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INTRODUCTION |
Activation of the transcription factor
NF-
B1 has been shown to be
a key component of innate immunity (1), promoting the expression by
macrophages of a set of genes involved in host defense, such as
pro-inflammatory cytokines (2, 3), NOS-2, cyclooxygenase 2, cell
adhesion molecules, and various matrix metalloproteinases (4-6).
NF-
B is present constitutively in the cytosol of the cells, where it
is retained through the interaction with inhibitory I
B proteins that
mask the nuclear localization domain of the complex (2).
NF-
B-dependent gene transcription requires the phosphorylation of I
B
by IKK2, which releases this inhibitory component from the dimer of Rel proteins (mainly p50, p65, and c-Rel),
and is followed by degradation of the phospho-I
B by the proteasome
(3, 7).
Biochemical, pharmacological, and genetic data indicate that the
control of NF-
B activation constitutes a relevant target for the
treatment of inflammatory diseases (2). For this reason, the research
on molecules endowed with the capacity to inhibit the consecutive steps
leading to NF-
B activation has been a subject of current interest
(2, 3). In this regard, among the natural products assayed, various
terpenoids have been described as potent inhibitors of NF-
B
activation in response to proinflammatory stimulation. Andalusol, a
labdane diterpene from Sideritis exerted anti-inflammatory
effects in vivo and in vitro by inhibiting
NF-
B activation (8, 9). Triterpenes also inhibited NOS-2 expression in RAW 264.7 cells (IC50, 0.2-0.3 µM) and
impaired NF-
B activity (10). In the case of sesquiterpenes, it has
been described that some of these compounds induce HSP72, which in turn
prevents NF-
B activation and NOS-2 expression (11, 12).
More recently, attention has been paid to the study of the biological
effects of tetracyclic ent-kaurene diterpenes, because the
number of these molecules isolated and characterized is continuously increasing (13, 14). Ent-kaurene is the main diterpene
intermediate involved in the biosynthesis of gibberellins, a widespread
family of plant hormones with isoprenoid structure that control various physiological plant functions such as growth, germination, and flowering (15). Some kaurenes exhibit cytotoxic activity against different cancer cell lines such as HL-60, K562, and MKN-28
(16, 17), whereas others exert trypanosomidal activity (18)
and inhibit human immunodeficiency virus replication in
vitro (19). We have compared a series of kaurene and clerodane
diterpenes as likely candidates to modulate NF-
B activity.
Clerodanes were used because these diterpenes, with lactone and epoxy
groups adjacent to ring A, offer the possibility of performing Michael
addition reactions, and recent work has indicated that this mechanism
is important in the physiological inhibition of IKK and NF-
B
activities by cyclopentenone prostaglandins (20, 21). Our data show
that molecules based on the kaurene structure potently inhibit NF-
B activation by interfering with steps preceding IKK, presumably NIK activity. In addition to this, kaurenes delayed the activation of
ERK1 and 2 and p38 MAPK, but not that of JNK, suggesting that the
alteration in the coordinate temporal sequence of events triggered after LPS activation plays an important role in the transient activation of NF-
B.
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MATERIALS AND METHODS |
Chemicals--
Reagents were obtained from Sigma, Roche
Molecular Biochemicals, and Merck. Series of clerodane and kaurene
diterpenes were obtained as described (22), and their structures are
shown in Fig. 1. Antibodies were from
Santa Cruz Biotechnology (Santa Cruz, CA), anti-CD14 and anti-CD11b
monoclonal antibodies were from PharMingen (Heidelberg, Germany), and
anti-phosphoprotein antibodies were from New England Biolabs (Beverly,
MA). LPS was from Salmonella typhimurium
(Sigma). Serum and media were from BioWhittaker (Walkersville, MD).

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Fig. 1.
Structures of kaurene and clerodane
diterpenes. The tetracyclic kaurene diterpenes constitute a large
family of hydroxylated molecules derived from the
ent-kaur-16-en-19-oic acid (compounds
1-3). The bicyclic clerodane diterpenes used
have a furan ring in the side chain and a 17 12 or 20 12 lactone group (compounds 4-6).
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Macrophage Cultures--
J774 cells were grown in RPMI 1640 medium containing 2 mM glutamine, 10% fetal calf serum,
and 50 µg/ml penicillin, streptomycin, and gentamicin. Subconfluent
cultures (2 days after seeding) were maintained in phenol red-free RPMI
1640 supplemented with 0.5 mM arginine and 1% fetal calf
serum, followed by treatment with the indicated diterpenes added 30 min
prior to activation with LPS.
Description of Plasmids--
A (
B)3ConA.LUC
plasmid containing three copies of the
B motif of the human
immunodeficiency virus long terminal repeat enhancer fused to
the minimal conalbumin A promoter and linked to the luciferase gene was
used to measure
B activation (23, 24). The ConA.LUC vector, lacking
the
B tandem, was used as a control and was not modulated by the
diterpenes assayed. A pRK5-Myc-NIK expression vector (Dr. W. C. Greene, The J. David Gladstone Institute, San Francisco, CA) and
kinase-deficient (K429A/K430A, NIK-KD) NIK vector (Dr. D. Wallach, The Weizmann Institute of Science, Israel) were used for
transient expression of NIK. An expression vector encoding FLAG-IKK2
and the kinase-deficient form (IKK2-KD) were used. Plasmids were
purified using EndoFree Qiagen columns (Hilden, Germany).
Transfection of J774 Cells and Assay of Luciferase
Activity--
Subconfluent cells were transfected for 6 h with
FuGENE following the instructions of the supplier (Roche Molecular
Biochemicals) and kept overnight with 2 ml of RPMI plus 1% fetal calf
serum prior to stimulation. Equal amounts of DNA were used in the
transfection experiments. Luciferase activity was assayed using the
reagents and protocol prepared by Promega (Madison, WI).
Expression and Purification of GST Fusion
Proteins--
GST-c-Jun-(1-79), GST-I
B
-(1-317), and
GST-I
B
-(1-54), wild type and mutant (S32A and S36A), were
expressed in DH5
F' Escherichia coli and purified by
glutathione-Sepharose 4B chromatography (Amersham Pharmacia
Biotech), as described (23).
Preparation of Cytosolic and Nuclear Extracts--
The cell
layers (3 × 106) were washed with ice-cold
phosphate-buffered saline, scraped off the dishes, and collected by
centrifugation. Cell pellets were homogenized in 100 µl of buffer A
(10 mM Hepes, pH 7.9, 1 mM EDTA, 1 mM EGTA, 100 mM KCl, 1 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 10 µg/ml leupeptin, 2 µg/ml TLCK, 5 mM NaF, 1 mM NaVO4, and 10 mM Na2MoO4). After 10 min at
4 °C Nonidet P-40 was added (0.5% v/v), and the tubes were vortexed
(15 s) and centrifuged at 8,000 × g for 15 min. The
supernatants were stored at
80 °C (cytosolic extracts), and the
pellets were resuspended in 50 µl of buffer A supplemented with 20%
glycerol, 0.4 M KCl, and shaken for 30 min at 4 °C.
After centrifugation for 15 min at 13,000 × g, the
supernatants (nuclear protein extracts) were stored at
80 °C (25).
Protein was determined using the Bio-Rad protein assay. All steps of
fractionation were carried out at 4 °C.
Electrophoretic Mobility Shift Assays--
The oligonucleotide
sequence 5'-TGCTAGGGGGATTTTCCCTCTCTCTGT-3', corresponding to the
consensus
B site (nucleotides
978 to
952) of the murine NOS-2
promoter (26) was annealed with the complementary DNA and end-labeled
with Klenow enzyme and 50 µCi of [
-32P]dCTP. The DNA
probe (5 × 104 dpm) was incubated for 15 min at
4 °C with 3 µg of nuclear protein, 2 µg of poly(dI·dC), 5%
glycerol, 1 mM EDTA, 100 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 10 mM Tris-HCl, pH 7.8, in a final volume of 20 µl. The
DNA-protein complexes were separated on native 6% polyacrylamide gels
in 0.5% Tris borate-EDTA buffer. Supershift assays were carried out
after incubation of the nuclear extracts for 30 min at 4 °C
with 2 µg of Ab (anti-p50, anti-c-Rel, and anti-p65), followed by
electrophoretic mobility shift assay (data not shown). When the effect
of diterpenes and 15dPGJ2 on the binding of Rel proteins to
the
B motif was assayed in vitro, nuclear extracts from
LPS-activated cells were treated for 5 min with these ligands prior to
the addition of the DNA probe. Normalization for lane charge of the
blots was accomplished by measuring the binding to the peroxysomal
proliferator-activated receptor-
sequence as described (23).
Characterization of Proteins by Western Blot--
Cytosolic
protein extracts were size-separated via 10% SDS-polyacrylamide gel
electrophoresis. The gels were blotted onto a polyvinylidene difluoride
membrane (Amersham Pharmacia Biotech) and incubated with anti-NOS-2,
anti-I
B
, anti-IKK2, and anti-
-actin Abs (Santa Cruz
Laboratories), anti-FLAG and anti-Myc Abs (Sigma), and
anti-cyclooxygenase 2 Ab (Cayman, Ann Arbor, MI). The levels of
phosphorylated and total p38 and ERK1 and 2 were determined by Western
blot using cytosolic extracts and specific commercial Abs (New England
Biolabs). In experiments using
anti-phospho-Ser32-I
B
Ab, the blot incubation
solution contained 50 ng/ml GST-I
B
-(1-317) treated previously
with alkaline phosphatase-agarose (23). The blots were submitted to
sequential reprobing with Abs after treatment with 100 mM
-mercaptoethanol and 2% SDS in Tris-buffered saline and
heated at 60 °C for 30 min. The blots were revealed by ECL (Amersham
Pharmacia Biotech). Different exposure times of the films were used to
ensure that bands were not saturated. Quantification of the films was
performed by laser densitometry (Molecular Dynamics, Kemsing, UK).
Determination of NO Synthesis--
NO release was determined
spectrophotometrically by the accumulation of nitrite in the medium
(phenol red-free). Nitrite was determined with Griess reagent. The
absorbance at 548 nm was compared with a standard of NaNO2.
Results were expressed as the amount of nitrite released per mg of cell protein.
Assay of TNF-
Secretion--
The accumulation of TNF-
in
the culture medium was measured per duplicate using a commercial kit
(Biotrak, Amersham Pharmacia Biotech).
Measurement of IKK2 and JNK Activities--
Cells
(107) were homogenized in 1 ml of buffer A and centrifuged
for 10 min in a microcentrifuge. The supernatant was precleared, and
IKK2 or JNK were IP with 1 µg of anti-IKK2 or anti-JNK Abs, respectively. After washing the immunoprecipitates with 4 ml of buffer A, the pellet was resuspended in kinase buffer (20 mM Hepes, pH 7.4, 0.1 mM EDTA, 100 mM NaCl, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 10 µg/ml leupeptin, 2 µg/ml TLCK, 5 mM NaF, 1 mM NaVO4, 10 mM
Na2MoO4, and 10 nM okadaic acid).
The kinase activity was assayed in 100 µl of kinase buffer containing
100 ng of IP protein, 50 µM
[
-32P]ATP (0.5 µCi) and using as substrate 100 ng of
GST-I
B
or GST-c-Jun. Aliquots of the reaction were stopped with 1 ml of ice-cold buffer A supplemented with 5 mM EDTA. The
same protocol was used when the activity of IKK2 was followed by
Western blot using anti-phospho-Ser32-I
B
Ab,
except that 1 mM MgATP was used instead of
[
-32P]ATP. GST-I
B
and GST-c-Jun were purified by
glutathione-agarose chromatography and analyzed via 10%
SDS-polyacrylamide gel electrophoresis. The linearity of the kinase
reaction was confirmed over a period of 30 min (23).
Fluorescence-activated Cell Sorter Analysis of
Cells--
J774 cells were incubated for 30 min with diterpenes, and
the recognition of CD11b and CD14 was accomplished with
phycoerythrin-anti-CD11b Ab (Mac-1) and fluorescein
isothiocyanate-anti-CD14 Ab (PharMingen) by flow cytometry (FACSscan).
The percentage of positive cells and the mean channel fluorescence were quantified.
Data Analysis--
The number of experiments analyzed is
indicated in the figures. Statistical differences (p < 0.05) between mean values were determined by one-way analysis of the
variance followed by Student's t test. In experiments using
x-ray films (Hyperfilm) different exposure times were employed to avoid
saturation of the bands.
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RESULTS |
Kaurene Diterpenes Inhibited J774 Activation by LPS--
To
analyze the anti-inflammatory effects of the diterpenes with kaurene
structure (compounds 1-3), J774 macrophages were
stimulated for 24 h with LPS, and the levels of NOS-2 and cyclooxygenase 2 were determined as markers of the activation process.
As Fig. 2A shows, kaurene
diterpenes assayed at 50 µM potently inhibited the
expression of NOS-2 and were less efficient regarding the effects on
cyclooxygenase 2. However, diterpenes with a clerodane structure
(compounds 4-6) were unable to modify the
steady-state levels of both proteins. A dose-dependent effect of diterpenes on NOS-2 expression is shown in Fig.
2B, and good correlation between NOS-2 expression and
inhibition of nitrite synthesis was observed. The apparent
IC50 values for NO synthesis of diterpenes 1 and
3 were 3-5 µM, whereas diterpene 2 exhibited an IC50 of 9 µM (Fig. 2,
C and D).

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Fig. 2.
Inhibition of NOS-2 expression and NO
synthesis by kaurene diterpenes. J774 cells were treated for 30 min with the indicated concentrations of kaurene (compounds
1-3, described in the legend to Fig. 1) and clerodane
diterpenes (compounds 4-6, described in the legend to Fig.
1), followed by stimulation with 500 ng/ml LPS. After 24 h of
incubation, the levels of NOS-2 and cyclooxygenase 2 (COX-2) protein were determined by Western blot. The
content of -actin was used for normalization of the blot
(A and B). The accumulation of nitrite in the
culture medium was measured with Griess reagent (diterpenes were used
at 50 µM) (C). The dose-dependent
effect of diterpenes on NO synthesis was measured at 24 h
(D). Results show a representative experiment of three
(Western blot) or the mean ± S.E. of three experiments (nitrite
determination).
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Inhibition of NF
B Activity by Kaurene Diterpenes--
To
investigate the mechanism of action of kaurenes on macrophage function,
the effects on NF-
B activity, as a key step in the inflammatory
onset, were analyzed. Treatment of macrophages with kaurenes impaired
the activation of NF-
B as determined by electrophoretic mobility
shift assay (Fig. 3, A and
B). In addition to this, J774 cells were transiently
transfected with a plasmid containing a tandem of three copies of a
B motif linked to the luciferase gene and with an epitope-tagged
IKK2 expression vector. As Fig. 3C shows, LPS increased
luciferase activity, but this effect was inhibited by kaurene
diterpenes. Similar results were obtained with cells transfected in the
absence of FLAG-IKK2, and transfection with a ConA.LUC plasmid, lacking
the
B motif, failed to show differences in the transcription of the
reporter gene (data not shown). To evaluate the possibility of a direct
interaction between kaurenes and the proteins present in the NF-
B
complex, an in vitro assay was performed by incubating
nuclear protein extracts from LPS-activated cells with the diterpenes,
followed by the addition of the
B DNA probe. As Fig.
4A shows, kaurenes did not
influence the formation of the NF-
B-DNA complexes, using the
B
motif of the NOS-2 promoter. As a positive control, nuclear extracts
were treated with the cyclopentenone PG 15dPGJ2, and a
dose-dependent inhibition of the binding of NF-
B to the
DNA motif was observed (21, 23, 27). Moreover, the characteristic degradation of I
B
dependent on LPS activation was impaired by kaurenes (Fig. 4B). Because phosphorylation of I
B
in
Ser32 and Ser36 was required for
targeting of the complex, we inhibited the degradation of I
B
by
the proteasome by incubating the cells with MG-132 and then analyzed
the extent of the phosphorylation by using a specific
anti-phospho-Ser32-I
B
Ab. As shown in Fig.
4C, the phosphorylation of I
B
was inhibited when cells
were treated with kaurenes or 15dPGJ2, which indicates that
IKK activity was impaired in LPS-activated cells treated under these
conditions.

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Fig. 3.
Inhibition of NF- B
activation by kaurene diterpenes. J774 cells were treated as
described in the legend to Fig. 2, and 30 or 60 min after LPS challenge
cell extracts were prepared (the numbering of each terpene is the same
as used in Fig. 1). The binding of nuclear proteins to the B motif
of the NOS-2 promoter was analyzed by electrophoretic mobility shift
assay. The binding of nuclear proteins to peroxysomal
proliferator-activated receptor- (PPAR- ) was used to ensure equal
loading of the lanes (A). The densitometry corresponding to
the band b (p50.p65 complexes, as determined by supershift
assays) was plotted (B). Alternatively, cells were
transfected with a ( B)3ConA.LUC plasmid and a FLAG-IKK2
expression vector and stimulated for 24 h with 20 µM
diterpene and 500 ng/ml LPS. Cell extracts were prepared, and
luciferase activity was measured. Western blot of cytosolic proteins
with anti-FLAG Ab was carried out to ensure equal efficiencies in the
transfection (C). Results show the mean ± S.E. of four
experiments and a representative blot of four.
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Fig. 4.
Kaurenes fail to inhibit
NF- B activity in vitro.
The ability of kaurenes to influence the binding to a B DNA probe of
nuclear proteins from cells treated for 30 min with 500 ng/ml LPS was
assayed in vitro. As a positive control, 15dPGJ2
was used (A). The cytosolic levels of I B were
determined by Western blot of extracts prepared from cells activated
for 30 min as indicated in the legend to Fig. 3B. The
specific phosphorylation in Ser32 of I B was
determined 15 min after LPS activation by Western blot using a specific
anti-phospho-Ser32-I B antibody and inhibiting its
degradation by the proteasome by treating the cells with 20 µM MG-132 (C). Results show a representative
experiment of three.
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Kaurene Diterpenes Inhibited IKK2 Activity--
To evaluate the
effect of kaurenes on IKK2 activity, the IKK complex was IP from
LPS-activated cells and assayed in vitro with GST-I
B
as substrate. As Fig. 5A
shows, the phosphorylation of I
B
was significantly inhibited
(70-80%, on average) when cells were treated with 25 µM
kaurene diterpenes. The inhibitory effect of these diterpenes on IKK
activity exhibited some degree of specificity, because JNK, which is
rapidly activated in macrophages after treatment with LPS (28, 29), was
unaffected by kaurenes having a total concentration of 50 µM (Fig. 5A). Moreover, using as substrate
GST-I
B
-(1-54) and the corresponding protein with S32A and S36A
mutations, it was concluded that these serine residues were the
specific targets of phosphorylation (Fig. 5B). As described for the inhibitory action of 15dPGJ2 on NF-
B activity,
this prostaglandin also inhibited IKK2 via the formation of Michael
adducts involving cysteine 179 (21). To evaluate the capacity of
kaurenes to directly inhibit IKK2 activity, J774 cells were transfected
with an expression vector encoding FLAG-IKK2, and after activation with
LPS, IKK2 was IP with anti-FLAG Ab. As Fig. 5C shows,
kaurene diterpenes assayed up to 50 µM did not affect the
kinase activity. However, 15dPGJ2 exhibited the expected
inhibitory effect on IKK2 activity. Similar results were obtained when
the endogenous IKK complex was IP with anti-IKK2 Ab and the effect of
kaurenes was assayed in vitro (data not shown). Because
these results suggest that kaurenes inhibit a step preceding IKK
activation, cells were transfected with a Myc-NIK expression vector
that, after overexpression, triggers IKK2 activation per se
(15, 30). Under these conditions, kaurenes inhibited the
Myc-NIK-dependent activation of IKK2, in the absence of LPS
stimulation (Fig. 6A).
Expression of a kinase-deficient NIK (K429A/K430A mutant) abrogated the
LPS-induced IKK2 activity in these cells (data not shown; see below).
Moreover, co-transfection of Myc-NIK and (
B)3ConA.LUC
was sufficient to direct the expression of the reporter gene; under
these conditions, kaurenes significantly inhibited the activation of
NF-
B, whereas clerodanes failed to influence the reporter activity
(Fig. 6B). Moreover, expression of a kinase-deficient IKK2
inhibited (82%) the expression of the NF-
B-dependent
luciferase gene upon LPS challenge, whereas expression of a
kinase-deficient NIK inhibited 62% of the effect. Taken together, these results indicate that kaurenes inhibit NIK activity.

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Fig. 5.
Inhibition of IKK2 activation by kaurene
diterpenes. J774 cells (6-cm dishes) were transfected with 0.5 µg of a FLAG-IKK2 expression vector as indicated in the legend to
Fig. 3. The next day, cells were treated with diterpenes or
15dPGJ2 and activated for 15 min with 500 ng/ml LPS, and
cell extracts were prepared. The IKK complex was IP with anti-FLAG Ab,
and the kinase activity was assayed using the indicated GST-I B
and [ -32P]ATP as substrates. The incorporation of
[32P]phosphate into I B was determined by
SDS-polyacrylamide gel electrophoresis (A and B).
JNK activity was IP and assayed using GST-c-Jun-(1-79) as substrate
(A). The in vitro effect of diterpenes and
15dPGJ2 on IKK2 activity was determined after incubation
for 5 min with the immunoprecipitated enzyme from cells treated with
LPS, followed by assay of the activity using GST-I B as substrate.
A blot with anti-IKK2 Ab was used to ensure that the assays contained
similar amounts of kinase (C). Results show a
representative experiment of three. IP,
immunoprecipitation; WB, Western blot.
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Fig. 6.
Inhibition of NIK by kaurene diterpenes.
Cells were transfected with 0.5 µg of pRK5-Myc-NIK and
( B)3ConA.LUC as indicated in the legend to Fig. 3. The
transfection medium was removed, and cells were treated with 20 µM diterpenes, 5 µM 15dPGJ2, or
vehicle (Me2SO). After 18 h in culture, cell extracts
were prepared, IKK was IP, and the kinase activity was assayed using
GST-I B as substrate. A blot of IKK2 was performed to ensure
similar lane charge (A). The expression of luciferase was
determined enzymatically. The levels of expression of Myc-NIK were
determined by Western blot with anti-Myc Ab (B).
Transfection with ( B)3ConA.LUC and 1 µg of IKK2, NIK,
or the corresponding kinase-deficient forms (KD) followed by
treatment with 500 ng/ml LPS was carried out, and luciferase activity
was measured and expressed as a percentage versus
the NIK condition (C). Results show a representative
experiment of three (A) and the mean ± S.D.
(n = 3) of luciferase activity assayed per duplicate
(B and C). *, p < 0.001 versus the condition in the absence of diterpene or
15dPGJ2. IP, immunoprecipitation;
WB, Western blot; n.s., nonspecific band.
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Various reports suggest that activation of p38 MAPK was required for
the activation of NF-
B in macrophages stimulated with TNF-
and
LPS (31). Therefore, the phosphorylation of p38 and p42/p44 ERK was
also investigated as potential targets to mediate the action of
kaurenes. As Fig. 7A shows,
kaurenes impaired the LPS-dependent phosphorylation of
p42/p44 at 5 min, but the response was recovered significantly at 15 min. In the case of p38, the phosphorylation was delayed for at least
15 min and recovered at 30 min (Fig. 7B).

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Fig. 7.
Delayed phosphorylation of p42, p44, and p38
MAPK in LPS-activated cells treated with kaurene diterpenes.
Macrophages were treated as indicated in the legend to Fig. 2, and the
phosphorylation of p42, p44, and p38 was determined by Western blotting
cytosolic extracts prepared at 5 and 15 min (A) and 30 min
(B) after stimulation and using specific anti-phosphoprotein
Abs. The same blots were reprobed with Abs against the full p42, p44,
and p38 proteins. Results show a representative experiment of
three.
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The Release of TNF-
by LPS-activated Cells Was Inhibited by
Kaurene Diterpenes--
The aforementioned results show an inhibition
of early pro-inflammatory signaling by kaurenes. To evaluate the effect
of these diterpenes on the release of inflammatory cytokines that
contribute to sustained activation of the macrophage, the levels of
TNF-
were determined at 4 and 24 h after LPS challenge. As Fig.
8 shows, the accumulation of TNF-
was
abolished when cells were treated with kaurenes, which indicates that
the inhibition of the early signaling plays an important role in the
commitment of macrophage activation, which cannot be recovered at later
times.

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Fig. 8.
Kaurene diterpenes inhibit the release of
TNF- by activated macrophages. Cells were
treated for 4 and 24 h as described in the legend to Fig. 2, and
the accumulation of TNF- in the culture medium was determined by
enzyme immunoassay. Internal standards of TNF- in the
culture medium containing diterpenes were performed to ensure
that these compounds did not affect the enzyme immunoassay. Results
show the mean ± S.E. of three experiments assayed per
duplicate.
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Diterpenes Did Not Affect the Percentage of CD14-positive
Cells--
Because the effect of kaurene diterpenes appears to be
mediated by inhibition of very early steps after LPS challenge, we evaluated the capacity of these molecules to modify the exposure of
CD14 and CD11b on the macrophage cell surface as markers of differentiation. As Fig. 9A
shows, kaurenes did not affect the percentage of CD14-positive cells
but significantly reduced the number of CD11b-positive cells. In
addition to this, kaurenes dose-dependently increased the
average mean channel fluorescence of CD14-positive cells, at the time
that showed decreased mean channel fluorescence of cells
positive for CD11b (Fig. 9B). These data suggest that these
diterpenes exert multiple effects on the differentiation of
macrophages, although the relevance of these changes on the
LPS-dependent signaling requires further work.

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Fig. 9.
Analysis of CD14- and CD11b-positive cells by
flow cytometry. J774 macrophages were incubated for 30 min with
the indicated diterpenes. After treatment for 15 min at 4 °C with
fluorescein isothiocyanate-anti-CD14 and
phycoerythrin-anti-Mac-1 Abs, cells were analyzed in a FACSscan
cytometer. The percentage of positive cells was determined
(A). The mean fluorescence intensity of CD14- and
Mac-1-positive J774 cells determined by flow cytometry was modified
after incubation with diterpenes. The dose-dependent value
of the mean fluorescence was expressed as a percentage with respect to
cells in the absence of diterpenes (B). Results show the
mean ± S.D. of three experiments.
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DISCUSSION |
In this work we have analyzed the effect of ent-kaurene
diterpenes on the activation of NF-
B in LPS-treated J774
macrophages. The action of these compounds was compared with that of
teucrine (compound 3) and related clerodane diterpenes
possessing
/
-unsaturated carbonyl groups that offer the
possibility of reacting with nucleophiles, such as cysteines,
by means of Michael-type addition (9, 20, 32, 33). In this regard, it
has been shown that physiological anti-inflammatory molecules, such as the cyclopentenone prostaglandin 15dPGJ2, efficiently
inhibit IKK activity and the interaction of the p65 subunit of NF-
B
with DNA
B motifs via formation of Michael addition derivatives on key cysteine residues (21, 23, 34); this is in addition to other
effects dependent on peroxysomal proliferator-activated receptor-
engagement (35, 36). However, in all the experiments performed in this
work the clerodanes were unable to modify the inflammatory response to
LPS, suggesting that these molecules cannot access the cysteines
relevant for the biological function of key proteins.
Our data show that kaurene diterpenes inhibit NF-
B activation
through a mechanism that involves an impairment of IKK activity, without any direct effect on the binding of the NF-
B complex to the
B motif or on the phosphorylation and targeting of the IKK substrate
I
B
. Interestingly, the activity of the IKK complex, which
constitutes a common point for the regulation of the pathway (7, 37,
38), was not inhibited after incubation with kaurenes, indicating the
presence of targets upstream of this step (39). Indeed, activation of
IKK integrates the signaling pathway triggered by LPS in monocytes (28,
39) and by CD28 and CD40 engagement in lymphocytes (40, 41). In the
same vein, most of the work on the anti-inflammatory action of
flavonoids, terpenes, polyphenols, salicylates, and other natural
products has shown that these compounds exert their effects by
inhibiting IKK/NF-
B activation (42-46).
The mechanism by which IKK activity is impaired by terpenes and
flavones has been poorly characterized. The effect on steps upstream of
IKK of the diterpenes used in this work was analyzed, taking advantage
of the fact that transient expression of NIK was sufficient for the
activation of IKK and therefore of NF-
B (15, 30, 47). Under these
conditions, kaurenes persistently inhibited ~50% of NF-
B activity
as deduced from cells co-transfected with NIK and
(
B)3ConA.Luc plasmids, although IKK activity was more inhibited by kaurenes (>75%). Because the effect of kaurenes on
NF-
B activity was more potent in intact cells treated with LPS than
after NIK overexpression, it might be suggested that other pathways
activated in response to LPS (in particular members of the MAPK
family), also affected by these diterpenes, participate in the overall
activation of NF-
B (48-50). NIK can be considered as a member of
the MAPK kinase superfamily and, indeed, it was originally
described as a MAP3K-related kinase (15). Both NIK and MEK kinase 1, a
MAPK kinase, act as shuttle kinases relaying the signaling from
the membrane to the high molecular weight IKK complex (30, 51). NIK and
MEK kinase 1 can phosphorylate IKK1 and IKK2 with some specificity;
whereas MEK kinase 1 preferentially phosphorylates IKK2, NIK can act
over both IKKs (30, 49, 52). According to this mechanism of
activation, it has been proposed that depending on the MAPK kinase
present in the IKK complex, the cell specifically responds to
inflammatory (NIK) or mitogenic stimulation (MEK kinase 1). In the
context of inflammation, the inhibition of NIK by kaurenes allows a
deactivation of the signaling mediated through Traf2 and Traf6,
the adapter molecules that activate NF-
B via NIK, in response to
stimuli using the p75 TNF-
receptor and the Il-1
receptor,
respectively (15, 53), suggesting their action as general
anti-inflammatory molecules.
In addition to the effect of kaurenes on NIK, the activation by LPS of
p38 and ERK1 and ERK2 MAPKs, but not that of JNK, was notably
delayed in cells treated with these diterpenes, although the activity
recovered at later periods of time. These results suggest that the loss
of efficiency in the signaling coming from the MAPK pathway might
contribute, at least in part, to the impairment of IKK activity.
Indeed, the coordinate activation of p38 and ERK appears to be critical
for the inflammatory response and for the activation of NF-
B
(2, 50, 54, 55). For example, LPS promotes TNF-
expression through
the Ras/Raf-1/MEK/MAPK pathway in macrophages, and constitutively
active forms of Raf-1 increase notably, although they cannot replace,
the requirements of LPS (31, 48); the selective inhibition of ERK1 and
ERK2 with PD-098059 was sufficient to abolish TNF-
synthesis by
human monocytes in response to LPS (48). In agreement with these data,
kaurenes were very efficient at inhibiting TNF-
secretion by J774
cells activated with LPS, at least for 24 h, which reflects the
persistent inhibition of the pathway. Taken together, these results
suggest that the lack of ERK and p38 phosphorylation at appropriate
times notably reduces the engagement of subsequent signaling pathways involved in the full activation of NF-
B in response to LPS.
The ability of diterpenes to increase the exposure of CD14 was
unexpected. Indeed, one possibility for their mechanism of action was a
direct interaction with the membrane receptor, but this appears not to
be the case, in view of the absence of alteration of JNK activation
(28). Interestingly, the exposure of CD11b, which was used as a marker
of differentiation and as a control for the studies on CD14 modulation,
exhibited an opposite behavior, with a reduction in the binding when
the concentration of the diterpenes increased. Interestingly, aspirin
and heparin have been described as molecules that decrease the exposure
of CD11b and other adhesion molecules in monocyte/macrophages,
involving the inhibition of ERK (42, 56).
Many terpenes, including diterpenes, triterpenes, and sesquiterpenes
have proved to possess anti-inflammatory activity both in
vivo and in vitro, and most of them inhibited NF-
B
activity, although the precise mechanisms of action have not been fully characterized. In the case of kaurenes we have identified relevant targets for this inhibition, showing that the inhibition of NIK and the
lack of a coordinate activation of p38 and/or ERK1 and ERK2 lead to the
abrogation of the inflammatory response, in particular, of genes
depending on NF-
B activation. The fact that kaurenes are
intermediates in the biosynthesis of plant hormones, such as
gibberellins, offers the possibility of envisaging further analysis of
the interaction of these molecules in several aspects of mammalian cell biology.