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J. Biol. Chem., Vol. 278, Issue 34, 31825-31830, August 22, 2003
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B Kinase, and NFB, Do Not Reduce Amyloid 
42 Production*
¶
From the
Department of Neurosciences, University
of California San Diego, La Jolla, California 921093 and the
Department of Neuroscience and Pharmacology,
Mayo Clinic, Jacksonville, Jacksonville, Florida 32224
Received for publication, April 7, 2003 , and in revised form, June 6, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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42 peptide in both cultured cells and transgenic mice. Although this
activity does not appear to require the action of cyclooxygenases in cultured
cells, it is not known whether other NSAID-sensitive targets contribute to
this A42 effect. In this study, we have used both pharmacological and
genetic means to determine if other known cellular targets of NSAIDs could
mediate the reduction in A42 secretion from cultured cells. We find that
altered arachidonic acid metabolism via NSAID action on cyclooxygenases and
lipoxygenases does not alter A42 production. Furthermore, we demonstrate
that alterations in activity of peroxisome proliferator-activated receptors,
I
B kinase or nuclear factor B do not affect A42
production. Thus, NSAIDs do not appear to alter A42 production
indirectly through previously identified cellular targets and may interact
directly with the 
-secretase complex itself to affect amyloid
production. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-protein
(A), derived from the amyloid precursor protein (APP), in senile plaques
in brain. There are two predominant isoforms of A peptide, A40 and
A42, differing in their C termini: A42, the longer isoform is more
amyloidogenic and toxic to cultured cells. Virtually all mutations associated
with familial AD preferentially increase amount of A42 produced, and
hence the ratio of A42:A40
(1). Thus, decreasing
production or increasing the clearance of A42 may be an effective way to
either prevent the development of or treat AD.
Epidemiological studies demonstrate that persons with a history of
non-steroidal anti-inflammatory drug (NSAID) use have a reduced risk of AD
(2,
3). It is generally assumed
that the inflammatory responses seen in brains of AD individuals play a key
role in neurodegeneration (4,
5), but it has not been
established that the anti-inflammatory properties of NSAIDs underlie their
apparent neuroprotective effects
(6,
7). In line with the
epidemiological findings, treatment of APP transgenic mice, which develop
A deposits and associated pathology, with these compounds has proven to
be beneficial. Specifically, ibuprofen, curcumin, and a nitric oxide
derivative of flurbiprofen were recently shown to decrease the formation of
amyloid plaques and reduce inflammatory markers in a transgenic mouse model of
AD
(8–10).
These and other observations have led to a number of clinical trials to
determine if NSAIDs or immunosuppression may be useful in the treatment of
AD.
In our recent studies (11)
we found that some NSAIDs, including sulindac, ibuprofen, and indomethacin,
lowered the levels of the amyloidogenic A42 isoform, hence reducing the
ratio of A42:A40, in medium from a variety of cultured cells as
well as in brains of APP transgenic mice. The effective NSAIDs did not grossly
affect the production or processing of the amyloid precursor protein (APP).
While these NSAIDs decreased the ratio of A42:A40, they appear to
increase the production of shorter A peptides, such as A38. This
suggested that NSAIDs might subtly alter the production of various A
species. To our knowledge, the NSAIDs are the first class of compounds that
specifically reduce A42 production without significant alteration in
A40 levels.
The focus of these studies was to further investigate the cellular
mechanisms responsible for the A42:A40 reduction by certain
NSAIDs. Our initial report established that inhibition of the cyclooxygenase
enzymes COX1 and COX2, the canonical targets of NSAIDs, was not sufficient to
reduce the A42 levels
(11). We therefore proposed
that NSAIDs affect the amyloid pathology by lowering the amyloidogenic
A42 peptide through a COX-independent pathway. This hypothesis is not
unparalleled, as a dual-action model has been suggested for the effectiveness
of NSAIDs in colorectal cancer studies where both COX-dependent and
COX-independent mechanisms have been described. To further understand the
mechanism whereby NSAIDs lower A42 levels, we examined whether other
NSAID-sensitive targets including arachidonic acid, lipoxygenases, peroxisome
proliferator-activated receptors (PPAR), and nuclear factor B influence
the generation of A42 (Fig.
1). In addition, we examined several other compounds that have
been reported to lower risk of AD or to reduce amyloid pathology in transgenic
mice to determine whether they also have a previously unrecognized activity
against A42.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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1 x 105 cells/ml. Embryonic fibroblasts from
wild-type, IKK2 knockout (kindly provided by I. Verma) or p65/RelA knockout (a
gift from E. Shaulian) mice were maintained as above. Primary cultures of
ALOX5 and ALOX15 knockout fibroblasts were generated from neonatal mice
resulting from homozygous ALOX5 or ALOX15 knockout (mice purchased from The
Jackson Laboratory) breeding. Fibroblasts were infected with 60 pfu per cell
of adenovirus encoding wild-type APP751 for 2 h before treatment with NSAIDs
(11).
Drugs and Chemicals—Compounds were obtained from Sigma
Chemical Co except as noted here. Arachidonic acid, carbaprostacyclin
(cyclic-PgI2), and naproxen from Cayman Chemicals; baicalein, caffeic acid,
MK-886, 8-S-HETE, and 15-deoxy-
-prostaglandin J2 (PgJ2) from
Calbiochem; ibuprofen, indomethacin, sulindac sulfide from Biomol; melittin
from ICN Biosciences; S-flurbiprofen from Aldrich; A1–40,
1–42 standard peptides from American Peptide Inc. Complete protease
inhibitor pellet from Roche Applied Science; and
-cyano-4-hydroxycinnamic acid solution from Agilent Technologies,
GW9662 was provided by GlaxoSmithKline. LG10305 and BRL49653 were kind gifts
from R. Evans. R-flurbiprofen was supplied by Encore
Pharmaceuticals.
Enzyme-linked Immunosorbent Assays—For most experiments
human A ELISA were performed as described
(12,
13). For each experiment
duplicate or triplicate samples were analyzed. Each drug was tested at several
doses in a minimum of two independent experiments with sulindac sulfide as a
positive control. Selective reduction in A42 was determined by
calculating the A42:A40 ratio for each sample. These ratios were
normalized to vehicle control in each experiment. These normalized ratios were
pooled and analysis of variance used to determine if the A42:A40
ratio was significantly different from vehicle with p < 0.05.
Error bars in figures represent standard errors.
Matrix-assisted Laser Desorption/Ionization Time-Of-Flight
Mass Spectrometry—MALDI-TOF was performed on A peptides
immunoprecipitated from conditioned medium of CHO cells as described
(14) with the following
modifications. Complete protease inhibitor, phosphoramidon and a synthetic
A1–22 peptide, that served as an internal control, were added, and
all A1-x were immunoprecipitated from conditioned medium by overnight
incubation with anti-mouse IgG agarose beads and 26D6, which recognizes the N
terminus of A. Extraction from the beads was with formic
acid/water/isopropyl alcohol 1:4:4 (v/v/v). Eluted material was mixed 1:1 with
-cyano-4-hydroxycinnamic acid solution prior to spotting for
spectrometry. Spectra shown are representative of at least two experiments
performed with duplicate samples. Treatment-induced changes in A species
distribution were determined by normalization of peak heights to
A40.
Bicine/Urea SDS-PAGE and Western Blots—Conditioned
medium was immunoprecipitated as for MALDI-TOF analysis, except without the
addition of synthetic A22. Bicine/urea gels were performed as described
in Wiltfang et al.
(15) with the following
modifications. The final acrylamide concentration in the separating gel was
10% T, 5% C. The comb gel was eliminated from our protocol. In addition, the
30% sucrose and 5% 2-mercaptoethanol were replaced with glycerol and
dithiothreitol. Proteins were transferred to nitrocellulose in 10
mM CAPS, pH 11, with 10% methanol and boiled for 5 min.
Immunoblotting was with 26D6 APP antibody and the signal detected by
horseradish peroxidase-conjugated goat anti-mouse IgG followed by enhanced
chemiluminescence. Gels shown are representative of a minimum of three
experiments with 2–3 replicates per experiment. Bands intensities were
quantified using a CCD camera and software (Syngene).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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42 Is Not Generalized Anti-inflammatory
Actions—To confirm that the A42-reducing activity was
specific to NSAIDs and not mimicked by other anti-inflammatory compounds, we
investigated the effects of glucocorticoids on A42 levels in CHO cells
stably transfected with wild-type human APP (APP-WT). In clinical trials,
these compounds did not slow the rate of cognitive decline in AD individuals
(16,
17). At the concentrations
tested, neither prednisone nor dexamethasone reduced the amount of A42
or the A42:A40 ratio (Table
I).
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Epidemiological studies
(18) have also suggested that
antihistamine usage reduces the incidence of AD. Since histamine responses
trigger inflammatory changes, we wanted to determine if blocking histamine
action altered A42 levels. Therefore, we treated A-producing PS1ML
cells with 0.5–20 µM of the H-2 histamine receptor
antagonist cimetidine. This treatment did not alter the levels of A
detectable by ELISA (Table I)
or bicine/urea gel analysis of conditioned medium.
Recent studies indicated that the NSAID-like compound curcumin might be
beneficial in a mouse model of AD
(9). Curcumin has both
anti-inflammatory and anti-oxidant properties. APP-WT CHO cells were treated
with curcumin to determine if it affected the production of A species.
Between 1 and 100 µM, there was no reduction in A levels
or A42:A40 (Table
I). Counter to the effect of NSAIDs, there was a slight increase
(20%) in A42:A40 at doses between 20 and 100 µM.
This increase in A42:A40 could be attributed to a reduction in
A40 levels. Higher doses were toxic to the cells so A could not be
evaluated.
COX inhibition Is Not Required for NSAID Action on
A42—We previously reported that the reduction in
A42 levels secondary to NSAID treatment was accompanied by an increase
in the amount of A38 species
(11). However, several NSAIDs,
such as naproxen and aspirin, did not reduce A42 levels. Whether these
NSAIDs affect shorter A species was not investigated as the inactivity
of naproxen in our previous study had only been determined by ELISA. Here we
confirmed the lack of effect on A peptides by both MALDI-TOF
spectrometry and bicine/urea SDS-PAGE methods. In these assays, naproxen did
not reduce A1–42 or increase A1–38, whereas sulindac
sulfate, ibuprofen, flurbiprofen, and indomethacin were all effective
(Fig. 2 and data not shown). In
our earlier work, we used COX-deficient cells to demonstrate that COX enzymes
are not required for sulindac sulfide to reduce A42 levels. However,
both of these approaches only concluded that COX inactivation is insufficient
to reduce A42 levels. To address whether COX inhibition is required to
alter A production we used R-flurbiprofen, which does not
inhibit COX and is not efficiently isomerized to S-flurbiprofen.
R-flurbiprofen potently reduced A42 levels and increased
production of shorter As (Fig.
2C), in agreement with a recent report
(19).
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Arachidonic Acid and Lipoxygenases Do Not Affect A42
Levels—NSAIDs inhibit not only COX but can alter the activities of
several lipoxygenases, which could increase arachidonic acid levels and effect
the processing of arachidonic acid into HETEs and leukotrienes
(Fig. 1). To mimic the effect
of NSAIDs on increasing arachidonic acid levels, we treated the PS1ML cells
with exogenous arachidonic acid and assayed the levels of the various A
species by ELISA and bicine/urea gels. We also treated cells with the
phospholipase A2 activator, melittin
(20). This treatment mobilizes
endogenous arachidonic acid from cellular membranes. Arachidonic acid, either
added to the extracellular medium or induced by melittin, increased the total
amount of A generated by 20% as compared with control cells. In the
case of arachidonic acid there was a slight reduction in the
A42:A40 ratio, because of an increase in A40 and not a
decrease in A42 (Table
II). Melittin treatment had no effect on A42:A40
ratio.
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Several NSAIDs, including ibuprofen and indomethacin, can inhibit the
activity of 5-lipoxygenase
(21). 5-lipoxygenase
expression may be elevated in aging human brains
(22), so we wanted to
determine if the NSAID effect on A42 was mediated by inhibition of this
enzyme. We examined whether other 5-lipoxygenase inhibitors could reduce the
A42 levels detected by our sensitive ELISA assay
(Table II). The doses chosen
for these compounds were above those published to inhibit 5-lipoxygenase, but
below toxic concentrations. The inhibitors nordihydroguiaretic acid, MK-886,
and caffeic acid, which preferentially inhibit 5-LOX, were without effect on
total A or A42 production. Since at elevated doses these
pharmacological inhibitors have multiple activities, which could mask the
desired effect, we also used a genetic approach to eliminate 5-lipoxygenase
activity. Neonatal fibroblasts from 5-lipoxygenase-deficient mice were
infected with an adenovirus encoding APP695 and then treated with sulindac
sulfide. As shown in Fig.
3A, sulindac retained the ability to inhibit A42
production in these cells, verifying that 5-LOX is not necessary for the NSAID
effect on A42.
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Recent reports indicate that some NSAIDs activate the murine leukocyte-type
12-lipoxygenase and its human homologue 15-lipoxygenase
(23,
24), so we examined the effect
of modulating 15-lipoxygenase on A42:A40. The 15-lipoxygenase
inhibitor baicalein had no effect on A42 levels
(Table II). As above, we
cultured fibroblasts from L-12-lipoxygenase-deficient mice and assayed A
levels after adenoviral infection. Once again we saw a marked reduction in
A42 with sulindac treatment (Fig.
3B), indicating that this enzyme is also not required for
the amyloid-altering effects of NSAIDs.
Peroxisome Proliferator-activated Receptors Do Not Alter
A42 levels—The peroxisome proliferator-activated
receptors (PPARs) are sensitive to NSAIDs and the eicosanoids produced by
arachidonic acid metabolism. Recent studies have also indicated that PPAR
activation inhibits microglial activation and the production of
proinflammatory molecules and may attenuate A toxicity
(25,
26). NSAIDs activate
PPAR and PPAR
(27), and inhibit PPAR
(28). Each or these PPARs must
heterodimerize with the RXR nuclear transcription factor to bind DNA and alter
transcription. If NSAIDS reduce A42 by activating PPAR then the
PPAR agonist 8(S)-HETE should also reduce A42 levels
from APP-expressing cells. However, 8(S)-HETE did not reduce
A42 levels secreted from APP-WT cells. Likewise the PPAR agonists
ciglitazone, 15-deoxy--prostaglandin J2 or BRL49653
(29) failed to reduce
A42 levels (Table III).
In addition, we examined the ability of a PPAR antagonist, GW9662
(30), to block the response to
sulindac sulfide. This antagonist had no effect on the ability of sulindac to
reduce A42 levels (Fig. 4, A
and B). We also tested the A42-reducing potential
of the RXR agonist LG10305, which was ineffective in reducing A42
levels.
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To confirm the pharmacological approach, APP-WT CHO cells were transfected
with either PPAR or PPAR cDNAs and then stimulated with
15-deoxy--prostaglandin J2 or cyclic-prostaglandin I2,
respectively. Overexpression of these receptors, with or without activator,
did not alter the ratio of A42:A40
(Fig. 4C). Because
pharmacologic and genetic manipulations of the various PPARs had no affect on
A42: A40, these receptors are unlikely to mediate the NSAID
A42 response.
NFB Is Not Required to Mediate the Sulindac
Sulfide-induced Reduction in A42—Several NSAIDs can
inhibit the activity of the NFB transcription complex at one or more
levels: the inhibition of IKK and the prevention of NFB DNA binding
(31,
32). Inhibition of IKK would
prevent IB phosphorylation and degradation and prevent translocation of
NFB to the nucleus. The NFB complex is a dimer that can be
composed of several proteins. The most common NFB complex contains
p65/RelA and p50. Tomita et al.
(33) reported that
overexpression of p65/RelA increased production of A42 but not A40
indicating a potential A42-specific signaling pathway that can be
modulated by NSAIDs. Because elimination of p65/RelA by genetic knockout
prevents induction of most NFB-responsive genes
(34), we used mouse embryonic
fibroblasts from IKK or p65-knockout mouse embryos to test whether the
NFB pathway was required for the NSAID-mediated reduction in A42.
As before, the MEFs were infected with APP695 adenovirus and then treated with
sulindac sulfide. In both cell lines, sulindac inhibited the production of
A42 similar to control fibroblasts
(Fig. 3), indicating that these
proteins are not required to mediate the NSAID response.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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42
in vitro in cell culture medium and in vivo in brains of
transgenic mice opened two key questions. The first question was whether the
effect was mediated by the anti-inflammatory action of these compounds, even
if not due to COX inhibition. The second is the mechanism by which NSAIDs
lower A42 levels. The goal of this study is to examine a number of
candidate cellular pathways that may be responsible for this NSAID effect on
A42 generation.
NSAIDs are potent anti-inflammatories that are primarily known to inhibit
COX enzymes. However, our initial report
(11) demonstrated that
A42 was reduced by sulindac in cells lacking COX enzymes. Furthermore,
several NSAIDs that inhibited COX could not reduce A42:A40. These
findings indicate that inhibition of COX was not sufficient to reduce
A42 levels. Further confirmation of this hypothesis was obtained from a
study treating cultured cells with the R-enantiomers of flurbiprofen
and ibuprofen (19) and from
our results with R-flurbiprofen using both MALDI-TOF and Western
blotting analyses. The fact that R-enantiomers of these NSAIDs, which
are inactive against COX, were able to reduce A42 production argues
against COX inhibition as the basis for A42 reduction.
To examine whether NSAIDs may act through a general anti-inflammatory
mechanism, we screened several compounds unrelated to conventional NSAIDs with
known anti-inflammatory activity. One of the proposed bases for the
neuroprotective benefits of NSAIDs is to inhibit the inflammatory responses
seen in brains of AD individuals, thus we thought it is pertinent to analyze
this potential mechanism. This notion led to a treatment trial in AD
individuals with the corticosteroid, prednisone, which was found to be without
benefit (17). In our cultured
cell system, we found that glucocorticoids, including prednisone and
dexamethasone, did not reduce A42 levels. On the other hand, our finding
that curcumin does not alter A42 levels is notable in light of the
recent report showing a significant reduction in amyloid pathology in
transgenic mice treated with curcumin chronically
(9). As curcumin has multiple
properties, these findings taken together suggest that curcumin may reduce
A load in brain by a mechanism distinct from the A42 property seen
in NSAIDs. Although there was initial suggestion that H2 antagonists, such as
cimetidine, lowered the risk of AD, a recent analysis of the Cache county
epidemiological data and a placebo-controlled study concluded that use of
these drugs did not affect AD risk or progression
(35,
36). These more recent
analyses are more aligned with our finding that cimetidine had no effect on
A42. However, as our cell culture experiments are performed under basal
conditions, we cannot draw any conclusion as to the benefit of the
afore-mentioned anti-inflammatory compounds in cells already challenged by
inflammatory stimuli. It is possible that mediation of inflammatory responses
may be beneficial in treating AD, and this benefit could be dependent or
independent of A42 production. These findings therefore highlight the
fact that there likely are multiple activities from these anti-inflammatory
compounds and that multiple mechanisms can account for the reduction in
A levels, amyloid pathology, and/or ameliorate AD risk.
A major focus of this study is to test candidate cellular pathways that may
be responsible for the observed reduction in A42 by examining the
non-COX pathways known to be affected by NSAIDs. Although the primary target
of NSAIDs is COX inhibition, NSAIDs are pleiotropic compounds with other known
effects. These pathways are summarized in the schematic diagram in
Fig. 1. For example, one such
pathway is through altered AA metabolism. Inhibition of COX blocks the
conversion of AA to prostaglandin H2 and results in a build up of AA that has
been mobilized from the plasma membrane by the action of phospholipase A2
(37). Phospholipase A2 has
been reported to increase APPs secretion, which presumably would preclude
A release (38). In
addition, NSAIDs regulate arachidonic acid not only by inhibiting COX enzymes
but also via interaction with lipoxygenases. 5-Lipoxygenase activity increases
in the brain with age and is inhibited by indomethacin, ibuprofen, or sulindac
(21,
22). On the other hand, some
NSAIDs increase activity and expression of 15-lipoxygenase. Consequently, we
investigated the role of lipoxygenases and AA metabolism in A42
generation via pharmacologic and genetic means. Mobilization of AA by
supplementation of culture medium with exogenous AA or by activation of PLA2
by melittin did not reduce A42 levels but showed a slight increase in
total A levels. In our studies, neither 5- nor 15-LOX appeared to
influence A42:A40 in conditioned medium. We cannot exclude the
possibility that inflammatory leukotrienes generated by lipoxygenases are an
exacerbating factor in Alzheimer's disease, but this does not appear to be
manifested in A42 levels.
PPARs are another important class of NSAID targets that have been the focus
of some attention, especially in the cancer-related literature. The PPAR
family of nuclear receptors consists of PPAR, PPAR, and
PPAR (also denoted as PPAR). The NSAIDs indomethacin, flufenamic
acid, fenoprofen, and ibuprofen appear to directly activate the
transcriptional activity of PPAR and PPAR
(27). PPARs also have
important roles in modulating lipid and glucose metabolism. In this regard,
-secretase activity can be strongly influenced by the cholesterol
content of the plasma membrane
(39), and insulin affects not
only glucose metabolism but also APP processing and A levels
(40). In addition,
PPAR- agonists have anti-inflammatory properties, and in particular,
these agonists inhibited A stimulated release of proinflammatory
products from microglia (25).
Despite interesting potential links to amyloid or NSAID activity, our studies
described here were not able to observe any selective effects of PPAR
overexpression, activation, or inhibition on lowering A42 levels.
Lastly, PPARs also regulate transcriptional activation by STAT, AP-1, and
NFB transcription factors. In this context, overexpression of
NFB/p65 has been shown to increase the proportion of A1–42
(33). Moreover, there is
evidence that NSAIDs can antagonize NFB more directly. NFB may
have profound effects in Alzheimer's disease progression by elevating levels
of inducible nitric-oxide synthase, tumor necrosis factors, interleukins, and
complement factors, all of which are known to be elevated in AD brains
(3). Our results showed that
NSAID-induced A42 effects were intact in IKK or p65-deficient
fibroblasts, much as has been seen in COX-deficient MEFs, thereby arguing
against this pathway as a major contributor to the A42 reduction.
In summary, our studies failed to identify a potential candidate cellular
pathway, known to be affected by NSAIDs, that mediates the A42 effect.
Our studies therefore reinforce the concept that the ability to reduce
A42 production is not shared by all NSAIDs, but rather it is due to a
novel secondary activity. Our studies did not address whether NSAIDs can
directly target either APP or the -secretase complex itself. In view of
our results, it is tempting to suggest that NSAIDs may conformationally alter
-secretase activity, much as presenilin mutations preferentially
increase A42 levels. This notion would be consistent with the subtle
switch in A peptides following treatment of cultured cells with certain
NSAIDs to favor the shorter species. In related studies we have observed that
the A42 lowering activity of NSAIDs can be modified by presenilin
mutations and that A42 generation from an in vitro
-secretase assay can be modified by NSAIDs
(42). A recent report by
Takahashi et al. (41)
using in vitro assay also suggested that NSAIDs alter the activity of
-secretase. Taken together, these observations lead us to favor the
notion that NSAIDs alter A42 production by subtly modifying APP cleavage
either at the level of the substrate or a component of the -secretase
complex, rather than indirectly, such as by modulating cellular signaling
pathways. Identification of the molecular target, however, will be difficult
for two reasons. First, the precise nature of -secretase remains to be
elucidated and second, NSAIDs reduce A42 production with low affinity.
Nevertheless, the findings presented here are an important first step in the
long term goal of identifying compounds that selectively lower A42
in vivo but have negligible COX inhibition to limit the well known
gastrointestinal side effects as potential candidates for AD treatment.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Dept. of Neurosciences, University of California San Diego, La Jolla, CA 92093-0691. E-mail: edkoo{at}ucsd.edu.
1 The abbreviations used are: AD, Alzheimer's disease; AA, arachidonic acid;
A, amyloid ; APP, amyloid precursor protein; CHO, Chinese hamster
ovary; COX, cyclooxygenase; ELISA, enzyme-linked immunosorbent assay; IKK,
inhibitor of B kinase; LOX, lipoxygenase; MALDI-TOF, matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry; MEF, mouse
embryonic fibroblast; NFB, nuclear factor B; NSAID,
non-steroidal anti-inflammatory drug; PLA2, phospholipase A2; PPAR, peroxisome
proliferator-activated receptor; STAT, signal transducer and activator of
transduction; CAPS, 3-(cyclohexylamino)propanesulfonic acid.
|
ACKNOWLEDGMENTS |
|---|
knockout MEFs, Drs. C. Glass and M. Ricote kindly provided PPAR plasmids, Dr.
Numa Gottardi-Littell for APP695 adenovirus, W. Wechter of Encore
Pharmaceuticals for R-flurbiprofen. Compounds LG10305 and BRL49653
were kindly provided by Dr. R. Evans. Compound GW9662 was kindly provided by
Glaxo Smith Kline Pharmaceuticals. We are grateful to R. Wang and E. Komives
for providing training and advice for MALDI-TOF. We also wish to thank T.
Souder and T. Monnier for their efforts in performing A ELISA
assays. |
REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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