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Originally published In Press as doi:10.1074/jbc.M004045200 on September 8, 2000

J. Biol. Chem., Vol. 275, Issue 47, 36703-36707, November 24, 2000
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Induction of Ikappa Balpha Expression as a Mechanism Contributing to the Anti-inflammatory Activities of Peroxisome Proliferator-activated Receptor-alpha Activators*

Philippe DeleriveDagger, Philippe Gervois, Jean-Charles Fruchart, and Bart Staels§

From the INSERM U325, Département d'Athérosclérose, Institut Pasteur de Lille, 1 rue Pr. Calmette 59019 Lille and Faculté de Pharmacie, Université de Lille II, 59000 Lille, France

Received for publication, May 11, 2000, and in revised form, September 6, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Chronic inflammation is a hallmark of degenerative diseases such as atherosclerosis. Peroxisome proliferator-activated receptors (PPARs) are transcription factors belonging to the nuclear receptor superfamily, which are expressed in the cells of the atherosclerosic lesion. PPARalpha ligands have been reported to exert anti-inflammatory activities in different cell types by antagonizing the transcriptional activity of NF-kappa B. In the present study, the influence of PPARalpha activators on the NF-kappa B signaling pathway was investigated. Our results show that fibrates, synthetic PPARalpha activators, induced the expression of the inhibitory protein Ikappa Balpha in human aortic smooth muscle cells as well as in primary human hepatocytes, whereas neither Ikappa B-kinase activity nor the degradation rate of Ikappa Balpha were affected. Using PPARalpha -null mice, we demonstrated that fibrates induced Ikappa Balpha in liver in vivo and that this action required PPARalpha . Furthermore, fibrate treatment induced Ikappa Balpha protein expression in the cytoplasm and also enhanced IL-1beta -induced accumulation of Ikappa Balpha protein in the nucleus. These actions of fibrates on Ikappa Balpha expression were accompanied by a decrease in NF-kappa B DNA binding activity as demonstrated by electrophoretic mobility shift assays. Taken together, these data provide an additional molecular mechanism for the anti-inflammatory activity of PPARalpha agonists and reinforce their potential use in the treatment of inflammatory diseases.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The NF-kappa B family of transcription factors plays a major role in the regulation of the expression of a number of genes implicated in cell growth, inflammation, and apoptosis (1, 2). This NF-kappa B/Rel family consists of five members, c-Rel, p65, Rel B, p50, and p52, which form heterodimeric complexes that are most frequently composed of p50 and p65 proteins. In most non-activated cells, NF-kappa B remains in a cytoplasmic inactive complex through its association with the inhibitory proteins Ikappa Bs (3). Inducers of NF-kappa B, which include inflammatory cytokines, reactive oxygen species, and viral products, activate a dimeric Ikappa B kinase (IKK)1 complex (4-6), which phosphorylates Ikappa Balpha on Ser-32 and Ser-36 leading to subsequent ubiquitination and degradation of Ikappa Balpha and release of NF-kappa B proteins (1, 2). Free NF-kappa B dimers translocate to the nucleus where they regulate target gene transcription. NF-kappa B has been suggested to play a crucial role in the pathogenesis of atherosclerosis (7). NF-kappa B was reported to be essential for the proliferation of vascular smooth muscle cells (SMC) (8), and activated NF-kappa B heterodimers are detected in human atherosclerotic lesions (9).

Peroxisome proliferator-activated receptors (PPARs), transcription factors belonging to the nuclear receptor superfamily, have also been reported to be expressed in vascular cells in vitro and in vivo (10-19). To date, three different PPAR subtypes have been identified: PPARalpha , PPARbeta /delta , and PPARgamma . PPARs regulate gene expression by binding with the retinoid receptor RXR as a heterodimeric partner to specific DNA sequence elements termed PPAR response elements (PPRE) (20). In addition to regulating gene transcription via PPREs, PPARs have recently been shown to modulate gene transcription also by negatively interfering with other transcription factor pathways in a DNA binding-independent manner (18, 21). Among the three different PPARs, PPARalpha activation has been shown to repress cytokine-induced activation of a number of inflammatory genes such as VCAM-1, COX-2, and IL-6 by negatively interfering with NF-kappa B transcriptional activity (14, 22).

However, the influence of PPARalpha activators on the different components of the NF-kappa B signaling cascade have not yet been explored. In the present study, we show that fibrates, synthetic PPARalpha ligands, induce Ikappa Balpha expression in a PPARalpha -dependent manner. By contrast, PPARalpha agonists do not influence Ikappa Balpha degradation nor IKK activity. This induction results in an inhibition of NF-kappa B DNA binding leading to a sharp reduction of the p65-mediated gene activation. These actions may contribute to the anti-inflammatory activities of PPARalpha ligands.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Cell Culture and Chemical Reagents-- Human aortic SMC (Cascade Biologics, Portland, OR) were cultured in SMC basal medium containing 5% SMC growth supplement (Cascade Biologics). Cells from passages 5-8 were used for the experiments. Human primary hepatocytes were prepared as described previously (23). Wy-14643 was from Chemsyn, Lenexa, KS; fenofibric acid from Laboratoires Fournier, Dijon, France, IL-1beta from Genzyme, Cambridge, MD; and ciprofibrate from Sigma, Saint Quentin, France.

IKK Assays-- IKK assays were performed as described previously (24). Briefly, total protein extracts were subjected to immunoprecipitation with anti-NEMO antibody (a kind gift of Dr Israël, Institut Pasteur, France) in TNT buffer (200 mM NaCl, Tris 20 mM, pH 7.5, 1% Triton X-100) and collected on protein A-Sepharose beads. The beads were then washed three times with TNT buffer and three times with kinase buffer (20 mM HEPES, 10 mM MgCl2, 100 µM Na3VO4, 20 mM beta -glycerophosphate, 2 mM dithiothreitol, 50 mM NaCl, pH 7.5). Kinase reactions were performed for 30 min at 30 °C using 5 µCi of [gamma -32P]ATP and GST·Ikappa Balpha -(1-72) as substrate. The reaction products were analyzed on 10% SDS-polyacrylamide gels and detected by autoradiography.

RNA Analysis-- RNA preparation and Northern blot hybridizations were performed as described previously (25). Human IL-6, Ikappa Balpha (26), and 36B4 cDNA fragments were used as probes.

Western Blot Analysis-- Protein extracts were fractionated on 10% polyacrylamide gels under reducing conditions (sample buffer containing 10 mM dithiothreitol, transferred onto nitrocellulose membranes, and probed with various antibodies as stated in figure legends). After incubation with a secondary peroxidase-conjugated antibody, signals were visualized by chemiluminescence (Amersham Pharmacia Biotech).

Nuclear Extracts and Electrophoretic Mobility Shift Assay (EMSA)-- SMC cells (3 × 106) were cultured for 2 or 24 h in SMC medium with Wy-14643 (100 µM) or vehicle (Me2SO, 0.1%) and were subsequently treated with IL-1beta (10 ng/ml) for 1 h. Cells were then harvested, and nuclear extracts were obtained as described previously (27). Nuclear proteins were quantified using the bicinchonic acid assay and stored at -80 °C. For EMSA, an NF-kappa B and an OCT1 double-stranded oligonucleotide (Promega) were end-labeled with [gamma -32P]ATP using T4 polynucleotide kinase according to standard protocols. 5 µg of nuclear extracts from SMC cells were incubated with 50,000 cpm of labeled probes for 20 min at room temperature in 20 µl of buffer containing 10 mM Tris pH 7.5, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, 0.3 µg bovine serum albumin, and 2 µg of poly(dIdC). The reactions were analyzed by electrophoresis in a non-denaturing 5% polyacrylamide gel in 0.5× Tris-borate-EDTA buffer. The gels were then dried and exposed at -80 °C for autoradiography.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Recently, it was demonstrated that PPARalpha activators inhibit IL-1beta -induced IL-6 secretion by human aortic SMC (HASMC) in a dose-dependent manner (14). Furthermore, PPARalpha activators negatively regulate IL-1beta -induced-IL-6 production at the gene expression level by inhibiting NF-kappa B transcriptional activity (21). Activation of the transcription factor NF-kappa B is controlled by a cytokine-activated protein kinase complex, which phosphorylates Ikappa Balpha , triggering its polyubiquitination (5). Because PPARalpha agonists negatively interfere with IL-1beta -induced NF-kappa B transcriptional activity in HASMC, we investigated whether PPARalpha activators influence IL-1beta -induced IKK activity in these cells. IKK activity was very low in non-stimulated HASMC treated with or without the PPARalpha ligand Wy-14643 (Fig. 1A). Treatment with IL-1beta for 10 min resulted in a strong induction of IKK activity. This induction was not affected by preincubation with the PPARalpha activator Wy-14643 for 2 h, the time period previously reported to inhibit IL-1beta -induced COX-2 gene expression (14). This result indicates that PPARalpha activators do not regulate NF-kappa B transcriptional activity by modulating IKK function. Furthermore, we investigated the influence of Wy-14643 on IL-1beta -induced-Ikappa Balpha degradation. Western blot analysis showed that Ikappa Balpha protein was degraded within 30 min after IL-1beta stimulation, followed by reappearance of the protein at 1 h as previously reported (28) (Fig. 1B). Fibrate treatment did not affect Ikappa Balpha protein degradation, consistent with the absence of any effect on IKK. Taken together, these data indicate that fibrates do not impair NF-kappa B transcriptional activation by modulating IKK activity or Ikappa Balpha degradation.


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  		Fig. 1.   PPARalpha activators do not influence Ikappa Balpha degradation. A, human aortic SMC (80% confluence) were incubated for 2 h in standard medium with Wy-14643 (100 µM) or vehicle (Me2SO, 0.1%) and were subsequently stimulated with IL-1beta (10 ng/ml) for different time periods (min). Cytoplasmic extracts were prepared and subjected to IKK assay as described previously (46). B, human aortic SMC (80% confluence) were incubated for 2 h in standard medium with Wy-14643 (100 µM) or vehicle (Me2SO, 0.1%) and were subsequently stimulated with IL-1beta (10 ng/ml) for different time periods (min). Total protein extracts were prepared and proteins (50 µg) were separated by SDS-PAGE, transferred to a Hybond membrane, and probed with antibodies to Ikappa Balpha and beta -actin (Santa Cruz Biotechnology).

Next, it was investigated whether PPARalpha agonists influence Ikappa Balpha expression. Incubation of HASMC with IL-1beta resulted in the induction of both IL-6 and Ikappa Balpha mRNA (Fig. 2A), which is consistent with the previous demonstration that NF-kappa B controls the expression of Ikappa Balpha by an inducible autoregulatory loop (28). Wy-14643 alone induced Ikappa Balpha mRNA (Fig. 2A), and this effect was further enhanced by IL-1beta suggesting that PPARalpha and IL-1beta regulate Ikappa Balpha gene expression by different mechanisms. By contrast, Wy-14643 treatment inhibited the induction of IL-6 mRNA levels by IL-1beta as described previously (21). Similarly, when incubations were done with fenofibric acid, another PPARalpha ligand, Ikappa Balpha mRNA was significantly induced (Fig. 1B). This induction occurred rapidly with a maximum reached after 1 h. Ikappa Balpha mRNA levels declined thereafter but remained elevated to increase again at 24 h (Fig. 2B). Moreover, Wy-14643 increased Ikappa Balpha mRNA levels in a dose-dependent manner (Fig. 2C). Results from actinomycin D transcription inhibition experiments demonstrated that fibrates induce Ikappa Balpha expression at the transcriptional level (data not shown). In HASMC, Wy-14643 also increased Ikappa Balpha protein with a maximum of induction reached after 24 h of treatment (2.25 ± 0.19-fold; p < 0.05), demonstrating that the induction of Ikappa Balpha gene expression results in increased protein levels (Fig. 2D).


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  		Fig. 2.   PPARalpha activators induce Ikappa Balpha mRNA and protein levels in human aortic SMC. A, human aortic SMC (80% confluence) were incubated for 2 h in standard medium with Wy-14643 (250 µM) or vehicle (Me2SO, 0.1%) and were subsequently stimulated with IL-1beta (10 ng/ml) for 3 h. IL-6, Ikappa Balpha , and 36B4 mRNA were measured by Northern blot analysis. B, SMC were cultured in standard medium with fenofibric acid (100 µM) for different times as indicated. RNA (10 µg) was extracted, separated on a 1% agarose-formaldehyde gel, transferred to a nylon membrane, and analyzed by hybridization to Ikappa Balpha and 36B4 cDNA probes. C, SMC were cultured in standard medium with increasing concentrations of Wy-14643 for 1 h. RNA (10 µg) was extracted, separated on a 1% agarose-formaldehyde gel, transferred to a nylon membrane, and analyzed by hybridization to Ikappa Balpha and 36B4 cDNA probes. D, human aortic SMC were cultured in standard medium with Wy-14643 (100 µM) or vehicle (Me2SO, 0.1%) for different lengths of time (h). Protein extracts were prepared, and 30 µg of each extract were separated by SDS-PAGE, transferred to Hybond membrane, and probed with antibodies to Ikappa Balpha and beta -actin.

To determine whether this induction of Ikappa Balpha expression by fibrates occurs in other PPARalpha - expressing cell types, Ikappa Balpha regulation was next studied in the liver, where PPARalpha is highly expressed (29, 30). In line with previous studies (31), human primary hepatocytes express Ikappa Balpha mRNA under basal conditions (Fig. 3A). Interestingly, treatment with Wy-14643 increased Ikappa Balpha mRNA (approximately 3-fold)(Fig. 3A), indicating that PPARalpha ligands regulate Ikappa Balpha expression also in human primary hepatocytes. Western blot analysis confirmed that Wy-14643 treatment led to Ikappa Balpha protein induction in human primary hepatocytes (Fig. 3C). To determine whether Ikappa Balpha mRNA induction by fibrates in liver is mediated by PPARalpha , further studies were performed in PPARalpha -null mice (32). In livers of PPARalpha wild-type mice, treatment with the PPARalpha agonist ciprofibrate resulted in a significant increase (>4-fold) of Ikappa Balpha mRNA, whereas in PPARalpha -null mice no induction was observed (Fig. 3B). This result indicates that fibrates regulate Ikappa Balpha in liver in a PPARalpha -dependent manner. Because the liver constitutes a major organ implicated in the synthesis of numerous acute phase proteins such as cytokines and hemostatic factors, whose transcription is under the control of NF-kappa B transcription factors (33, 34), we next studied NF-kappa B target gene regulation by fibrates in human primary hepatocytes. Primary human hepatocytes express basal COX-2 protein levels, which were modestly induced by IL-1beta (Fig. 3D). Fibrate treatment dose-dependently inhibited COX-2 expression similarly as described previously in human aortic SMC (14), suggesting a similar role for PPARalpha in the control of the hepatic inflammatory response.


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  		Fig. 3.   Fibrates induce Ikappa Balpha mRNA and protein expression in human primary hepatocytes and in livers of PPARalpha +/+ but not of PPARalpha -/- mice. A, human primary hepatocytes were cultured with standard medium supplemented with Wy-14643 (50 µM) or vehicle (Me2SO, 0.1%) for 24 h. RNA (10 µg) was extracted, separated on a 1% agarose-formaldehyde gel, transferred to a nylon membrane, and analyzed by hybridization to Ikappa Balpha and 36B4 cDNA probes. B, male PPARalpha -/- and +/+ mice (n = 3/group) were fed with rodent chow or rodent chow supplemented with 0.05% ciprofibrate for 2 weeks. At the end of the treatment period, livers were isolated and total RNA was extracted. RNA (10 µg) was separated on 1% agarose, transferred to a nylon membrane, and analyzed by hybridization to Ikappa Balpha and 36B4 cDNA probes. Statistical analysis was assessed by analysis of variance (p < 0.05). Statistical significant differences between groups were then evaluated by the Student's t test. C, human primary hepatocytes were cultured with standard medium supplemented with Wy-14643 (50 µM) or vehicle (Me2SO, 0.1%) for 24 h. Protein extracts were prepared and 50 µg of each extract were separated by SDS-PAGE, transferred to Hybond membrane, and probed with antibodies to Ikappa Balpha and beta -actin. D, human primary hepatocytes were cultured with standard medium supplemented with Wy-14643 (50 µM or 100 µM) or vehicle (Me2SO, 0.1%) for 24 h and subsequently stimulated with IL-1beta (10 ng/ml) for 16 h. Protein extracts were then prepared, and 50 µg of each extract were separated by SDS-PAGE, transferred to Hybond membrane, and probed with antibodies to COX-2 (a kind gift from Dr Habib, Paris, France) and beta -actin.

After Ikappa Balpha degradation, p50/p65 dimers translocate to the nucleus and activate gene transcription. To explore the functional consequences of Ikappa Balpha up-regulation, EMSAs, using a NF-kappa B consensus site probe, were performed. Extracts from non-activated SMC contained a basal NF-kappa B binding activity, which was inhibited by fibrate treatment (Fig. 4A). The presence of NF-kappa B proteins in the complex was verified by shifting the complex using an anti-p65 antibody (data not shown). Treatment with IL-1beta resulted in a drastic increase of NF-kappa B binding, which was reduced by Wy-14643 treatment for 2 h and completely abolished after 24-h fibrate pretreatment, whereas binding of OCT1 proteins to their respective consensus site was unaffected (Fig. 4A). As a control, Western blot analysis demonstrated that PPARalpha activators do not affect either p50 or p65 protein levels (Fig. 4B and data not shown). Poynter and Daynes (35) and Marx et al. (22) showed that PPARalpha activators significantly reduced NF-kappa B DNA binding activities in aged murine splenocytes and in TNFalpha -stimulated endothelial cells, respectively. Therefore, our data are in line with the findings of these studies and suggest that the inhibition of NF-kappa B DNA binding activities depends on the time of PPARalpha agonist exposure.


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  		Fig. 4.   Wy-14643 reduces IL-1beta -induced NF-kappa B DNA binding activities in cultured HASMC and increases nuclear Ikappa Balpha protein. A, human aortic SMC were cultured for 2 or 24 h with Wy-14643 (100 µM) or vehicle (Me2SO, 0.1%) and were subsequently treated with IL-1beta (10 ng/ml) for 1 h. Nuclear proteins were extracted and 5 µg of each sample were subjected to EMSAs using NF-kappa B consensus site or consensus OCT1 site radiolabeled probes (Promega). Complexes were visualized by autoradiography. B, SMC were cultured in standard medium with fenofibric acid (100 µM) or vehicle (Me2SO, 0.1%) for 24 h and subsequently treated with IL-1beta (10 ng/ml) for different time intervals (min). Nuclear and cytoplasmic protein extracts were prepared and proteins (20 µg) were separated by SDS-PAGE, transferred to a Hybond membrane, and probed with antibodies to p65. C and D, SMC were cultured in standard medium with Wy-14643 (100 µM) or vehicle (Me2SO, 0.1%) for 24 h and were subsequently treated with or without IL-1beta for 1 h. Nuclear (C) and cytoplasmic protein extracts (D) were prepared and proteins (50 µg) were separated by SDS-PAGE, transferred to a Hybond membrane, and probed with antibodies to Ikappa Balpha , histone H1, and beta -actin.

In resting cells, Ikappa Balpha sequesters p50/p65 heterodimers in a cytoplasmic inactive complex. To determine whether Ikappa Balpha induction modulates NF-kappa B protein translocation, nuclear and cytoplasmic extracts from cultured SMC were isolated. IL-1beta induced a rapid but transient p65 translocation to the nucleus (Fig. 4B). Surprisingly, fenofibric acid treatment did not inhibit IL-1beta -induced p65 translocation suggesting that Ikappa Balpha up-regulation by fibrates does not result in an increased sequestration of NF-kappa B in the cytoplasm (Fig. 4B). This result constitutes a major difference between fibrates and glucocorticoids, which were reported to induce Ikappa Balpha in T cells and to block p65 translocation (36, 37). Because newly synthesized Ikappa Balpha protein accumulates not only in the cytoplasm but also in the nucleus reducing thereby NF-kappa B binding (38, 39), Ikappa Balpha protein localization was analyzed in cytoplasmic and nuclear extracts from human aortic SMC after fibrate treatment by Western blot analysis using a monoclonal anti-Ikappa Balpha antibody. As a control, fibrate treatment resulted in an increase of the Ikappa Balpha cytoplasmic content (Fig. 4D), which is consistent with our findings in Fig. 2D. Surprisingly, in non-stimulated cells, Ikappa Balpha protein was also present in the nucleus but its levels were not affected by fibrate treatment (Fig. 4C). IL-1beta treatment for 1 h did not result in an increased nuclear content of Ikappa Balpha . This finding is in line with a previous study demonstrating that newly synthesized Ikappa Balpha protein is only detectable in the nucleus after 2 h of IL-1beta or TNFalpha exposure in HeLa S3 cells (39). Interestingly, fibrate treatment in the presence of IL-1beta for 1 h led to an increase of nuclear Ikappa Balpha protein content (Fig. 4C), which occurs concomitantly with the loss of NF-kappa B DNA binding activity observed in IL-1beta treated cells (Fig. 4A).

Several concurring mechanisms may explain the overall anti-inflammatory activities of PPARalpha ligands. First, PPARs have been shown to down-regulate inflammatory response genes by negatively interfering with the STAT, AP-1, and NF-kappa B transcriptional pathways (14, 18, 21, 40). Direct protein-protein interactions between PPARalpha and AP-1 and NF-kappa B proteins have been invoked as mechanisms of transrepression (21). Second, by regulating anti-oxidant enzyme activities such as catalase (41), PPARalpha activators reduce the oxidative stress, and, as such, may inhibit NF-kappa B activation. Finally, the results of the present study provide an additional mechanism through which PPARalpha activators may antagonize NF-kappa B activation (Fig. 5). Induction of Ikappa Balpha mRNA by fibrates may contribute to the inhibition of inflammatory gene activation such as COX-2 or IL-6. The induction of Ikappa Balpha by fibrates in cytokine-activated cells should result in an acceleration of NF-kappa B nuclear desactivation. This is consistent with a previous report in which PPARalpha ligands were shown to affect the duration of the inflammatory response in a PPARalpha -dependent manner (42). Consistent with this observation, the increase of Ikappa Balpha protein after treatment with PPARalpha activators would lead to a halt in p65-mediated gene activation thereby reducing the duration of the inflammatory response.


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  		Fig. 5.   Model of NF-kappa B signaling pathway inhibition by PPARalpha activators.

The Ikappa Balpha promoter contains several regulatory regions among which are sites for NF-kappa B (43, 44). The results using the PPARalpha -deficient mice indicate that Ikappa Balpha activation by fibrates occurs in a PPARalpha -dependent manner. Our EMSA experiments indicate that PPARalpha activators do not activate Ikappa Balpha transcription in a NF-kappa B-dependent manner because basal NF-kappa B binding activity present in SMC was not increased by Wy-14643 but was conversely lowered. Furthermore, Ikappa Balpha mRNA appeared to be synergistically induced by fibrates in the presence of IL-1beta (Fig. 2A) suggesting that PPARalpha activators regulate Ikappa Balpha gene expression via a distinct signaling pathway. In addition, PPARalpha was reported to play a major role in the control of the cellular redox status (35). Klucis et al. (41) reported that administration of PPARalpha activators results in a drastic increase in the activity of catalase, an anti-oxidant enzyme. This induction occurs in the absence of oxidative stress as demonstrated by the absence of increase of F2-isoprostanes after Wy-14643 treatment (45). Taken together, these observations allow us to exclude that PPARalpha activators induce an oxidative stress resulting in NF-kappa B activation and induction of Ikappa Balpha .

In conclusion, the demonstration that PPARalpha activators modulate NF-kappa B activation by inducing Ikappa Balpha provides an additional, complementary action mechanism contributing to the overall anti-inflammatory properties of PPARalpha agonists.

    ACKNOWLEDGEMENTS

We thank O. Vidal, B. Derudas, and P. Poulain for technical contributions. We thank Dr. Frank Gonzalez for providing us the PPARalpha -null mice and Dr. Alain Israël (Pasteur Institute, Paris) who provided us the materials necessary to perform IKK assays.

    FOOTNOTES

* This work was supported in part by grants from the Institut Pasteur de Lille, INSERM, ARCOL, Laboratoires Fournier, and the Région Nord-Pas-de-Calais/Feder.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.

Dagger Supported by a grant from the Région Nord-Pas-de-Calais.

§ To whom correspondence should be addressed. Tel.: 33-3-2087-7388; Fax: 33-3-2087-7360; E-mail: Bart.Staels@pasteur-lille.fr.

Published, JBC Papers in Press, September 8, 2000, DOI 10.1074/jbc.M004045200

    ABBREVIATIONS

The abbreviations used are: IKK, Ikappa B kinase; SMC, smooth muscle cells; PPAR, peroxisome proliferator-activated receptors; PPRE, PPAR response element; IL, interleukin; GST, glutathione S-transferase; EMSA, electrophoretic mobility shift assay; HASMC, human aortic SMC; STAT, signal transducers and activators of transcription; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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