Tyrosine kinase inhibitors and antioxidants modulate NF-kappa B and NOS-II induction in retinal epithelial cells

Violaine Faure, Yves Courtois, and Olivier Goureau

Développement, Vieillissement, et Pathologie de la Rétine, Unité 450 Institut National de la Santé et de la Recherche Médicale, Association Claude Bernard, 75016 Paris, France

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Bovine retinal pigmented epithelial (RPE) cells express an inducible nitric oxide synthase (NOS-II) after activation with interferon-gamma (IFN-gamma ) and lipopolysaccharide (LPS). Experiments were performed to investigate the effects of tyrosine kinase inhibitors (genistein and herbimycin A) and antioxidants [pyrrolidine dithiocarbamate (PDTC) and butyl hydroxyanisol] on NOS-II induction. The LPS-IFN-gamma -induced nitrite release was inhibited in a concentration-dependent manner by these compounds. Analysis by Northern blot showed that this inhibitory effect correlated with a decrease in NOS-II mRNA accumulation. Analysis by electrophoretic mobility shift assay of the activation of the transcription factor nuclear factor-kappa B (NF-kappa B) involved in NOS-II induction demonstrated that LPS alone or combined with IFN-gamma induced NF-kappa B binding. NF-kappa B activation was not changed by the presence of tyrosine kinase inhibitors but was totally prevented by PDTC pretreatment. Immunocytochemistry experiments confirmed the reduction of the nuclear translocation of NF-kappa B only by PDTC. Our results demonstrated the existence in retinal pigmented epithelial cells of different intracellular signaling pathways in NOS-II induction, since tyrosine kinase inhibitors blocked NOS-II mRNA accumulation without inhibiting NF-kappa B activation. Furthermore, the LPS-IFN-gamma -induced NOS-II mRNA accumulation was sensitive to cycloheximide, suggesting that, in addition to NF-kappa B, transcriptional factors that require new protein synthesis are involved in NOS-II induction.

interferon-gamma ; lipopolysaccharide; nitric oxide; transcription factor

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE ENZYME NITRIC OXIDE (NO) synthase (NOS) transforms L-arginine into NO and L-citrulline in the presence of oxygen, NADPH, tetrahydrobiopterin, flavin mononucleotide, and FAD (12, 28). Three isoforms of NOS have been identified. Two isoforms are continuously expressed and are called constitutive NOS: NOS-I (nNOS) is present essentially in neurons of the central and peripheral nervous system (6), whereas NOS-III (eNOS) is originally localized in the plasmic membrane of vascular endothelial cells (12). These two enzymes are calcium and calmodulin dependent. On the other hand, the inducible isoform, NOS-II (iNOS), is calcium and calmodulin independent and is expressed in different cell types only after a transcriptional activation by endotoxins or cytokines (25, 28, 29). Different roles have been attributed to NO, such as vasorelaxation and neurotransmission (6, 28). NO produced by NOS-II plays a role in immunological defenses (28, 29) as antitumoral, antimicrobicidal, and antiviral agents. NO is also considered to be a mediator of autoimmune and inflammatory responses (25).

In the retina, NOS-II is expressed in retinal pigmented epithelial (RPE) and Müller glial cells in some pathological disorders (10) as well as in vitro after stimulation by cytokines (13, 15-17). In this context, we previously demonstrated that, in bovine RPE cells, NOS-II was induced by treatment with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma ) added together, but not individually (14, 17).

NOS-II regulation is dependent on signal transduction activated by endotoxin, cytokines, and growth factors (13, 28). These signals are the results of the activation of serine-threonine or tyrosine kinases (9, 36). It has been established that LPS interacts with its membrane receptor and activates mitogen-activated protein (MAP) kinases (38) and reactive oxygen species, which activate the nuclear transcription factor nuclear factor-kappa B (NF-kappa B) (20, 32). The activation of NF-kappa B has been implicated in the induction of NOS-II in different cell types, such as murine macrophages (27, 39), vascular smooth muscle cells (33), or 3T3 fibroblasts (22). IFN-gamma interacts with its receptor and uses cytosolic kinases (e.g., Janus kinases) and transcriptional factors (e.g., STAT proteins), which activate different genes (9).

In this study we have attempted to elucidate the mechanisms involved in the induction of NOS-II by investigating the effect of tyrosine kinase inhibitors and antioxidants on the induction of the NOS-II mRNA by LPS and IFN-gamma . We have demonstrated that tyrosine kinase inhibitors and antioxidants decrease nitrite release and NOS-II mRNA accumulation, whereas only antioxidants reduced the nuclear translocation of NF-kappa B and the formation of NF-kappa B-DNA complexes (or binding of NF-kappa B to DNA).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell cultures. Bovine RPE cells were prepared as previously reported (17) and cultured in DMEM supplemented with 10% FCS (GIBCO BRL, Cergy-Pontoise, France), fungizone (2.5 µg/ml), gentamicin (50 µg/ml), and L-glutamine (2 mM). Cultures were homogenous and contained only RPE cells, characterized by immunohistochemistry with anticytokeratin monoclonal antibody KL-1 (4). Cells at passages 1-5 were used for experiments.

Chemicals, cytokines, and antibodies. LPS from Salmonella typhymurium, pyrrolidine dithiocarbamate (PDTC), butyl hydroxyanisol (BHA), actinomycin D, cycloheximide, genistein, and herbimycin A were obtained from Sigma Chemical. NF-kappa B p65, p50, and C-rel antibodies were purchased from Santa Cruz Biotechnology. Bovine recombinant IFN-gamma was generously provided by Dr. T. Ramp (Ciba-Geigy, Basel, Switzerland). The rabbit antiliver NOS-II antibody was a generous gift of Dr. Ohshima [Centre International de Recherche Sur le Cancer (CIRC), Lyon, France].

Assay for nitrite. Confluent RPE cells in 12-well culture plates (averaging 105 cells/well) were treated with LPS and IFN-gamma in the absence or presence of tyrosine kinase inhibitors or antioxidants in fresh DMEM-10% FCS. After 72 h of incubation, nitrite concentration was determined in cell-free culture supernatants with use of the spectrophotometric method based on the Griess reaction, as previously described (17).

RNA isolation and Northern blot analysis. Total RNA was extracted from RPE cells in 90-mm petri dishes treated for 24 h with LPS and IFN-gamma , in the absence or presence of tyrosine kinase inhibitors or antioxidants, by lysis of cells in guanidinium isothiocyanate followed by phenol extraction (8). RNA were denatured by heating (65°C) in 50% formamide-6% formaldehyde, electrophoresed (25 µg/lane) in 1% agarose gel containing 6% formaldehyde, and then transferred to a nylon membrane. An Sma I/EcoR I digestion of a plasmid containing the murine macrophage NOS-II cDNA cloned into pGEM (24) yielded a 950-bp fragment, which was labeled using the random priming method. Blots were hybridized overnight at 42°C with this 32P-labeled fragment (106 cpm/ml) and washed at 60°C in 1× NaCl-sodium citrate (0.15 M NaCl-0.015 M sodium citrate) and 0.1% SDS. Autoradiography was performed by exposure to Kodak X-omat film at -70°C. Membranes were stripped and rehybridized with a full-length glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe as a means of checking the amounts of RNA added for each sample. Northern blot results were quantified with a scanning densitometer (Biocom). All values obtained with the specific NOS-II probe were corrected for slight differences in RNA loading by normalization to the values obtained with the GAPDH probe.

Western blot analysis. After treatment with LPS and IFN-gamma with or without PDTC, BHA, or genistein for 72 h, cells were washed with PBS and scraped in lysis buffer containing protease inhibitors. Samples were centrifuged, and after one freeze-thaw cycle, 100 µg of supernatant proteins were subjected to SDS-PAGE. Proteins were then transferred to an Immobilon membrane (Millipore, Saint Quentin en Yvelines, France) by electroblotting. Western blot analysis with use of a polyclonal antibody specific for liver inducible NOS was performed as previously described (14). The intensity of the bands was quantified using densitometric measurements with densitometric software (One Descan, Scanalytics, Billerica, MA).

Electrophoretic mobility shift assay. RPE cells were pretreated for 2 h with or without PDTC (10 µM), genistein (90 µM), or herbimycin A (2 µM), washed, and treated for 30 min with LPS (1 µg/ml) and IFN-gamma (100 U/ml) in the presence or absence of antioxidants or tyrosine kinase inhibitors. Cells were washed three times in cold PBS, and whole cell extracts were prepared from cells lysed at 4°C in HEPES (10 mM, pH 7.9) containing 0.1 mM EDTA, 5% glycerol, 0.4 M NaCl, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.1 µg/ml leupeptin, 4 µg/ml aprotinin, 1.5 µg/ml pepstatin, 1 µg/ml chymostatin, and 2 µg/ml antipain. After 15 min of centrifugation at 37,000 rpm, the supernatants were stored at -80°C. Protein concentrations of extracts were determined by the Bradford analysis (Bio-Rad). Double-stranded consensus oligonucleotides for NF-kappa B (5'-CTAGACAGAGGGGATTTCCGATTCCGAGAGGT-3'; Genset, Paris, France) were end labeled by Klenow polymerase (Appligene, France) and gamma -[32P]CTP. The binding reactions were carried out by incubating extracts (25 µg) with gamma -32P-labeled NF-kappa B consensus oligonucleotides (104 cpm) in a buffer containing 40% Ficoll, 200 mM HEPES, pH 7.5, 600 mM KCl, 20 mM dithiothreitol, 0.1% NP-40, 1 mg/ml BSA, and 10 µg of salmon sperm DNA for 20 min at room temperature. For supershift and competition binding experiments, antiserum against p50, p65, or C-rel NF-kappa proteins or a 100-fold molar excess of unlabeled oligonucleotide was added to the reaction mixture along with the labeled probe. The reaction mixture was loaded onto a 5% polyacrylamide gel made in 0.5× Tris borate-EDTA (25 mM Tris, 44 mM borate, 0.5 mM EDTA) and electrophoresed at 200 V at room temperature.

Immunocytochemistry. RPE cells were plated onto Labtek slides (Nunc, PolyLabo) and pretreated for 2 h with or without antioxidants or tyrosine kinase inhibitors. Fresh medium (DMEM-10% FCS) containing LPS (1 µg/ml) and IFN-gamma (100 U/ml) was then added to cell cultures. After a 30-min incubation, coverslips were washed three times with PBS, dried for 15 min at room temperature, and fixed for 30 min in 4% paraformaldehyde. After they were washed with PBS, the cells were permeabilized with methanol for 5 min at -20°C, washed, and saturated with PBS-5% skim milk for 30 min. Coverslips were then incubated for 1 h at room temperature with the rabbit anti-p65 antibody (diluted 1:50) in PBS-1% skim milk. Coverslips were washed five times with PBS-1% skim milk, incubated for 1 h at room temperature with biotinylated sheep anti-rabbit IgG (Amersham; 1:100 in PBS-1% skim milk), and then incubated with extravidin-fluorescein isothiocyanate (1:100 in PBS-1% skim milk). Coverslips were extensively washed in PBS and, during the last wash, treated for 5 min with the fluorescent nuclear stain 4,6-diamidino-2-phenylindole. Sections were mounted in Fluoprep and viewed under a Leitz Aristoplan microscope equipped with an epi-illuminator mercury vapor arc lamp (HBO) and filters for rhodamine and 4,6-diamidino-2-phenylindole fluorescence.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Inhibition of nitrite release and NOS-II mRNA accumulation by antioxidants. We first studied the effect of two different types of antioxidants, PDTC and BHA, on nitrite production by LPS-IFN-gamma . PDTC and BHA decreased the nitrite production after 72 h of stimulation in a dose-dependent manner with IC50 values of 0.4 and 70 µM and a maximal inhibitory effect of 5-10 and 250 µM, respectively (Fig. 1). Trypan blue viability test confirmed that the inhibitory effect of PDTC and BHA was not due to a cytotoxic effect of these compounds (data not shown). The expression of NOS-II mRNA was investigated by Northern blot to determine whether inhibition of nitrite release corresponded to a decrease in NOS-II mRNA accumulation. Total RNA was extracted from RPE cells after 24 h of treatment, corresponding to the maximal expression of NOS-II mRNA (14). Only one detectable mRNA signal at 4.4 kb was detected after LPS-IFN-gamma treatment, whereas in the unstimulated RPE cells, NOS-II mRNA was not detectable (Fig. 2), as we recently reported (14). Figure 2 shows that antioxidant treatment inhibited to a large extent the expression of NOS-II mRNA in RPE cells stimulated with LPS-IFN-gamma . This decreased mRNA accumulation correlated with NOS activity, which was evaluated by the nitrite level in the culture medium (Fig. 2).


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Fig. 1.   Effect of different concentrations of antioxidants on nitrite production induced by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma ) in bovine RPE cells. Cells were incubated with LPS (1 µg/ml) and IFN-gamma (100 U/ml) and different concentrations of pyrrolidine dithiocarbamate (PDTC, A) or butyl hydroxyanisol (BHA, B). After 72 h, nitrite accumulated in culture medium was measured by Griess reaction. Data are presented as a percentage of nitrite determined in culture medium of LPS-IFN-gamma -stimulated cells without antioxidant treatment (100% corresponds to 20-25 µM nitrite). Values are means ± SE of 3 independent experiments.


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Fig. 2.   Effect of antioxidants on inducible nitric oxide synthase (NOS-II) mRNA accumulation. Confluent cells were incubated in medium alone (lane 1) or in medium containing 1 µg/ml LPS and 100 U/ml IFN-gamma (lane 2) or LPS-IFN-gamma with 250 µM BHA (lane 3) or with 10 µM PDTC (lane 4). After 24 h, nitrite in culture medium was measured by Griess reaction (C), then total RNA was isolated, and levels of NOS-II and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs were assessed by Northern analysis (A and B). Experiment represents 1 of 3 independent experiments that gave similar results. AU, arbitrary units.

Inhibition of nitrite release and NOS-II mRNA accumulation by tyrosine kinase inhibitors. To establish the transduction mechanisms involved in NOS-II induction and, in particular, the role of tyrosine kinases, we have studied the effect of two different tyrosine kinase inhibitors: genistein and herbimycin A. Incubation with either compound for 72 h diminished nitrite production measured after stimulation with LPS-IFN-gamma (Fig. 3). The effect of genistein was superior to that of herbimycin A: maximal inhibition of LPS-IFN-gamma -induced nitrite production was only 40% with 2 µM herbimycin A. Elevating the concentration of herbimycin A (>5 µM) did not increase inhibition but led to cell toxicity, as evaluated by trypan blue viability test (data not shown). The presence of genistein with LPS-IFN-gamma induced a decrease of NOS-II mRNA accumulation that correlated with the activity of NOS as determined by release of nitrite into culture media (Fig. 4). The same decrease of NOS-II mRNA could be observed with herbimycin A (data not shown). In the presence of 0.25% DMSO, which was used to make genistein soluble in the aqueous media, there was no decrease in NOS-II mRNA accumulation due to LPS-IFN-gamma (Fig. 4), demonstrating that DMSO did not play a role in the inhibition due to genistein.


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Fig. 3.   Effect of different concentrations of tyrosine kinase inhibitors on nitrite production induced by LPS-IFN-gamma . Confluent cells were incubated with LPS (1 µg/ml) and IFN-gamma (100 U/ml) and different concentrations of genistein (A) or herbimycin A (B). After 72 h, nitrite accumulation in culture medium was measured by Griess reaction. Data are presented as a percentage of nitrite determined in culture medium of LPS-IFN-gamma -stimulated cells without tyrosine kinase inhibitor treatment (100% corresponds to 20-25 µM nitrite). Values are means ± SE of 3 independent experiments.


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Fig. 4.   Effect of a tyrosine kinase inhibitor on NOS-II transcription. Confluent cells were incubated in medium alone (lane 1) or in medium containing 1 µg/ml LPS and 100 U/ml IFN-gamma (lane 2) or LPS-IFN-gamma with 0.25% DMSO (lane 3) or with 90 µM genistein (lane 4). After 24 h, nitrite accumulation in culture medium was measured by Griess reaction (C), then total RNA was isolated, and levels of NOS-II and GAPDH mRNAs were assessed by Northern analysis (A and B). Experiment represents 1 of 3 independent experiments that gave similar results.

Inhibition of NOS-II protein accumulation by antioxidants and tyrosine kinase inhibitors. The expression of RPE NOS-II protein was investigated by Western blot analysis after 72 h with LPS-IFN-gamma with or without PDTC, BHA, and genistein. As we previously reported (14), a band at 130 kDa was detected in LPS-IFN-gamma -treated cells but not in untreated cells (data not shown). Densitometric analysis revealed that simultaneous treatment with PDTC or BHA greatly decreased the 130-kDa protein signal by 88 and 81%, respectively, compared with LPS-IFN-gamma (Table 1). Furthermore, a decrease of 84% of the signal intensity was observed with the addition of genistein (Table 1).

                              
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Table 1.   Decrease of NOS-II protein by PDTC, BHA, and genistein

Activation of NF-kappa B binding activity by LPS-IFN-gamma in RPE cells and effect of pretreatment with PDTC or tyrosine kinase inhibitors. Because PDTC, described as an inhibitor of the transcription factor NF-kappa B (32), inhibited NOS-II mRNA accumulation, we used electrophoretic mobility shift assay (EMSA) to investigate the involvement of NF-kappa B in the induction of NOS-II. Bovine RPE cells were stimulated for 30 min with LPS alone or LPS-IFN-gamma with or without tyrosine kinase inhibitors or antioxidants. With LPS alone, two bands appeared that were absent in unstimulated cells (Fig. 5, lane 2). The addition of excess unlabeled consensus oligonucleotide completely prevented complex formation (lane 8), demonstrating the specificity of the DNA-protein interaction. Stimulation with IFN-gamma alone did not induce the complexes (lane 3), and the addition of IFN-gamma did not seem to increase the intensity of the signal induced by LPS (lane 4). When cells were pretreated with PDTC (lane 7) and stimulated with LPS-IFN-gamma , a marked decrease of the intensity of both bands was observed. However, the addition of inhibitors of tyrosine kinases had no significant effect on NF-kappa B activation (lanes 5 and 6). The use of antibody against p65 supershifted both complexes induced by LPS-IFN-gamma stimulation, whereas antibody against p50 subunit supershifted only the lower complex (Fig. 6). Antibody directed against C-rel failed to supershift the complexes (data not shown).


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Fig. 5.   Effect of antioxidants and tyrosine kinase inhibitors on nuclear factor-kappa B (NF-kappa B)-specific DNA-protein complex formation. Retinal pigmented epithelial cells were stimulated for 30 min with different combinations of cytokines as follows: no stimulation (lane 1), 1 µg/ml LPS (lane 2), 100 U/ml IFN-gamma (lane 3), LPS-IFN-gamma (lane 4), LPS-IFN-gamma plus 90 µM genistein (lane 5), LPS-IFN-gamma plus 2 µM herbimycin A (lane 6), or LPS-IFN-gamma plus 10 µM PDTC (lane 7). Whole cell extracts were subjected to electrophoretic mobility shift assay by use of an NF-kappa B oligonucleotide probe. Specificity was determined by competition with an excess of cold probe (lane 8). Experiment represents 1 of 4 independent experiments that gave similar results.


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Fig. 6.   Supershift of 2 NF-kappa B complexes. Confluent cells were stimulated for 30 min with LPS (1 µg/ml) and IFN-gamma (100 U/ml), then incubated with specific radioactive probe alone (lane 1) or with 0.1 µg anti-p50 subunit antibody (lane 2) or 0.1 µg of anti-p65 antibody (lane 3) for 20 min. Experiment represents 1 of 3 independent experiments that gave similar results.

Differential effects of PDTC and genistein on the nuclear translocation of p65 subunits induced by LPS-IFN-gamma . To confirm the translocation of NF-kappa B to the nucleus after stimulation of the cells, we performed immunocytochemistry with antibody to NF-kappa B p65, which was present in both complexes observed by EMSA. In unstimulated cells, cytoplasmic staining was prominent (Fig. 7). When cells were stimulated with LPS or LPS-IFN-gamma , which activated NF-kappa B by EMSA, we observed an important nuclear staining (Fig. 7). Addition of IFN-gamma alone, which failed to activate NF-kappa B, did not induce translocation of p65 in the nucleus (data not shown). The presence of genistein did not impair translocation of p65 induced by LPS-IFN-gamma . However, pretreatment with PDTC blocked translocation of p65 to the nucleus (Fig. 7), confirming its effect on NF-kappa B binding activity.


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Fig. 7.   Immunocytochemistry for NF-kappa B p65 in bovine retinal pigmented epithelial cells. Coverslips were untreated (A and B) or treated for 30 min with 1 µg/ml LPS (C) or LPS-IFN-gamma (100 U/ml) without (D) or with 10 µM PDTC (E) or 90 µM genistein (F). Cells were then fixed and stained with antibody against p65. B: nuclear staining with 4,6-diamidino-2-phenylindole from untreated cells. Experiment represents 1 of 3 independent experiments that gave similar results.

Accumulation of NOS-II mRNA required protein synthesis. When cycloheximide at 2 µg/ml was added simultaneously with LPS-IFN-gamma , a marked decrease of NOS-II mRNA was observed (Fig. 8), suggesting that NOS-II mRNA accumulation was dependent on protein synthesis. This inhibitory effect was not due to a cytotoxic effect of cycloheximide, as demonstrated by trypan blue viability test (data not shown).


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Fig. 8.   Effect of cycloheximide on NOS-II mRNA accumulation induced by LPS and IFN-gamma . Confluent cells were not stimulated (lane 1) or treated with LPS (1 µg/ml) and IFN-gamma (100 U/ml) for 24 h without (lane 2) or with (lane 3) 2 µg/ml cycloheximide. Total RNA was extracted, and levels of NOS-II and GAPDH mRNAs were assessed by Northern blot analysis. Experiment represents 1 of 3 independent experiments that gave similar results.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have demonstrated that induction of NOS-II activity in RPE cells by LPS-IFN-gamma can be modulated by antioxidants and tyrosine kinase inhibitors. This study of NOS-II regulation allows elucidation of some of the different regulatory elements involved in the induction of NOS-II: tyrosine kinase(s) and oxygen free radicals, which seem to act at the level of NOS-II transcription.

A decrease of nitrite release and NOS-II mRNA accumulation was observed with genistein and herbimycin A, as previously observed in different cell types, such as murine macrophages (11, 25). The use of these inhibitors demonstrates the important role of tyrosine kinase(s) in the induction of NOS-II, although they are not specific for a given protein kinase. Because JA kinases and MAP kinase kinase (MEK)/MAP kinases are activated by IFN-gamma and LPS, respectively, in different cell types (9, 36), it would be interesting to determine the potential involvement of these kinases in the induction of NOS-II by LPS-IFN-gamma in bovine RPE cells. In this context, preliminary experiments showed that PD-98059, a specific MEK-1 inhibitor, blocked LPS-IFN-gamma -induced nitrite release, suggesting the role of the MEK/MAP kinases in NOS-II induction. Concerning the JAK-STAT pathway, the involvement of STAT-1 and interferon regulatory factors (IRF-1 and IRF-2) is under investigation by EMSA studies.

A decrease of nitrite release and NOS-II mRNA production was also obtained by treatment with two antioxidants (PDTC and BHA). Some differences between these components were observed, since PDTC was more potent than BHA. This difference might be related to the dual ability of PDTC to act as a metal chelator and as a strong antioxidant (34). Bovine RPE cells react similarly to other cell types in which PDTC is often described as an inhibitor of NOS-II induction (5, 23, 30, 31, 39). Our results reported some differences in terms of the percentage of inhibition by antioxidants and genistein between nitrite release or NOS-II protein level and mRNA accumulation. The uptake of antioxidants and genistein into cells could be a slow process. Because transcription of NOS-II mRNA occurred within a few hours after stimulation with LPS-IFN-gamma , in contrast to NOS-II protein translation and nitrite release, which required >24 h, it would be envisaged that effective concentrations of antioxidants or genistein intracellularly could be not totally attained. This phenomenon could explain the differences in the extent of inhibition between mRNA accumulation and NOS-II protein level or nitrite release. However, we cannot exclude that these compounds could also have an inhibitory effect on NOS-II induction at a posttranscriptional level, as suggested in adenocarcinoma EM-T6 cells with antioxidants (7). Indeed, the large inhibition observed by Western blot on NOS-II protein level with antioxidants and genistein seems to confirm an effect of these compounds on NOS-II translation or on NOS-II mRNA stability.

The presence of NF-kappa B binding sequences in the NOS-II promoter (40) and the requirement of nuclear translocation of NF-kappa B for NOS-II induction (39) indicate the importance of this factor in NOS-II induction. NF-kappa B is not synthesized de novo in the cell but is present in the cytoplasm in an inactivated form complexed by the protein Ikappa B-alpha (2). Stimuli activate NF-kappa B by phosphorylating Ikappa B-alpha on serines in its regulatory NH2 terminus, an event leading to Ikappa B conjugation with ubiquitin and subsequent proteasome-mediated degradation (37). Another possibility is that Ikappa B-alpha is phosphorylated on tyrosine, releasing NF-kappa B, but then not degraded (18).

In bovine RPE cells we demonstrated that LPS, but not IFN-gamma , induced the translocation of NF-kappa B into the nucleus and the formation of DNA-NF-kappa B complexes observed by EMSA. Furthermore, IFN-gamma , which is required for NOS-II induction, did not seem to potentiate LPS-induced NF-kappa B activation. By using the tyrosine kinase inhibitors and antioxidants, we demonstrated that nuclear translocation of NF-kappa B and formation of DNA-NF-kappa B complexes involved the action of oxygen free radicals but that tyrosine kinase(s) was not necessary for NF-kappa B activation. This latter result was surprising, because activation of tyrosine kinases on LPS stimulation (38) has been reported and the involvement of this pathway in NF-kappa B activation has been demonstrated (19). However, Tetsuka et al. (35) recently reported that activation of tyrosine kinase may not be required for interleukin-1beta -induced NF-kappa B activation in rat mesangial cells. It is likely that, in bovine RPE cells, LPS can activate NF-kappa B independently of tyrosine kinase activation. However, we cannot exclude that the inhibitors used in this study failed to block activation of some specific tyrosine kinase(s).

An important finding in the present study is that although LPS alone induced binding of NF-kappa B, it failed to induce NOS-II mRNA and nitrite production. However, inhibition of NF-kappa B binding with PDTC partially prevented NOS-II induction by LPS-IFN-gamma treatment. These results indicate that activation of NF-kappa B may not be sufficient to induce NOS-II in bovine RPE cells but is required for this process. NF-kappa B has been reported to be necessary, but not sufficient, for the induction of NOS-II in other cell types (1, 3). Our study suggests the existence of different intracellular signaling pathways in NOS-II induction in RPE cells, since tyrosine kinase inhibitors blocked NOS-II mRNA accumulation without affecting NF-kappa B binding. We propose that the tyrosine kinase pathway converges with the NF-kappa B pathway, downstream of the activation of NF-kappa B, for NOS-II induction. Furthermore, the LPS-IFN-gamma -induced NOS-II mRNA accumulation was sensitive to cycloheximide. This result is similar to those generally obtained in cytokine-NOS-II induction models (28) and suggests that, in addition to NF-kappa B, transcriptional factors that depend on de novo protein synthesis are required for NOS-II expression. Effectively, induction of NO production in RPE cells requires both LPS and IFN-gamma . In this context, IRF-1, which is transcriptionally activated on IFN-gamma stimulation, has been involved in NOS-II induction (26). Furthermore, in mice genetically deficient in IRF-1, NOS-II induction is markedly decreased (21). The role of IRF-1 and of its repressor IRF-2 in the induction of NOS-II by LPS and IFN-gamma in bovine RPE cells is under investigation in our laboratory.

    ACKNOWLEDGEMENTS

We thank Dr. J. M. Cunningham (Hematology-Oncology Division, Harvard Medical School, Boston, MA) for the kind gift of murine macrophage NOS-II cDNA, Dr. H. Oshima (CIRC, Lyon, France) for the kind gift of antiserum against NOS-II, F. Régnier-Ricard for technical assistance, Dr. S. Michelson for critical reading, and H. Coet for photographic work.

    FOOTNOTES

This work was supported by grants from Association Française Retinitis Pigmentosa.

Address for reprint requests: O. Goureau, Développement, Vieillissement, et Pathologie de la Rétine, U450 INSERM, 29 rue Wilhem, 75016 Paris, France.

Received 1 December 1997; accepted in final form 3 April 1998.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Amoah-Aparaku, B., L. J. Chandler, J. K. Harrison, S. Tang, J. R. Ingelfinger, and N. J. Guzman. NFkappa B and transcriptional control of renal epithelial-inducible nitric oxide synthase. Kidney Int. 48: 674-682, 1995[Medline].

2.   Baeuerle, P. A., and D. Baltimore. Ikappa B: a specific inhibitor of the NF-kappa B transcription factor. Science 242: 540-546, 1988[Medline].

3.   Beck, K., and R. B. Sterzel. Cloning and sequencing of the proximal promoter of the rat iNOS gene: activation of NFkappa B is not sufficient for transcription of the iNOS gene in rat mesangial cells. FEBS Lett. 394: 263-267, 1996[Medline].

4.   Becquet, F., O. Goureau, G. Soubrane, G. Coscas, Y. Courtois, and D. Hicks. Superoxide inhibits proliferation and phagocytic internalization of photoreceptor outer segments by bovine retinal pigment epithelium in vitro. Exp. Cell Res. 212: 374-382, 1994[Medline].

5.   Bedoya, F. J., M. Flodström, and D. L. Eizirik. Pyrrolidine dithiocarbamate prevents IL-1-induced nitric oxide synthase mRNA, but not superoxide dismutase mRNA, in insulin producing cells. Biochem. Biophys. Res. Commun. 210: 816-822, 1995[Medline].

6.   Bredt, D. S., and S. H. Snyder. Nitric oxide: a physiologic messenger molecule. Annu. Rev. Biochem. 63: 175-195, 1994[Medline].

7.   Chénais, B., P. Bobé, G. Lemaire, and J. P. Tenu. Inhibition of NO synthase induction by antioxidant molecules: transcriptional and post-transcriptional regulation (Abstract). Endothelium 1: S20, 1993.

8.   Chirgwin, J. M., A. E. Pryzyla, R. J. McDonald, and W. J. Rutter. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294-5295, 1978.

9.   Darnell, J. E., I. M. Kerr, and G. R. Stark. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264: 1415-1421, 1994[Medline].

10.   Dighiero, P., I. Reux, J. J. Hauw, A. M. Fillet, Y. Courtois, and O. Goureau. Expression of inducible nitric oxide synthase in cytomegalovirus-infected glial cells of retinas from AIDS patients. Neurosci. Lett. 166: 31-34, 1994[Medline].

11.   Dong, Z., X. Qi, K. Xie, and I. J. Fidler. Protein tyrosine kinase inhibitors decrease induction of nitric oxide synthase activity in lipopolysaccharide-responsive and lipopolysaccharide-nonresponsive murine macrophages. J. Immunol. 151: 2717-2724, 1993[Abstract/Free Full Text].

12.   Fösrstermann, U., E. I. Closs, J. S. Pollock, M. Nakane, P. Schwarz, I. Gath, and H. Kleiner. Nitric oxide synthase isozymes. Hypertension 23: 1121-1131, 1994[Abstract].

13.   Goureau, O., F. Becquet, and Y. Courtois. Nitric oxide in the retina: potential involvement in retinal degeneration and its control by growth factors and cytokines. In: Degenerative Diseases of the Retina, edited by R. E. Anderson, M. M. LaVail, and J. G. Hollyfield. New York: Plenum, 1995, p. 61-68.

14.   Goureau, O., V. Faure, and Y. Courtois. Fibroblast growth factors decrease inducible nitric oxide synthase mRNA accumulation in bovine retinal pigmented epithelial cells. Eur. J. Biochem. 230: 1046-1052, 1995[Abstract].

15.   Goureau, O., D. Hicks, Y. Courtois, and Y. de Kozak. Induction and regulation of nitric oxide synthase in retinal Müller glial cells. J. Neurochem. 63: 310-317, 1994[Medline].

16.   Goureau, O., M. Lepoivre, F. Becquet, and Y. Courtois. Differential regulation of inducible nitric oxide synthase by fibroblast growth factors and transforming growth factor-beta . Inverse correlation with cellular proliferation. Proc. Natl. Acad. Sci. USA 90: 4276-4280, 1993[Abstract].

17.   Goureau, O., M. Lepoivre, and Y. Courtois. Lipopolysaccharide and cytokines induce a macrophage-type of nitric oxide synthase in bovine retinal pigmented epithelial cells. Biochem. Biophys. Res. Commun. 186: 854-859, 1992[Medline].

18.   Imbert, V., R. A. Rupec, A. Livolsi, H. L. Pahl, E. B. Traenckner, C. Mueller-Dieckmann, D. Farahifar, B. Rossi, P. Auberger, P. A. Baeuerle, and J. Peyron. Tyrosine phosphorylation of Ikappa Balpha activates NF-kappa B without proteolytic degradation of Ikappa Balpha . Cell 86: 787-798, 1996[Medline].

19.   Ishikawa, Y., N. Mukaida, K. Kuno, N. Rice, S. Okamoto, and K. Matsushima. Establishment of lipopolysaccharide-dependent nuclear factor kappa B activation in a cell-free system. J. Biol. Chem. 270: 4158-4164, 1995[Abstract/Free Full Text].

20.   Israel, N., M. Gougerot-Pocidalo, F. Aillet, and J. Virelizier. Redox status of cells influences constitutive or induced NF-kappa B translocation and HIV long terminal repeat activity in human T and monocytic cell lines. J. Immunol. 149: 3386-3393, 1992[Abstract/Free Full Text].

21.   Kamijo, R., H. Harada, T. Matsuyama, M. Bosland, J. Gerecitano, D. Shapiro, S. I. Koh, T. Kimura, S. J. Green, T. W. Mak, T. Taniguchi, and J. Vilcek. Requirement for transcription factor IRF-1 in NO synthase induction in macrophages. Science 263: 1612-1615, 1994[Medline].

22.   Kleinert, H., C. Euchenhofer, I. Ihrig-Biedert, and U. Förstermann. In murine 3T3 fibroblasts, different second messenger pathways resulting in the induction of NO synthase II (iNOS) converge in activation of transcription factor NF-kappa B. Mol. Pharmacol. 49: 15-21, 1996[Abstract].

23.   Kwon, G., J. A. Corbett, C. P. Rodi, P. Sullivan, and M. L. McDaniel. Interleukin-1beta -induced nitric oxide synthase expression by rat pancreatic beta -cells: evidence for the involvement of nuclear factor kappa B in the signaling mechanism. Endocrinology 136: 4790-4795, 1995[Abstract].

24.   Lyons, C. R., G. J. Orloff, and J. M. Cunningham. Molecular cloning and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line. J. Biol. Chem. 207: 6370-6374, 1992.

25.   MacMicking, J., Q. Xie, and C. Nathan. Nitric oxide and macrophage function. Annu. Rev. Immunol. 15: 323-350, 1997[Medline].

26.   Martin, E., C. Nathan, and Q. Xie. Role of interferon regulatory factor 1 in induction of nitric oxide synthase. J. Exp. Med. 180: 977-984, 1994[Abstract].

27.   Murphy, W. J., M. Muroi, C. X. Zhang, T. Suzuki, and S. W. Russell. Both basal and enhancer kappa B elements are required for full induction of the mouse inducible nitric oxide synthase gene. J. Endotoxin Res. 3: 381-393, 1996.

28.   Nathan, C., and Q. Xie. Nitric oxide synthases: roles, tolls and controls. Cell 78: 915-918, 1994[Medline].

29.   Nussler, A. K., and T. R. Billiar. Inflammation, immunoregulation, and inducible nitric oxide synthase. J. Leukoc. Biol. 54: 171-178, 1993[Abstract].

30.   Oddis, C. V., and M. S. Finkel. NF-kappa B and GTP cyclohydrolase regulate cytokine-induced nitric oxide production by cardiac myocytes. Am. J. Physiol. 270 (Heart Circ. Physiol. 39): H1864-H1868, 1996[Abstract/Free Full Text].

31.   Saura, M., S. Lopez, M. R. Puyol, D. R. Puyol, and S. Lamas. Regulation of inducible nitric oxide synthase expression in rat mesangial cells and isolated glomeruli. Kidney Int. 47: 500-509, 1995[Medline].

32.   Schreck, R., B. Meier, D. N. Mannel, W. Droge, and P. A. Baeuerle. Dithiocarbamates as potent inhibitors of nuclear factor kappa B activation in intact cells. J. Exp. Med. 175: 1181-1194, 1992[Abstract].

33.   Spink, J., J. Cohen, and T. J. Evans. The cytokine responsive vascular smooth muscle cell enhancer of inducible nitric oxide synthase. J. Biol. Chem. 270: 29541-29547, 1995[Abstract/Free Full Text].

34.   Sunderman, F. W. Forty-five years of proficiency testing. Ann. Clin. Lab. Sci. 21: 143-144, 1991[Medline].

35.   Tetsuka, T., S. K. Srivastava, and A. R. Morrison. Tyrosine kinase inhibitors, genistein and herbimycin A, do not block interleukin-1beta -induced activation of NF-kappa B in rat mesangial cells. Biochem. Biophys. Res. Commun. 218: 808-812, 1996[Medline].

36.   Ulevitch, R. J., and P. S. Tobias. Recognition of endotoxin by cells leading to transmembrane signaling. Curr. Opin. Immunol. 6: 125-130, 1994[Medline].

37.   Verma, I. M., J. K. Stevenson, E. M. Schwarz, D. Van Antwerp, and S. Miyamoto. Rel/NF-kappa B/Ikappa B family: intimate tales of association and dissociation. Genes Dev. 9: 2723-2735, 1996[Medline].

38.   Weinstein, S. L., S. Sanghera, K. Lemke, A. L. De France, and S. L. Pelech. Bacterial lipopolysaccharide induces tyrosine phosphorylation and activation of mitogen-activated protein kinases in macrophages. J. Biol. Chem. 267: 14955-14962, 1992[Abstract/Free Full Text].

39.   Xie, Q., Y. Kashiwabara, and C. Nathan. Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase. J. Biol. Chem. 269: 4705-4708, 1994[Abstract/Free Full Text].

40.   Xie, Q. W., R. Whisnant, and C. Nathan. Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon-gamma and bacterial lipopolysaccharide. J. Exp. Med. 177: 1779-1784, 1993[Abstract].


Am J Physiol Cell Physiol 275(1):C208-C215
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