COMMUNICATION
Rapid Inactivation of NOS-I by Lipopolysaccharide Plus
Interferon-
-induced Tyrosine Phosphorylation*
Marco
Colasanti
,
Tiziana
Persichini
,
Elisabetta
Cavalieri§,
Cinzia
Fabrizi
,
Sofia
Mariotto§,
Marta
Menegazzi§,
Giuliana M.
Lauro
, and
Hisanori
Suzuki§¶
From the
Department of Biology, University
of Rome, ROMA TRE, Viale Marconi 446, I-00146 Rome, Italy and the
§ Department of Neuroscience and Vision, Laboratory of
Biological Chemistry, University of Verona, Strada Le Grazie 8, I-37134 Verona, Italy
 |
ABSTRACT |
Human astrocytoma T67 cells constitutively
express a neuronal NO synthase (NOS-I) and, following administration of
lipopolysaccharide (LPS) plus interferon-
(IFN
), an inducible NOS
isoform (NOS-II). Previous results indicated that a treatment of T67
cells with the combination of LPS plus IFN
, 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 IFN
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/IFN
-induced tyrosine
phosphorylation provides the best conditions for NOS-II expression in
human astrocytoma T67 cells.
 |
INTRODUCTION |
Nitric oxide is a major messenger molecule that is generated by a
family of enzymes, termed NO synthases (NOS) (for recent reviews see
Refs. 1-5).1 There are at
least three distinct isoforms 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.
 |
EXPERIMENTAL PROCEDURES |
Materials
N
-nitro-L-arginine methyl ester
(L-NAME) and E. coli lipopolysaccharide (LPS;
Serotype 0127:B8) were obtained from Sigma (Milan, Italy). Human
recombinant interferon-
(IFN
) was supplied by Biogen SA (Geneva,
Switzerland; specific activity, 2 × 107 IU mg
protein
1) and
L-2,3,4,5-[3H]arginine by Amersham.
Recombinant rat neuronal NOS was purchased from Calbiochem (La Jolla, CA).
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 × 106
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 µg/ml aprotinin, and 10 µg/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, 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-[3H]arginine to
L-2,3-[3H]citrulline according to the
modification of the method described by Bredt and Snyder (10). 2 × 106 human astrocytoma T67 cells were homogenized with
Ultra-Turrax homogenizer (5-mm blade) for 60 s in 200 µl of a
buffer containing 50 mM HEPES, pH 7.4, 1 mM
dithiothreitol, 1 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml
soybean trypsin inhibitor, 10 µg/ml antipain, and 1 mM
phenylmethylsulfonyl fluoride. For
Ca2+-dependent activity, after centrifugation
(10,000 × g for 30 min at 4 °C), an aliquot of the
supernatant was added to a reaction mixture of a final volume of 100 µl containing 50 mM HEPES, pH 7.4, 20 nM
[3H]arginine, 1 µM arginine, 1 mM NADPH, 1 mM EDTA, 1.2 mM
CaCl2, 1 µg/ml calmodulin, 10 µM FAD, 0.1 mM (6R)-5,6,7,8-tetrahydro-1-biopterin, and 1 mM dithiothreitol. For Ca2+-independent
activity, 1.2 mM CaCl2 and 1 µg/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+ 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 × 106 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 virus-reverse 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 µl 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-STM 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, Düsseldorf, Germany). The nucleotide sequence was
determined using the AmpliCycleTM sequencing kit
(Perkin-Elmer) by direct cycle sequencing with Taq DNA
polymerase and 35S-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
(GenBankTM 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
Ca2+-dependent NOS activity but not NOS-I
mRNA expression (8). Recently, it has been reported that in
endothelial cells, the rapid inhibition of the
Ca2+-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 µg/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).

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Fig. 1.
Western blot analysis of NOS-I in human
astrocytoma T67 cells. A, NOS-I was immunoprecipitated
from 1 × 106 cells/sample and Western blotted using a
polyclonal anti-NOS-I antibody. As expected, the resulting band (about
150 kDa) corresponded to that of recombinant rat NOS
(RecNOS), used as a positive control. B,
immunoprecipitated NOS-I was Western blotted using a specific antiserum
against phosphotyrosine. NOS-I from untreated cells was weakly and
partially tyrosine-phosphorylated (lane 1). Treatment of
cells with LPS (10 µg/ml) plus IFN (1000 units/ml) for 30 min
increased tyrosine phosphorylation of NOS-I (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 (lane 3). Vanadate was able to increase
LPS/IFN -induced tyrosine phosphorylation of NOS-I (lane
4). Preincubation of both LPS/IFN -treated (lane 5)
and untreated (lane 6) T67 cells with an inhibitor of
protein-tyrosine kinases, genistein (1 mM for 2 h),
completely abolished tyrosine phosphorylation of NOS-I.
|
|
As described previously, homogenates of unstimulated T67 cells
exhibited a basal NOS-I activity (8). The elimination of free
Ca2+ by EGTA (1 mM) from the reaction mixture
almost totally abolished this activity (Fig.
2), confirming that the isoform of the
enzyme was Ca2+-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 µg/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).

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Fig. 2.
Effect of tyrosine phosphorylation on NOS-I
activity in human astrocytoma T67 cells. Homogenates of
unstimulated T67 cells exhibited a basal NOS activity
(control). The presence of EGTA (1 mM) in the
assay mixture abolished this activity, demonstrating that the enzyme
isoform was a Ca2+-dependent NOS. A 30-min
treatment of T67 cells with LPS (10 µg/ml) plus IFN (1000 units/ml) strongly inhibited NOS-I activity. A preincubation (for
2 h) of LPS/IFN -stimulated T67 cells with 1 mM
genistein, an inhibitor of protein-tyrosine kinases, completely
restored LPS/IFN -decreased NOS-I activity. A pretreatment (for
2 h) of T67 cells with 1 mM vanadate, an inhibitor of
protein-tyrosine phosphatases, reduced the basal level of NOS-I
activity in T67 cells. Values are expressed as the percentage of NOS
activity versus untreated T67 cells.
|
|
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 µg/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 µg/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 overexpression (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).

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Fig. 3.
Effect of tyrosine phosphorylation on NOS-II
mRNA expression in human astrocytoma T67 cells. Lane
1, untreated cells. Treatment of cells with a suboptimal
concentration of a mixture of LPS (10 µg/ml) plus IFN (100 units/ml) for 8 h induced a less pronounced NOS-II mRNA
expression (lane 2) than treatment with the optimal
concentration of LPS (10 µg/ml) plus IFN (1000 units/ml)
(lane 3). The same sample as in lane 2 preincubated with vanadate (1 mM) for 2 h showed a
NOS-II mRNA overexpression (lane 4), the same effect
being obtained by preincubation of the cells with the NOS inhibitor
L-NAME (1 mM) (lane 6). The same
sample as in lane 2 preincubated with genistein (1 mM for 2 h) completely suppressed NOS-II mRNA
expression (lane 5).
|
|
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 (17-24), 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.
 |
ACKNOWLEDGEMENTS |
We thank Prof. Paolo Ascenzi and Prof.
Giorgio Venturini for helpful discussion and Luisella Mattace for
editorial assistance.
 |
FOOTNOTES |
*
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. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dipartimento di
Scienze Neurologiche e della Visione, Sezione di Chimica Biologica, Università di Verona, Strada Le Grazie 8, I-37134 Verona, Italy. Tel.: 39-45-8098167; Fax: 39-45-8098170.
 |
ABBREVIATIONS |
The abbreviations used are:
NOS, NO synthase(s);
cNOS, constitutive Ca2+-dependent
NOS isoform;
LPS, lipopolysaccharide;
IFN
, interferon-
;
L-NAME, N
-nitro-L-arginine
methyl ester;
PCR, polymerase chain reaction.
 |
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