). The blot is representative of three experiments.

from either exogenous or endogenous sources, inhibits ribosome http://www.jimmunol.org/ fraction-associated peptidyl transferase activity.

Discussion In this model of LPS-mediated iNOS expression in ANA-1 murine macrophages, we demonstrate that NO, both from endogenous and exogenous sources, inhibits total protein synthesis and cell proliferation. This inhibition parallels total NO production beginning after ϳ12 h of exposure to NO and reaches its maximum following by guest on October 2, 2021 ϳ20 h of exposure. In addition, 28S and 18S rRNA cleavage occur at all concentrations of LPS examined during a standard 20-h incubation, suggesting that rRNA cleavage is both NO- and time- dependent. Transcription, as determined by nuclear run-on analysis of the standard “housekeeping” genes, ␤-actin, GAPDH, and cyclophillin, is not altered by LPS-mediated NO production. Northern blot analysis demonstrated the presence of two cleavage sites in both 18S and 28S rRNA. The time course and NO concentration dependence of decreased total protein synthesis correlates with appearance of this distinctive rRNA cleavage pattern. We hypothesized that NO-mediated inhibition of cellular protein synthesis may be the result of fragmentation of the rRNA components essential to 60S ribosome-mediated protein elongation. Sub- sequent studies found that levels of 60S ribosome and ribosomal peptidyl transferase activity were significantly decreased in ANA-1 murine macrophages exposed to endogenous and exogenous sources of NO. We conclude that NO-mediated inhibition of total protein synthesis is the result of depressed 60S ribosome- associated peptidyl transferase activity. Eukaryotic protein synthesis is catalyzed by ribosomes, which FIGURE 8. A, Ribosomal peptidyl transferase activity in ANA-1 mu- are large complexes of proteins and rRNA (8). The smaller 40S rine macrophages. Peptidyl transferase activity was determined in the ri- rRNA subunit binds both mRNA and tRNA, while the larger 60S bosomal fraction from ANA-1 murine macrophages exposed to 1, 5, 10, rRNA subunit catalyzes peptide bond formation. Together the 60S 25, 50, 100, and 1000 ng/ml LPS, 400 ␮M SNAP, and/or 4 mM L-NAME and 40S ribosomes compromise the larger functional unit of the for 20 h. Activity is presented as fmol/mg protein/min. The results repre- Ͻ 80S ribosome. The process of protein translation centers upon sent data from three experiments. p 0.05; SNAP vs LPS at all concen- ϩ Ͻ binding of an aminoacyl-tRNA molecule to the vacant A site on trations, LPS L-NAME, and control. p 0.05; 5, 10, 25, 50, 100, and 1000 ng/ml LPS vs LPS ϩ L-NAME and control. B, IC of ribosomal the 40S ribosomal subunit. The carboxyl end of the polypeptide 50 peptidyl transferase activity in ANA-1 murine macrophages and LPS chain is uncoupled from the tRNA molecule linked to the growing concentration. end of the polypeptide chain at the adjacent P site; a peptide bond is formed to the amino acid linked to the tRNA molecule in the A 3984 NO AND RIBOSOMAL RNA DEGRADATION site. This central reaction, termed peptidyl transferase, requires an mechanism may reside in enzymatic endonuclease or ribozyme intact 28S rRNA in combination with four ribosomal proteins in activity. Finally, it will be important to determine the signaling the 60S ribosome. Work based upon the prokaryotic model indi- pathway for the induction of this cleavage pattern. cates that the 28S rRNA equivalent is a critical component of the The functional correlate of NO-mediated inhibition of total pro- basic catalytic unit (9, 10). The effect of NO on peptidyl trans- tein synthesis in the setting of LPS stimulation is unknown. Is it a ferase activity has not been previously examined. However, a sin- cytotoxic or cytoprotective response? It has been hypothesized that gle conserved histidine residue associated with the catalytic site of inhibition of protein synthesis and mitochondrial respiration may peptidyl transferase is sensitive to oxidative stress induced by sin- serve as a stress response (5). The purpose may be to transiently glet oxygen generation (8). Although our data suggests that NO shut-down nonessential cellular functions and conserve cellular induces a specific pattern of rRNA degradation, the sensitivity of resources in the presence of proinflammatory cytokines and/or en- the active site of peptidyl transferase to oxidative stress suggests dotoxemia. However, when chronically maintained, this process another possible mechanism by which NO may modulate peptidyl may lead to ultimate loss of cell viability. In our study, there were transferase function and ultimately, total protein synthesis. no discernible differences in cell viability based upon trypan blue In our model, we hypothesize that NO mediates a specific pat- exclusion or LDH release. tern of 18S and 28S rRNA cleavage. As a result of depletion of These considerations notwithstanding, we have demonstrated intact components of the 60S ribosome, peptidyl transferase activ- that LPS-mediated NO production is associated with decreased ity is decreased, total protein synthesis is depressed, and inhibition total protein synthesis without change in global gene transcription of cell proliferation ensues. Alternatively, it is possible that these programming. This process is paralleled by a specific, reproduc- RNA bands are the result of altered maturation or cleavage of ible, NO- and time-dependent cleavage pattern in rRNA constitu- rRNA precursors rather than or in addition to that of the mature ents of the 60S ribosome, 28S and 18S rRNA. This results in Downloaded from rRNA forms. NO-mediated inhibition of protein synthesis has been decreased expression of both the 60S ribosome and the complete previously observed in multiple experimental models using hepa- 80S ribosome, both of which are essential for protein synthesis, tocytes and macrophages. However, the underlying mechanism and significant inhibition of 60S ribosome-associated peptidyl has not been not extensively studied. Recently, Kim et al. (5) ex- transferase. The time course of decreased peptidyl transferase ac- amined NO-mediated inhibition of total protein synthesis in RAW tivity closely parallels that noted with cleavage of 28S and 18S 264.7 murine macrophages. They found that exposure to NO was rRNA. In this model of LPS-treated ANA-1 murine macrophages, http://www.jimmunol.org/ associated with phosphorylation of the protein synthesis initiation we conclude that one potential mechanism underlying the inhibi- factor, eIF-2␣. 80S ribosome formation was inhibited following a tion of total protein synthesis is NO-mediated degradation of con- 14-h incubation with LPS. They postulated that NO impairs pro- stituent rRNA components of the 60S ribosome and decreased tein translation by phosphorylation of the ␣ subunit of eIF-2␣ and 60S-associated peptidyl transferase activity, an essential central inhibits binding of the initiator methionyl-tRNA to 40S rRNA. step in the protein synthetic pathway. These investigators did not specifically address the role of NO in rRNA processing. References In a similar fashion, our results also indicate diminution in quan- 1. Nathan, C. 1992. Nitric oxide as a secretory product of mammalian cells. FASEB by guest on October 2, 2021 tities of both 80S and 60S ribosome, which are essential structural J 6:1157. 2. Stamler, J. S., D. I. Simon, J. A. Osborne, M. E. Mullins, O. Jaraki, T. Michel, scaffolds upon which translation occurs. In addition, 60S ribo- D. J. Singel, and J. Loscalzo. 1992. S-nitrosylation of proteins with nitric oxide: somal peptidyl transferase activity paralleled the time course of synthesis and characterization of biologically active compounds. Proc. Natl. 28S and 18S rRNA degradation. We did not specifically address Acad. Sci. USA 89:444. 3. Stamler, J. S. 1994. Redox signaling: nitrosylation and related target interactions eIF-2␣ activity. However, NO may certainly act at multiple points of nitric oxide. Cell 78:931. in the protein synthetic pathway, including eIF-2␣ phosphorylation 4. Curran, R. D., T. R. Billiar, D. J. Stuehr, J. B. Ochoa, B. G. Harbrecht, S. G. Flint, and R. L. Simmons. 1990. Multiple cytokines are required to induce hepatocyte and inhibition of 60S ribosome formation with consequent ablation nitric oxide production and inhibit total protein synthesis. Ann. Surg. 212:462. of peptidyl transferase activity. 5. Kim, Y. M., K. Son, S. J. Hong, A. Green, J. J. Chen, E. Tzeng, C. Hierholzer, In this experimental model, NO mediates a specific cleavage and T. R. Billiar. 1998. Inhibition of protein synthesis by nitric oxide correlates with cytostatic activity: nitric oxide induces phosphorylation of initiation factor pattern in rRNA that is not paralleled by apoptosis. The effect of eIF-2␣. Mol. Med. 4:179. NO on rRNA metabolism has not been previously addressed. 6. Snell, F. D., and C. T. Snell. 1984. Colorimetric Methods of Analysis. Van Nos- However, work in the early 1980’s by Varesio and colleagues (11– trand, New York. 7. Khaitovich, P., A. S. Mankin, R. Green, L. Lancaster, and H. F. Noller. 1999. 14) examined murine macrophages activated by individual expo- Characterization of functionally active subribosomal particles from Thermus sure to IFN-␣, IFN-␥, and LPS. Similar to that found in our study, aquaticus. Proc. Natl. Acad. Sci. USA 96:85. 8. Green, R., and H. F. Noller. 1997. Ribosomes and translation. Annu. Rev. Bio- they demonstrated a specific cleavage pattern in 28S rRNA with chem. 66:679. decreased accumulation of 28S rRNA and associated ribosomal 9. Holmberg, L., Y. Melander, and O. Nygard. 1994. Probing the conformational particles. This pattern was associated with a cytotoxic functional changes in 5.8S, 18S and 28S rRNA upon association of derived subunits into complete 80S ribosomes. Nucleic Acids Res. 22:2776. profile rather than a suppressive profile. Given that this body of 10. Leviev, I., S. Levieva, and R. A. Garrett. 1995. Role for the highly conserved work predates the discovery of NO, the authors did not specifically region of domain IV of 23S-like rRNA in subunit-subunit interactions at the address the presence or absence of NO. However, the similarities peptidyl transferase centre. Nucleic Acids Res. 23:1512. 11. Varesio, L. 1984. Down-regulation of RNA labeling as a selective marker for with the results from our study are compelling. Varesio et al. (12) cytotoxic but not suppressor macrophages. J. Immunol. 132:2683. may have been the first to identify a specific rRNA cleavage pat- 12. Varesio, L., M. Clayton, D. Radzioch, and E. Bonvini. 1987. Selective inhibition of 28S ribosomal RNA in macrophages activated by interderon. J. Immunol. tern that is mediated by cytokine- or LPS-mediated NO synthesis 138:2332. in macrophages. The specific sites of rRNA cleavage in 18S and 13. Radzioch, D., M. Clayton, and L. Varesio. 1987. Interferon augments the levels 28S rRNA are the subject of ongoing investigation in our lab. It is of rRNA precursors in peritoneal macrophages but not in macrophage cell lines and fibroblasts. J. Immunol. 139:805. unclear whether the cleavage site is specific for a defined nucleo- 14. Varesio L. 1985. Imbalanced accumulation of ribosomal RNA in macrophages tide sequence or a rRNA tertiary structural motif. In addition, the activated in vivo or in vitro to a cytolytic stage. J. Immunol. 134:1262.