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Departments of
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Medicine and
Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1, IL-6, and nitric oxide
(NO)3 (1, 2). The signaling
pathways consequent upon stimulation are beginning to be unraveled;
however, understanding of these is far from complete. LPS is believed
to mediate its effects through the CD14 receptor (3) and requires
additionally a serum-derived LPS binding protein. The CD14 receptor
lacks intrinsic kinase activity and was proposed to transduce signaling
through the p53/56lyn protein tyrosine kinase
(4). However, recent work casts doubt on this step, as macrophages
without Hck, Fgr, and Lyn demonstrated unimpaired responses to LPS (5).
Normally, phosphorylation of receptor tyrosine kinases enables binding
with molecules containing SH2 groups such as the adaptor subunit of the
PI 3-kinase. These is an important class of lipid kinases that catalyze
the phosphorylation of phosphoinositides at the D-3 hydroxyl of the
inositol ring generating PI 3-phosphate, PI 3,4-biphosphate, and PI
3,4,5-triphosphate (6, 7). It is noteworthy that PI 3-kinase is
activated by LPS and appears to associate with
p53/56lyn in human monocytes (8). This
event would implicate the activation of downstream signaling molecules
such as p70 S6 kinase, but this has not been shown in the
monocyte/macrophage system. However, PKC
activation has been
demonstrated to occur by a PI 3-kinase-dependent mechanism using
specific inhibitors and a dominant-negative p85 mutant (9).
Multiple cytokines have been demonstrated to activate PI 3-kinase in a variety of systems, including IL-2, IL-4, IL-10, IL-13, granulocyte-macrophage CSF, and steel factor (10, 11, 12, 13). Investigation of the functional importance of this pathway for hemopoietic cells has revealed an interesting regulatory role for this kinase in the immune response. Pharmacologic intervention with wortmannin and Ly294002 (PI 3-kinase inhibitors) upon granulocyte-macrophage CSF/TNF/FMLP-activated neutrophils indicates a function for PI 3-kinase in superoxide generation, platelet-activating factor release, and migration (14). However, cytokines mentioned previously, such as IL-10, are clearly involved in negatively regulating immune cell function (11). Furthermore, activated PI 3-kinase inhibited T cell receptor-mediated NF-AT induction (15).
The downstream effector molecules for PI 3-kinase remain to be clarified, but the current literature favors a model in which PKB (the cellular homologue of the transforming oncogene, v-akt) lies below PI 3-kinase and upstream of p70 S6 kinase (16, 17). Activation of PKB is recognized to occur through a wortmannin-sensitive phosphorylation of PKB at the Thr308 and Ser473 sites (18) and appears to involve membrane translocation of the kinase (19, 20). Recently, the kinase responsible for Thr308 phosphorylation has been characterized and has been designated PDK1 for PI 3,4,5-triphosphate-dependent protein kinase 1 (21). From a functional perspective, PKB is itself an important molecule in insulin signaling as well as in prevention of apoptosis (22, 23).
The target of rapamycin (known as TOR or FRAP for FKBP12-rapamycin-associated protein) lies either below PKB or on a parallel pathway (24) and is also involved in the activation of p70 S6 kinase (25). It has demonstrated kinase activity in terms of both autophosphorylation and phosphorylating the PHAS-1 protein, which is also known as eIF-4E binding protein and functions as a translational repressor. Recent work indicates that FRAP is a proline-directed protein kinase (26, 27, 28).
The p70 S6 kinase is the kinase responsible for phosphorylation of the
S6 protein in response to various stimuli, including GH and insulin.
Its activation is complex, requiring multiple serine and threonine
phosphorylation events. Intriguingly, recent work has indicated that
PDK1 can phosphorylate and activate the p70 S6 kinase (29). Although
its functions remain to be clarified (demonstrated indirectly through
the use of rapamycin), they include cell cycle control, transcription,
and translation initiation (30). To date, the best evidence for a
direct functional role involves transcriptional regulation. Rapamycin
blocks the Ser117 phosphorylation of the cAMP-responsive
modulator (CREM
) in response to serum, which is the site
phosphorylated by p70 S6 kinase in vivo (31).
The biochemical regulation of the production of biologically active
molecules by hemopoietic cells is presently not well understood. This
is likely to be important, as some of the signaling proteins involved
may well be useful targets for therapeutic intervention. We have
previously shown that in Raw 264.7 cells LPS activates multiple
mitogen-activated protein kinase family members, such as p44 Erk1, p42
Erk2, p46/p54 JNK/SAPK, and p38 Hog (32). JNK/SAPK has been
demonstrated to have a role in the translation of TNF- (33).
Additionally, p38 Hog has been demonstrated to be involved in the
expression of the inducible nitric oxide synthase (iNOS) gene in mouse
astrocytes and primary glial cultures (34, 35). However, there are
limited data concerning the involvement of the PI 3-kinase and its
putative effector molecules in similar systems. One such example is
that histamine secretion in basophils has been demonstrated to be PI
3-kinase dependent (36). Additionally, rapamycin has been demonstrated
to inhibit IFN-
induced by IL-1 in the murine T cell lymphoma, YAC-1
(37). We therefore hypothesized that activation of the PI
3-kinase/PKB/p70 S6 kinase pathway is involved in the production of NO.
The latter is produced by the catalytic transformation of
L-arginine to citrulline by iNOS, which in Raw
macrophage-like cells is a process requiring protein tyrosine
phosphorylation (38). Posttranslational modification of iNOS through
its tyrosine phosphorylation has recently been demonstrated to be an
early event (39). NO is an important biologic molecule with
tissue-specific effects that in macrophages include microbicidal and
tumoricidal activities as well as apoptosis (3, 40). It also has been
demonstrated to be directly correlated with the severity of pathologic
conditions, such as AIDS dementia, bronchial asthma, and inflammatory
bowel disease (41).
In this study we provide evidence for the activation of individual components of this pathway by LPS in a macrophage cell line and the involvement of an Ly294002- and rapamycin-sensitive protein kinase in the generation of nitric oxide through a possible phosphorylation-dependent mechanism of iNOS itself.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LPS (Escherichia coli serotype 055:B5) and MTT were
obtained from Sigma (St. Louis, MO). Abs for p70 S6 kinase (NT), p85 of
PI 3-kinase, and the pleckstrin homology domain of PKB as well as the
S6 protein peptide substrate and histone H2B were obtained from Upstate
Biotechnology (Lake Placid, NY). Ab to iNOS was obtained from Santa
Cruz Biotechnology (Santa Cruz, CA). The lipid substrate
phosphatidylinositol was obtained from Avanti Polar Lipids (Alabaster,
Atlanta, GA). Goat anti-rabbit IgG was obtained from Bio-Rad
(Richmond, CA). The inhibitors used were wortmannin, Ly290042, and
rapamycin (all from Calbiochem, San Diego, CA). Protein A-Sepharose
beads were obtained from Pharmacia (Piscataway, NJ).
[-32P]ATP was purchased from DuPont (Wilmington, DE).
The TLC plates were obtained from EM Science (Gibbstown, NJ).
Cell culture
Raw 264.7 cells were cultured and stimulated as previously described with minor modifications (32). Briefly 1 x 106 cells were seeded into 100-mm petri dishes containing 10 ml of DMEM supplemented with 10% heat-inactivated FCS and grown at 37°C. After 40 h, by which time they had reached 90% confluence, the cells were switched to serum-free medium and were stimulated with either LPS or PMA 6 h later. For PI 3-kinase activity determinations, the cells were serum starved overnight. Where inhibitors were employed, a preincubation time of 1 h was allowed. Following stimulation the cells were washed with ice-cold PBS, scraped into a 15-ml Falcon tube using a rubber policeman, sedimented by centrifugation, and sonicated for 20 s after the addition of homogenization buffer containing 20 mM MOPS, 50 mM ß-glycerophosphate, 5 mM EGTA, 50 mM NaF, 1 mM sodium vanadate, and 1 mM PMSF. For the PI 3-kinase assays the lysis buffer consisted of 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 1% Triton X-100, 2 mM EDTA, 1 mM sodium vanadate, 1 mM sodium molybdate, and 10% glycerol. Before use of either buffer the following protease inhibitors were added: 40 µg/ml PMSF, 0.5 µg/ml leupeptin, and 2 µg/ml aprotinin.
Western blotting
Aliquots of crude extracts were resolved using 11% SDS-PAGE (42) and transferred to nitrocellulose membrane using a Bio-Rad transblot apparatus at 300 mA for 3 h. The blots were blocked with 5% BSA overnight and washed three times with TTBS (20 mM Tris-HCl (pH 7.4), 250 mM NaCl, and 0.05% Tween 20), the appropriate primary Ab was applied for 4 h, blots were rewashed with TTBS, the secondary Ab was applied for 1 h, and blots were rewashed and developed using enhanced chemiluminescence for horseradish peroxidase-conjugated secondary Abs according to the manufacturer’s recommendations (ECL kit, Amersham, Arlington Heights, IL).
Immunoprecipitation
Five hundred micrograms of soluble protein were added to 200 µl of an IP buffer containing 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 50 mM NaF, 5 mM EDTA, and 3% Nonidet P-40. Five microliters of Ab was added, and the tubes were placed on a Labquake shaker for 1 h at 4°C. Then, 30 µl of a 1/1 slurry of protein A-Sepharose beads was added, and the rotation was continued for 2 h at 4°C. Following this the beads were washed twice with IP buffer and twice with 12.5 mM ß-glycerol phosphate, 20 mM MOPS (pH 7.2), 5 mM EGTA, 7.5 mM MgCl2, 50 mM NaF, and 0.25 mM DTT. Then the beads were resuspended and subjected to an immune complex kinase assay as described below.
Immune complex assays
Ten microliters of the substrate mixture, either histone H2B (1
mg/ml) or the S6 protein-based peptide substrate (0.5 mg/ml)
AKRRRLSSL-RASTSKSESSQK (43) in assay dilution buffer (20 mM MOPS
(pH 7.2), 25 mM ß-glycerophosphate, 20 mM MgCl2, 5 mM
EGTA, 2 mM EDTA, 1 mM DTT, and 1 mM sodium vanadate), were added to the
washed beads, and the reaction was conducted as described above for 20
min using 5 µl of the ATP mixture (250 µM ATP and 1 µCi of
[-32P]ATP). Twenty microliters were then spotted onto
P81 filter paper, washed extensively with 1% phosphoric acid, and
analyzed in a scintillation counter after addition of 250 µl of
scintillation fluid. Thirty microliters of 2x sample buffer were added
to the beads, and after boiling for 5 min the sample was resolved by
11% SDS-PAGE and subjected to immunoblotting.
PI 3-kinase assays
This was performed essentially as previously described (13).
Briefly detergent lysates were immunoprecipitated with the p85-PI
3-kinase Ab for 3 h, and the beads were washed twice with lysis
buffer and three times with Tris-HCl (pH 7.4). Subsequently, the PI was
prepared by drying with nitrogen and resuspending in 10 µl of 30 mM
HEPES. This was added to the washed beads, and the tube was left on ice
for 10 min. Then 40 µl of kinase buffer (30 mM HEPES, 30 mM
MgCl2, 50 µM ATP, 200 µM adenosine, and 10 µCi of
[-32P]ATP) were added to each tube, and the reaction
was allowed to proceed at room temperature for 15 min. The reaction was
stopped with 0.1 N HCl, and the lipids were extracted with 200 µl of
chloroform/methanol (1/1). The products were separated on potassium
oxalate-pretreated TLC plates by developing with chloroform, methanol,
water, and 30% ammonium hydroxide (112/88/19/6). After drying, the
plates were exposed to autoradiography, and the phosphorylated products
were quantified by excising the spot and scintillation counting.
NO assay
Raw 264.7 cells were treated with the indicated inhibitors in the absence and the presence of LPS, and culture supernatants collected at 24, 48, and 72 h were centrifuged at 600 x g for 10 min. Nitrite levels were measured by addition of 50 µl of the Griess reagent (1.5% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2.5% H3PO4) to 50 µl of culture supernatant in 96-well plates, leaving in the dark for 10 min, and measuring the color intensity with an automated microtiter plate reader at 550 nm.
Inducible NOS immunoprecipitation
Raw cells were cultured in 60-mm petri dishes for 24 h in the presence and the absence of LPS, Ly294002, rapamycin, and wortmannin. After cell lysis as described above the iNOS protein was immunoprecipitated from equal amounts of crude lysate using a rabbit polyclonal Ab. The samples were then resolved on 9% SDS-PAGE and probed with the same Ab, and the bands were detected using ECL.
MTT and [3H]thymidine uptake assays
Raw cells were grown in 96-well plates at a concentration of 5000 cells/well for 24 h, and then MTT or [3H]]thymidine was added. The plates were washed with PBS 4 h later, and scintillation fluid was added for the DNA synthesis assay and quantitated for radioactivity in a scintillation counter (1450 Microbeta, Wallac, Gaithersburg, MD). For the MTT assay 0.1 N HCl was added in absolute propanol, and the absorbance was measured spectrophotometrically at a wavelength of 570 nm. The results are representative of three experiments performed in quadruplicate and are expressed as absorbance units for the MTT assay and counts per minute for the [3H]thymidine uptake assay.
In vivo labeling of iNOS
Raw 264.7 cells were cultured under the conditions outlined above. They were switched to phosphate-free medium containing 0.5 mCi of [32P]phosphate on the second day, and the protein kinase inhibitors were added 30 min before adding LPS. After 18 h at 37°C the cells were lysed in homogenization buffer, and immunoprecipitations were conducted for the iNOS protein as outlined above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although activation of p70 S6 kinase has been previously
demonstrated in the liver of endotoxic rabbits (44), this has not been
described in LPS-stimulated macrophages. The initial objective was
therefore to ascertain whether LPS activated p70 S6 kinase in Raw 264.7
cells. The phosphotransferase activity of p70 S6 kinase toward a
peptide substrate was assayed following immunoprecipitation from the
crude lysates of cells incubated for 0–60 min with LPS. Optimal
activation of p70 S6 kinase was observed after 30 min of LPS treatment
as shown in Fig. 1
. This corresponded to
the timing of the maximal electromobility band shift shown in Fig. 1B. These results were obtained with two different Abs
directed at the N and C termini of p70 S6 kinase.
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PMA treatment of diverse cells induces a robust activation of p70
S6 kinase via PKC. As shown in Fig. 2B, LPS and PMA both elicited
a fivefold activation of S6 kinase after a 30-min stimulation. This was
accompanied by a retardation of the electromobility of the S6 kinase
consistent with its hyperphosphorylation (Fig. 2A). When the
FRAP inhibitor rapamycin and the PI 3-kinase inhibitor Ly294002 were
coincubated with LPS or PMA in the medium of the Raw cells, there was a
complete inhibition of both the phosphorylation and the activation of
S6 kinase. Intriguingly, wortmannin, an alternative PI 3-kinase
inhibitor, did not completely abolish the activation of p70 S6 kinase
by LPS (Fig. 2B), effecting approximately a 50% reduction
of the kinase activity. This indicated that wortmannin and Ly294002 may
act via additional targets besides PI 3-kinase. Previous work has
indicated that wortmannin (in supra-PI 3-kinase inhibitory
concentrations) and Ly294002 are capable of inhibiting the autokinase
activity of FRAP (26). Our observations are compatible with a FRAP
isoform in Raw cells that is more sensitive to the effects of Ly294002
than to wortmannin. Significantly, in another study (25) FRAP was found
to be resistant to the effects of wortmannin, lending further weight to
this possibility. Ultimately, characterization of the FRAP isoform in
macrophages will clarify this problem. The existence of a PI 3-kinase
isoform that is more sensitive to inhibition by Ly294002 than to
wortmannin is unlikely, since novel isoforms, such as the PI 3-kinase
activated by Gß subunits (45), retain sensitivity to these
inhibitors. The PMA activation of p70 S6 kinase was also relatively
insensitive to intervention with wortmannin (Fig. 2), yet it was
clearly inhibited by Ly294002, lending further support to Ly294002
possibly behaving like a FRAP inhibitor (in addition to its PI 3-kinase
inhibitory activity).
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To clarify whether the p85-associated PI 3-kinase was, in fact,
sensitive to inhibition by the standard inhibitors, the next step was
to assess the activation of PI 3-kinase in this system. This was
conducted by immunoprecipitation and assessment of the in vitro lipid
phosphotransferase activity. As shown in Fig. 3, A and B, there
was a rapid and sustained activation of PI 3-kinase with LPS. This
finding was in accordance with previous results in human monocytes,
where LPS led to an activation of PI 3-kinase and its association with
the tyrosine kinase Lyn (8). Wortmannin and Ly294002 both inhibited PI
3-kinase activity following 10 min of LPS (Fig. 3C). These
findings indicated that PI 3-kinase did exhibit activation in response
to LPS in Raw 264.7 cells and that this activation was sensitive to
both of the standard PI 3-kinase inhibitors. Therefore, the incomplete
inhibition of p70 S6 kinase with wortmannin was unlikely to be due to a
differential sensitivity of the PI 3-kinase to this agent and Ly294002.
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The current model of p70 S6 kinase activation has implicated the
involvement of PKB upstream, so its role was investigated. The Ab used
for this analysis was directed at the pleckstrin homology domain
present at the N terminus, which is conserved among all the known
isoforms of PKB. Fig. 4 shows that while
LPS was able to activate PKB approximately twofold, the effects of PI
3-kinase inhibitors on this activation were surprisingly modest. In
control experiments with murine keratinocytes using insulin, a fivefold
activation of PKB was observed, with attenuation of this response when
the cells were pretreated with wortmannin (data not shown). Although
the magnitude of the LPS-mediated activation was modest, PKB activation
was PI 3-kinase independent in this system and probably did not
contribute significantly to activation of p70 S6 kinase. Two recent
observations make these data compatible with current understanding of
the regulation of this kinase. Firstly, PKB can be activated following
cellular stresses in a PI 3-kinase-independent fashion (46). Secondly,
cAMP also activates PKB via a wortmannin-insensitive mechanism (47).
Furthermore, taken in conjunction with the data in Fig. 2
, a model in
which FRAP, rather than PKB, lies upstream of p70 S6 kinase appears
more plausible. The observation of the absence of PKB activation by PMA
makes it highly unlikely that PKC is involved in its regulation.
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Having demonstrated that biochemical activation of the signaling
molecules under investigation does indeed occur in macrophages in
response to LPS, and that their regulation occurred somewhat
differently from the generally accepted model for insulin signaling,
the next step was to investigate the effects of wortmannin, Ly294002
and rapamycin on NO production. NO is a functionally important molecule
that can be deleterious in certain situations when produced in excess.
Its production has been well characterized in Raw cells and correlates
very well with the appearance of iNOS (39). The effects of the
inhibitors on NO production by Raw cells were assessed using the Griess
reagent. Fig. 5 shows that LPS effects a
robust production of NO over a period of 72 h. Examining the
effects of the inhibitors revealed that while Ly294002 and rapamycin
were able to abolish this response almost completely, wortmannin was
ineffective, even when it was added again after 12 h of
incubation, because of its relative instability in aqueous solutions.
In fact, wortmannin paradoxically led to a small increase in its
production. The observed changes were maintained for a total duration
of 72 h without further addition of inhibitor for either Ly294002
or rapamycin and daily addition of wortmannin. This inhibitory effect
on NO production by Ly294002 and rapamycin was not due to killing of
the cells by an apoptosis-promoting effect, as cell viability, assessed
by trypan blue exclusion, at 24 h was unchanged among the various
treatment groups.
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,
confirm that the effects of the inhibitors could not be attributed
simply to a nonspecific toxicity. More specifically, LPS induced an
increase in mitochondrial activity (MTT) that was not influenced by any
of the inhibitors (a vs b, p < 0.005). Predictably,
LPS mediated a negative influence on cell proliferation as assessed by
[3H]thymidine uptake (a vs b, p <
0.0005), which persisted to an extent with Ly294002, but was abolished
using either rapamycin or wortmannin. Furthermore, the relative
insensitivity of PKB activity to these inhibitors argues against an
apoptosis-promoting effect. These data indicate a specific effect of
both Ly294002 and rapamycin on pathways directly involved in the
production of NO.
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The role of iNOS protein induction was investigated by directly
immunoprecipitating the protein from macrophage extracts stimulated for
24 h with LPS in the absence and the presence of Ly294002,
rapamycin, and wortmannin. Fig. 6 shows
that LPS stimulation resulted in a predictable increase in the amount
of iNOS protein, and that this was largely unaffected by any of the
inhibitors used in the study. This was a surprising finding given that
the production of NO is currently believed to be due to induction of
the iNOS protein. This invoked a posttranslational mechanism as being
the step critically modulated by both Ly294002 and rapamycin.
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To address whether a phosphorylation step was involved in the
regulation of iNOS activity, an in vivo labeling study was performed.
Raw 264.7 cells were cultured in medium containing 0.5 mCi of
orthophosphate with the appropriate inhibitors and LPS. After
harvesting the cells, immunoprecipitation of the iNOS protein was
conducted, and the samples were resolved on 9% SDS-PAGE. Fig. 7 indicates that the protein was
phosphorylated upon stimulation of Raw 264.7 cells with LPS. To our
knowledge this is the first time that this has been demonstrated and
implicates iNOS phosphorylation as an important potential means of its
regulation. Of further significance is the observation that
phosphorylation of iNOS was abolished in response to pretreatment with
both Ly294002 and rapamycin in concert with the inhibition of NO
production by these agents. Wortmannin pretreatment had no effect on
the iNOS phosphorylation, in keeping with its inability to inhibit NO
production. These observations indicate that FRAP or its downstream
effector(s) is involved in the regulation of iNOS activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role of LPS in the activation of the PI 3-kinase PKB and p70 S6 kinase and the influence of rapamycin and PI 3-kinase inhibitors on this activation and on nitric oxide production have never been formally investigated. The findings of our study indicate that all the components of this pathway are activated in response to stimulation with LPS; however, the use of pharmacologic inhibitors indicates that this activation does not proceed along the previously accepted model established for insulin signaling. This is exemplified by two observations. Firstly, PKB activation with LPS is uninfluenced by PI 3-kinase inhibitors. Secondly, p70S6 kinase activation is only partially abolished by wortmannin in response to both LPS and PMA, whereas both Ly294002 and rapamycin completely inhibited this activation with both agonists. When the effects of these inhibitors on NO production were examined, a similar pattern was observed, in that both Ly294002 and rapamycin completely inhibited NO production, but wortmannin did not have any effect. The explanation for the differential response to wortmannin and Ly294002 is not immediately clear, but it indicates that different kinases may be involved. In this regard, the incomplete inhibition of p70 S6 kinase activation by wortmannin indicates that PI 3-kinase-independent mechanisms may be playing a role. In support of this, cardiomyocytes exhibit a relative insensitivity to wortmannin of p70 S6 kinase activation in response to sodium arsenite (51), and additionally, endothelin-mediated activation of p70 S6 kinase in bovine airway smooth muscle cells is wortmannin insensitive (52).
The discovery that there are multiple regulatory subunits of the PI
3-kinase (53) with different responses to insulin activation indicates
that these (i.e., p50 and p55) may play a role in LPS signaling.
Additionally, the identification of the p101 regulatory subunit
mediating signaling through Gß subunits (45) serves to emphasize
that the regulation of these lipid signaling molecules will probably be
more complex than is generally appreciated. In the current study,
however, the p85-associated in vitro PI 3-kinase activity was clearly
sensitive to inhibition by both inhibitors.
Although the question of wortmannin stability is an important one,
several lines of evidence in different cell systems indicate that this
may have been overemphasized. A study in chicken macrophages clearly
showed an inhibitory effect of wortmannin on LPS-induced NO production
(54). In an intestinal cell line, it was clearly able to attenuate the
inhibitory effect of IL-13 on TNF/IFN-/IL-1-mediated NO production
(12).
PKC is also an important downstream effector of PI 3-kinase (24, 55). It has been implicated in both the LPS and the IFN--mediated
production of NO (56, 57, 58). In Raw cells, PMA is unable to stimulate NO
production unless there is overexpression of PKC
(59). As PMA
produced a robust activation of p70 S6 kinase in the present study
(Fig. 1), our findings indicate that this activation is insufficient
for production of NO. An upstream kinase is probably required for this
response in a manner analogous to the mechanism proposed for the
insulin-mediated phosphorylation of PHAS-1 (60).
Intriguingly, a strong case is made for the involvement of FRAP in the
LPS-mediated production of NO. A differential sensitivity of FRAP to
Ly294002 and wortmannin in this particular system could be an
explanation for the lack of a functional effect for the latter. This is
possible, as one study clearly demonstrated that FRAP kinase activity
was wortmannin insensitive (25). More importantly, however, the
findings from this study indicate that rapamycin and Ly294002 affect NO
production at a site distal to the induction of the iNOS protein. A
schematic diagram (Fig. 8) summarizes the
novel findings of this study, the most pertinent of which are the
unlikelihood that PI 3-kinase regulates FRAP in this system and that
FRAP or a downstream effector is responsible for the phosphorylation
and consequently the activation of iNOS leading to the production of
NO.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. S. Pelech, Department of Medicine, University of British Columbia, 1779 West 75th Ave., Vancouver, British Columbia, Canada V6P 6P2. E-mail address:
3 Abbreviations used in this paper: NO, nitric oxide; PI 3-kinase, phosphatidylinositol 3-kinase; PKC, protein kinase C; PKB, protein kinase B; FRAP, FKBP12-rapamycin-associated protein; iNOS, inducible nitric oxide synthase; MTT, 3-(4,5,-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Received for publication April 14, 1998. Accepted for publication August 31, 1998.
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References |
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D. Aki, R. Mashima, K. Saeki, Y. Minoda, M. Yamauchi, and A. Yoshimura Modulation of TLR signalling by the C-terminal Src kinase (Csk) in macrophages Genes Cells, April 1, 2005; 10(4): 357 - 368. [Abstract] [Full Text] [PDF]
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S. Noubir, Z. Hmama, and N. E. Reiner Dual Receptors and Distinct Pathways Mediate Interleukin-1 Receptor-associated Kinase Degradation in Response to Lipopolysaccharide: INVOLVEMENT OF CD14/TLR4, CR3, AND PHOSPHATIDYLINOSITOL 3-KINASE J. Biol. Chem., June 11, 2004; 279(24): 25189 - 25195. [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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D. Shegogue and M. Trojanowska Mammalian Target of Rapamycin Positively Regulates Collagen Type I Production via a Phosphatidylinositol 3-Kinase-independent Pathway J. Biol. Chem., May 28, 2004; 279(22): 23166 - 23175. [Abstract] [Full Text] [PDF]
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 S. Uthaisangsook, N. K. Day, R. Hitchcock, A. Lerner, M. James-Yarish, R. A. Good, and S. Haraguchi Negative Regulation of Interleukin-12 Production by a Rapamycin-Sensitive Signaling Pathway: A Brief Communication Experimental Biology and Medicine, October 1, 2003; 228(9): 1023 - 1027. [Abstract] [Full Text] [PDF]
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J. Y. Lee, J. Ye, Z. Gao, H. S. Youn, W. H. Lee, L. Zhao, N. Sizemore, and D. H. Hwang Reciprocal Modulation of Toll-like Receptor-4 Signaling Pathways Involving MyD88 and Phosphatidylinositol 3-Kinase/AKT by Saturated and Polyunsaturated Fatty Acids J. Biol. Chem., September 26, 2003; 278(39): 37041 - 37051. [Abstract] [Full Text] [PDF]
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J. A. Lehman, V. Calvo, and J. Gomez-Cambronero Mechanism of Ribosomal p70S6 Kinase Activation by Granulocyte Macrophage Colony-stimulating Factor in Neutrophils: COOPERATION OF A MEK-RELATED, THR421/SER424 KINASE AND A RAPAMYCIN-SENSITIVE, mTOR-RELATED THR389 KINASE J. Biol. Chem., July 18, 2003; 278(30): 28130 - 28138. [Abstract] [Full Text] [PDF]
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 S. O. Kim, K. Ono, P. S. Tobias, and J. Han Orphan Nuclear Receptor Nur77 Is Involved in Caspase-independent Macrophage Cell Death J. Exp. Med., June 2, 2003; 197(11): 1441 - 1452. [Abstract] [Full Text] [PDF]
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 T. Ronni, V. Agarwal, M. Haykinson, M. E. Haberland, G. Cheng, and S. T. Smale Common Interaction Surfaces of the Toll-Like Receptor 4 Cytoplasmic Domain Stimulate Multiple Nuclear Targets Mol. Cell. Biol., April 1, 2003; 23(7): 2543 - 2555. [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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 S. O. Rizzoli and W. J. Betz Effects of 2-(4-Morpholinyl)-8-Phenyl-4H-1-Benzopyran-4-One on Synaptic Vesicle Cycling at the Frog Neuromuscular Junction J. Neurosci., December 15, 2002; 22(24): 10680 - 10689. [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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R. A. Zirngibl, Y. Senis, and P. A. Greer Enhanced Endotoxin Sensitivity in Fps/Fes-Null Mice with Minimal Defects in Hematopoietic Homeostasis Mol. Cell. Biol., April 15, 2002; 22(8): 2472 - 2486. [Abstract] [Full Text] [PDF]
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 R. van Crevel, T. H. M. Ottenhoff, and J. W. M. van der Meer Innate Immunity to Mycobacterium tuberculosis Clin. Microbiol. Rev., April 1, 2002; 15(2): 294 - 309. [Abstract] [Full Text] [PDF]
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 A. KOTANIDOU, A. XAGORARI, E. BAGLI, P. KITSANTA, T. FOTSIS, A. PAPAPETROPOULOS, and C. ROUSSOS Luteolin Reduces Lipopolysaccharide-induced Lethal Toxicity and Expression of Proinflammatory Molecules in Mice Am. J. Respir. Crit. Care Med., March 15, 2002; 165(6): 818 - 823. [Abstract] [Full Text] [PDF]
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T. Kawai, O. Takeuchi, T. Fujita, J.-i. Inoue, P. F. Muhlradt, S. Sato, K. Hoshino, and S. Akira Lipopolysaccharide Stimulates the MyD88-Independent Pathway and Results in Activation of IFN-Regulatory Factor 3 and the Expression of a Subset of Lipopolysaccharide-Inducible Genes J. Immunol., November 15, 2001; 167(10): 5887 - 5894. [Abstract] [Full Text] [PDF]
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 S. H. Diks, S. J.H. van Deventer, and M. P. Peppelenbosch Invited review: Lipopolysaccharide recognition, internalisation, signalling and other cellular effects Innate Immunity, October 1, 2001; 7(5): 335 - 348. [Abstract] [PDF]
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B. W. Jones, T. K. Means, K. A. Heldwein, M. A. Keen, P. J. Hill, J. T. Belisle, and M. J. Fenton Different Toll-like receptor agonists induce distinct macrophage responses J. Leukoc. Biol., June 1, 2001; 69(6): 1036 - 1044. [Abstract] [Full Text] [PDF]
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M. M. Monick, A. B. Carter, P. K. Robeff, D. M. Flaherty, M. W. Peterson, and G. W. Hunninghake Lipopolysaccharide Activates Akt in Human Alveolar Macrophages Resulting in Nuclear Accumulation and Transcriptional Activity of {{beta}}-Catenin J. Immunol., April 1, 2001; 166(7): 4713 - 4720. [Abstract] [Full Text] [PDF]
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 E. Hiraoka, S. Kawashima, T. Takahashi, Y. Rikitake, T. Kitamura, W. Ogawa, and M. Yokoyama TNF-{alpha} induces protein synthesis through PI3-kinase-Akt/PKB pathway in cardiac myocytes Am J Physiol Heart Circ Physiol, April 1, 2001; 280(4): H1861 - H1868. [Abstract] [Full Text] [PDF]
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O. Equils, E. Faure, L. Thomas, Y. Bulut, S. Trushin, and M. Arditi Bacterial Lipopolysaccharide Activates HIV Long Terminal Repeat Through Toll-Like Receptor 4 J. Immunol., February 15, 2001; 166(4): 2342 - 2347. [Abstract] [Full Text] [PDF]
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 A. Xagorari, A. Papapetropoulos, A. Mauromatis, M. Economou, T. Fotsis, and C. Roussos Luteolin Inhibits an Endotoxin-Stimulated Phosphorylation Cascade and Proinflammatory Cytokine Production in Macrophages J. Pharmacol. Exp. Ther., January 1, 2001; 296(1): 181 - 187. [Abstract] [Full Text]
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J. Lee, L. Mira-Arbibe, and R. J. Ulevitch TAK1 regulates multiple protein kinase cascades activated by bacterial lipopolysaccharide J. Leukoc. Biol., December 1, 2000; 68(6): 909 - 915. [Abstract] [Full Text]
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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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