(Received for publication, September 8, 1994; and in revised form, November 30, 1994)
From the
Two isoforms of cyclooxygenase (COX) have been identified in
eukaryotic cells: a constitutively expressed COX-1 and
mitogen-inducible COX-2, which is selectively expressed in response to
various inflammatory stimuli. Thus, COX-2 instead of COX-1 is
implicated to produce prostanoids mediating inflammatory responses.
Major efforts have been focused on identifying nonsteroidal
anti-inflammatory drugs (NSAIDS) which can selectively inhibit the
enzyme activity of COX-2. Such NSAIDS would be more desirable
anti-inflammatory agents in comparison to NSAIDS which inhibit both
COX-1 and COX-2. Other than glucocorticoids, pharmacological agents
which can selectively suppress the expression of COX-2 without
affecting that of COX-1 have not been identified. We report here that
radicicol, a fungal antibiotic, is a potent protein tyrosine kinase
inhibitor, and that it inhibits the expression of COX-2 without
affecting COX-1 expression in lipopolysaccharide (LPS)-stimulated
macrophages with the IC value of 27 nM. Radicicol
inhibited tyrosine phosphorylation of p53/56
, a Src
family tyrosine kinase and one of the major tyrosine-phosphorylated
proteins in LPS-stimulated macrophages. Radicicol also inhibited COX-2
expression in vivo in glomeruli of rats with experimental
glomerulonephritis induced by the anti-glomerular basement membrane
antibodies, in which COX-2 expression is known to be enhanced. The
enzyme activity of COX-1 or COX-2 was not affected by radicicol in
macrophages. Radicicol also suppressed the COX-2 expression induced by
IL-1
in rat smooth muscle cells. Other protein tyrosine
kinase inhibitors suppressed the LPS-induced COX-2 expression in
macrophages but at much higher concentrations than needed for
radicicol. Radicicol did not inhibit the COX-2 expression induced by
phorbol 12-myristate 13-acetate in macrophages. These results suggest
that the activation of tyrosine-specific protein kinases is the
proximal obligatory step in the LPS-induced signal transduction pathway
leading to the induction of COX-2 expression in macrophages. The
magnitude of the inhibition of COX-2 protein synthesis by radicicol was
much greater than that of the steady state levels of COX-2 mRNA. These
results suggest that radicicol inhibits COX-2 expression mainly at
post-transcriptional steps.
Cyclooxygenase (COX; prostaglandin endoperoxide
synthase, EC 1.14.99.1) catalyzes the conversion of arachidonic acid to
prostaglandin (PG) endoperoxide (PGH
). This is the
rate-limiting step in PG and thromboxane biosynthesis. Two isoforms of
COX have been cloned from various animal cells: constitutively
expressed COX-1 (1, 2, 3, 4, 5) and
mitogen-inducible COX-2 (6, 7, 8, 9, 10, 11) .
Prostaglandins produced as a result of the activation of COX-1 may have
some physiological functions, such as the antithrombogenic action of
prostacyclin released by the vascular endothelium, and cytoprotective
effect of PGs produced by the gastric mucosa(12) . COX-2 is
expressed following the activation of cells by various proinflammatory
agents including cytokines(9, 11, 13) ,
endotoxin(14) , and other
mitogens(7, 8, 9) . These observations lead
to the suggestion that COX-2 instead of COX-1 may be responsible for
producing prostanoids involved in inflammation and/or mitogenesis.
Furthermore, the ability of nonsteroidal anti-inflammatory drugs
(NSAIDS) to inhibit COX-2 may well explain their therapeutic efficacy
as anti-inflammatory drugs, whereas inhibition of COX-1 may explain
their unwanted side effects(15) .
For this reason,
considerable interest has been focused on finding NSAIDS which can
selectively inhibit COX-2 as more desirable anti-inflammatory agents
with minimum side effects. While these NSAIDS inhibit the enzyme
activity of endogenous COX, glucocorticoids are known to suppress COX-2
expression at transcriptional and/or translational steps without
significant effect on COX-1 expression (14, 15, 16, 17) . Other than
glucocorticoids, pharmacological agents that can suppress the
expression of COX-2 without affecting that of COX-1 have not been
reported. Neoplastic transformation of chicken embryo fibroblasts by
Rous sarcoma virus results in the activation of a set of early response
genes encoding growth factors and transcription factors involved in the
regulation of cell division(18, 19) . One of these
genes encodes the mitogen-inducible cyclooxygenase (COX-2). Induction
of these genes is dependent on the activity of the v-Src oncogene
product p60, a tyrosine kinase. Radicicol, a fungal
antibiotic with a macrocyclic ring structure(20, 21) ,
was reported as an inhibitor of p60
tyrosine
kinase(22) . It has been demonstrated that lipopolysaccharide
(LPS) rapidly increases protein tyrosine phosphorylation in
macrophages, and that this early signaling event appears to mediate
some downstream macrophage responses to
LPS(23, 24, 25) . For this reason, we
investigated whether radicicol inhibits the expression of COX-2 in rat
alveolar macrophages stimulated with LPS. We report here that radicicol
suppresses protein tyrosine phosphorylation and the expression of COX-2
(IC
, 27 nM) without affecting COX-1 expression in
LPS-stimulated alveolar macrophages in rats.
Figure 1: Chemical structure of radicicol.
To locate Src family kinases and mitogen-activated protein kinases, the same membrane used for antiphosphotyrosine immunoblot was stripped in the buffer (2% SDS, 50 mM Tris-HCl, 100 mM 2-mercaptoethanol, pH 6.5 at 50 °C for 30 min). The stripped membrane was reprobed with polyclonal anti-mitogen-activated protein kinase antibodies (UBI, 1 µg/2 ml in PBS containing 0.1% Tween 20 and 3% nonfat dry milk) or polyclonal anti-Src family kinase antibodies (Santa Cruz, 1 µg/ml), followed by goat anti-rabbit IgG coupled to horseradish peroxidase (1 µg/10 ml). The immunoreactive protein bands were visualized by the ECL detection system.
Effects of radicicol on enzyme activities of COX-1 and COX-2
purified from ram seminal vesicle and sheep placenta, respectively,
(Cayman Chemicals) were determined by the conversion of
[C]arachidonic acid (Dupont, specific activity,
57 mCi/mmol) to PGE
after separation by thin layer
chromatography (TLC) as described by Mitchell et
al.(30) . The reaction mixture (1 ml) in 50 mM Tris buffer (pH 8.0) contained arachidonic acid (10.88
µM) together with [
C]arachidonic
acid, glutathione (5 mM), epinephrine (5 mM), and
hematin (1 µM). The reactions were initiated by adding the
purified enzymes and incubated at 37 °C for 10 min. PGs were
separated in TLC in a solvent of the organic phase of ethyl
acetate/trimethylpentane/acetic acid/water, 110:50:20:100 (v/v). The
PGE
band was identified by a cold standard run side by side
and visualized in an iodine tank. Radioactive PGE
bands
were identified by superimposing the autoradiograph run on TLC plates
and then scraped off. The radioactivity of the PGE
band was
determined by scintillation counting.
Figure 2:
Time course of protein tyrosine
phosphorylation and its inhibition by radicicol in LPS-stimulated
macrophages. A, cells were pretreated with radicicol (200
ng/ml) for 4 h and then stimulated with LPS (10 µg/ml) containing
radicicol or vehicle, dimethyl sulfoxide (DMSO, 5 µl/ml)
for the various time periods indicated. Solubilized proteins were
analyzed by antiphosphotyrosine immunoblotting as described under
``Materials and Methods.'' Molecular size markers run on the center lane between dimethyl sulfoxide, and radicicol lanes
are shown on the left. An arrow on the right indicates tyrosine-phosphorylated protein bands superimposed with
p53/56 bands shown below. B, the same membrane
used for antiphosphotyrosine immunoblot in A was stripped and
reprobed with polyclonal antimitogen-activated protein kinase
antibodies (UBI) recognizing both MAPK-1 and MAPK-2. The time scale is
the same as that for A. C, the same sample used for A was immunoblotted with antiphosphotyrosine antibody, stripped, and
reprobed with polyclonal anti-p53/56
antibodies. The time
scale is the same as that for A.
Figure 3:
Time course for the expression of COX
activity induced by LPS and its inhibition by radicicol in macrophages
pretreated with aspirin. Rat alveolar macrophages were allowed to
attach for 2.5 h in the presence of aspirin (500 µM) to
inactivate endogenous cyclooxygenase, washed three times, and then
incubated in RPMI with LPS (10 µg/ml) in the presence or absence of
radicicol (200 ng/ml). After removing the media, cells were incubated
with arachidonic acid (30 µM) for 10 min. The levels of
PGE produced from exogenous arachidonic acid were measured
by radioimmunoassay to determine the activity of de novo synthesized cyclooxygenase. The value for 2.5 h indicates the
endogenous COX activity prior to aspirin treatment. Values for each
time point are the mean of three samples.
Figure 5:
Time course for levels of mRNA for COX-1,
COX-2, and glyceraldehyde-3-phosphate dehydrogenase (GAP) in
LPS-treated macrophages in the presence or absence of radicicol. Rat
alveolar macrophages were incubated with LPS in the presence or absence
of radicicol (100 ng/ml) for specified times. Total RNA was extracted
and hybridized with riboprobes, as described under ``Materials and
Methods.'' The [-
P]UTP-labeled COX-1,
COX-2, and glyceraldehyde-3-phosphate dehydrogenase were protected with
the complementary mRNA from macrophages. The gel was scanned (AMBIS),
and the radioactivities of COX-1 and COX-2 bands were factored relative
to that of the glyceraldehyde-3-phosphate dehydrogenase
band.
Figure 6:
Western blot analyses of pooled cell
lysates used in Fig. 2for individual Src family tyrosine
kinase. Membranes used for antiphosphotyrosine immunoblot were stripped
and reprobed with polyclonal anti-p60,
p58/64
, p53/56
, or p59
antibodies. P, antiphosphotyrosine immunoblot of pooled
lysates; S, stripped membrane reprobed with anti-p60
antibodies; H, stripped membrane reprobed with
anti-p58/64
antibodies; L, stripped membrane
reprobed with anti-p53/56
antibodies; F,
stripped membrane reprobed with anti-p59
antibodies.
Molecular size markers are in kilodaltons.
Antiphosphotyrosine immunoblot of
immunoprecipitates of cell lysates indicated that p53/56 and p59
were the major tyrosine phosphorylated
Src family tyrosine kinases (Fig. 7). When the cell lysates
after the immunoprecipitation were analyzed by antiphosphotyrosine
immunoblot, the lower band superimposing with p53/56
disappeared completely in the sample for which p53/56
was removed by immunoprecipitation with anti-p53/56
antibodies (Fig. 8). It has been demonstrated that LPS
induces rapid tyrosine phosphorylation of isoforms of mitogen-activated
protein kinases in elicited murine peritoneal exudate macrophages and
RAW 264.7 cells(24) . Both MAPK-1 (42 kDa) and MAPK-2 (44 kDa)
were detected in cell lysates derived from the time course study as
determined by Western blot analysis using polyclonal anti-rat MAP
kinase antibodies recognizing both MAPK-1 and MAPK-2 (UBI, Fig. 2B). However, tyrosine-phosphorylated
mitogen-activated protein kinases were not detected by the
antiphosphotyrosine immunoblotting procedure used in our studies (Fig. 2A).
Figure 7:
Antiphosphotyrosine immunoblot analyses
of individual Src tyrosine kinase immunoprecipitated from pooled cell
lysates used in Fig. 2. P, pool cell lysate; S, immunoprecipitate with anti-p60 antibodies; H, immunoprecipitate with p58/64
antibodies; L, immunoprecipitate with p53/56
antibodies; F, immunoprecipitate with p59
antibodies.
Molecular size markers are in kilodaltons.
Figure 8:
Antiphosphotyrosine immunoblot analyses
of cell lysates after immunoprecipitating individual Src family
tyrosine kinase as described in the legend for Fig. 7. P, pooled cell lysate without immunoprecipitation; S,
cell lysate after depleting p60 by immunoprecipitation; H, cell lysate after depleting p58/64
by
immunoprecipitation; L, cell lysate after depleting
p53/56
by immunoprecipitation; F, cell lysate
after depleting p59
by immunoprecipitation. Molecular
size markers are in kilodaltons.
Pretreatment of macrophages with
radicicol in our studies resulted in suppression of levels of
tyrosine-phosphorylated proteins in time-dependent and dose-dependent
fashions (Fig. 2A and 9A). Pretreatment of
macrophages with radicicol or herbimycin A resulted in a significant
reduction of levels of p53/56 (Fig. 9B).
This reduction in levels of p53/56
was correlated to
suppressed levels of tyrosine-phosphorylated proteins by radicicol or
herbimycin A both in the time course (Fig. 2A) and the
dose-response studies (Fig. 9A). Pretreatment of
macrophages with radicicol not only suppressed the basal levels (zero
time) of tyrosine-phosphorylated proteins, but also attenuated the
stimulatory effect of LPS on protein tyrosine phosphorylation (Fig. 2A). Some Src family tyrosine kinases, in
association with cell surface proteins, participate in normal signaling
pathways in hemopoietic cells including monocytes and
macrophages(35) . It has been demonstrated that initial
interaction of LPS with monocytes and macrophages involves a
LPS-binding protein that binds to LPS and a
glycosylphosphatidylinositol-anchored cell-surface glycoprotein,
CD14(25) . It was also shown that p53/56
was
co-immunoprecipitated with CD14 in human monocytes(23) . This
suggests a critical role of Src family tyrosine kinases in the
LPS/CD14-mediated signal transduction pathway in monocytes and
macrophages. Our results shown in Fig. 6Fig. 7Fig. 8indicate that Src family
tyrosine kinases are the major tyrosine-phosphorylated proteins, and
that p53/56
is one of the major tyrosine-phosphorylated
Src family tyrosine kinases in rat alveolar macrophages. These results
are consistent with the results showing that p53/56
is
the major Src family tyrosine kinase which is associated with CD14 in
human monocytes(23) . Furthermore, radicicol inhibited tyrosine
phosphorylation and levels of p53/56
in a time-dependent
and dose-dependent fashion (Fig. 2, A and B,
and 9, A and B). Inhibitory effects of radicicol on
protein tyrosine phosphorylation required pretreatment of cells with
radicicol for 4 h prior to LPS stimulation. Such pretreatment of cells
was also required for herbimycin A in our studies and in studies by
other investigators(24, 25) .
Figure 9:
Dose-response by radicicol in inhibiting
protein tyrosine kinase in LPS-stimulated macrophages. Inhibitory
effects of radicicol and herbimycin A on tyrosine-specific protein
phosphorylation in LPS-stimulated macrophages were determined by
antiphosphotyrosine immunoblotting as described under ``Materials
and Methods.'' A, cells were pretreated with indicated
concentrations of radicicol or herbimycin A for 4 h and then stimulated
with LPS (10 µg/ml) containing radicicol or herbimycin A. The
control was pretreated with vehicle, dimethyl sulfoxide (5 µl/ml)
only. An arrow on the right indicates
tyrosine-phosphorylated protein bands superimposed with p53/56 bands shown below. Rad, radicicol; Herb,
herbimycin A. B, Western blot analysis of p53/56
in the same samples used in A as described in the legend
for Fig. 2C. LYN, p53/56
.
Molecular size markers are shown in
kilodaltons.
In vitro kinase assay using purified p60 kinase (UBI) and
p60
kinase substrate peptide revealed that radicicol
inhibits the kinase activity at much higher concentrations (IC
= 8.2 µM, Fig. 10) than those required
to suppress protein tyrosine phosphorylation in LPS-stimulated
macrophages (Fig. 9A) or those required to suppress the
expression of COX-2 (Fig. 11). Similarly, concentrations of
herbimycin A needed to suppress COX-2 expression and protein tyrosine
phosphorylation in LPS-stimulated macrophages were much lower than
those known to be required to inhibit p60
kinase
activity in vitro. The IC
value of herbimycin A
for the in vitro kinase activity was reported as 12
µM(36) .
Figure 10:
Inhibition of p60
tyrosine kinase activity by radicicol. In vitro kinase assay
was carried out using purified p60
, p60
kinase substrate peptide, and [
-
P]ATP
as described under ``Materials and Methods.'' Values are mean
± S.E. of three samples.
Figure 11:
The dose-response by radicicol in
inhibiting the recovery of COX activity and 5-lipoxygenase (5-LO) activity in LPS-stimulated macrophages. Rat alveolar
macrophages pretreated with aspirin as described in the legend for Fig. 3were incubated in RPMI with LPS and various concentrations
of radicicol for 16 h. The activity of de novo synthesized COX
was determined by measuring the levels of PGE produced from
exogenous arachidonic acid as described in the legend for Fig. 3. The activity of 5-lipoxygenase was determined by
measuring the levels of 5-hydroxyeicosatetraenoic acid by
radioimmunoassay. Values for each dose are the means of three to six
samples.
Together, these results suggest that
radicicol suppresses tyrosine phosphorylation of Src family tyrosine
kinases in LPS-stimulated macrophages both by decreasing levels of
enzyme proteins and inhibiting the enzyme activity. It has been shown
that herbimycin A increased the degradation of
p60(37) . However, the possibility that
radicicol and herbimycin A inhibit expression of the tyrosine kinases
during the pretreatment period cannot be ruled out.
The mechanism by
which radicicol inhibits protein tyrosine kinases is not known. It has
been speculated that herbimycin A inactivates p60 kinase by irreversibly binding to SH group(s) of
p60
. This speculation was based on the fact that
herbimycin A is readily inactivated by sulfhydryl compounds. These
compounds abolished the inhibitory effect of herbimycin A on
p60
kinase activity. It was postulated that this
inactivation occurs through conjugation between highly polarized double
bonds in the benzoquinone moiety of herbimycin A and the SH group of
sulfhydryl compounds or Src kinases. Similarly, conjugation of SH group
can occur at C-9 with a conjugated double bond or at C-5 and C-6
bearing an epoxide in the radicicol molecule as shown in Fig. 1.
It was observed that dithiothreitol abolished the ability of radicicol
to block p60
kinase activity(22) . However,
there is no evidence as to whether sulfhydryl compounds also abolish
effects of radicicol propagated to steps downstream of Src tyrosine
kinases.
The recovery of COX activity in aspirin-pretreated and LPS-stimulated macrophages started only after 6 h of incubation (Fig. 3). This time course paralleled the time course of de novo synthesized COX-2 protein as shown in our previous studies(14) . COX activity recovered after 16 h of incubation was always greater than COX activity of unstimulated cells prior to aspirin treatment, although the magnitude of the difference varies with batches of cells. This indicates that the activity of COX-2 expressed as a result of LPS stimulation is much greater than that of COX-1 present in unstimulated cells. This suggests that COX-2 but not COX-1 plays a major role in producing prostanoids in response to inflammatory stimuli in macrophages. The inhibition of COX-2 expression by radicicol was not reversed during the 24-h incubation period.
The
dose-response to radicicol in inhibiting the recovery of COX activity
showed that the IC is 10 ng/ml (27 nM) as shown
in Fig. 11. These doses of radicicol did not significantly
affect 5-lipoxygenase activity as determined by measuring
5-hydroxyeicosatetraenoic acid (5-HETE) produced from exogenous
arachidonic acid (30 µM). Radicicol suppressed the
expression of COX-2 protein but not COX-1 and
glyceraldehyde-3-phosphate dehydrogenase (GAP) proteins. The rate of
COX-2 protein synthesis, as determined by the immunoprecipitation assay
using the specific COX-2 antibodies(14) , showed a parallel
dose-dependent inhibition by radicicol in macrophages which were
metabolically labeled with [
S]methionine in the
presence of LPS (Fig. 4A). When the same samples were
precleared with COX-2 antiserum to remove COX-2 protein and then
immunoprecipitated with COX antibodies which preferentially recognize
COX-1 protein, no COX bands were detected in the autoradiograph (figure
not shown). This result indicates that COX-1 is not synthesized in
significant amounts during the 2-h labeling period in LPS-treated
macrophages.
Figure 4:
Effects of radicicol on expressions of
COX-2, COX-1, and glyceraldehyde-3-phosphate dehydrogenase (GAP). A, rat alveolar macrophages were preincubated
for 14 h with LPS (10 µg/ml) and various concentrations of
radicicol and then further incubated in methionine-free RPMI containing
200 µCi of [S]methionine for 2 h. Cells were
lysed in the lysing buffer. Aliquots of the samples with equal amounts
of radioactivity were precleared with the preimmune serum,
immunoprecipitated with COX-2 antibodies, and subjected to
SDS-polyacrylamide gel electrophoresis and fluorography as described
under ``Materials and Methods.'' B, rat alveolar
macrophages were incubated in RPMI with 3% serum for 16 h with or
without LPS and/or radicicol. For Western blot analysis, microsomes
from lysed cells were used for COX-1, and whole lysate was used for
glyceraldehyde-3-phosphate dehydrogenase. Lane 1, cells
incubated without LPS and radicicol; lane 2, cells incubated
with LPS (10 µg/ml) only; lane 3, cells incubated with LPS
and radicicol (50 ng/ml); lane 4, cells incubated with LPS and
radicicol (200 ng/ml).
Levels of COX-1 and glyceraldehyde-3-phosphate dehydrogenase proteins were assessed by Western blot analysis (Fig. 4B). Levels of these enzymes in macrophages were not affected by either LPS or radicicol. Both COX-1 and glyceraldehyde-3-phosphate dehydrogenase are products of housekeeping genes whose expression, normally, is not stimulated by mitogens.
Radicicol also inhibited the LPS-induced expression of COX-2 in rat
smooth muscle cells and human peripheral blood monocytes. Furthermore,
radicicol inhibited the expression of COX-2 induced by IL-1 in rat smooth muscle cells, although the IC
value of
radicicol for the inhibition of IL-1
-induced COX-2
expression was much greater than that for the inhibition of COX-2
expression induced by LPS (Table 1).
Radicicol at
concentrations up to 1000 ng/ml did not significantly affect the
expression of COX-2 induced by phorbol 12-myristate 13-acetate (PMA) in
rat alveolar macrophages (Table 1). It was also shown that
another protein tyrosine kinase inhibitor, herbimycin A, only weakly
inhibits PMA-induced release of [H]arachidonic
acid metabolites in macrophage-like cell line (RAW 264.7), whereas it
dramatically inhibits LPS-induced release of
[
H]arachidonic acid metabolites(24) . It
has been demonstrated that PMA-stimulated c-raf activity in
human T cells and some of functional effects of PMA were resistant to
the inhibitory effects of herbimycin A(38) . These results
suggest that protein tyrosine phosphorylation is proximal to protein
kinase C activation in these signal transduction pathways.
It has
been well documented that endotoxin LPS induces rapid protein tyrosine
phosphorylation in macrophages(23, 24, 25) .
In addition to radicicol, other tyrosine kinase inhibitors also
suppressed LPS-induced expression of COX-2, although IC values of these inhibitors were much greater than that of
radicicol (22 µM, 52 nM, and 357 µM for genistein, herbimycin A, and tyrphostin (AG-494),
respectively). Earlier, it was shown that PMA-induced expression of
COX-2 was not inhibited by radicicol. Together, these results suggest
that tyrosine protein phosphorylation is the proximal step in the
LPS-induced signal transduction pathway leading to the induction of
COX-2 expression in macrophages, and that the inhibitory effect of
radicicol on LPS-induced COX-2 expression is due at least in part to
the suppression of activities of tyrosine kinases.
Figure 12: Dose-response of COX-1, COX-2, and glyceraldehyde-3-phosphate dehydrogenase mRNA levels to radicicol. Rat alveolar macrophages were incubated with LPS in the presence of various concentrations of radicicol for 2 h. Levels of mRNA were determined by RNase protection assay, as described in the legend for Fig. 11.
Figure 13:
The effect of radicicol on the stability
of COX-1 and COX-2 mRNA. The effect of radicicol on COX-1 and COX-2
mRNA stability was examined by stimulating macrophages with LPS for 2 h
in the presence or absence of radicicol (100 ng/ml). Actinomycin D at a
final concentration of 2 µg/ml was added to block further
transcription, and the levels of COX-1 and COX-2 mRNA were monitored at
specified time intervals over a period of 6 h after the addition of
actinomycin D by RNase protection assay. Glyceraldehyde-3-phosphate
dehydrogenase mRNA, which is known to have a long half-life, was used
as an internal control for RNA loading. The radioactivity was
quantitated by AMBIS as shown in Fig. 5. Percents of initial
levels (cpm of COX-1 or COX-2 10
cpm of
glyceraldehyde-3-phosphate dehydrogenase) were plotted against the
incubation time after the addition of actinomycin D. A, COX-1
mRNA stability. B, COX-2 mRNA
stability.
To determine
the direct effect of radicicol on the enzyme activity of COX-2,
macrophages were pretreated with aspirin (500 µM) for 4 h
to inactivate the endogenous COX, washed three times, and then further
incubated with LPS for 16 h in order to maximally stimulate COX-2
expression. After removing the medium, cells were incubated with
radicicol (100 ng/ml) or with the vehicle (dimethyl sulfoxide) for 1 h,
and then COX activity was determined as described above. COX activity
in these cells reflects specifically that of COX-2 because endogenous
COX-1 was inactivated by aspirin and LPS induces selective expression
of COX-2 in macrophages as shown in our previous study(14) .
The results showed that the level of PGE produced from
exogenous arachidonic acid in cells treated with radicicol was 150.2
± 9.3 (pg/µg of protein, n = 3), whereas
that in cells treated with the vehicle was 121.1 ± 8.8
(pg/µg of protein, n = 3). The enzyme activities of
purified COX-1 and COX-2 were also not affected by radicicol (1000
ng/ml). COX-1 activities were 3.82 ± 0.42 and 5.40 ± 0.78
nmol/µg of protein/10 min (n = 4) for the vehicle
and radicicol-treated samples, respectively, whereas COX-2 activities
were 5.59 ± 0.46 and 5.18 ± 0.90 nmol/µg of
protein/10 min (n = 4) for the vehicle and
radicicol-treated samples, respectively. These results indicate that
radicicol does not directly affect the enzyme activities of COX-1 and
COX-2 but it specifically suppresses the expression of COX-2 in
LPS-stimulated alveolar macrophages.
The protein tyrosine kinase
inhibitor genistein has been shown to inhibit platelet-activating
factor-stimulated PGE production in LPS-primed p388D1
macrophage-like cells(39) . Genistein and another protein
tyrosine kinase tyrphostin-25 inhibited PGE
production in
murine resident peritoneal macrophages stimulated with zymosan, calcium
ionophore A23187, and PMA(39) . It was shown that these
inhibitors had no inhibitory effect on cyclooxygenase activity in the
intact macrophages(40) . Therefore, it was speculated that the
inhibition of PGE
production by protein tyrosine kinase
results from reduced arachidonic acid release from membrane lipids by
phospholipase A
. Specific effects of these inhibitors on
the expression of COX were not determined in these studies. Our results
showing that protein tyrosine kinase inhibitors suppress the expression
of COX-2 suggest that the inhibition of PG production in LPS-stimulated
macrophages by these inhibitors is due at least in part to the
suppression of COX-2 expression.
Figure 14: In vivo effects of radicicol on expressions of COX-1, COX-2, and glyceraldehyde-3-phosphate dehydrogenase in rats with experimental glomerulonephritis. Glomerular injury was induced in rats with nephritogenic doses of anti-glomerular basement membrane (GBM) antibodies, as described under ``Materials and Methods.'' Radicicol or vehicle was infused through the renal artery for 60 min at the dose of 10 µg/100 g body weight per min. The rats were sacrificed after 4 h and 24 h following anti-GBM antibody injection. The total RNA extracted from whole kidneys and glomeruli was used for RNase protection assay to determine levels of COX-1, COX-2, and glyceraldehyde-3-phosphate dehydrogenase mRNA.
In summary, Src family tyrosine kinases were the major tyrosine-phosphorylated proteins in LPS-stimulated macrophages. Radicicol suppressed tyrosine phosphorylation of these kinases. Radicicol suppressed the expression of COX-2, but not COX-1, in LPS-stimulated macrophages and in glomeruli of rats with experimentally induced glomerulonephritis in which COX-2 expression is known to be enhanced. Thus, radicicol is a potent inhibitor for the expression of COX-2 both in vitro and in vivo. Radicicol did not inhibit PMA-induced expression of COX-2. Other protein tyrosine kinases inhibited the COX-2 expression in LPS-stimulated macrophages. These results suggest that the inhibition of COX-2 expression by radicicol is at least in part mediated through the inhibition of protein tyrosine kinases in LPS-stimulated macrophages. The magnitude of the inhibition of COX-2 protein synthesis by radicicol was much greater than that of the steady state levels of COX-2 mRNA. The rate of COX-2 mRNA degradation was slightly increased by radicicol. However, this does not account for the drastic inhibition of the activity and protein levels of COX-2 by radicicol. Taken together, these results suggest that the inhibition of COX-2 expression by radicicol occurs mainly at post-transcriptional steps.