Rapid Inactivation of NOS-I by Lipopolysaccharide Plus Interferon-γ-induced Tyrosine Phosphorylation*

Human astrocytoma T67 cells constitutively express a neuronal NO synthase (NOS-I) and, following administration of lipopolysaccharide (LPS) plus interferon-γ (IFNγ), an inducible NOS isoform (NOS-II). Previous results indicated that a treatment of T67 cells with the combination of LPS plus IFNγ, by affecting NOS-I activity, also inhibited NO production in a very short time. Here, we report that under basal conditions, a NOS-I protein of about 150 kDa was weakly and partially tyrosine-phosphorylated, as verified by immunoprecipitation and Western blotting. Furthermore, LPS plus IFNγ increased the tyrosine phosphorylation of NOS-I, with a concomitant inhibition of its enzyme activity. The same effect was observed in the presence of vanadate, an inhibitor of phosphotyrosine-specific phosphatases. On the contrary, genistein, an inhibitor of protein-tyrosine kinases, reduced tyrosine phosphorylation of NOS-I, enhancing its enzyme activity. Finally, using reverse transcriptase-polymerase chain reaction, we have observed that a suboptimal induction of NOS-II mRNA expression in T67 cells was enhanced by vanadate (or l-NAME) and inhibited by genistein. Because exogenous NO has been found to suppress NOS-II expression, the decrease of NO production that we have obtained from the inactivation of NOS-I by LPS/IFNγ-induced tyrosine phosphorylation provides the best conditions for NOS-II expression in human astrocytoma T67 cells.

Nitric oxide is a major messenger molecule that is generated by a family of enzymes, termed NO synthases (NOS) (for recent reviews see Refs. [1][2][3][4][5]. 1 There are at least three distinct iso-forms of NOS; two enzymes (NOS-I and NOS-III, also called cNOS) are constitutively expressed in neurons, endothelial cells, and glial cells, and one inducible NOS (NOS-II) is expressed after stimulation with endotoxin and/or cytokines in a number of cells including macrophages, neutrophils, hepatocytes, and glial cells.
Recently, we have reported that in human microglial cells, which do not express cNOS, low concentrations of exogenous NO suppressed the induction of NOS-II expression (6), suggesting an involvement of physiological NO levels, as produced by cNOS, in preventing the accidental or unfavorable induction of NOS-II expression. Paradoxically, in cell types (e.g. astroglial cells) expressing both constitutive and inducible NOS, the induction of the latter should be a rare event, unless the NO produced by constitutive NOS is preventively and quickly down-regulated. In this respect, we have further demonstrated that in human astroglial cells, Escherichia coli lipopolysaccharide (LPS) plus interferon-␥ (IFN␥), two common inducers of NOS-II expression (7), elicited a decrease in NO synthesis. This effect was mediated by a very fast inhibition of NOS-I activity, without affecting the NOS-I mRNA transcription (8).
Here, we used human astrocytoma T67 cells, as an astroglial model, for a better understanding of the biochemical mechanism of the fast NOS-I inactivation and successive NOS-II induction. In this respect, we hypothesize that the rapid inhibition of NOS-I activity by NOS-II inducers (i.e. LPS plus IFN␥) in astroglial cells may be mediated by tyrosine phosphorylation of NOS-I.

Methods
Preparation of Astrocytoma Cells-Human astrocytoma T67 cells were obtained from explant of III WHO gemistocytic astrocytoma and were characterized in our laboratory, as described previously (9).
Immunoprecipitation and Western Blot Analysis-For the immunoprecipitation of NOS-I, T67 cells (1 ϫ 10 6 cells/sample) were washed with phosphate-buffered saline and lysed on ice in 10 mM Tris-HCl buffer, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 1 mM EDTA, 1 mM EGTA, 1 mM vanadate, 10 mM NaF, 10 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, and 10 g/ml leupeptin. Lysates were cleared by centrifugation, and equal amounts of proteins were incubated with an anti-NOS-I polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), diluted 1:30, for 3 h at 4°C. Protein A-agarose was added, and the mixture was incubated for additional 30 min. After three washes, the samples were boiled in SDS-polyacrylamide gel electrophoresis sample buffer for 5 min. Immunoprecipitated samples were subjected to electrophoresis in 7.5% polyacrylamide gels and blotted to nitrocellulose. Membranes were blocked with 5% nonfat milk for 1 h and incubated for 2 h, with the anti-NOS-I polyclonal antibody being diluted (1:1000) in the blocking buffer. After extensive washing, a goat anti-rabbit IgG horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology, Inc.) was added. In some experiments, a monoclonal anti-phosphotyrosine horseradish peroxidase-conjugated antibody (RC20 from Transduction Laboratories, Lexington, * This work was supported by a Consiglio Nazionale delle Ricerche project grant (to M. C.) and Consiglio Nazionale delle Ricerche target oriented project Biotechnology grant and a Ministero dell'Università el della Ricerca Scientifica e Tecnologica project grant (to H. S. and G. M. L.). 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 with 18 U.S.C. Section 1734 solely to indicate this fact.
KY) was used according to the manufacturer's instructions. Antibody binding was detected by chemiluminescence (ECL, Amersham Italia S.r.l., Milan, Italy).
Assay of Astrocytoma NOS Activity-NOS activity was estimated by measuring the conversion of L-2,3,4,5-[ 3 H]arginine to L-2,3-[ 3 H]citrulline according to the modification of the method described by Bredt and Snyder (10). 2 ϫ 10 6 human astrocytoma T67 cells were homogenized with Ultra-Turrax homogenizer (5-mm blade) for 60 s in 200 l of a buffer containing 50 mM HEPES, pH 7.4, 1 mM dithiothreitol, 1 mM EDTA, 10 g/ml leupeptin, 10 g/ml soybean trypsin inhibitor, 10 g/ml antipain, and 1 mM phenylmethylsulfonyl fluoride. For Ca 2ϩ -dependent activity, after centrifugation (10,000 ϫ g for 30 min at 4°C), an aliquot of the supernatant was added to a reaction mixture of a final volume of 100 l containing 50 mM HEPES, pH 7.4, 20 nM [ 3 H]arginine, 1 M arginine, 1 mM NADPH, 1 mM EDTA, 1.2 mM CaCl 2 , 1 g/ml calmodulin, 10 M FAD, 0.1 mM (6R)-5,6,7,8-tetrahydro-1-biopterin, and 1 mM dithiothreitol. For Ca 2ϩ -independent activity, 1.2 mM CaCl 2 and 1 g/ml calmodulin were omitted from and 1 mM EGTA was added to the reaction mixture. The reactions were stopped by adding 0.4 ml (1:1) of slurry of Dowex AG50WX-8 (Bio-Rad, Na ϩ form) in 50 mM HEPES, pH 5.5. and after 15 min of shaking, radioactivity in the supernatant was measured. The enzyme activity was linear up to 15 min of incubation. Specific enzyme activity was calculated as pmol of citrulline formed in 1 min by 1 mg of protein. Values are expressed as the percentage of NOS activity versus untreated T67 cells. Protein concentration in the samples was determined by the method of Bradford (11).
Reverse Transcriptase-PCR-Total cellular RNA was purified from 1 ϫ 10 6 human astrocytoma T67 cells by the method of Chomczynski and Sacchi (12). Briefly, a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture was performed. Whole RNA was reverse transcribed into cDNA by Moloney murine leukemia virusreverse transcriptase using oligo(dT) 12-18 as primer. PCR was carried out with Taq DNA polymerase in an automatic DNA thermal cycler (GeneAmp 2400, Perkin-Elmer). NOS-II cDNA amplification was obtained as described previously (6). 10 l of each final PCR product (450 base pairs) were electrophoresed on 1.5% agarose gel and then visualized after ethidium bromide staining. The bands so obtained were quantified with Fluor-S TM MultImager (Bio-Rad). In addition, the NOS-II-amplified DNA fragment was purified after gel extraction (using Qiaquick gel extraction kit, purchased from Qiagen GmbH, Dü sseldorf, Germany). The nucleotide sequence was determined using the Ampli-Cycle TM sequencing kit (Perkin-Elmer) by direct cycle sequencing with Taq DNA polymerase and 35 S-labeled dCTP according to the manufacturer's instructions. As expected, we found that the nucleotide sequence of the PCR product from T67 cells displayed 99% identity with NOS-II cDNA (from 3131 to 3580) of human hepatocytes (GenBank TM accession number L09210). The mRNA for the constitutive glycerol-3-phosphate dehydrogenase was examined as the reference cellular transcript. Glycerol-3-phosphate dehydrogenase mRNA amplification products (195 base pairs) were present at equivalent levels in all cell lysates. The reaction was performed using specific primers as described elsewhere (13).

RESULTS AND DISCUSSION
As described previously, a treatment of human astrocytoma T67 cells with the combination of LPS plus IFN-␥ strongly inhibited NO production in a very short time, by affecting the Ca 2ϩ -dependent NOS activity but not NOS-I mRNA expression (8). Recently, it has been reported that in endothelial cells, the rapid inhibition of the Ca 2ϩ -dependent NOS activity (e.g. NOS-III) seems to be associated with the phosphorylation of tyrosine residue(s) of the enzyme (14). Here, we have evaluated the tyrosine phosphorylation state of NOS-I protein in human astrocytoma T67 cells. As shown in Fig. 1A, a NOS-I protein from the soluble fractions of T67 cell homogenates was evidenced. The molecular mass was about 150 kDa, as was that from recombinant rat neuronal NOS, used as a positive control. Moreover, under basal conditions, NOS-I protein immunoprecipited from T67 cells was weakly and partially tyrosine-phosphorylated (Fig. 1B, lane 1). When T67 cells were treated with LPS (10 g/ml) plus IFN␥ (1000 units/ml) for 30 min, tyrosine phosphorylation of NOS-I was enhanced (Fig. 1B, lane 2). The same effect, even more marked, was observed in the presence of vanadate (1 mM for 2 h), an inhibitor of protein-tyrosine phosphatases (Fig. 1B, lane 3). Moreover, a pretreatment with vanadate for 2 h was able to increase LPS/IFN␥-induced tyrosine phosphorylation of NOS-I (Fig. 1B, lane 4). On the contrary, preincubation of both LPS/IFN␥-treated and untreated T67 cells with an inhibitor of protein-tyrosine kinases, genistein (1 mM for 2 h), completely abolished tyrosine phosphorylation of NOS-I (Fig. 1B, lanes 5 and 6, respectively).
As described previously, homogenates of unstimulated T67 cells exhibited a basal NOS-I activity (8). The elimination of free Ca 2ϩ by EGTA (1 mM) from the reaction mixture almost totally abolished this activity (Fig. 2), confirming that the isoform of the enzyme was Ca 2ϩ -dependent. To verify the effect of tyrosine phosphorylation state of NOS-I on its enzyme activity, we used genistein, which decreases the tyrosine phosphorylation of NOS-I protein (Fig. 1). In this respect, a preincubation of T67 cells with 1 mM genistein for 2 h enhanced basal NOS-I activity (Fig. 2), the weak, constitutive tyrosine phosphorylation of the enzyme being likely reduced. On the contrary, a 30-min treatment of T67 cells with LPS (10 g/ml) plus IFN␥ (1000 units/ml), which increases the tyrosine phosphorylation of NOS-I protein (Fig. 1), strongly inhibited NOS-I activity (Fig. 2). When LPS/IFN␥-stimulated T67 cells were preincubated with 1 mM genistein for 2 h, NOS-I activity was completely restored to reach the same effect as obtained with genistein alone (Fig. 2). Also, the involvement of tyrosine phosphorylation in modulating NOS-I activity was confirmed using vanadate, which increased tyrosine phosphorylation of NOS-I protein (Fig. 1B). In this respect, the treatment of T67 cells with vanadate alone (1 mM for 2 h) reduced the basal level of NOS-I activity, in the same manner as LPS plus IFN␥ (Fig. 2).
Taken together, our results suggest that the balance between a native and a tyrosine-phosphorylated form as well as the enzymatic activity of NOS-I can be controlled by the activity of protein-tyrosine kinase(s) and phosphotyrosine-specific phosphatase(s). Moreover, by affecting tyrosine phosphorylation, NOS-II inducers (e.g. LPS and IFN␥) quickly inhibit NOS-I activity, thereby providing the best conditions for NOS-II induction. To verify this hypothesis, we have analyzed the effect of the tyrosine phosphorylation on NOS-II mRNA expression as induced by suboptimal concentrations of LPS plus IFN␥ in human astrocytoma T67 cells. As shown in Fig. 3, incubation of T67 cells with a suboptimal concentration of LPS (10 g/ml) plus IFN␥ (100 units/ml) for 8 h induced a less pronounced NOS-II mRNA expression (lane 2) than observed in the treatment with the optimal concentration of LPS (10 g/ml) plus IFN␥ (1000 units/ml) (lane 3). When T67 cells were incubated with the suboptimal concentration of LPS plus IFN␥, a NOS inhibitor L-NAME (1 mM) caused a NOS-II mRNA overexpres-sion (see lane 6), indicating that a rapid NOS inactivation facilitated the induction of NOS-II expression. The present results are in agreement with our previous reports indicating that NO exerts a suppressive effect on NOS-II expression (6,15,16).
Furthermore, when T67 cells were stimulated by the suboptimal concentration of LPS plus IFN␥, preincubation (for 2 h) with 1 mM genistein, which inhibited the tyrosine phosphorylation of NOS-I (Fig. 1B) causing a NOS-I hyper-activation (Fig.  2), completely suppressed NOS-II mRNA expression (Fig. 3,  lane 5). On the other hand, when T67 cells were treated with the suboptimal concentration of LPS plus IFN␥, preincubation (for 2 h) with 1 mM vanadate, which enhanced the tyrosine phosphorylation of NOS-I (Fig. 1B) causing a NOS-I inhibition (Fig. 2), induced a NOS-II mRNA overexpression (Fig. 3, lane  4). Our observations are in agreement with previous results indicating a requirement for protein-tyrosine kinase activation as part of the process of NOS-II induction in several cell types (17)(18)(19)(20)(21)(22)(23)(24), and this may reflect the need to switch off cNOS enzyme activity.
As a whole, our results confirm our previous reports suggesting that a change in endogenous NO levels may be a key factor in regulating the induction of NOS-II expression in human astrocytoma cells as well as in rat neutrophils (8,15). In this respect, affecting the NOS-I tyrosine phosphorylation, NOS-II inducers (e.g. LPS and IFN␥) elicit a rapid inactivation of the enzyme, leading to a decrease of basal NO levels (8). Thus, our findings can explain how NOS-II inducers are able to provide the best conditions for the induction of NOS-II expression, thereby resolving an apparent paradox.