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Vesnarinone Suppresses TNF-Induced Activation of NF-B, c-Jun Kinase, and Apoptosis¹

Cytokine Research Laboratory, Department of Bioimmunotherapy, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vesnarinone, a synthetic quinolinone derivative used in the treatment of cardiac failure, exhibits immunomodulatory, anti-inflammatory, and cell growth regulatory properties. The mechanisms underlying these properties are not understood, but due to the critical role of nuclear transcription factor NF-B in these responses, we hypothesized that vesnarinone must modulate NF-B activation. We investigated the effect of vesnarinone on NF-B activation induced by inflammatory agents. Vesnarinone blocked TNF-induced activation of NF-B in a concentration- and time-dependent manner. This effect was mediated through inhibition of phosphorylation and degradation of IB, an inhibitor of NF-B. The effects of vesnarinone were not cell type specific, as it blocked TNF-induced NF-B activation in a variety of cells. NF-B-dependent reporter gene transcription activated by TNF was also suppressed by vesnarinone. The TNF-induced NF-B activation cascade involving TNF receptor 1-TNF receptor associated death domain-TNF receptor associated factor 2 NF-B-inducing kinase-IKK was interrupted at the TNF receptor associated factor 2 and NF-B-inducing kinase sites by vesnarinone, thus suppressing NF-B reporter gene expression. Vesnarinone also blocked NF-B activation induced by several other inflammatory agents, inhibited the TNF-induced activation of transcription factor AP-1, and suppressed the TNF-induced activation of c-Jun N-terminal kinase and mitogen-activated protein kinase kinase. TNF-induced cytotoxicity, caspase activation, and lipid peroxidation were also abolished by vesnarinone. Overall, our results indicate that vesnarinone inhibits activation of NF-B and AP-1 and their associated kinases. This may provide a molecular basis for vesnarinone’s ability to suppress inflammation, immunomodulation, and growth regulation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vesnarinone (3,4-dihydro-6-[4-(3, 4)-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone; also called OPC-8212) is a synthetic quinolinone derivative with inotropic effects, used clinically for the treatment of chronic congestive heart failure (CHF)³ (1). This agent exhibits potent immunomodulatory activity, as indicated by the suppression of NK cell activity (2, 3), endotoxin-induced production of inflammatory cytokines (4, 5, 6, 7, 8, 9, 10), and nitric oxide synthase from macrophages (11, 12) and cardiac myocytes (11, 13), and it abrogate E-selectin expression in endothelial cells (5). These immunosuppressive effects of vesnarinone have been suggested to contribute to its activity against CHF (9), prolongation of cardiac allograft survival (14, 15), and suppression of systemic inflammation (16). Vesnarinone also causes agranulocytosis by impairing stromal functions and cytokine inhibition (17, 18) and inhibits immune-mediated hepatic injury (19). In addition, vesnarinone has been shown to inhibit the production of HIV-1 (20) and reduces the lethality of endotoxemia (21). Besides immunomodulatory effects, vesnarinone has been found to suppress the growth of a wide variety of tumor cell lines, including those derived from human gastric cancer (22, 23), lung cancer (24, 25), hepatocellular carcinoma (26), adenoid squamous carcinoma (27, 28), glioma (29), acute myeloid leukemia (30, 31), and prostate cancer (32).

The mechanism by which vesnarinone inhibits cytokine production and viral replication, regulates the immune system, or suppresses cell growth is not known. Nuclear factor-B is a ubiquitous transcription factor conserved from Drosophila to man and has been shown to regulate the immune system, inflammatory cytokine production, cell growth, and inflammation (33). It also regulates the expression of various genes (34) that are known to be suppressed by vesnarinone. Therefore, we postulated that vesnarinone mediates this wide variety of effects by suppressing NF-B activation. We found that indeed vesnarinone inhibits NF-B activation by a variety of agents in different cell types. This suppression occurs through inhibition of degradation of IB, an inhibitor of NF-B. The activation of various kinases of the mitogen-activated protein kinase kinase (MAPK) family needed for the activation of NF-B and a similar transcription factor, AP-1, was also inhibited, and vesnarinone blocked TNF-induced apoptosis.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials

Vesnarinone was provided by Dr. Hideo Yoshida (Otsuka America Pharmaceutical, Palo Alto, CA). Four milligrams of vesnarinone was dissolved in 1 ml of 1 N HCl, and then 1 ml of freshly prepared 1 N NaOH was added to neutralize the solution. It was then immediately diluted 1/1 with RPMI 1640 medium containing 10% FBS and was diluted further in the same medium as needed. Bacteria-derived recombinant human TNF, purified to homogeneity with a sp. act. of 5 x 10⁷ U/mg, was provided by Genentech (South San Francisco, CA). Penicillin, streptomycin, RPMI 1640, and FCS were obtained from Life Technologies (Grand Island, NY). Glycine, PMA, LPS, ceramide, NaCl, and BSA were obtained from Sigma (St. Louis, MO). Ab against IB p65, c-Rel, cyclin D1, and single- and double-stranded oligonucleotide with consensus sequences of NF-B and AP-1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-PARP Ab was obtained from PharMingen (San Diego, CA). Expression plasmids encoding FLAG-tagged NF-B-inducing kinase (NIK) (35) were provided by D. Wallach (Weizmann Institute of Science, Rehovot, Israel). The expression plasmid encoding Myc-tagged TNF receptor associated factor 2 (TRAF2) has been previously described (36).

Cell lines

The cell lines employed in this study included U937 (human histiocytic lymphoma), HeLa (human epithelial cells), H4 (glioma cells), and T-Jurkat (T cells); all were obtained from the American Type Cell Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml).

NF-B activation assays

To measure NF-B activation EMSAs were conducted essentially as previously described (37). Briefly, nuclear extracts prepared from TNF-treated cells (2 x 10⁶/ml) were incubated with ³²P end-labeled 45-mer double-stranded NF-B oligonucleotide (4 µg of protein with 16 fmol of DNA) from the HIV long terminal repeat, 5'-TTGTTACAAGGGACTTTCCGCTG GGGACTTTCCAGGGA GGCGT GG-3' (underline indicates NF-B binding sites) for 15 min at 37°C, and the DNA-protein complex formed was separated from free oligonucleotide on 4.5% native polyacrylamide gels. A double-stranded mutated oligonucleotide, 5'-TTGTTACAACTCACTTTCCGCTGCTCACTTTCCAGGGAGG CGTGG-3', was used to examine the specificity of binding of NF-B to the DNA. The specificity of binding was also examined by competition with the unlabeled oligonucleotide. The dried gels were visualized, and radioactive bands were quantitated by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using ImageQuant software.

NF-B-dependent reporter gene transcription

The effects of vesnarinone on TNF-, TRAF-2-, NIK-, and p65 (trans-activation subunit of NF-B)-induced NF-B-dependent reporter gene transcription was measured as previously described (36). Briefly, HeLa cells (0.1 x 10⁶ cells/well) were plated in six-well plates, pretreated with 30 µg/ml vesnarinone for 1 h, and then transfected by the calcium phosphate method. Cells were transfected with medium (1 ml) containing plasmid DNAs for TRAF2, NIK, or p65 (1 µg each) along with 0.5 µg of NF-B promoter DNA linked to heat-stable secretory alkaline phosphatase (SEAP) gene. The total final amount of DNA was maintained at 3 µg by the addition of the control plasmid pCMVFLAG1 DNA.

To examine TNF-induced reporter gene expression, cells were transfected with the SEAP expression plasmid for 10 h before treatment with TNF (1 nM). Treatment with vesnarinone was continued during the transfection reaction. Twelve hours after transfection, conditioned medium was harvested and analyzed (25 µl) for alkaline phosphatase activity essentially as described by the protocol of Clontech (Palo Alto, CA). The activity of SEAP was assayed on a 96-well fluorescent plate reader (Fluoroscan II, Labsystems, Chicago, IL) with excitation set at 360 nm and emission at 460 nm. This reporter system was specific, since TNF-induced NF-B SEAP activity was inhibited by overexpression of an IBa mutant lacking Ser^32/36, a kinase inactive NIK, or a dominant-negative TRAF2 mutant (36).

AP-1 activation assay

To determine the activation of AP-1, 4–5 µg of nuclear extract prepared as indicated above was incubated with 16 fmol of the ³²P end-labeled AP-1 consensus oligonucleotide 5'-CGCTTGATGACTCAGCCGGAA-3' (Santa Cruz Biotechnology; underline indicates NF-B binding sites) for 15 min at 37°C and analyzed by EMSA (38). The specificity of binding was examined by competition with unlabeled oligonucleotide. Visualization and quantitation of radioactive bands were conducted with a PhosphorImager (Molecular Dynamics) using ImageQuant software.

Western blot for IB and p65

To determine the levels of IB, postnuclear (cytoplasmic) extracts were prepared (38) from TNF-treated cells and resolved on 10% SDS-polyacrylamide gels. To determine the levels of IB and p65 proteins, nuclear and postnuclear extracts prepared from TNF-treated cells were resolved on 10% SDS-PAGE. After electrophoresis, the proteins were electrotransferred to nitrocellulose filters, probed with rabbit polyclonal Abs against IB or p65, and detected by chemiluminescence (ECL, Amersham, Piscataway, NJ). The bands obtained were quantitated with Personal Densitometer Scan version 1.30 using ImageQuant software version 3.3 (Molecular Dynamics).

Cytotoxicity assay by the 3-(4,5-dihydro-6-(4-(3,4-dimethoxybenzoyl)-1-piperazinyl)-2(1H)-quinolinone (MTT) method

Cytotoxicity was also measured by the modified tetrazolium salt MTT assay (38). Briefly, 5000 cells/well were incubated in the presence or the absence of the indicated test sample in a final volume of 0.1 ml for 72 h at 37°C. Thereafter, 0.025 ml of MTT solution (5 mg/ml in PBS) was added to each well. After a 2-h incubation at 37°C, 0.1 ml of the extraction buffer (20% SDS and 50% dimethylformamide) was added. After an overnight incubation at 37°C, the ODs at 590 nm were measured using a 96-well multiscanner autoreader (MR 5000, Dynatech, Chantilly, VA), with the extraction buffer as a blank.

Immunoblot analysis of PARP degradation

TNF-induced apoptosis was examined by proteolytic cleavage of PARP (38). Briefly, cells (2 x 10⁶/ml) were treated with various concentrations of vesnarinone at 37°C for 1 h and then stimulated with 1 nM TNF with cycloheximide (2 µg/ml) for 2 h at 37°C. The cells were then washed and extracted by incubation for 30 min on ice in 0.05 ml of buffer containing 20 mM HEPES (pH 7.4), 2 mM EDTA, 250 mM NaCl, 0.1% Nonidet P-40, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM PMSF, 0.5 µg/ml benzamidine, 1 mM DTT, and 1 mM sodium vanadate. The lysate was centrifuged, and the supernatant was collected. Cell extract protein (50 µg) was resolved on 7.5% SDS-PAGE, electrotransferred onto a nitrocellulose membrane, blotted with mouse anti-PARP Ab, and then detected by chemiluminescence (ECL, Amersham). Apoptosis was represented by the cleavage of 116-kDa PARP into an 85-kDa product.

MAPKK assay

Jurkat cells, pretreated with different concentrations of vesnarinone for 1 h, were stimulated with 1 nM TNF for 30 min at 37°C, washed with Dulbecco’s PBS, and then lysed on ice for 15 min with buffer containing 20 mM HEPES (pH 7.4), 2 mM EDTA, 250 mM NaCl, 0.1% Nonidet P-40, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM PMSF, 0.5 µg/ml benzamidine, 1 mM DTT, and 1 mM sodium orthovanadate. A 50-µg aliquot of protein was resolved on 10% SDS-PAGE, electrotransferred to nitrocellulose filters, and probed with the phospho-specific anti-p44/42 MAP kinase (Thr²⁰²/Tyr²⁰⁴) Ab (New England Biolabs, Beverley, CA) raised in rabbits (1/3000 dilution). Then the membrane was incubated with peroxidase-conjugated anti-rabbit IgG (1/3000 dilution), and the bands were detected by chemiluminescence (ECL, Amersham).

c-Jun kinase assay

The c-Jun kinase assay was performed by a modified method as described previously (38). Briefly, after treatment of cells (3 x 10⁶/ml) with TNF for 10 min, cell extracts were prepared by lysing cells in buffer containing 20 mM HEPES (pH 7.4), 2 mM EDTA, and 250 mM NaCl), 1% Nonidet P-40, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM PMSF, 0.5 µg/ml benzamidine, and 1 mM DTT. Cell extracts (150–250 µg/sample) were immunoprecipitated with 0.3 µg of anti-JNK Ab for 60 min at 4°C. Immune complexes were collected by incubation with protein A/G-Sepharose beads for 45 min at 4°C. The beads were extensively washed with lysis buffer (four times, 400 µl) and kinase buffer (twice, 400 µl; 20 mM HEPES (pH 7.4), 1 mM DTT, and 25 mM NaCl). Kinase assays were performed for 15 min at 30°C with GST-Jun_1–79 as a substrate in 20 mM HEPES (pH 7.4), 10 mM MgCl₂, 1 mM DTT, and 10 µCi [-³²P]ATP. Reactions were stopped with the addition of 15 µl of 2x SDS sample buffer, boiled for 5 min, and subjected to SDS-PAGE (9%). GST-Jun_1–79 was visualized by staining with Coomassie Blue, and the dried gel was analyzed with a PhosphorImager (Molecular Dynamics).

Determination of lipid peroxidation

TNF-induced lipid peroxidation was determined by detection of thiobarbituric acid-reactive malondialdehyde (MDA), an end product of the peroxidation of polyunsaturated fatty acids and related esters, as previously described (39). Jurkat (3 x 10⁶/ml) cells pretreated with either medium or different concentrations of vesnarinone for 1 h were stimulated with TNF (1 nM) for 30 min. Then cells were washed with PBS and subjected to three cycles of freeze-thawing in 200 µl of water. After protein determination, we added 300 µg of protein (in 0.1 ml) in 800 µl of assay mix containing 0.4% (w/v) thiobarbituric acid, 0.5% (w/v) SDS, and 9.4% (v/v) acetic acid, pH 3.5. After incubation for 1 h at 95°C, samples were cooled to room temperature and centrifuged at 14,000 x g for 10 min, and the absorbance of the supernatants was read at 532 nm. Results were normalized with the amount of MDA equivalents per milligrams of protein and were expressed as a percentage of thiobarbituric acid-reactive substances above control values. Untreated cells showed 0.571 ± 0.126 nmol of MDA equivalents/mg of protein.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
In preliminary experiments the concentration and duration of treatment with vesnarinone used in these studies had no effect on either TNF receptors or cell viability (data not shown). For most studies human Jurkat T cells were used because these cells express both types of TNF receptor, and the cellular responses examined are well characterized in our laboratory (40). Additionally, since vesnarinone is used to treat cardiac failure, we used TNF, a major mediator of CHF (9), to activate the cells.

Vesnarinone inhibits TNF-induced NF-B activation

Jurkat cells were pretreated for 1 h with different concentrations of vesnarinone and then stimulated with 100 pM TNF for 30 min; nuclear extracts were prepared and assayed for NF-B by EMSA. As shown in Fig. 1A, TNF activated NF-B, whereas vesnarinone did not. Vesnarinone did, however, block TNF-mediated NF-B activation, with optimum suppression occurring at 30 µg/ml (Fig. 1A). We next tested the length of incubation with vesnarinone required to suppress NF-B activation. For this, cells were incubated with vesnarinone for various times in relation to the addition of TNF for 30 min. Only when the cells were pretreated for 60 min with vesnarinone was the NF-B activation almost completely inhibited, and the inhibition decreased gradually with decreased preincubation time. Cotreatment or post-treatment with vesnarinone was less inhibitory than pretreatment (Fig. 1B).

View larger version (42K): [in this window] [in a new window]   FIGURE 1. Vesnarinone inhibits TNF-dependent NF-B. A, Jurkat cells (2 x 106/ml) were preincubated at 37°C for 1 h with different concentrations (0–100 µg/ml) of vesnarinone followed by 30-min incubation with 0.1 nM TNF. After these treatments nuclear extracts were prepared and then assayed for NF-B as described in Materials and Methods. B, Cells were preincubated at 37°C with 30 µg/ml vesnarinone for the indicated times and then treated with 0.1 nM TNF at 37°C for 30 min. After these treatments nuclear extracts were prepared and then assayed for NF-B. C, Cells pretreated with 30 µg/ml vesnarinone for 1 h at 37°C were exposed to different concentrations of TNF for 30 min at 37°C and then assayed for NF-B. D, Cells pretreated with 30 µg/ml vesnarinone for 1 h at 37°C were exposed to TNF for different times at 37°C and then assayed for NF-B.

Next, both untreated and vesnarinone-pretreated cells were incubated with TNF (100 pM), and the kinetics of TNF activation were measured. TNF activation increased as time of incubation increased, whereas in vesnarinone (30 µg/ml)-pretreated cells, no activation of NF-B was detected after up to 60 min of TNF stimulation (Fig. 1D).

Activated NF-B inhibited by vesnarinone consists of p50 and p65 subunits

Various combinations of Rel/NF-B proteins can constitute an active NF-B heterodimer that binds to specific sequences in DNA. To show that the retarded band visualized by EMSA in TNF-treated cells was indeed NF-B, we incubated nuclear extracts from TNF-activated cells with Ab to either p50 (NF-BI) or p65 (Rel A) subunits and then conducted EMSA. Abs to either subunit of NF-B shifted the band to a higher m.w. (Fig. 2A), suggesting that the TNF-activated complex consisted of p50 and p65 subunits. Neither preimmune serum nor irrelevant Abs such as anti-cRel or anti-cyclin DI had any effect on the mobility of NF-B. An excess (100-fold) of unlabeled NF-B oligo prevented formation of the band, indicating the specificity of NF-B binding. Specificity was indicated by the observation that the labeled oligonucleotide with a mutated NF-B binding site failed to bind the NF-B protein.

View larger version (64K): [in this window] [in a new window]   FIGURE 2. A, Supershift and specificity of the NF-B. Nuclear extracts were prepared from untreated or TNF (0.1 nM)-treated Jurkat cells (2 x 106/ml), incubated for 15 min with different Abs and cold and mutated NF-B, and then assayed for NF-B. PIS, preimmune serum. Competition indicates inhibition of binding by unlabeled wild type NF-B oligonucleotide. B, Vesnarinone does not interfere with DNA binding of NF-B protein. Cytoplasmic extracts (CE) from untreated Jurkat cells (10 µg protein/sample) were treated with 0.8% DOC for 15 min at room temperature, incubated with different concentrations of vesnarinone for 1 h at room temperature, and then assayed for DNA binding by EMSA. Competition indicates inhibition of binding by unlabeled wild-type NF-B oligonucleotide. C, Nuclear extracts were prepared from 0.1 nM TNF-treated U-937 cells; 5 µg/sample NE protein was treated with the indicated concentration of vesnarinone for 1 h at room temperature and then assayed for DNA binding by EMSA. Competition indicates inhibition of binding by unlabeled wild type NF-B oligonucleotide.

Vesnarinone does not interfere with the DNA-binding ability of NF-B proteins

Certain NF-B inhibitors, such as L-p-tosylamino-2-phenylethyl chloromethyl ketone, a serine protease inhibitor; herbimycin A, a protein tyrosine kinase inhibitor; or caffeic acid phenylethyl ester, down-regulate NF-B activation by modifying the NF-B subunits so that they cannot bind to DNA (41, 42, 43). To determine whether vesnarinone also directly modifies NF-B proteins, we incubated cytoplasmic extracts with deoxycholate (DOC; 0.8%) for 15 min at room temperature or nuclear extracts from TNF-triggered cells and then treated them with various concentrations of vesnarinone. Then DNA-binding activity was detected using EMSA. The DOC treatment has been shown to dissociate the IB subunit, thus releasing NF-B for binding to the DNA. Vesnarinone did not modify the DNA-binding ability of NF-B proteins prepared by treatment with either DOC or TNF (Fig. 2, B and C). Therefore, vesnarinone inhibits NF-B activation through a mechanism different from that of L-p-tosylamino-2-phenylethyl chloromethyl ketone, herbimycin A, or caffeic acid phenylethyl ester.

Inhibition of NF-B activation by vesnarinone is not cell type specific

All the effects of vesnarinone described above were examined in Jurkat cells. There are reports suggesting that the mechanism of NF-B activation varies in different cell types (44), so whether vesnarinone affects other cell types was also investigated. In our experiments vesnarinone blocked TNF-induced NF-B activation in myeloid (U-937), epithelial (HeLa), and glioma (H4) cells (Fig. 3). Almost complete inhibition was observed with all these cells, suggesting that this effect was not cell type specific. NF-B binding in all cells was abrogated by a 25-fold molar excess of unlabeled oligonucleotide.

View larger version (46K): [in this window] [in a new window]   FIGURE 3. The effect of vesnarinone on TNF-induced NF-B activation is not cell type specific. U-937, HeLa, and H4 cells (2 x 106/ml) were incubated with 30 µg/ml vesnarinone for 1 h and then treated with 100 pM TNF at 37°C for 30 min. After these treatments nuclear extracts were prepared and assayed for NF-B.

Vesnarinone blocks phorbol ester-, LPS-, okadaic acid-, ceramide-, and H₂O₂-mediated activation of NF-B

Besides TNF, NF-B is also activated by a wide variety of other inflammatory agents, including phorbol ester, H₂O₂, LPS, okadaic acid, and ceramide (33, 34). However, the signal transduction pathway induced by these agents may differ (44, 45, 46). We therefore examined the effect of vesnarinone on activation of the transcription factor by these various agents. All the agents tested activated NF-B, and vesnarinone completely blocked the activation of NF-B induced by all five agents (Fig. 4). These results suggest that vesnarinone may act at a step where all these agents converge in the signal transduction pathway leading to NF-B activation.

View larger version (98K): [in this window] [in a new window]   FIGURE 4. Vesnarinone inhibits NF-B activation induced by PMA, serum-activated LPS, H2O2, okadaic acid, ceramide, and TNF. Jurkat cells (2 x 106/ml) were preincubated with 30 µg/ml vesnarinone for 1 h at 37°C, then treated with PMA (25 ng/ml), SA-LPS (10 µg/ml), H2O2 (250 µM), okadaic acid (500 nM), ceramide (10 µM), and TNF (0.1 nM) for 30 min and tested for NF-B activation.

Vesnarinone inhibits TNF-dependent phosphorylation and degradation of IB and translocation of p65

The translocation of NF-B to the nucleus is preceded by the phosphorylation and proteolytic degradation of IB (33). To determine whether the inhibitory action of vesnarinone was due to its effect on IB degradation, cytoplasmic levels of IB proteins were examined by Western blot analysis. IB degradation started by 5 min after TNF treatment of Jurkat cells began and then disappeared within 10 min. The band reappeared by 30 min. The presence of vesnarinone produced no change in the band intensity (Fig. 5A). Thus, vesnarinone completely blocked TNF-mediated degradation of IB.

View larger version (55K): [in this window] [in a new window]   FIGURE 5. Vesnarinone blocks TNF-induced phosphorylation and degradation of IB and the level of p65. Jurkat cells (2 x 106/ml) pretreated with 30 µg/ml vesnarinone for 1 h at 37°C were incubated for different times with TNF (0.1 nM) and then assayed in cytosolic fractions for IB (A) and phosphorylated IB (B) by Western blot analysis. C, The levels of p65 in cytoplasmic and nuclear extracts were measured by Western blot analysis.

Because NF-B activation also requires nuclear translocation of the p65 subunit of NF-B, we measured the level of p65 in the cytoplasm and nucleus. Upon TNF treatment, the level of p65 declined in the cytoplasm, as expected with a concurrent increase in the nucleus (Fig. 5C). The treatment of the cells with vesnarinone abolished the TNF-dependent change in nuclear and cytoplasmic p65 levels. These results show that vesnarinone inhibits the TNF-induced translocation of p65 to the nucleus, and this is consistent with the inhibition of TNF-dependent degradation of IB.

Vesnarinone represses MDR-NF-B-CAT gene expression

To date we have shown that vesnarinone blocks the DNA binding of NF-B protein to its consensus sequence. DNA binding alone does not always correlate with NF-B-dependent gene transcription, suggesting that there are additional regulatory steps (47). To determine the effect of vesnarinone on TNF-induced NF-B-dependent reporter gene expression, the promoter of the rat mdr1b gene containing the NF-B binding site linked to the CAT reporter gene was used. Jurkat cells were transiently transfected with the CAT reporter construct and then stimulated with TNF in the presence or the absence of vesnarinone. CAT activity increased almost 6-fold upon stimulation with TNF (Fig. 6A). However, TNF-induced CAT activity was completely inhibited when the cells transfected with the wild-type NF-B sequence were pretreated with vesnarinone for 1 h before TNF treatment. Transfection with the MDR gene containing the mutated NF-B binding site did not result in induction of CAT by TNF. These results demonstrate that vesnarinone also represses NF-B-dependent gene expression induced by TNF.

View larger version (40K): [in this window] [in a new window]   FIGURE 6. A, Vesnarinone blocks TNF-induced NF-B reporter CAT gene expression. Cells were transiently transfected with MDR-NF-B-CAT (-243RMICAT) containing either wild-type or mutant NF-B site, pretreated with 30 µg/ml vesnarinone for 1 h, and then exposed to 1 nM TNF for 2 h. Thereafter, CAT activity was assayed as described in Materials and Methods. Results are expressed as fold activity over the nontransfected control. B, Vesnarinone inhibits the NF-B-dependent reporter gene expression induced by TNF, TRAF-2, and NIK. HeLa cells were either untreated or treated with vesnarinone (30 µg/ml) for 1 h and then transiently transfected with the indicated plasmids along with an NF-B-containing plasmid linked to the SEAP gene. Where indicated, cells were exposed to 1 nM TNF for 12 h. Cells were assayed for SEAP activity as described in Materials and Methods. Results are expressed as fold activity over the nontransfected control.

TNF-induced NF-B activation is mediated through sequential interaction of the TNF receptor with TNF receptor associated death domain (TRADD), TRAF2, NIK, and IKK-ß, resulting in phosphorylation of IB (48, 49). To delineate the site of action of vesnarinone in the TNF-signaling pathway leading to NF-B activation, cells were transfected with TRAF2, NIK, and p65 plasmids, and then NF-B-dependent SEAP expression was monitored in untreated and vesnarinone-treated cells. As shown in Fig. 6B, TRAF2, NIK, and p65 plasmids induced gene expression, and vesnarinone suppressed TRAF-2- and NIK-induced expression but had little effect on p65-induced NF-B reporter expression. Receptor activator of NF-B, another NF-B-inducing receptor, was minimally affected by vesnarinone, indicating the specificity. The specificity of the assay results is also demonstrated by the suppression of TNF-induced NF-B reporter activity by the dominant-negative IB plasmid. Thus, vesnarinone must act at a step downstream from NIK. Since NIK is known to activate IKK-ß, which, in turn, phosphorylates IB, it appears that vesnarinone must block the activity of IKK-ß, a kinase that phosphorylates IB directly.

Vesnarinone inhibits TNF-induced AP-1 activation

Besides NF-B, TNF potently activates another transcription factor, AP-1 (50). This transcription factor is activated through a series of steps, some of which overlap with NF-B. Therefore, we also examined the effect of vesnarinone on TNF-induced AP-1 activation. AP-1 activation occurred with increasing concentrations of TNF, and this activation was specific, as the unlabeled AP-1 oligonucleotide prevented binding to the DNA (Fig. 7). Pretreatment of cells with vesnarinone for 1 h inhibited the TNF-induced activation of AP-1 (Fig. 7).

View larger version (104K): [in this window] [in a new window]   FIGURE 7. Vesnarinone inhibits TNF-dependent AP-1 activation. Jurkat cells (2 x 106) were pretreated with 30 µg/ml vesnarinone for 1 h at 37°C. Then cells were stimulated with different concentrations of TNF for 1 h, and AP-1 was assayed by EMSA as described in Materials and Methods. FP, free probe incubated without cell extracts.

The activation of AP-1 requires the activation of c-Jun kinase (50). Whether vesnarinone blocks AP-1 activation through suppression of JNK was examined. Jurkat cells were pretreated with different concentrations of vesnarinone for 1 h and then stimulated with TNF (1 nM) for 10 min. About 17-fold activation of JNK was detected in TNF-treated cells, which gradually decreased with increasing concentrations of vesnarinone (Fig. 8A). A 30 µg/ml concentration of vesnarinone totally suppressed TNF-induced JNK activation (Fig. 8A).

View larger version (44K): [in this window] [in a new window]   FIGURE 8. Vesnarinone inhibits TNF induced c-Jun kinase and MAPKK activation. Jurkat cells were pretreated with different concentrations of vesnarinone as indicated and then stimulated with 1 nM TNF at 37°C for 10 min (JNK) or 30 min (MEK). Thereafter, the cells were washed, pellets were extracted, and JNK (A) and MEK (B) activation was detected from the extract as described in Materials and Methods.

Activation of JNK occurs through activation of an upstream kinase, JNKK-1 (also called MEK4), whereas activation of MAPK requires MAPKK (or MEK1 and MEK2) (50). The possibility that vesnarinone blocks the activation of MEK1 and MEK2 was explored. Jurkat cells were pretreated with different concentrations of vesnarinone for 1 h and then stimulated with 1 nM TNF for 30 min. The phosphorylated form of MAP kinase was detected by Western blot. The results in Fig. 8B show that TNF activated this kinase, and vesnarinone suppressed the activation in a dose-dependent manner, with the maximum effect occurring at 30 µg/ml.

Vesnarinone blocks TNF-induced apoptosis

Among the cytokines, TNF is one of the most potent inducers of apoptosis. We first investigated the effects of vesnarinone on TNF-induced cytotoxicity against Jurkat cells. Cells were incubated with various concentrations of TNF for 72 h in the presence or the absence of vesnarinone (30 µg/ml) and then examined for cell viability by the MTT method. As shown in Fig. 9A, TNF induced cytotoxicity in Jurkat in a dose-dependent manner, and this effect was completely abolished by the presence of vesnarinone. These results indicate that vesnarinone is cytoprotective. TNF induces cytotoxic effects through activation of various caspases, which can cleave several cellular proteins, including PARP; therefore, whether vesnarinone affects TNF-induced PARP cleavage was also examined. As shown in Fig. 9B, TNF (1 nM) induced cleavage of PARP, and this cleavage was abolished by pretreatment of cells with vesnarinone in a dose-dependent manner. Thus, these results suggest that vesnarinone is a potent inhibitor of TNF-induced apoptosis.

View larger version (16K): [in this window] [in a new window]   FIGURE 9. Vesnarinone blocks TNF-induced MTT conversion and PARP degradation. A, Jurkat cells, pretreated with 30 µg/ml vesnarinone for 1 h, were incubated with different concentrations of TNF as indicated for 72 h at 37°C in a CO2 incubator and then cell viability was examined by the MTT method. A mean of triplicate determinations is shown. B, Jurkat cells were incubated with different concentrations of vesnarinone for 1 h at 37°C, then treated with 2 µg/ml cycloheximide and TNF (1 nM) for 2 h at 37°C and assayed for PARP cleavage by Western blot. C, Vesnarinone inhibits TNF-induced lipid peroxidation. Jurkat cells (3 x 106 in 1 ml) were pretreated with different concentrations of vesnarinone for 1 h, then incubated with TNF for 30 min and assayed for lipid peroxidation as described in Materials and Methods.

Previous reports have shown that lipid peroxidation is involved in the activation of NF-B by TNF (45). As shown in Fig. 9C, TNF induced lipid peroxidation in Jurkat cells, and this was completely suppressed by vesnarinone. Thus, it is quite likely that vesnarinone blocks TNF signaling through suppression of lipid peroxidation.

Uridine reverses the suppressive effects of vesnarinone on TNF signaling

Previous studies have shown that vesnarinone inhibits nucleotide transport (51, 52); whether this plays a role in the effects of vesnarinone described here was investigated. To determine whether uridine reverses the suppression of TNF-induced NF-B activation in Jurkat cells, we pretreated cells with vesnarinone in the presence of various concentrations of uridine and then treated them with TNF. As shown in Fig. 10, TNF-induced NF-B activation was suppressed by vesnarinone, and uridine reversed the suppression in a dose-dependent manner. Uridine or vesnarinone alone did not activate NF-B, and uridine alone had a minimal effect on TNF-mediated NF-B activation. This suggests that vesnarinone mediated its effects on TNF-induced NF-B activation by interfering with the pyrimidine biosynthesis pathway.

View larger version (121K): [in this window] [in a new window]   FIGURE 10. Uridine reverses vesnarinone-mediated inhibition of NF-B activation. Jurkat cells (2 x 106/ml) were pretreated with different concentrations of uridine for 1 h and then treated with 30 µg/ml vesnarinone for 1 h at 37°C. Thereafter, cells were activated with TNF (100 pM for 30 min), and nuclear extracts were prepared and analyzed by EMSA.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

There are various ways that vesnarinone might inhibit TNF-induced NF-B activation. We showed that it does not interfere with the binding of NF-B to the consensus DNA binding site. Inasmuch as NF-B activation requires sequential phosphorylation, ubiquitination, and degradation of IB, and vesnarinone blocks IB phosphorylation and degradation, vesnarinone’s effect on NF-B may be due to inhibition of phosphorylation and thus the proteolysis of IB. The lack of a visible IB band when Abs against the phosphorylated form of IB were used after treatment of cells with the quinolinone suggests that vesnarinone blocked IB phosphorylation. IB phosphorylation is regulated by many kinases, including IB- kinase (IKK)-, IKK-ß, IKK-, NIK, TGF-ß-activated kinase-1, and MEKK1 (53, 54, 55, 56, 57). Besides MEKK1, MEKK2 and MEKK3, through IKK, have been implicated in NF-B activation (58), whereas MEKK4, through MEK4, activates JNK (50). We found that vesnarinone inhibited the activation of MEK1, JNK, and IKK. Thus, it is possible that vesnarinone inhibited IB phosphorylation by inhibiting the activity of MEKK1. TNF-induced NF-B activation involves the sequential interaction of TNF receptor with TRADD, TRAF2, and NIK, which then activates IKK (48, 49). Our findings that vesnarinone blocks NF-B-dependent reporter gene expression induced by TNFR1, TRAF2, and NIK, but not by p65, also suggests that vesnarinone acts at a step downstream from NIK. How vesnarinone inhibits the activation of MEK, JNK, and IKK is not clear. Because all these kinases are redox-sensitive kinases, it is possible that vesnarinone inhibits these kinases through the antioxidant mechanism. Additionally, inhibition of nucleoside transport by vesnarinone may affect the activation of these kinases.

We found that vesnarinone blocked NF-B activation induced by a wide variety of agents, including TNF, okadaic acid, ceramide, LPS, H₂O₂, and PMA, in U-937 cells. These results indicate that vesnarinone is a broad inhibitor of NF-B activation and acts at a step where most of these pathways converge. Our studies also show that suppression of NF-B by vesnarinone is not cell type specific, as TNF-induced NF-B activation in U-937 (myeloid), HeLa (epithelial), and H4 (glioma) cells was inhibited by vesnarinone.

Vesnarinone blocked TNF-induced cytotoxicity and inhibited apoptosis by suppressing caspase activation. How vesnarinone suppresses caspase activation is not clear. The redox regulation of caspase activation (59, 60) suggests that vesnarinone may suppress caspases through its antioxidant activity. Recently, a role for JNK activation in TNF-induced apoptosis was reported (61, 62, 63). Thus, it is possible that vesnarinone inhibits apoptosis by inhibiting JNK activation. Indeed, studies clearly demonstrate that TNF-induced JNK activation is completely blocked by vesnarinone. Because JNK activation is sensitive to the redox status of the cell (64), the inhibition of JNK by vesnarinone may also be due to its antioxidant properties.

The role of NF-B in the regulation of apoptosis is controversial. Several studies indicate that NF-B activation blocks apoptosis, while others show that NF-B activation has no effect on TNF-induced apoptosis (63, 64, 65, 66), and then there are reports indicating that NF-B activation is required for apoptosis (67, 68). The inhibition of NF-B by vesnarinone did not potentiate the apoptotic effects of TNF but, rather, suppressed it, suggesting that either inhibition of apoptosis by vesnarinone is dependent on inhibition of NF-B activation or that NF-B and apoptosis are inhibited independently of each other.

Since vesnarinone inhibited TNF-induced activation of both NF-B and apoptosis simultaneously, vesnarinone may inhibit a common step upstream in the TNF signaling pathway. Recent studies from our laboratory showed that overexpression of cells with either superoxide dismutase (38) or -glutamylcysteine synthetase, a rate-limiting enzyme in the glutathione biosynthesis pathway (69), blocks both apoptosis and TNF-induced activation of NF-B, AP-1, and JNK. Thus, it is possible that the effects of vesnarinone are mediated through quenching of reactive oxide intermediates. Indeed, our results demonstrate the inhibitory effect of vesnarinone on TNF-induced lipid peroxidation. A role for lipid peroxidation in TNF signaling has been reported (39, 45). Previously, we have shown that leflunomide inhibits NF-B activation and cell proliferation (40), and these effects are mediated through suppression of nucleoside transport (70). Vesnarinone is also known to block nucleoside transport (51, 52), and our results suggest that nucleoside transport plays a role in vesnarinone’s ability to suppress NF-B activation. How nucleoside transport is involved in NF-B activation is not clear, but it is possible that it interferes with the translocation of p50-p65 subunits of NF-B from the cytoplasm to the nucleus.

We found that vesnarinone blocks NF-B-dependent reporter gene expression. Several genes that require NF-B are known to be suppressed by vesnarinone, including such inflammatory cytokines as TNF and IL-6 (3, 4, 5, 6, 7, 8, 9), nitric oxide synthetase (11, 12, 13), and various adhesion molecules (5). It is quite likely that vesnarinone suppresses the expression of these genes by suppressing NF-B. Vesnarinone also blocks HIV-1 replication (20), which itself requires NF-B activation, so it, too, may be mediated through NF-B suppression. The effects of vesnarinone on allograft survival, systemic inflammation (16), immune-mediated hepatic injury (19), endotoxemia (21), and granulocytosis (18) could also be mediated through suppression of NF-B.

Several reports indicate that TNF, NF-B, apoptosis, and reactive oxide intermediates play a role in CHF (71, 72, 73, 74). The suppression of TNF production, NF-B activation, apoptosis, and lipid peroxidation as demonstrated may account for some of the effects of vesnarinone against CHF (1). Like vesnarinone, the anti-inflammatory drugs sodium salicylate and aspirin also block the activation of NF-B by preventing the degradation of IB (75). The effects of salicylate on NF-B activation were observed, however, at a suprapharmacologic concentration (>5 mM). In contrast, vesnarinone in our studies was effective at 1% of that concentration, suggesting that it is a potent inhibitor. Our results suggest that vesnarinone may also have applications for various other diseases, including cancer, inflammation, and AIDS, where NF-B activation plays a major role. These possibilities require further investigation in detail.

	Footnotes

 
¹ This study was conducted by The Clayton Foundation for Research.

² Address correspondence and reprint requests to Dr. Bharat B. Aggarwal, Cytokine Research Laboratory, Department of Bioimmunotherapy, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030.

³ Abbreviations used in this paper: CHF, congestive heart failure; MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; SEAP, secretory alkaline phosphatase; NIK, NF-B-inducing kinase; IKK, IB- kinase; SA-LPS, serum-activated LPS; JNK, c-Jun N-terminal kinase; DOC, deoxycholate; MTT, 3-(4,5-dihydro-6-(4-(3,4-dimethoxybenzoyl)-1-piperazinyl)-2(1H)-quinolinone; PARP, poly(ADP ribose) polymerase; MDA, malondialdehyde; MEK, mitogen-activated protein kinase kinase; CAT, chloroamphenicol acetyltransferase; TRAF2, TNF receptor associated factor 2; TRADD, TNF receptor associated death domain.

Received for publication January 21, 2000. Accepted for publication March 21, 2000.

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