As shown by the rightwards shiftand the reduced apparent efficacy of the inhibition curves described for both dexamethasone and budesonide, the glucocorticoid-dependent repression of COX/
Trang 1Differential effects of RU486 reveal distinct mechanisms for
Joanna E Chivers1, Lisa M Cambridge1, Matthew C Catley1, Judith C Mak1, Louise E Donnelly1,
Peter J Barnes1and Robert Newton2
1
Department of Thoracic Medicine, National Heart and Lung Institute, Imperial College London, Faculty of Medicine, London, UK; 2
Department of Biological Sciences, University of Warwick, Coventry, UK
In A549 pulmonary cells, the dexamethasone- and
budeso-nide-dependent repression of interleukin-1b-induced
pros-taglandin E2release was mimicked by the steroid antagonist,
RU486 Conversely, whereas dexamethasone and
budeso-nide were highly effective inhibitors of
interleukin-1b-induced cyclooxygenase (COX)/prostaglandin E synthase
(PGES) activity and COX-2 expression, RU486 (< 1 lM)
was a poor inhibitor, but was able to efficiently antagonize
the effects of dexamethasone and budesonide In addition,
both dexamethasone and RU486 repressed [3
H]arachido-nate release, which is consistent with an effect at the level
of phospholipase A2 activity By contrast, glucocorticoid
response element-dependent transcription was unaffected by
RU486 but induced by dexamethasone and budesonide,
whilst dexamethasone- and budesonide-dependent
repres-sion of nuclear factor-jB-dependent transcription was
maximally 30–40% and RU486 (< 1 lM) was without significant effect Thus, two pharmacologically distinct mechanisms of glucocorticoid-dependent repression of prostaglandin E2release are revealed First, glucocorticoid-dependent repression of arachidonic acid is mimicked by RU486 and, second, repression of COX/PGES is antagon-ized by RU486 Finally, whilst all compounds induced glucocorticoid receptor translocation, no role for glucocor-ticoid response element-dependent transcription is suppor-ted in these inhibitory processes and only a limisuppor-ted role for glucocorticoid-dependent inhibition of nuclear factor-jB in the repression of COX-2 is indicated
Keywords: corticosteroid; cyclooxygenase; epithelial cell; glucocorticoid receptor; prostaglandin E2
Synthetic glucocorticoids are potent repressors of
inflam-mation and are a first-line therapy for inflammatory diseases
[1] However, their clinical usage is limited by
immunosup-pression as well as by metabolic effects, including increased
gluconeogenesis, increased blood glucose, amino and fatty
acid mobilization, and loss of bone [2] In addition,
endogenous glucocorticoids participate in feedback
inhibi-tion of the hypothalamo-pituitary-adrenal axis, and
long-term high-dose synthetic glucocorticoid usage may cause
hypothalamo-pituitary-adrenal insufficiency and
glucocor-ticoid dependency
Glucocorticoids are believed to act primarily via the
glucocorticoid receptor (GR), which is maintained as an
inactive cytoplasmic complex with heat shock proteins (hsp)
and immunophilins [3] Following ligand binding and complex dissociation, the GR translocates to the nucleus where it binds glucocorticoid response elements (GREs), as
a dimer, to promote the transcription of responsive genes [2] However, the GR may also act as a monomer to inhibit key inflammatory transcription factors, such as nuclear factor-jB (NF-jB) and activator protein-1, by direct interaction, competition for cofactors or by modifying the chromatin structure to prevent the expression of inflamma-tory genes [1,2]
Inflammatory prostaglandins, produced by the arachi-donic acid cascade, play a pathophysiological role in edema, bronchoconstriction, fever and hyperalgesia [4] Arachidonic acid, released from cell membranes by phospholipase A2 (PLA2), is converted to prostaglandin
H2 (PGH2) by cyclooxygenase enzymes (COX), and further modified by specific isomerases and reductases to produce biologically relevant prostaglandins, including prostaglandin E2 (PGE2), which is the major prostaglan-din product of both airway epithelial and A549 cells [5] In inflammation, the inducible COX, COX-2, is normally up-regulated and accounts for the elevated levels of prostaglandins [4] Conversely, COX-2 expression is highly sensitive to glucocorticoid inhibition, suggesting that inhibition of COX-2 is critical in the repression of prostaglandins by glucocorticoids As cytokine-induced COX-2 and PGE2release are highly NF-jB-dependent in A549 cells [6], and treatment with dexamethasone pro-foundly represses PGE release and COX-2 expression [7],
University of Warwick, Coventry CV4 7AL, UK.
Fax: +44 2476 523701; Tel.: +44 2476 574187;
E-mail: robert.newton@imperial.ac.uk
Abbreviations: COX, cyclooxygenase; CRE, cyclic AMP response
element; DAPI, 4¢,6¢-diamidino-2-phenylinole dihydrochloric hydrate;
EGF, epidermal growth factor; GR, glucocorticoid receptor;
GRE, glucocorticoid response element; hsp, heat shock protein;
SFM, serum-free media.
(Received 13 January 2004, revised 16 August 2004,
accepted 23 August 2004)
Trang 2we have used this system to further explore the
mecha-nisms of glucocorticoid action
Materials and methods
Cell culture
A549 cells were cultured to confluence, as described
previously [7] Following overnight incubation in
serum-free media (SFM), drugs (dexamethasone, budesonide,
ionomycin, RU486) were added 1 h before stimulation with
interleukin-1b (IL-1b) (R & D Systems, Oxon, UK)
Dexamethasone and budesonide (both Sigma, Poole, UK)
were dissolved in Hank’s balanced salt solution (Sigma)
Ionomycin and RU486 (both Sigma) were dissolved in
ethanol Final concentrations of ethanol were less than
0.1% (v/v)
PGE2release, COX/prostaglandin E synthase (PGES)
activity and COX-2 expression
PGE2 released into the medium was measured using a
commercially available PGE2 antibody (Sigma) [5,8] For
the assay of combined COX/PGES activity, cells were
rinsed with SFM prior to incubation at 37C for 10 min in
SFM supplemented with 30 lM arachidonic acid, and
released PGE2was taken as a index of COX/PGES activity
[5,8] Northern and Western blot analyses were performed
as described previously [7]
Reporter cell lines and luciferase assay
A549 cells containing the NF-jB-dependent reporter,
6jBtkluc, have been described previously [9] The
1·GRE-dependent and 2·GRE-1·GRE-dependent reporters,
pGL3.neo.-TATA.GRE and pGL3.neo.TATA.2GRE, respectively,
were based on the parent vector pGL3.neo.TATA, which
contains a modified minimal b-globin promoter, as
pre-viously described [10] This was digested at the SmaI
site, upstream of the minimal promoter, and
double-stranded oligonucleotides (sense strand: 5¢-GCTGTACAG
GATGTTCTAG-3¢ and 5¢-GCTGTACAGGATGTTC
TAGGCTGTACAGGATGTTCTAG-3¢), containing one
or two copies of a consensus GRE site (underlined) [11],
were inserted to produce pGL3.neo.TATA.GRE and
pGL3.neo.TATA.2GRE, respectively A 2·GRE(mut)
reporter was generated as described above, but using a
mutated 2·GRE oligonucleotide (sense strand
5¢-GCTcaACAGGATcaTCTAGGCTcaACAGGATcaT
CTAG-3¢) (mutated bases in lower case) The cyclic AMP
response element (CRE)-dependent reporter, which
con-tains six CRE sites, was as previously described [12] A549
cells, stably harboring the luciferase reporters, were
gener-ated as previously described [9] Prior to experiments,
confluent plates of reporter cells were incubated overnight in
serum-free, G-418-free, media Cells were subsequently
harvested in 1· reporter lysis buffer (200 ll) (Promega) 6 h
after treatment for luciferase activity assay (Promega)
As each well is confluent and all the cells contain the
reporter construct, we find reporter activity to be highly
reproducible, and normalization to a second reporter is
unnecessary [9]
[3H]Arachidonic acid release
As previously described [8], cells were incubated overnight
in 0.5 mL of SFM supplemented with 0.125 lCi [5,6,8,9,11,12,14,15-3H]arachidonic acid (Amersham Phar-macia) Cells were washed twice prior to treatment with dexamethasone or RU486 After 1 h, supernatants were changed to fresh SFM containing 2 mgÆmL)1fatty acid-free BSA (Sigma) plus drugs prior to stimulation Supernatants were collected and cells washed prior to harvesting in 1% (w/v) SDS Release of [3H]arachidonic acid, or its metabolites, was expressed as a percentage of the total incorporated
Ligand binding
At 80% confluence, A549 cells cultured in T175 flasks were transferred to SFM and harvested the following day in cell dissociation solution (C-5789; Sigma) Cells (1.5–4· 106cells per mL) were incubated overnight at
4C with increasing concentrations of [3H]dexamethasone,
in the presence of 10 lM dexamethasone, to determine nonspecific binding Free radioligand was removed by the rapid filtration of cells through glass-fibre filters (GF/B) presoaked in NaCl/Pi(PBS), 0.1% (v/v) polyethylenimine, using a cell harvester [M-24R Brandel, SEMAT Technical (UK) Ltd, St Albans, Hertfordshire, UK] Filters were combined with Filtron-X scintillant (National Diagnostics, Atlanta, GA, USA) and radioactivity was measured using a beta counter (2200CA Tri-carb Liquid Scintillation Ana-lyser; Canberra Packard, Berks., UK) Kdand Bmax.values were determined using saturation binding isotherms and Scatchard analysis, [Bound]/[Free] vs [Bound], where the x-intercept ¼ Bmax. and the gradient ¼ ) 1/Kd (Fig 1A) (PRISM 3; GraphPad, San Diego, CA, USA) Relative binding affinity was assessed by incubating cells with an increasing concentration of unlabelled steroid and 4 nM [3H]dexamethasone overnight at 4C Bound and free radioligand were separated as described above Specific binding was calculated by subtraction of nonspecific from total binding, and Cheng–Prusoff analysis was performed to determine the Kivalue: Ki¼ IC50/{1 + ([Free Count]/Kd)}, where IC50is the concentration that results in 50% inhibi-tion (Table 1) [13]
Immunocytochemistry Cells grown on coverslips were transferred at 70% conflu-ence to SFM for 24 h After incubation with steroid for the indicated times, cells were washed with NaCl/Pi(PBS) and fixed with 4% (w/v) paraformaldehyde before successive incubations in 0.5% (v/v) Nonidet P-40 and 100 mM glycine Coverslips were blocked in NaCl/Pi(PBS) contain-ing 0.1% (v/v) Tween-20, 0.1% (w/v) BSA and 10% (v/v) human serum prior to incubation for 1 h in 5 lgÆmL)1 rabbit anti-human GR (PA1–511A; Affinity Bioreagents Inc., Golden, CO, USA) or rabbit isotype control (Dako, Glostrup, Denmark) After washing with NaCl/Pi (PBS) containing 0.1% (v/v) Tween and incubation with biotin-ylated anti-rabbit immunoglobulins (Dako) for 1 h, cells were incubated with fluorescein isothiocyanate (FITC)-linked streptavidin (Dako) for 1 h Nuclei were then stained
Trang 3with 1 lM 4¢,6¢-diamidino-2-phenylinole dihydrochloric
hydrate (DAPI) (Sigma) and coverslips were mounted on
glass slides using Citifluor mounting fluid (Citifluor Ltd,
London, UK), prior to analysis using a Leica TCS 4D
confocal microscope (Leica Microsystems, Milton Keynes,
UK) equipped with argon, krypton, and ultraviolet lasers
Confocal images were acquired at·40 magnification using
TCS NTsoftware (Leica Microsystems)
Statistical analysis
Statistical analysis was performed using analysis of variance
( ) with a Dunn’s post-test, unless specifically stated
otherwise in the figure legends Significance was taken at P-values of < 0.05 (*), < 0.01 (**) and < 0.001 (***)
Results
Repression of PGE2release, COX/PGES activity and COX-2 expression
As reported previously [7,14], untreated A549 cells released low levels of PGE2(1.2 ± 0.2 ngÆmL)1) and showed low levels of combined COX/PGES activity (3.1 ± 0.6 ngÆ
mL)1Æmin)1), which were both increased upon stimulation with IL-1b (1 ngÆmL)1) (22.6 ± 3.7 ngÆmL)1 and
0 50 100
-7-6 -5 -10 -9 -8 -7 -6 -5
IL-1 Log Log [Bud] (M)
[RU486]
(M)
0 50 100
-7-6 -5 -10 -9 -8 -7 -6 -5
IL-1 Log Log [Bud] (M) [RU486]
(M)
0
50
100
-10 -9 -8 -7 -6 -5
Log [Steroid] (M)
0 50 100
0 50 100
Log [RU486] (M)
Log [RU486] (M)
0 50 100
0 50 100
-7-6 -5 -10 -9 -8 -7 -6 -5
IL-1 Log Log [Dex] (M) [RU486]
(M)
-7-6 -5 -10 -9 -8 -7 -6 -5
IL-1 Log Log [Dex] (M)
[RU486]
(M)
-11
0
50
100
-10 -9 -8 -7 -6 -5
Log [Steroid] (M)
-11
D
(COX)/prostaglandin E synthase (PGES) activity (A) A549 cells were cultured with various concentrations of dexamethasone (j), budesonide (h)
or no stimulation (NS) (C and D) Cells were treated with various concentrations of dexamethasone (C) or budesonide (D) in the absence (j) or
± SEM The following levels of significance were established, expressed as P-values of < 0.05 (*), < 0.01 (**) and < 0.001 (***) (A) (upper panel)
Trang 432.8 ± 2.0 ngÆmL)1Æmin)1, respectively) In each case the
IL-1b-induced release of PGE2and combined COX/PGES
activity were repressed in a concentration-dependent
man-ner to near-basal levels by dexamethasone [50% effective
concentration (EC50) values of 1.9 nMand 3.2 nM,
respect-ively) and budesonide (EC50values of 2.6 nMand 7.8 nM,
respectively) (Fig 1A, upper and lower panels) Similarly,
RU486 produced a concentration-dependent repression of
IL-1b-induced PGE2 release (EC50¼ 33.1 nM) (Fig 1A,
upper panel), yet was considerably less effective against
combined COX/PGES activity, with concentrations of less
than 1 lM being without significant effect (EC50¼ 5 lM) (Fig 1A, lower panel)
This effect was even more apparent when RU486 was used to antagonize the responses to dexamethasone and budesonide Thus, whereas the glucocorticoid-dependent inhibition of IL-1b-induced PGE2 release was not antag-onized (Fig 1B, upper panel), the inhibition of COX/PGES activity was effectively antagonized by RU486 (Fig 1B, lower panel) The abilities of dexamethasone and budeso-nide to inhibit both PGE2release and COX/PGES activity were further tested in the presence of various concentrations
Steroid
ligands
Radioligand
release
COX/PGES activity
GRE (23GRE)
NF-jB (6jBtk)
A
B
Fig 2 Effect of dexamethasone and RU486 on cyclooxygenase-2 (COX-2) expression (A) Cells were either not stimulated (NS) or pretreated with
harvested at 6 h for RNA, and Northern blot (NB) analysis was performed for COX-2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
in the presence of various concentrations of RU486 Cells were harvested as described in (A) for Northern and Western blot analyses In each case,
blots; lower panels, Northern blots) from the experiments in (A) were expressed as a percentage of IL-1b, treated and plotted as mean ± SEM.
Trang 5of RU486 (Fig 1C,D) As shown by the rightwards shift
and the reduced apparent efficacy of the inhibition curves
described for both dexamethasone and budesonide, the
glucocorticoid-dependent repression of COX/PGES activity
was clearly antagonized by increasing the concentration of
RU486 However, in marked contrast, RU486 primarily
resulted in an increased overall inhibition of the response
curves described for dexamethasone and budesonide on
PGE2release, as shown by the progressive flattening of the
respective lines (Fig 1C,D) These data are therefore
indicative of a primary inhibitory effect of RU486 on
PGE2release, but not on combined COX/PGES activity
Analysis of COX-2 mRNA and protein expression,
which is responsible for the inflammatory release of PGE2
from A549 cells [5,15], often revealed basal levels of
expression, as reported previously [16] However, in each
case, and as previously shown, COX-2 expression was
dramatically increased by treatment with IL-1b [7,14]
Consistent with the combined COX/PGES data, the
analysis of COX-2 mRNA and protein expression revealed
a concentration-dependent inhibition of COX-2 expression
by dexamethasone, whereas RU486 showed little effect
except at high doses (Fig 2A,B) Consistent with Fig 1B,
0.1 lM dexamethasone almost totally repressed both
mRNA and protein expression of COX-2, and this effect
was efficiently antagonized by RU486 (Fig 2B)
Effect of dexamethasone and RU486 on arachidonic
acid release
To investigate the possibility of an effect of steroids
upstream of COX-2, cells were loaded with [3H]arachidonic
acid prior to stimulation in the presence of dexamethasone
or RU486 As IL-1b alone is a poor activator of arachidonic
acid release [8], cells were also treated with ionomycin or
with IL-1b + ionomycin, which provides a Ca2+stimulus,
causing translocation and membrane association of
cyto-solic (c)PLA2to markedly enhance cPLA2activity [8,17,18]
IL-1b, ionomycin and IL-1b + ionomycin increased
[3H]arachidonic acid release by 1.6-fold, 3.2-fold and
7.2-fold, respectively (Fig 3A) In each case, dexamethasone
produced repressions of 50, 61 and 68%, whilst RU486
resulted in repressions of 58, 53 and 63%, respectively To
further characterize this inhibition, cells were treated with
various concentrations of either dexamethasone or RU486
prior to stimulation with IL-1b + ionomycin In each case,
a concentration-dependent inhibition of [3H]arachidonic
acid release (EC50¼ 18.7 ± 10.6 and 26.2 ± 11.6 nM,
respectively) was observed, thereby confirming the
inde-pendent inhibitory effect of RU486 acting at the level of
arachidonic acid release (Fig 3B)
Transactivation and transrepression by glucocorticoids
and RU486
The effect of dexamethasone and RU486 was analyzed on
GRE-dependent and NF-jB-dependent transcription
From the 1·GRE reporter, pGL3.neo.TATA.GRE,
GRE-dependent transcription was increased by 4.5-fold
(EC50¼ 46.7 ± 17.7) by dexamethasone and fivefold
(EC50¼ 53.5 ± 20.8 nM, respectively) by budesonide
(Fig 4A) Similarly the 2·GRE-driven reporter,
pGL3.neo.TATA.2GRE, gave rise to over a 15-fold (EC50¼ 54.5) induction by dexamethasone and a 20-fold induction (EC50¼ 65.3 nM) by budesonide (Fig 4B) No response was observed with reporters containing either mutated GRE elements (pGL3.neo.TATA.2GREmut) or
no GRE sites (pGL3.neo.TATA) (data not shown), which confirms the specificity of these reporter systems for the presence of GRE sites In each case, RU486 showed little
or no ability to activate GRE-dependent transcription (Fig 4A,B), but demonstrated a profound ability to antag-onize both 1·GRE and 2·GRE reporter activity induced by 0.1 lMof either dexamethasone or budesonide (Fig 4C,D) Analysis of IL-1b-induced NF-jB-dependent transcrip-tion revealed a modest 30–40% inhibitranscrip-tion (EC ¼
3 H arachidonic acid release (
Dex Ru486 0 5 10
*
**
** **
***
*** **
**
***
0 50 100
3 H arachidonic acid release (
-10 -9 -8 -7 -6 -5
Log [Steroid] (M)
A
B
Fig 3 Inhibition of arachidonic acid release by dexamethasone and
(Iono) or both together (IL + Iono), and the supernantants and cells
or 5) are shown as arachidonate release expressed as a percentage of the total incorporated ± SEM Significance was assessed using the Student’s t-test *P < 0.05, **P < 0.01 (B) Cells were treated as in (A) except that various concentrations of either dexamethasone (j) or
arachi-donate release as a fraction of the total incorporated was expressed as a percentage of the IL-1b + ionomycin stimulus and plotted as mean ± SEM Significance was assessed using analysis of variance ( ANOVA ) with a Dunn’s post-test **P < 0.01, ***P < 0.001.
Trang 63.2 ± 1.3 and 7.8 ± 1.9 nM) by dexamethasone and
budesonide, respectively, and just over a 50% inhibition
by 10 lMRU486 (Fig 5A) RU486 was without effect at
0.1 lM and required to be present at concentrations of
100-fold higher than either dexamethasone or budesonide
to achieve similar levels (30–40%) of inhibition It is worth
noting that the inhibition of NF-jB by RU486 correlates
very closely with the effects observed on COX activity and
COX-2 expression (Figs 1 and 2) In addition, the ability of
RU486 to antagonize the repressive effects of 0.1 lM
dexamethasone or budesonide was examined In each case,
a concentration-dependent antagonism was observed up to
a maximum of 0.1 lM RU486 (Fig 5B) Above this
concentration, increasing levels of inhibition were observed
owing to the repressive effect of RU486 acting alone (data
not shown and see Fig 5A)
The expression of COX-2 may also depend on activating
transcription factors (ATFs) and activator protein-1
(AP-1)-like factors acting at a CRE site located in the proximal
region of the COX-2 promoter [19–21] Consistent with this,
we have previously found that a CRE-driven reporter
construct was unresponsive to cAMP in A549 cells, but
responded to IL-1b [10] This was not believed to reflect a general problem with this reporter, as strong cAMP-indu-cibility has been demonstrated in other experimental systems [12] Consistent with these earlier findings, IL-1b was shown
to induce reporter activity twofold (Fig 5C) In each case, both dexamethasone (0.1 lM) and RU486 (10 lM) were found to produce marked repressive effects (Fig 5C)
Binding affinity of steroid ligands and effect
on GR translocation Saturation binding studies using [3H]dexamethasone dem-onstrated one-site binding in A549 cells and revealed
16 500 ± 2700 GR/cell with an affinity of 1.36 ± 0.10 nM, which is consistent with other reports, including primary epithelial cells, indicating an affinity in the low nM range (Fig 6A) [22–24] Competitive binding studies were performed to examine the relative GR-binding affinity of these steroid ligands, and the following rank order of affinity was observed: RU486 > budesonide > dexameth-asone (Fig 6B) The appropriate Ki values are given in Table 1
0 1 2 3 4 5 6
-11 -10 -9 -8 -7 -6 -5
0 5 10 15 20
-11 -10 -9 -8 -7 -6 -5
Log [Steroid] (M) Log [Steroid] (M)
0
20 40 60 80 100 120
20 40 60 80 100 120
-10 -9 -8 -7 -6 -5
Log [RU486] (M) Log [RU486] (M)
***
***
***
***
***
***
1 GRE
1 GRE
2 GRE
2 GRE
D C
Fig 4 Effect of dexamethasone, budesonide and RU486 on glucocorticoid response element (GRE)-dependent transcription (A) 1·GRE or (B) 2·GRE A549 reporter cells were either not stimulated (NS) or treated with various concentrations of dexamethasone (j), budesonide (h) or
various concentrations of RU486 Cells were harvested as described above, and luciferase activity, expressed as a percentage of the activity induced
both budesonide and dexamethasone In addition, the following levels of significance were established, expressed as P-values of < 0.05 (*), < 0.01
Trang 7Nuclear translocation of GR by dexamethasone
and RU486
Dexamethasone induced a rapid (within 15 min)
transloca-tion of GR from the cytoplasm to the nuclear compartment,
with complete translocation observed by 1 h (Fig 7A)
Similarly, and as expected, nuclear translocation of GR was
also induced by budesonide (Fig 7B) In addition, RU486
was also efficient at inducing GR nuclear translocation,
indicating that binding of the antagonist can result in
dissociation of the cytoplasmic hsp–GR complex (Fig 7B)
Analysis of an isotype-control antibody revealed no
signi-ficant immunoreactivity, suggesting that the observed signal
was GR-specific (Fig 7C)
Discussion
In the above studies, dexamethasone and budesonide
produced a near-total inhibition of both PGE2and COX/
PGES activity, and acted with similar efficacies (Table 1)
and potencies However, whilst the steroid receptor
antag-onist, RU486, showed reversal of both COX-2 expression
and COX/PGES activity, which is consistent with a
GR-dependent mechanism, RU486 was incapable of
ant-agonizing the repression of IL-1b-induced PGE2 release
produced by either dexamethasone or budesonide In fact,
RU486 resulted in the progressive repression of PGE2
release at increasing concentrations Analysis of RU486
alone on IL-1b-induced PGE2release revealed a
concentra-tion-dependent inhibition of PGE2release, yet showed little
or no effect on COX/PGES activity or COX-2 expression
until RU486 concentrations of 1 lM were reached This
clear discrepancy strongly suggests that RU486 may exert
an inhibitory effect upstream of COX-2, possibly at the level
of PLA2and arachidonic acid release
This proposal was confirmed by the analysis of
[3H]arachidonate release, which revealed
concentration-dependent inhibition by both dexamethasone and RU486 Interestingly, the EC50 values for repression of PGE2 release, and the repression of arachidonic acid release by RU486 (33.1 and 26.2 nM, respectively), correlate closely and therefore support the suggestion of a mechanistically distinct action for RU486 at the level of arachidonic acid release We therefore conclude that these data document the existence of at least two functionally distinct processes for the inhibition of inflammatory PGE2 release by steroids
In the first mechanism, glucocorticoids, such as dexameth-asone or budesonide, inhibit the expression of COX-2, and this response is antagonized efficiently by RU486 This contrasts with a second, and pharmacologically distinct mechanism, which occurs at the level of arachidonic acid release, in which the actions of glucocorticoids are mimicked
by RU486
Previous reports have also documented the inhibition of arachidonic acid release in A549 cells by dexamethasone [25] However, these authors did not report any inhibition
by RU486 (10 nM) alone [26], and showed a 50% antag-onism of the dexamethasone-dependent repression when using RU486 at 10 lM[25] In an attempt to reconcile the apparent differences between the results of these reports and those of the present study, it is noticeable that different mechanisms of stimulation were used in each of the studies, and this alone could account for any differences Further-more, inspection of our current data on the repression of both PGE2 release and arachidonic acid release, suggests that the effects of 10 nMRU486 could be at the margins of experimentally discernable repression (see Figs 1A and 3B)
We also note that Croxtall et al did not seemingly test higher concentrations of RU486 acting alone for an inhibitory effect on epidermal growth factor (EGF)-stimu-lated arachidonic acid release [25] This therefore leaves open the possibility that the incomplete antagonism of RU486 observed on dexamethasone-dependent repression
of EGF-stimulated arachidonic acid release is, in fact,
Dex RU486
80 70 60
-10 -9 -8 -7
Log [Steroid] (M)
-10 -9 -8 -7 -6 -5 0
100
50
IL-1 NS
0 1 2
3
*
**
90
Log [RU486] (M)
Fig 5 Transrepression by dexamethasone, budesonide and RU486 (A) 6jBtk reporter cells were either not treated or were treated with various
plotted as mean ± SEM Significance was established, expressed as P-values of < 0.05 (*), < 0.01 (**) and < 0.001 (***), for: budesonide at
mean ± SEM *P < 0.05, **P < 0.01.
Trang 8attributable to a partial agonistic effect of RU486 acting
alone [25]
It is well established that glucocorticoids can repress
the transcription of inflammatory genes via transcription
factors such as NF-jB [1,2] However, whilst some
degree (30–40% inhibition) of glucocorticoid-dependent
inhibition of NF-jB-dependent transcription was
observed in response to both dexamethasone and
budes-onide, this effect is clearly insufficient to account for the
near-complete repression of COX-2 expression or PGE2
release observed with each of these compounds As
PGE2 release and COX-2 expression in A549 cells is
highly NF-jB-dependent, and this level of inhibition of
NF-jB-dependent transcription correlates very well with
our previous observation that the IL-1b-induced COX-2
transcription rate was inhibited by 40% by
dexameth-asone, we are compelled to suggest that additional
mechanisms of glucocorticoid-dependent repression of
COX-2 must also exist [6,7] Similarly, whilst
GRE-dependent transcription was robustly increased following
dexamethasone and budesonide treatment, this
mechan-ism is unlikely to account for the repression of COX-2 or
COX/PGES activity, as the EC50 for this effect is greater than 10-fold more than that required for the inhibition
of PGE2 release or COX/PGES activity (Table 1) Interestingly, this shift in the concentration–response curve for transactivation effects at GREs (EC50 values
of 54.5 and 65.3 nM for dexamethasone and budesonide, respectively) when compared with transrepression, for example of NF-jB (EC50 values of 3.2 and 7.8 nM for dexamethasone and budesonide, respectively), has been previously reported, although the exact mechanistic explanation is currently lacking [27] Therefore, in respect
of COX-2, these data suggest that other, non-NF-jB-mediated and probably non-GRE-non-NF-jB-mediated, mechanisms
of dexamethasone-dependent inhibition must be in operation to account for the full repression of COX-2 and COX/PGES activities in these cells
By contrast, the inhibition of NF-jB-dependent tran-scription by high concentrations of RU486 correlated very closely, in terms of both apparent efficacy and potency, with the inhibition of COX/PGES activity, thereby providing further strength to the argument that additional mecha-nisms, other than the inhibition of NF-jB, account for the inhibition by dexamethasone However, the basis of this inhibition by RU486 is currently unclear to us because these levels of steroid are vastly in excess of that necessary to saturate GR, as suggested by our own, and previously reported [24,28], ligand-binding studies (Fig 6) It is pos-sible that at these high concentrations RU486 is acting in a GR-independent manner Notwithstanding the inhibition at high doses, it is clear that at concentrations of 1 or 0.1 lM, RU486 shows a limited or no effect on NF-jB-dependent transcription, yet is effective at inhibiting both PGE2and arachidonic acid release, suggesting that the inhibition of NF-jB plays no role in this response
Previous studies have suggested that, relative to dexamethasone, RU486 is a poor inducer of glucocorti-coid-dependent transcription [29–35] Similarly, in the present study, RU486-induced GRE-dependent transcrip-tion from either a 1·GRE or a 2·GRE reporter was virtually absent, and this is consistent with data from primary human bronchial epithelial cells [24] These data therefore raise the possibility that RU486 inhibits arachidonic acid release via a mechanism that is independent of transcription Indeed, the rapid dexa-methasone-dependent repression of EGF-induced release
of arachidonic acid was previously shown to be actino-mycin D insensitive and therefore independent of tran-scription [25] In this respect, RU486 has previously been shown to mimic other nongenomic glucocorticoid responses, including the down-regulation of GR itself [36,37] Certainly, our data indicate that RU486, can, like dexamethasone and budesonide, bind to and induce the nuclear translocation of GR We therefore speculate that binding of ligand, including antagonists such as RU486,
to GR, and complex dissociation, may be sufficient for the inhibition of arachidonic acid release and that this represents a mechanistically distinct event from the inhibition of inflammatory gene expression In this context it is notable that various nongenomic actions
of steroid hormones have been identified [38,39], which raises the possibility of ligand-dependent nongenomic anti-inflammatory functions for GR or for GR-associated
-11 -10 -9 -8 -7 -6 -5 0
25
50
75
100
Log [Steroid] (M)
0
2.5
5.0
7.5
10
Log [Dex] (nM)
3 (dpm)
(dpm)
5 7 10
0 1 2 3 4
A
B
Fig 6 Analysis of glucocorticoid receptor (GR) number and relative
affinity of ligands (A) A typical saturation–binding isotherm, showing
analysis (inset), where the ratio of free to bound radioligand is plotted
against log [steroid] to give a straight line with a gradient equal to
binding curves showing relative affinity in A549 cells, where
dexa-methasone (j), budesonide (h), RU24858 (d) or RU486 (.) compete
Trang 9proteins present in the GR–hsp complex Finally, we
should point out that a number of effects of
glucocor-ticoids, which are independent of the classical GR, are
also reported to occur and these could help to explain
our results [39] Thus, the mineralocorticoid receptor may
mediate glucocorticoid responsiveness in the brains of
GR knockout mice [40] In addition, a pharmacologically
distinct pool of membrane-localized glucocorticoid
recep-tors have been identified by various authors [39] For
example, a membrane glucocorticoid receptor has been
biochemically identified in amphibians [41] However, it
is currently unclear whether this represents a version of
the classical GR [42] or P-glycoprotein/multiple drug
resistance gene, a member of the ATP-binding cassette
(ABC) transporters [43,44], or some other receptor [45]
In this context, P-glycoprotein is of interest as it actively
exports certain steroids, and blocking its function has
been shown to promote glucocorticoid actions [46,47]
In conclusion, we present data which further confirm that
the inhibition of NF-jB-dependent transcription cannot
account for all the repressive effects of glucocorticoids on
inflammatory genes such as COX-2 Furthermore, we
present evidence that glucocorticoids and RU486 also inhibit
the release of arachidonic acid via a process that does not
involve either inhibition of NF-jB or the activation of
GRE-mediated transcription and which is mechanistically
distinct from the inhibition of COX-2 Taken together, these
data indicate the existence of pharmacologically distinct
processes that are collectively responsible for the repression
of inflammatory PGE2release by glucocorticoids
Acknowledgements
J.E.C and M.C.C were collaborative students with the BBSRC and the MRC, respectively, and both were supported by Aventis Pharma-ceuticals.
References
1 Barnes, P.J (1999) Therapeutic strategies for allergic diseases Nature 402 (Suppl.), B31–B38.
2 Newton, R (2000) Molecular mechanisms of glucocorticoid action: what is important? Thorax 55, 603–613.
3 Schaaf, M.J & Cidlowski, J.A (2002) Molecular mechanisms of glucocorticoid action and resistance J Steroid Biochem Mol Biol 83, 37–48.
4 Funk, C.D (2001) Prostaglandins and leukotrienes: advances in eicosanoid biology Science 294, 1871–1875.
5 Mitchell, J.A., Belvisi, M.G., Akarasereenont, P., Robbins, R.A., Kwon, O.J., Croxtall, J., Barnes, P.J & Vane, J.R (1994) Induction of cyclo-oxygenase-2 by cytokines in human pulmonary epithelial cells: regulation by dexamethasone Br J Pharmacol.
113, 1008–1014.
6 Catley, M.C., Chivers, J.E., Cambridge, L.M., Holden, N., Slater, D.M., Staples, K.J., Bergmann, M.W., Loser, P., Barnes, P.J & Newton, R (2003) IL-1beta-dependent activation of
A
for 15, 30 or 60 min and then probed with a fluorescein isothiocyanate (FITC)-conjugated GR immunoglobulin (green) prior to imaging by confocal microscopy Nuclei are indicated by 4¢,6¢-diamidino-2-phenylinole dihydrochloric hydrate (DAPI) staining of chromatin (blue) (B) Cells
Cells were treated as in (A) and then probed with an isotype control for the GR antibody used in (A) and (B) All images are representative of three experiments.
Trang 10cyclooxygenase-2 and microsomal prostaglandin E synthase.
FEBS Lett 547, 75–79.
7 Newton, R., Seybold, J., Kuitert, L.M.E., Bergmann, M &
Barnes, P.J (1998) Repression of cyclooxygenase-2 and
and post-transcriptional mechanisms involving loss of
poly-adenylated mRNA J Biol Chem 273, 32312–32321.
8 Newton, R., Cambridge, L., Hart, L.A., Stevens, D.A., Lindsay,
M.A & Barnes, P.J (2000) The MAP kinase inhibitors,
PD098059, UO126 and SB203580, inhibit IL-1beta-dependent
PGE(2) release via mechanistically distinct processes Br J.
Pharmacol 130, 1353–1361.
9 Newton, R., Hart, L.A., Stevens, D.A., Bergmann, M., Donnelly,
L.E., Adcock, I.M & Barnes, P.J (1998) Effect of dexamethasone
on interleukin-1beta (IL-1beta) induced nuclear factor-kappaB
(NF-kappaB) and kappaB-dependent transcription in epithelial
cells Eur J Biochem 254, 81–89.
10 Catley, M.C., Cambridge, L.M., Nasuhara, Y., Ito, K., Chivers,
J.E., Beaton, A., Holden, N.S., Bergmann, M.W., Barnes, P.J &
Newton, R (2004) Inhibitors of protein kinase C (PKC) prevent
activated transcription: role of events downstream of NF-kappaB
DNA binding J Biol Chem 279, 18457–18466.
11 Strahle, U., Schmid, W & Schutz, G (1988) Synergistic action of
the glucocorticoid receptor with transcription factors EMBO J 7,
3389–3395.
12 Meja, K.K., Catley, M.C., Cambridge, L.M., Barnes, P.J., Lum,
H., Newton, R & Giembycz, M.A (2004) Adenovirus-mediated
delivery and expression of a cAMP-dependent protein kinase
inhibitor gene to BEAS-2B epithelial cells abolishes the
anti-in-flammatory effects of rolipram, salbutamol, and prostaglandin E2:
a comparison with H-89 J Pharmacol Exp Ther 309, 833–844.
13 Cheng, Y & Prusoff, W.H (1973) Relationship between the
inhibition constant (K1) and the concentration of inhibitor which
causes 50 per cent inhibition (I50) of an enzymatic reaction.
Biochem Pharmacol 22, 3099–3108.
14 Newton, R., Kuitert, L.M., Bergmann, M., Adcock, I.M &
Barnes, P.J (1997) Evidence for involvement of NF-jB in the
transcriptional control of COX-2 gene expression by IL-1b
Bio-chem Biophys Res Commun 237, 28–32.
15 Newton, R., Eddleston, J., Haddad, E., Hawisa, S., Mak, J., Lim,
S., Fox, A.J., Donnelly, L.E & Chung, K.F (2002) Regulation of
kinin receptors in airway epithelial cells by inflammatory cytokines
and dexamethasone Eur J Pharmacol 441, 193–202.
16 Watkins, D.N., Peroni, D.J., Lenzo, J.C., Knight, D.A., Garlepp,
M.J & Thompson, P.J (1999) Expression and localization of
COX-2 in human airways and cultured airway epithelial cells Eur.
Respir J 13, 999–1007.
17 Schievella, A.R., Regier, M.K., Smith, W.L & Lin, L.L (1995)
Calcium-mediated translocation of cytosolic phospholipase A2 to
the nuclear envelope and endoplasmic reticulum J Biol Chem.
270, 30749–30754
18 Leslie, C.C (1997) Properties and regulation of cytosolic
phos-pholipase A2 J Biol Chem 272, 16709–16712
19 Xie, W., Fletcher, B.S., Andersen, R.D & Herschman, H.R.
(1994) v-src induction of the TIS10/PGS2 prostaglandin synthase
gene is mediated by an ATF/CRE transcription response element.
Mol Cell Biol 14, 6531–6539.
20 Inoue, H., Yokoyama, C., Hara, S., Tone, Y & Tanabe, T (1995)
Transcriptional regulation of human prostaglandin-endoperoxide
synthase-2 gene by lipopolysaccharide and phorbol ester in
vascular endothelial cells Involvement of both nuclear factor for
interleukin-6 expression site and cAMP response element J Biol.
Chem 270, 24965–24971.
21 Xie, W & Herschman, H.R (1996) Transcriptional regulation of
prostaglandin synthase 2 gene expression by platelet-derived
growth factor and serum J Biol Chem 271, 31742–31748.
22 Hammer, S., Spika, I., Sippl, W., Jessen, G., Kleuser, B., Holtje, H.D & Schafer-Korting, M (2003) Glucocorticoid receptor interactions with glucocorticoids: evaluation by molecular mod-eling and functional analysis of glucocorticoid receptor mutants Steroids 68, 329–339.
23 Robin-Jagerschmidt, C., Wurtz, J.M., Guillot, B., Gofflo, D., Benhamou, B., Vergezac, A., Ossart, C., Moras, D & Philibert, D (2000) Residues in the ligand binding domain that confer progestin
or glucocorticoid specificity and modulate the receptor transacti-vation capacity Mol Endocrinol 14, 1028–1037.
24 LeVan, T.D., Babin, E.A., Yamamura, H.I & Bloom, J.W (1999) Pharmacological characterization of glucocorticoid receptors in primary human bronchial epithelial cells Biochem Pharmacol 57, 1003–1009.
25 Croxtall, J.D., Choudhury, Q & Flower, R.J (2000) Gluco-corticoids act within minutes to inhibit recruitment of signalling factors to activated EGF receptors through a receptor-dependent, transcription-independent mechanism Br J Pharmacol 130, 289–298.
26 Croxtall, J.D., Paul-Clark, M & van Hal, P.T (2003) Differential modulation of glucocorticoid action by FK506 in A549 cells Biochem J 376, 285–290.
27 Jonat, C., Rahmsdorf, H.J., Park, K.K., Cato, A.C., Gebel, S., Ponta, H & Herrlich, P (1990) Antitumor promotion and anti-inflammation: down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone Cell 62, 1189–1204.
28 Gagne, D., Pons, M & Philibert, D (1985) RU 38486: a potent antiglucocorticoid in vitro and in vivo J Steroid Biochem 23, 247–251.
29 Vayssiere, B.M., Dupont, S., Choquart, A., Petit, F., Garcia, T., Marchandeau, C., Gronemeyer, H & Resche Rigon, M (1997) Synthetic glucocorticoids that dissociate transactivation and AP-1 transrepression exhibit antiinflammatory activity in vivo Mol Endocrinol 11, 1245–1255.
30 Heck, S., Bender, K., Kullman, M., Gottlicher, M., Herrlich,
P & Cato, A.C.B (1997) IkBa-independent downregulation of NF-kB activity by glucocorticoid receptor EMBO J 16, 4698– 4707.
31 Wissink, S., van-Heerde, E.C., vand-der, B.B & van-der-Saag, P.T (1998) A dual mechanism mediates repression of NF-kappaB activity by glucocorticoids Mol Endocrinol 12, 355–363.
32 Liden, J., Delaunay, F., Rafter, I., Gustafsson, J & Okret, S (1997) A new function for the C-terminal zinc finger of the glu-cocorticoid receptor Repression of RelA transactivation J Biol Chem 272, 21467–21472.
33 Fryer, C.J., Kinyamu, H.K., Rogatsky, I., Garabedian, M.J & Archer, T.K (2000) Selective activation of the glucocorticoid receptor by steroid antagonists in human breast cancer and os-teosarcoma cells J Biol Chem 275, 17771–17777.
34 Rogatsky, I., Hittelman, A.B., Pearce, D & Garabedian, M.J (1999) Distinct glucocorticoid receptor transcriptional regulatory surfaces mediate the cytotoxic and cytostatic effects of gluco-corticoids Mol Cell Biol 19, 5036–5049.
35 Pariante, C.M., Pearce, B.D., Pisell, T.L., Su, C & Miller, A.H (2001) The steroid receptor antagonists RU40555 and RU486 activate glucocorticoid receptor translocation and are not excreted
by the steroid hormones transporter in L929 cells J Endocrinol.
169, 309–320.
36 Hoeck, W., Rusconi, S & Groner, B (1989) Down-regulation and
cells Investigations with a monospecific antiserum against a bac-terially expressed receptor fragment J Biol Chem 264, 14396– 14402.
37 Burnstein, K.L., Jewell, C.M., Sar, M & Cidlowski, J.A (1994) Intragenic sequences of the human glucocorticoid receptor com-plementary DNA mediate hormone-inducible receptor messenger