Inhibition of the Nuclear Factor kappa B (NF-kappa B) Pathway by Tetracyclic Kaurene Diterpenes in Macrophages

SPECIFIC EFFECTS ON NF-kappa B-INDUCING KINASE ACTIVITY AND ON THE COORDINATE ACTIVATION OF ERK AND p38 MAPK*

Antonio CastrilloDagger , Beatriz de las Heras§, Sonsoles HortelanoDagger , Benjamín Rodríguez, Angel Villar§, and Lisardo BoscáDagger ||

From the Dagger  Instituto de Bioquímica (Centro Mixto Consejo Superior de Investigaciones Científicas-Universidad Complutense de Madrid) and § Departamento de Farmacología, Facultad de Farmacia, Universidad Complutense, 28040 Madrid and the  Centro de Química Orgánica "M. Lora-Tamayo," Juan de la Cierva 3, 28006 Madrid, Spain

Received for publication, January 2, 2001, and in revised form, February 1, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The anti-inflammatory action of most terpenes has been explained in terms of the inhibition of nuclear factor kappa B (NF-kappa B) activity. Ent-kaurene diterpenes are intermediates of the synthesis of gibberellins and inhibit the expression of NO synthase-2 and the release of tumor necrosis factor-alpha in J774 macrophages challenged with lipopolysaccharide. These diterpenes inhibit NF-kappa B and Ikappa B kinase (IKK) activation in vivo but failed to affect in vitro the function of NF-kappa B, the phosphorylation and targeting of Ikappa Balpha , and the activity of IKK-2. Transient expression of NF-kappa B-inducing kinase (NIK) activated the IKK complex and NF-kappa B, a process that was inhibited by kaurenes, indicating that the inhibition of NIK was one of the targets of these diterpenes. These results show that kaurenes impair the inflammatory signaling by inhibiting NIK, a member of the MAPK kinase superfamily that interacts with tumor necrosis factor receptor-associated factors, and mediate the activation of NF-kappa B by these receptors. Moreover, kaurenes delayed the phosphorylation of p38, ERK1, and ERK2 MAPKs, but not that of JNK, in response to lipopolysaccharide treatment of J774 cells. The absence of a coordinate activation of MAPK and IKK might contribute to a deficient activation of NF-kappa B that is involved in the anti-inflammatory activity of these molecules.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Activation of the transcription factor NF-kappa 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-kappa B is present constitutively in the cytosol of the cells, where it is retained through the interaction with inhibitory Ikappa B proteins that mask the nuclear localization domain of the complex (2). NF-kappa B-dependent gene transcription requires the phosphorylation of Ikappa Balpha 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-Ikappa B by the proteasome (3, 7).

Biochemical, pharmacological, and genetic data indicate that the control of NF-kappa 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-kappa 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-kappa 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-kappa B activation (8, 9). Triterpenes also inhibited NOS-2 expression in RAW 264.7 cells (IC50, 0.2-0.3 µM) and impaired NF-kappa B activity (10). In the case of sesquiterpenes, it has been described that some of these compounds induce HSP72, which in turn prevents NF-kappa 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-kappa 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-kappa B activities by cyclopentenone prostaglandins (20, 21). Our data show that molecules based on the kaurene structure potently inhibit NF-kappa 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-kappa B.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


View larger version (30K):
[in this window]
[in a new window]
 
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 right-arrow 12 or 20 right-arrow 12 lactone group (compounds 4-6).

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 (kappa B)3ConA.LUC plasmid containing three copies of the kappa 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 kappa B activation (23, 24). The ConA.LUC vector, lacking the kappa 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-Ikappa Balpha -(1-317), and GST-Ikappa Balpha -(1-54), wild type and mutant (S32A and S36A), were expressed in DH5alpha 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 kappa 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 [alpha -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 kappa 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-gamma 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-Ikappa Balpha , anti-IKK2, and anti-beta -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-Ikappa Balpha Ab, the blot incubation solution contained 50 ng/ml GST-Ikappa Balpha -(1-317) treated previously with alkaline phosphatase-agarose (23). The blots were submitted to sequential reprobing with Abs after treatment with 100 mM beta -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-alpha Secretion-- The accumulation of TNF-alpha 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 [gamma -32P]ATP (0.5 µCi) and using as substrate 100 ng of GST-Ikappa Balpha 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-Ikappa Balpha Ab, except that 1 mM MgATP was used instead of [gamma -32P]ATP. GST-Ikappa Balpha 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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


View larger version (28K):
[in this window]
[in a new window]
 
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 beta -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).

Inhibition of NF-kappa B Activity by Kaurene Diterpenes-- To investigate the mechanism of action of kaurenes on macrophage function, the effects on NF-kappa B activity, as a key step in the inflammatory onset, were analyzed. Treatment of macrophages with kaurenes impaired the activation of NF-kappa 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 kappa 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 kappa 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-kappa 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 kappa B DNA probe. As Fig. 4A shows, kaurenes did not influence the formation of the NF-kappa B-DNA complexes, using the kappa 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-kappa B to the DNA motif was observed (21, 23, 27). Moreover, the characteristic degradation of Ikappa Balpha dependent on LPS activation was impaired by kaurenes (Fig. 4B). Because phosphorylation of Ikappa Balpha in Ser32 and Ser36 was required for targeting of the complex, we inhibited the degradation of Ikappa Balpha 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-Ikappa Balpha Ab. As shown in Fig. 4C, the phosphorylation of Ikappa Balpha 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.


View larger version (47K):
[in this window]
[in a new window]
 
Fig. 3.   Inhibition of NF-kappa 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 kappa B motif of the NOS-2 promoter was analyzed by electrophoretic mobility shift assay. The binding of nuclear proteins to peroxysomal proliferator-activated receptor-gamma (PPAR-gamma ) 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 (kappa 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.


View larger version (52K):
[in this window]
[in a new window]
 
Fig. 4.   Kaurenes fail to inhibit NF-kappa B activity in vitro. The ability of kaurenes to influence the binding to a kappa 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 Ikappa Balpha 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 Ikappa Balpha was determined 15 min after LPS activation by Western blot using a specific anti-phospho-Ser32-Ikappa Balpha 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.

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-Ikappa Balpha as substrate. As Fig. 5A shows, the phosphorylation of Ikappa Balpha 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-Ikappa Balpha -(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-kappa 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 (kappa B)3ConA.LUC was sufficient to direct the expression of the reporter gene; under these conditions, kaurenes significantly inhibited the activation of NF-kappa 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-kappa 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.


View larger version (60K):
[in this window]
[in a new window]
 
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-Ikappa Balpha and [gamma -32P]ATP as substrates. The incorporation of [32P]phosphate into Ikappa Balpha 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-Ikappa Balpha 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.


View larger version (45K):
[in this window]
[in a new window]
 
Fig. 6.   Inhibition of NIK by kaurene diterpenes. Cells were transfected with 0.5 µg of pRK5-Myc-NIK and (kappa 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-Ikappa Balpha 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 (kappa 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.

Various reports suggest that activation of p38 MAPK was required for the activation of NF-kappa B in macrophages stimulated with TNF-alpha 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).


View larger version (58K):
[in this window]
[in a new window]
 
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.

The Release of TNF-alpha 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-alpha were determined at 4 and 24 h after LPS challenge. As Fig. 8 shows, the accumulation of TNF-alpha 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.


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 8.   Kaurene diterpenes inhibit the release of TNF-alpha by activated macrophages. Cells were treated for 4 and 24 h as described in the legend to Fig. 2, and the accumulation of TNF-alpha in the culture medium was determined by enzyme immunoassay. Internal standards of TNF-alpha 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.

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.


View larger version (27K):
[in this window]
[in a new window]
 
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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this work we have analyzed the effect of ent-kaurene diterpenes on the activation of NF-kappa B in LPS-treated J774 macrophages. The action of these compounds was compared with that of teucrine (compound 3) and related clerodane diterpenes possessing alpha /beta -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-kappa B with DNA kappa 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-gamma 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-kappa B activation through a mechanism that involves an impairment of IKK activity, without any direct effect on the binding of the NF-kappa B complex to the kappa B motif or on the phosphorylation and targeting of the IKK substrate Ikappa Balpha . 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-kappa 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-kappa B (15, 30, 47). Under these conditions, kaurenes persistently inhibited ~50% of NF-kappa B activity as deduced from cells co-transfected with NIK and (kappa B)3ConA.Luc plasmids, although IKK activity was more inhibited by kaurenes (>75%). Because the effect of kaurenes on NF-kappa 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-kappa 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-kappa B via NIK, in response to stimuli using the p75 TNF-alpha receptor and the Il-1beta 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-kappa B (2, 50, 54, 55). For example, LPS promotes TNF-alpha 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-alpha synthesis by human monocytes in response to LPS (48). In agreement with these data, kaurenes were very efficient at inhibiting TNF-alpha 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-kappa 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-kappa 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-kappa 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.

    ACKNOWLEDGEMENTS

We thank Dr. W. C. Greene and P. Wallach for providing us with critical plasmids. We thank E. Lundin for help in the preparation of the manuscript.

    FOOTNOTES

* This work was supported by Grants PM98-0120 and 2FD97-1432 from Comisión Interministerial de Ciencia y Tecnología (Spain).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Instituto de Bioquímica, Facultad de Farmacia, 28040 Madrid, Spain. Fax: 3491 544 7254 and 3491 394 1782; E-mail: boscal@eucmax.sim.ucm.es.

Published, JBC Papers in Press, February 9, 2001, DOI 10.1074/jbc.M100010200

    ABBREVIATIONS

The abbreviations used are: NF-kappa B, nuclear factor kappa B; NOS-2, NO synthase-2; IKK, Ikappa B kinase; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase; LPS, lipopolysaccharide; NIK, NF-kappa B-inducing kinase; GST, glutathione S-transferase; TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone; Ab, antibody; 15dPGJ2, 15-deoxy-Delta 12,14-prostaglandin J2; TNF-alpha , tumor necrosis factor-alpha ; IP, immunoprecipitated; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Hoffmann, J. A., Kafatos, F. C., Janeway, C. A., and Ezekowitz, R. A. (1999) Science 284, 1313-1318[Abstract/Free Full Text]
2. Baeuerle, P. A. (1998) Cell 95, 729-731[Medline] [Order article via Infotrieve]
3. Ghosh, S., May, M. J., and Kopp, E. B. (1998) Annu. Rev. Immunol. 16, 225-260[CrossRef][Medline] [Order article via Infotrieve]
4. MacMicking, J., Xie, Q. W., and Nathan, C. (1997) Annu. Rev. Immunol. 15, 323-350[CrossRef][Medline] [Order article via Infotrieve]
5. Li, J. J., Westergaard, C., Ghosh, P., and Colburn, N. H. (1997) Cancer Res. 57, 3569-3576[Abstract]
6. Mohan, R., Rinehart, W. B., Bargagna-Mohan, P., and Fini, M. E. (1998) J. Biol. Chem. 273, 25903-25914[Abstract/Free Full Text]
7. Karin, M. (1999) J. Biol. Chem. 274, 27339-27342[Free Full Text]
8. de las Heras, B., Navarro, A., Diaz-Guerra, M. J., Bermejo, P., Castrillo, A., Bosca, L., and Villar, A. (1999) Br. J. Pharmacol. 128, 605-612[Abstract/Free Full Text]
9. Navarro, A., de las Heras, B., and Villar, A. M. (1997) Z. Naturforsch. Sect. C J. Biosci. 52, 844-849
10. Dirsch, V. M., Kiemer, A. K., Wagner, H., and Vollmar, A. M. (1997) Eur. J. Pharmacol. 336, 211-217[CrossRef][Medline] [Order article via Infotrieve]
11. Matsuda, H., Kagerura, T., Toguchida, I., Ueda, H., Morikawa, T., and Yoshikawa, M. (2000) Life Sci. 66, 2151-2157[CrossRef][Medline] [Order article via Infotrieve]
12. Matsuda, H., Kagerurd, T., Toguchida, I., Murakami, T., Kishi, A., and Yoshikawa, M. (1999) Bioorg. Med. Chem. Lett. 9, 3081-3086[CrossRef][Medline] [Order article via Infotrieve]
13. Tudzynski, B. (1999) Appl. Microbiol. Biotechnol. 52, 298-310[CrossRef][Medline] [Order article via Infotrieve]
14. Yamaguchi, S., and Kamiya, Y. (2000) Plant Cell Physiol. 41, 251-257[Medline] [Order article via Infotrieve]
15. Malinin, N. L., Boldin, M. P., Kovalenko, A. V., and Wallach, D. (1997) Nature 385, 540-544[CrossRef][Medline] [Order article via Infotrieve]
16. Hou, A. J., Li, M. L., Jiang, B., Lin, Z. W., Ji, S. Y., Zhou, Y. P., and Sun, H. D. (2000) J. Nat. Prod. (Lloydia) 63, 599-601[CrossRef][Medline] [Order article via Infotrieve]
17. Jiang, B., Lu, Z. Q., Hou, A. J., Zhao, Q. S., and Sun, H. D. (1999) J. Nat. Prod. (Lloydia) 62, 941-945[CrossRef][Medline] [Order article via Infotrieve]
18. Batista, R., Chiari, E., and de Oliveira, A. B. (1999) Planta Med. 65, 283-284[Medline] [Order article via Infotrieve]
19. Chen, K., Shi, Q., Fujioka, T., Nakano, T., Hu, C. Q., Jin, J. Q., Kilkuskie, R. E., and Lee, K. H. (1995) Bioorg. Med. Chem. 3, 1345-1348[CrossRef][Medline] [Order article via Infotrieve]
20. Negishi, M., Koizumi, T., and Ichikawa, A. (1995) J. Lipid Mediat. Cell Signal. 12, 443-448[CrossRef][Medline] [Order article via Infotrieve]
21. Rossi, A., Kapahi, P., Natoli, G., Takahashi, T., Chen, Y., Karin, M., and Santoro, M. G. (2000) Nature 403, 103-108[CrossRef][Medline] [Order article via Infotrieve]
22. de Quesada, T. G., Rodríguez, B., and Valverde, S. (1972) Tetrahedron Lett. 2187-2190
23. Castrillo, A., Diaz-Guerra, M. J., Hortelano, S., Martin-Sanz, P., and Bosca, L. (2000) Mol. Cell. Biol. 20, 1692-1698[Abstract/Free Full Text]
24. Diaz-Guerra, M. J. M., Velasco, M., Martin-Sanz, P., and Bosca, L. (1996) J. Biol. Chem. 271, 30114-30120[Abstract/Free Full Text]
25. Schreiber, E., Matthias, P., Muller, M. M., and Schaffner, W. (1989) Nucleic Acids Res. 17, 6419[Medline] [Order article via Infotrieve]
26. Xie, Q. W., Whisnant, R., and Nathan, C. (1993) J. Exp. Med. 177, 1779-1784[Abstract]
27. Straus, D. S., Pascual, G., Li, M., Welch, J. S., Ricote, M., Hsiang, C. H., Sengchanthalangsy, L. L., Ghosh, G., and Glass, C. K. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4844-4849[Abstract/Free Full Text]
28. Hambleton, J., Weinstein, S. L., Lem, L., and DeFranco, A. L. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2774-2778[Abstract/Free Full Text]
29. Procyk, K. J., Rippo, M. R., Testi, R., Hofmann, F., Parker, P. J., and Baccarini, M. (2000) Blood 96, 2592-2598[Abstract/Free Full Text]
30. Nemoto, S., DiDonato, J. A., and Lin, A. (1998) Mol. Cell. Biol. 18, 7336-7343[Abstract/Free Full Text]
31. Geppert, T. D., Whitehurst, C. E., Thompson, P., and Beutler, B. (1994) Mol. Med. 1, 93-103[Medline] [Order article via Infotrieve]
32. Chen, Y., Morrow, J. D., and Roberts, L. J. (1999) J. Biol. Chem. 274, 10863-10868[Abstract/Free Full Text]
33. Lee, I. S., Shamon, L. A., Chai, H. B., Chagwedera, T. E., Besterman, J. M., Farnsworth, N. R., Cordell, G. A., Pezzuto, J. M., and Kinghorn, A. D. (1996) Chem. Biol. Interact. 99, 193-204[CrossRef][Medline] [Order article via Infotrieve]
34. Petrova, T. V., Akama, K. T., and Van Eldik, L. J. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4668-4673[Abstract/Free Full Text]
35. Jiang, C., Ting, A. T., and Seed, B. (1998) Nature 391, 82-86[CrossRef][Medline] [Order article via Infotrieve]
36. Ricote, M., Huang, J. T., Welch, J. S., and Glass, C. K. (1999) J. Leukocyte Biol. 66, 733-739[Abstract]
37. Delhase, M., Hayakawa, M., Chen, Y., and Karin, M. (1999) Science 284, 309-313[Abstract/Free Full Text]
38. Zandi, E., and Karin, M. (1999) Mol. Cell. Biol. 19, 4547-4551[Free Full Text]
39. O'Connell, M. A., Bennett, B. L., Mercurio, F., Manning, A. M., and Mackman, N. (1998) J. Biol. Chem. 273, 30410-30414[Abstract/Free Full Text]
40. Harhaj, E. W., and Sun, S. C. (1998) J. Biol. Chem. 273, 25185-25190[Abstract/Free Full Text]
41. Kosaka, Y., Calderhead, D. M., Manning, E. M., Hambor, J. E., Black, A., Geleziunas, R., Marcu, K. B., and Noelle, R. J. (1999) Eur. J. Immunol. 29, 1353-1362[CrossRef][Medline] [Order article via Infotrieve]
42. Pillinger, M. H., Capodici, C., Rosenthal, P., Kheterpal, N., Hanft, S., Philips, M. R., and Weissmann, G. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 14540-14545[Abstract/Free Full Text]
43. Natarajan, K., Singh, S., Burke, T. R. J., Grunberger, D., and Aggarwal, B. B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9090-9095[Abstract/Free Full Text]
44. Manna, S. K., Sah, N. K., Newman, R. A., Cisneros, A., and Aggarwal, B. B. (2000) Cancer Res. 60, 3838-3847[Abstract/Free Full Text]
45. Hehner, S. P., Hofmann, T. G., Droge, W., and Schmitz, M. L. (1999) J. Immunol. 163, 5617-5623[Abstract/Free Full Text]
46. Yin, M. J., Yamamoto, Y., and Gaynor, R. B. (1998) Nature 396, 77-80[CrossRef][Medline] [Order article via Infotrieve]
47. Nakano, H., Shindo, M., Sakon, S., Nishinaka, S., Mihara, M., Yagita, H., and Okumura, K. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3537-3542[Abstract/Free Full Text]
48. van der Bruggen, T., Nijenhuis, S., van Raaij, E., Verhoef, J., and van Asbeck, B. S. (1999) Infect. Immun. 67, 3824-3829[Abstract/Free Full Text]
49. Lee, F. S., Peters, R. T., Dang, L. C., and Maniatis, T. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9319-9324[Abstract/Free Full Text]
50. Zhao, Q., and Lee, F. S. (1999) J. Biol. Chem. 274, 8355-8358[Abstract/Free Full Text]
51. Baumann, B., Weber, C. K., Troppmair, J., Whiteside, S., Israel, A., Rapp, U. R., and Wirth, T. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4615-4620[Abstract/Free Full Text]
52. Ling, L., Cao, Z., and Goeddel, D. V. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3792-3797[Abstract/Free Full Text]
53. Baud, V., Liu, Z. G., Bennett, B., Suzuki, N., Xia, Y., and Karin, M. (1999) Genes Dev. 13, 1297-1308[Abstract/Free Full Text]
54. Carter, A. B., and Hunninghake, G. W. (2000) J. Biol. Chem. 275, 27858-27864[Abstract/Free Full Text]
55. Lee, Y. W., Han, S. H., Lee, M., Yang, K. H., Kim, H. M., and Jeon, Y. J. (2000) Cancer Lett. 156, 133-139[CrossRef][Medline] [Order article via Infotrieve]
56. Peter, K., Schwarz, M., Conradt, C., Nordt, T., Moser, M., Kubler, W., and Bode, C. (1999) Circulation 100, 1533-1539[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.