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Instituto de Bioquímica (Centro Mixto Consejo Superior de Investigaciones Cientificas-Universidad Compluteuse de Madrid, Facultad de Farmacia, Universidad Complutense, Madrid, Spain 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.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B in LPS-treated
cells as well as an increase in the promoter activity of the iNOS gene
as deduced from transfection experiments using a 1.7-kb fragment of the
5' flanking region of the iNOS gene. Cotransfection of cells with a
catalytically active p110 subunit of PI3-kinase impaired the
responsiveness of the iNOS promoter to LPS stimulation, whereas
transfection with a kinase-deficient mutant of p110 maintained the
up-regulation in response to wortmannin. These results indicate that
PI3-kinase plays a negative role in the process of macrophage
activation and suggest that this enzyme might participate in the
mechanism of action of antiinflammatory
cytokines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1ß, and IL-6,
which favor the amplification of the original response. LPS interacts
with the macrophage through CD14, a glycophosphatidylinositol-anchored
molecule (4). Activation through this receptor promotes the stimulation
of several protein tyrosine kinases of the Src family favoring the
association of p53/p56lyn to the receptor (5, 6). The activation of this kinase facilitates the association
with PI3-kinase as well as the involvement of several serine/threonine
protein kinases, among them several members of protein kinase C family
(6, 7, 8). Activated macrophages release oxygen and nitrogen radicals that are important bactericidal and cytostatic molecules (1, 2, 9). However, massive production of these mediators can exert detrimental effects in the organism as occurs during septic shock or persistent local inflammatory processes (10, 11). For this reason, the study of the mechanism of action of antiinflammatory cytokines and drugs has constituted a subject of current interest (10, 11, 12, 13, 14).
One of the aspects most studied in stimulated macrophages is the
induction of the inducible type of NO synthase (iNOS)3 and
the increase of NO synthesis by these cells (1, 15, 16). It is well
known that iNOS expression is regulated mainly at the transcription
level due to the activation of several transcription factors that bind
to the promoter region of the iNOS gene, such as NF-B, STAT1 and
IRF-1 (15, 16, 17). The activation of NF-B depends on the degradation of
the corresponding inhibitory proteins B and Bß that retain
inactive the NF-B complex in the cytosol (18, 19). Several data
point to NF-B activation as a critical event in the expression
of iNOS (17, 20, 21), and most studies focused on the analysis of
antiinflammatory mechanisms suggested a prominent role for the
inhibition of this transcription factor in their mode of action
(22, 23).
More recently, several groups have shown that in the course of
macrophage activation various inhibitory mechanisms are engaged
favoring a controlled regulation of the process to avoid the harmful
effects of an exacerbated activation. Regarding iNOS expression, a
negative regulation by NO has been described (24), as well as a
competition between type I and type II IFNs in the synergistic action
with LPS (14). Moreover, it has been shown that LPS increases the
levels of PPAR
and this nuclear factor exerts an inhibitory effect
on macrophage activation, including the inhibition of iNOS and
gelatinase B transcription (12, 25).
In this work, we show that activation with LPS of the macrophage cell
line RAW 264.7 activates PI3-kinase. However, inhibition of this kinase
by wortmannin and LY294002 (5, 7, 26) results in an up-regulation of
iNOS expression, mainly through a mechanism that involves a sustained
activation of NF-B. Moreover, expression of a constitutively active
p110 subunit of the PI3-kinase attenuates the promoter activity of
cells cotransfected with a plasmid containing a 1.7-kb fragment of the
5' flanking region of the murine iNOS gene. These results indicate that
PI3-kinase plays a negative role in the expression of iNOS and might
contribute to understanding the mechanism of action of antiinflammatory
cytokines (IL-13 and IL-10) that activate PI3-kinase in the course of
their intracellular signaling (26, 27).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Biochemicals and reagents were from Sigma (St Louis, MO) or from Boehringer Mannheim (Mannheim, Germany). Wortmannin and LY294002 were from BioMol (Plymouth Meeting, PA). Electrophoresis equipment and reagents were from Bio-Rad (Richmond, CA) or from Amersham (Amersham Bucks., U.K.). Serum and media were from BioWhittaker (Walkersville, MD).
Cell culture
RAW 264.7 cells were obtained from American Type Culture Collection (Manassas, VA) and were seeded at 0.8–1 x 105/cm2 in RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% FCS, and antibiotics (50 µg/ml of penicillin, streptomycin, and gentamicin). After 2 days in culture, the cell layers were washed with PBS and the culture medium was replaced by phenol red-free RPMI 1640 containing 0.5 mM arginine and 5% FCS.
Plasmid constructs and preparation
The 1749-bp HincII fragment corresponding to the 5'
flanking region of iNOS (14, 15, 16) fused to a promoterless
chloramphenicol acetyltransferase (CAT) reporter gene (p1NOS.CAT) was a
generous gift from Drs. Q.-w. Xie and C. Nathan (Cornell University,
Ithaca, NY). A (B)3ConA.CAT plasmid construct, which
contains three copies of the B motif from the HIV long terminal
repeat enhancer with the conalbumin promoter, was used to measure B
transactivation capacity (21). A ConA.CAT vector lacking the B
tandem was used as control. rCD2p110, which encodes a constitutively
active molecule, including the extracellular and transmembrane domains
of the rat CD2 cell surface Ag and the p110 catalytic subunit of
PI3-kinase, rCD2p110kd, a kinase-deficient mutant, and p85d, which is
unable to bind p110 and therefore inhibits the recruitment of p110 to
the membrane, were a generous gift from Dr. D. A. Cantrell (Imperial
Cancer Research Fund, London, U.K.), and have been previously described
(28, 29). A rCD2 vector was used as control of specificity in the
response to stimuli, and its expression was not affected by LPS or
wortmannin. The level of expression of CD2 was determined by flow
cytometry after labeling with FITC-OX-34 (PharMingen, San Diego, CA). A
KSV2.CAT plasmid was used as a reference for maximal
efficiency of the transfection (21). Plasmids were purified using
EndoFree Qiagen columns (Hilden, Germany).
Transfection of RAW 264.7 cells and assay of CAT activity
The cell layer was washed twice with PBS and the plates (6 cm
diameter) were filled with 1.5 ml of RPMI 1640 medium without FCS.
Cells were transfected for 8 h by lipofection with
N-(1-[2,3-dioleoyloxy]propyl)-N,N,N-trimethylammonium
methylsulfate following the instructions of the supplier (Boehringer
Mannheim). After transfection, the cells were maintained for 24 h
prior to stimulation with RPMI 1640 medium containing 5% FCS. Equal
amounts of DNA were used in the transfection experiments, using a basic
p.CAT plasmid (Promega, Madison, WI) to normalize the DNA content. CAT
activity corresponding to the p1NOS.CAT promoter or
(B)3.CAT construct (21) was determined after 24 or
18 h of treatment of the cells with the indicated stimuli, as
follows: the cells were washed twice with PBS at 4°C, scraped off the
dishes, and centrifuged. The cell pellets were resuspended in 0.2 ml of
0.2 M Tris-HCl (pH 7.8), and after gently mixing, the extract was
submitted to three cycles of freezing and thawing, followed by
centrifugation at 12,000 x g for 10 min, and the
soluble protein was measured. Aliquots of the supernatant were
normalized for protein (200 µg) and heated at 65°C for 10 min. CAT
activity was measured in a final volume of 150 µl by the synthesis of
acetylated [14C]chloramphenicol following the TLC method
(21). The amount of acetylated substrate was quantified in a FUJI BAS
1000 radioactivity-detection system.
Characterization of iNOS expression by Northern blot
Total RNA (2–4 x 106 cells) was extracted
using the guanidinium thiocyanate method (30). After electrophoresis in
a 0.9% agarose gel containing 2% formaldehyde, the RNA was
transferred to a Nytran membrane (NY 13-N; Schleicher & Schüll,
Dassel, Germany), and the levels of iNOS mRNA were determined by using
an EcoRI-HindII fragment from the iNOS cDNA (21)
labeled with [-32P]dCTP using the Rediprime labeling
kit (Amersham). The membranes were exposed to x-ray films (Hyperfilm,
Amersham) and the intensity of the bands was measured by laser
densitometry (Molecular Dynamics, Sunnyvale, CA). Hybridization with an
18S ribosomal probe was used as an internal standard.
Determination of NO synthesis
NO was measured as the accumulation of nitrite in the incubation medium. Nitrite was determined spectrophotometrically with Griess reagent (21) by adding 100 µl of 10 mM sulfanilic acid (in 1 M HCl) to 850 µl of culture medium. After incubation for 5 min, the absorbance at 548 nm was measured and 50 µl of 20 mM naphthylenediamine was added. The reaction was completed after 15 min of incubation and the absorbance at 548 nm was compared with a standard of NaNO2. The amount of nitrate produced from NO was determined after reduction to nitrite and was below 15% of the nitrite measured.
Assay of PI3-kinase activity
The cell layers were washed twice with ice-cold buffer A (10 mM
HEPES (pH 7.9), 1 mM EDTA, 1 mM EGTA, 10 mM KCl, 1 mM DTT, 0.5 mM PMSF,
2 µg/ml aprotinin, 10 µg/ml leupeptin, 2 µg/ml
N-[alpha]-tosyl-L-lysine chloromethyl ketone, 5 mM NaF, 1
mM NaVO4, and 10 mM Na2MO4)
containing 120 mM NaCl. Lysis of the cells was performed at 4°C with
1 ml of buffer A supplemented with 0.5% Nonidet P-40 (NP-40) and under
continuous shaking. PI3-kinase was immunoprecipitated with
anti-PI3-kinase Ab following the instructions of the supplier
(Upstate Biotechnology, Lake Placid, NY). PI3-kinase activity was
determined using phosphatidylinositol (20 µg) and
[-32P]ATP (31). After TLC, the amount of
phosphorylated lipids was evaluated using a FUJI BAS 1000 detector.
Characterization of proteins by Western blot
Cultured RAW 264.7 cells (3–4 x 106) were
washed twice with PBS, scraped off the dishes, transferred to a 1.5-ml
tube and centrifuged. The cell pellets were homogenized in buffer A
containing 0.5% NP-40. After centrifugation of the cell extracts in a
microcentrifuge for 15 min, the proteins present in the supernatant
were size-separated in 10% SDS-PAGE. The gels were blotted onto a
polyvinylidene difluoride membrane (Millipore, Bedford, MA) and
incubated with several anti-iNOS, anti-p85, anti-IB,
or anti-IBß Abs (Santa Cruz Laboratories, Santa Cruz, CA). The
blots were revealed by enhanced chemiluminescence following the
manufacturer’s instructions (Amersham).
Preparation of cytosolic and nuclear extracts
A modified procedure based on the method of Diaz-Guerra et al. (21) Schreiber et al. (32) was used. Cells (1.5 x 106) were washed with PBS and collected by centrifugation. Cell pellets were homogenized with 100 µl of buffer A. After 10 min at 4°C, NP-40 was added to reach a 0.5% concentration. The tubes were gently vortexed for 15 s and nuclei were collected by centrifugation at 8,000 x g for 15 min. The supernatants were stored at -80°C (cytosolic extracts) and the pellets were resuspended in 50 µl of buffer A supplemented with 20% glycerol, 0.4 M KCl, and gently shaken for 30 min at 4°C. Nuclear protein extracts were obtained by centrifugation at 13,000 x g for 15 min, and aliquots of the supernatant were stored at -80°C. Protein content was assayed using the Bio-Rad protein reagent. All steps of cell fractionation were carried out at 4°C.
EMSAs
Oligonucleotides were synthesized in a Pharmacia (Piscataway,
NJ) oligonucleotide synthesizer and the sequence
5'-TGCTAGGGGGATTTTCCCTCTCTCTGT-3'
corresponding to the consensus NF-B-binding site (nt -978 to -952)
of the murine iNOS promoter was used (15); boldface type corresponds to
the binding sequence. Oligonucleotides were annealed with their
complementary sequence by incubation for 5 min at 85°C in 10 mM
Tris-HCl, pH 8.0; 50 mM NaCl, 10 mM MgCl2, and 1 mM DTT.
Aliquots of 50 ng of these annealed oligonucleotides were end-labeled
with Klenow enzyme fragment in the presence of 50 µCi of
[-32P]dCTP and the other unlabeled dNTPs in a final
volume of 50 µl. A total of 5 x 104 dpm of the DNA
probe were used for each binding assay of nuclear extracts as follows:
3 µg of protein were incubated for 15 min at 4°C with the DNA and 2
µg of poly(dL:dC), 5% glycerol, 1 mM EDTA, 100 mM KCl, 5 mM
MgCl2, 1 mM DTT, and 10 mM Tris-HCl, pH 7.8, in a final
volume of 20 µl. The DNA-protein complexes were separated on native
6% polyacrylamide gels in 0.5% Tris-borate-EDTA buffer (21).
Supershift assays were carried out after incubation of the nuclear
extracts with the Ab (0.5 µg) for 1 h at 4°C, followed by EMSA
(not shown). Anti-p50 (human) and anti-c-Rel (human) were a
generous gift of Dr N. R. Rice (National Cancer Institute, Frederick,
MD); anti-p65 (murine) Ab was from Santa Cruz.
Data analysis
The number of experiments analyzed is indicated in the corresponding figure. Statistical differences (p < 0.05) between mean values were determined by one-way analysis of the variance followed by Student’s t test. In experiments using x-ray films (Hyperfilm), different exposure times were used to ensure that bands were not saturated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cultured RAW 264.7 cells were incubated with wortmannin (100 nM)
and LY294002 (10 µM) followed by stimulation with LPS, IFN-,
IL-1ß, TNF-, or combinations of these, and the accumulation of
nitrite in the culture medium was determined. As Fig. 1
A shows, in the presence of
wortmannin or LY294002 a 4- and 2.7-fold increase in the concentration
of nitrite was measured in cells treated with 200 ng/ml of LPS. The
effect of these drugs was less important when cells were activated with
TNF-, IL-1ß, IFN-, or IFN- plus LPS acting in concert.
Agreement was observed between the synthesis of nitrite and the levels
of iNOS. Higher amounts of iNOS were observed when cells were treated
with LPS plus these inhibitors (Fig. 1B). These results
indicated that the maximal effectiveness of wortmannin on iNOS
expression was observed in cells stimulated with LPS. As Fig. 2A shows, low doses of LPS
fail to induce NO synthesis. However, when wortmannin was present, a
dose-dependent increase of nitrite accumulation was measured. This
potentiation of NO synthesis was still evident in cells treated with
LPS and IFN-, and the apparent Ka value for
wortmannin was similar in both cases (20 nM). The dose-dependent curve
for LPS is shown in Fig. 2B and saturation in the presence
of 100 nM wortmannin was obtained at concentrations of LPS higher than
200 ng/ml. Moreover, to establish the optimal period of wortmannin
treatment to increase NO synthesis, the drug was added at several times
with respect to LPS challenge. As Fig. 2C shows, an almost
linear fall in the response was observed after LPS challenge, with 50%
of the effect obtained when added at 2.5 h. This restricted effect
of wortmannin to early times of LPS activation is compatible with a
main effect of this molecule at the transcription level. Since
wortmannin appears to be unstable, sequential additions (1-h periods)
were performed although this did not modify the pattern of response
(not shown). Indeed, when the iNOS mRNA levels were determined at
6 h after LPS stimulation, wortmannin notably increased iNOS
expression, exhibiting an inhibition at concentrations higher than 200
nM (Fig. 3).
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B activation in LPS-treated
macrophages
To further study the mechanism of wortmannin enhancement of iNOS
expression, we investigated the effect of this substance on NF-B
activity. As Fig. 4A and
B show, in LPS-treated cells wortmannin did not
significantly increase this activity at 30 min but promoted a
time-sustained activation. Both p50 dimers and p50/p65 complexes
persisted up to 4 h after LPS stimulation, whereas in cells
treated without wortmannin the fall of both complexes was evidenced
after 2 h. To have more accurate information about the effect of
wortmannin on NF-B activity, cells were transfected with a
(B)3ConA.CAT plasmid and the activity of the reporter
was measured after 18 h. As Fig. 4C shows, wortmannin
did not affect the basal CAT activity; however, in LPS-treated cells,
wortmannin increased 3.9-fold the reporter activity with respect to the
LPS condition. The effect of LPS and wortmannin on CAT activity were
specific since in cells transfected with a ConA.CAT plasmid these
stimuli failed to promote a significant transcription of the reporter
gene (not shown). Because of these results on NF-B activity, the
effect of both wortmannin and LY294006 on IB and IBß levels,
the inhibitory subunits that retain NF-B inactive in the cytosol,
were measured. As Fig. 4D shows, after 1 h of
stimulation of cells with LPS, both LY294002 and wortmannin exhibited
low levels of IB and IBß in the cytosol, suggesting a
potentiation of the effect of LPS on IB degradation.
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Wortmannin and LY294002 are known inhibitors of PI3-kinase
activity (7, 33). To determine whether this enzyme is activated after
LPS stimulation of macrophages, PI3-kinase was immunoprecipitated, and
after resuspension, the lipid kinase activity was measured in vitro. As
Fig. 5 shows, treatment of cells with LPS
rapidly increased the phosphatidylinositol kinase activity in the
immunoprecipitated extracts. However, this response was absent in
LPS-treated cells incubated with 200 nM of wortmannin.
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Incubation of macrophages with wortmannin had no effect on the CAT
activity of cells transfected with p1NOS.CAT, a vector that contains a
1.7-kb fragment of the murine iNOS promoter. However, this drug
increased the reporter activity when cells were stimulated with LPS,
but not when activation was performed with LPS and IFN- (Fig. 6). To gain insight into the mechanism of
action of wortmannin in this system, cells were cotransfected with
p1NOS.CAT and either a plasmid encoding a p110 catalytically active
PI3-kinase subunit (rCD2p110), a kinase-deficient mutant (rCD2p110kd),
or a dominant negative form of p85 (p85d). As Fig. 7 shows, cotransfection with rCD2p110
resulted in a decrease of the reporter activity with respect to the
p1NOS.CAT alone (Fig. 6) or when compared with the effect of
transfection with rCD2p110kd. Indeed, in cells cotransfected with
rCD2p110 plus p1NOS.CAT, wortmannin was unable to increase CAT activity
as occurred in cells transfected with p1NOS.CAT or with the p110
kinase-deficient mutant and stimulated with LPS. Interestingly, these
plasmids encoding p110 had only minimal effects on the reporter
activity measured in cells stimulated with IFN- and LPS acting
synergistically. Cotransfection of p1NOS.CAT with a vector encoding a
dominant negative form of p85 had profound effects on the reporter
activity, suggesting that p85 might be involved at different levels in
the signaling pathway triggered by LPS. Divergent responses to dominant
negative p85 and p110 active subunits have been observed in other
experimental systems (29), and probably indicate that p85 has
additional adapter functions for other proteins different from p110.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stimulation of RAW 264.7 cells with LPS fails to significantly induce
iNOS. In these macrophages, LPS promotes a transient activation of
PI3-kinase as determined by the increase of phosphatidylinositol (PI)
phosphorylation using immunoprecipitated enzyme. Therefore, the
mechanism by which PI3-kinase inhibitors enhanced iNOS transcription
and NO synthesis after LPS-stimulation implies the suppression of a
LPS-dependent negative signaling. The effect of PI3-kinase inhibitors
in RAW 264.7 cells appears to include a sustained activation of NF-B
that extends for a longer period of time than if cells are treated with
LPS alone. It is possible that this persistent activation of NF-B
might contribute to favor the signaling by autocrine factors released
in response to LPS challenge (TNF- and several proinflammatory ILs).
In this regard, it should be noted that the maximal efficiency of
wortmannin was obtained when added immediately following LPS
stimulation, suggesting an effect over early responses activated by
LPS. The observation of lower levels of I-B proteins in the cytosol
of cells treated with LPS and PI3-kinase inhibitors is compatible with
the sustained activation of NF-B, and suggests a possible role for
wortmannin in the regulation of IB-kinase or proteasome activities
(18, 19). The abrogation of the effect of wortmannin on NO synthesis
when cells are activated with LPS and IFN- acting synergistically
suggests that the negative effect of PI3-kinase on this process can be
suppressed by other stimuli.
PI3-kinase is a complex enzyme that includes different isotypes (7, 33, 37, 38). At least three isoenzymes of PI3-kinase have been identified,
including the "classic" p85/p110 heterodimer, as well as a G
protein-coupled PI3-kinase-, and a PI3-kinase with narrow
specificity for PI (37, 38, 39). This diversity in the isoforms might
contribute to the regulation of specific responses (7). Since
PI3-kinase is involved in early steps of intracellular signaling, we
investigated other downstream PI3-kinase targets (7). PI3-kinase
signaling pathways include the activation of the serine/threonine
kinase, PKB, which activates p70 S6-kinase through a mechanism
controlled by the rapamycin target (mTOR) (7, 40). Treatment of RAW
cells with rapamycin (from 10 to 200 nM) did not affect NO synthesis,
which suggests that the PKB/mTOR pathway is not responsible for
these effects. Moreover, in human monocytes and in other cell types,
LPS activates PI3-kinase, which in turn stimulates protein kinase C-
(6). However, the effect of PI3-kinase on iNOS expression cannot be
attributed to protein kinase C- activation since in preliminary
experiments of cotransfection of RAW 264.7 cells with a plasmid
encoding a constitutively active protein kinase C- and a plasmid
containing the iNOS promoter an important increase of the iNOS promoter
activity was observed. These data suggest the existence of a
compensatory mechanism of the PI3-kinase inhibition downstream from the
pathway (work in progress). Indeed, overexpression in these cells of
protein kinase C-
, an isoenzyme activated by PI3-kinase-derived
lipids, also potentiates iNOS transcription (41). Taken together, these
results suggest that PI3-kinase might exert a modulatory role on early
steps of the LPS-dependent macrophage activation.
As previously discussed, wortmannin is not a PI3-kinase-specific
inhibitor, and at the low doses used it inhibits phospholipase
A2 (42) and, therefore, the synthesis of arachidonic
acid-derived metabolites (for example, PGs). Since some PGs synthesized
by cyclooxygenase, such as 15-deoxy-PG J2, could bind to
the PPAR and inhibit iNOS expression (25), we investigated this
possibility by adding exogenous arachidonate to the cells. However, NO
synthesis was not affected under these conditions (not shown),
suggesting that this mechanism is not relevant for the action of
wortmannin in RAW 264.7 cells.
Different inhibitory pathways have been described for the regulation of
iNOS transcription: NO and glucocorticoids decrease NF-B activation
mainly through an up-regulation of I-B levels (22, 23, 24); PPAR
ligands seem to block the transactivating activity of different
transcription factors including NF-B, AP-1, and STAT1, without
affecting their binding capacity (25). Antiinflammatory cytokines such
as IL-10 or IL-13 appear to activate PI3-kinase (26, 43, 44). Moreover,
the inhibition of iNOS expression after RON receptor engagement was
abolished in macrophages treated with PI3-kinase inhibitors
(45).
Regarding the possible physiological relevance of the results
described, we can speculate that in LPS-activated cells PI3-kinase
constitutes a switch of macrophage activation by LPS, favoring the
cooperation of other stimuli to initiate iNOS transcription. In line
with this, it has been shown that LPS stimulation of macrophages
increases the levels of PPAR, and this nuclear factor exerts an
important inhibition of the activation process, including a decreased
iNOS expression (25). Additionally, it has been proposed that
PI3-kinase participates in the process of monocyte/macrophage
proliferation in the case of activation by oxidized low density
lipoprotein (46). In this sense, the PI3-kinase inhibition of iNOS
expression might protect from the high output release of NO that,
because of its cytostatic and cytotoxic effects, may preclude the
expansion of the precursors. The analysis of this LPS-dependent
PI3-kinase activation in other cells might contribute to better
understanding of the contribution of PI3-kinase to the regulation of
iNOS expression. Finally, this work increases the number of inhibitory
mechanisms engaged in the process of macrophage activation, and is
intended to avoid the harmful effects of an exacerbated activation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Lisardo Boscá, Instituto de Bioquímica, Facultad de Farmacia, 28040 Madrid, Spain. E-mail address:
3 Abbreviations used in this paper: iNOS, inducible type of NO synthase; PI, phosphatidylinositol; PPAR, peroxisomal proliferator-activated receptor-; NP-40, Nonidet P-40; CAT, chloramphenicol acetyltransferase.
Received for publication December 9, 1998. Accepted for publication February 24, 1999.
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 J. Sun, R. D. Ramnath, R. Tamizhselvi, and M. Bhatia Role of protein kinase C and phosphoinositide 3-kinase-Akt in substance P-induced proinflammatory pathways in mouse macrophages FASEB J, April 1, 2009; 23(4): 997 - 1010. [Abstract] [Full Text] [PDF]
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K. Tsukamoto, K. Hazeki, M. Hoshi, K. Nigorikawa, N. Inoue, T. Sasaki, and O. Hazeki Critical Roles of the p110{beta} Subtype of Phosphoinositide 3-Kinase in Lipopolysaccharide-Induced Akt Activation and Negative Regulation of Nitrite Production in RAW 264.7 Cells J. Immunol., February 15, 2008; 180(4): 2054 - 2061. [Abstract] [Full Text] [PDF]
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K. Sakai, H. Suzuki, H. Oda, T. Akaike, Y. Azuma, T. Murakami, K. Sugi, T. Ito, H. Ichinose, S. Koyasu, et al. Phosphoinositide 3-Kinase in Nitric Oxide Synthesis in Macrophage: CRITICAL DIMERIZATION OF INDUCIBLE NITRIC-OXIDE SYNTHASE J. Biol. Chem., June 30, 2006; 281(26): 17736 - 17742. [Abstract] [Full Text] [PDF]
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E. Monsalve, M. A. Perez, A. Rubio, M. J. Ruiz-Hidalgo, V. Baladron, J. J. Garcia-Ramirez, J. C. Gomez, J. Laborda, and M. J. M. Diaz-Guerra Notch-1 Up-Regulation and Signaling following Macrophage Activation Modulates Gene Expression Patterns Known to Affect Antigen-Presenting Capacity and Cytotoxic Activity J. Immunol., May 1, 2006; 176(9): 5362 - 5373. [Abstract] [Full Text] [PDF]
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 K. Hazeki, S. Kinoshita, T. Matsumura, K. Nigorikawa, H. Kubo, and O. Hazeki Opposite Effects of Wortmannin and 2-(4-Morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one Hydrochloride on Toll-Like Receptor-Mediated Nitric Oxide Production: Negative Regulation of Nuclear Factor-{kappa}B by Phosphoinositide 3-Kinase Mol. Pharmacol., May 1, 2006; 69(5): 1717 - 1724. [Abstract] [Full Text] [PDF]
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 Z. Yu and B. C. Kone Targeted histone H4 acetylation via phosphoinositide 3-kinase- and p70s6-kinase-dependent pathways inhibits iNOS induction in mesangial cells Am J Physiol Renal Physiol, February 1, 2006; 290(2): F496 - F502. [Abstract] [Full Text] [PDF]
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V. B. Mehta and G. E. Besner Heparin-Binding Epidermal Growth Factor-Like Growth Factor Inhibits Cytokine-Induced NF-{kappa}B Activation and Nitric Oxide Production via Activation of the Phosphatidylinositol 3-Kinase Pathway J. Immunol., August 1, 2005; 175(3): 1911 - 1918. [Abstract] [Full Text] [PDF]
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 S. Ghisletti, C. Meda, A. Maggi, and E. Vegeto 17{beta}-Estradiol Inhibits Inflammatory Gene Expression by Controlling NF-{kappa}B Intracellular Localization Mol. Cell. Biol., April 15, 2005; 25(8): 2957 - 2968. [Abstract] [Full Text] [PDF]
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 K. Kitagawa, Y. Hamada, Y. Kato, K. Nakai, M. Nishizawa, S. Ito, and T. Okumura Epidermal growth factor and interleukin-1{beta} synergistically stimulate the production of nitric oxide in rat intestinal epithelial cells Am J Physiol Gastrointest Liver Physiol, December 1, 2004; 287(6): G1188 - G1193. [Abstract] [Full Text] [PDF]
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J. L. Oliva, N. Zarich, N. Martinez, R. Jorge, A. Castrillo, M. Azanedo, S. Garcia-Vargas, S. Gutierrez-Eisman, A. Juarranz, L. Bosca, et al. The P34G Mutation Reduces the Transforming Activity of K-Ras and N-Ras in NIH 3T3 Cells but Not of H-Ras J. Biol. Chem., August 6, 2004; 279(32): 33480 - 33491. [Abstract] [Full Text] [PDF]
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W.-K. Kim, S.-Y. Hwang, E.-S. Oh, H. Z. Piao, K.-W. Kim, and I.-O. Han TGF-{beta}1 Represses Activation and Resultant Death of Microglia via Inhibition of Phosphatidylinositol 3-Kinase Activity J. Immunol., June 1, 2004; 172(11): 7015 - 7023. [Abstract] [Full Text] [PDF]
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P. H. Correll, A. C. Morrison, and M. A. Lutz Receptor tyrosine kinases and the regulation of macrophage activation J. Leukoc. Biol., May 1, 2004; 75(5): 731 - 737. [Full Text] [PDF]
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A. S. Kristof, J. Marks-Konczalik, E. Billings, and J. Moss Stimulation of Signal Transducer and Activator of Transcription-1 (STAT1)-dependent Gene Transcription by Lipopolysaccharide and Interferon-{gamma} Is Regulated by Mammalian Target of Rapamycin J. Biol. Chem., September 5, 2003; 278(36): 33637 - 33644. [Abstract] [Full Text] [PDF]
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L. Connelly, A. T. Jacobs, M. Palacios-Callender, S. Moncada, and A. J. Hobbs Macrophage Endothelial Nitric-oxide Synthase Autoregulates Cellular Activation and Pro-inflammatory Protein Expression J. Biol. Chem., July 11, 2003; 278(29): 26480 - 26487. [Abstract] [Full Text] [PDF]
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A. M. Valverde, M. Arribas, C. Mur, P. Navarro, S. Pons, A.-M. Cassard-Doulcier, C. R. Kahn, and M. Benito Insulin-induced Up-regulated Uncoupling Protein-1 Expression Is Mediated by Insulin Receptor Substrate 1 through the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway in Fetal Brown Adipocytes J. Biol. Chem., March 14, 2003; 278(12): 10221 - 10231. [Abstract] [Full Text] [PDF]
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A. Castrillo, P. G. Traves, P. Martin-Sanz, S. Parkinson, P. J. Parker, and L. Bosca Potentiation of Protein Kinase C {zeta} Activity by 15-Deoxy-{Delta}12,14-Prostaglandin J2 Induces an Imbalance between Mitogen-Activated Protein Kinases and NF-{kappa}B That Promotes Apoptosis in Macrophages Mol. Cell. Biol., February 15, 2003; 23(4): 1196 - 1208. [Abstract] [Full Text] [PDF]
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D.-W. Park, J.-R. Kim, S.-Y. Kim, J.-K. Sonn, O.-S. Bang, S.-S. Kang, J.-H. Kim, and S.-H. Baek Akt as a Mediator of Secretory Phospholipase A2 Receptor-Involved Inducible Nitric Oxide Synthase Expression J. Immunol., February 15, 2003; 170(4): 2093 - 2099. [Abstract] [Full Text] [PDF]
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K. Cieslik, Y. Zhu, and K. K. Wu Salicylate Suppresses Macrophage Nitric-oxide Synthase-2 and Cyclo-oxygenase-2 Expression by Inhibiting CCAAT/Enhancer-binding Protein-beta Binding via a Common Signaling Pathway J. Biol. Chem., December 13, 2002; 277(51): 49304 - 49310. [Abstract] [Full Text] [PDF]
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B.-C. Chen, W.-T. Wu, F.-M. Ho, and W.-W. Lin Inhibition of Interleukin-1beta -induced NF-kappa B Activation by Calcium/Calmodulin-dependent Protein Kinase Kinase Occurs through Akt Activation Associated with Interleukin-1 Receptor-associated Kinase Phosphorylation and Uncoupling of MyD88 J. Biol. Chem., June 28, 2002; 277(27): 24169 - 24179. [Abstract] [Full Text] [PDF]
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 C. Zeng and A. R. Morrison Disruption of the actin cytoskeleton regulates cytokine-induced iNOS expression Am J Physiol Cell Physiol, September 1, 2001; 281(3): C932 - C940. [Abstract] [Full Text] [PDF]
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S.-H. Baek, T. K. Kwon, J.-H. Lim, Y.-J. Lee, H.-W. Chang, S.-J. Lee, J.-H. Kim, and K.-B. Kwun Secretory Phospholipase A2-Potentiated Inducible Nitric Oxide Synthase Expression by Macrophages Requires NF-{kappa}B Activation J. Immunol., June 15, 2000; 164(12): 6359 - 6365. [Abstract] [Full Text] [PDF]
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 R. Muniyappa, R. Xu, J. L. Ram, and J. R. Sowers Inhibition of Rho protein stimulates iNOS expression in rat vascular smooth muscle cells Am J Physiol Heart Circ Physiol, June 1, 2000; 278(6): H1762 - H1768. [Abstract] [Full Text] [PDF]
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Q.-P. Liu, K. Fruit, J. Ward, and P. H. Correll Negative Regulation of Macrophage Activation in Response to IFN-{gamma} and Lipopolysaccharide by the STK/RON Receptor Tyrosine Kinase J. Immunol., December 15, 1999; 163(12): 6606 - 6613. [Abstract] [Full Text] [PDF]
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C. A. Learn, M. S. Boger, L. Li, and C. E. McCall The Phosphatidylinositol 3-Kinase Pathway Selectively Controls sIL-1RA Not Interleukin-1beta Production in the Septic Leukocytes J. Biol. Chem., June 1, 2001; 276(23): 20234 - 20239. [Abstract] [Full Text] [PDF]
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S. Blum, K. Issbruker, A. Willuweit, S. Hehlgans, M. Lucerna, D. Mechtcheriakova, K. Walsh, D. von der Ahe, E. Hofer, and M. Clauss An Inhibitory Role of the Phosphatidylinositol 3-Kinase-signaling Pathway in Vascular Endothelial Growth Factor-induced Tissue Factor Expression J. Biol. Chem., August 31, 2001; 276(36): 33428 - 33434. [Abstract] [Full Text] [PDF]
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