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Carcinogenesis Advance Access originally published online on January 3, 2008
Carcinogenesis 2008 29(3):536-543; doi:10.1093/carcin/bgm293
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Jun D cooperates with p65 to activate the proximal {kappa}B site of the cyclin D1 promoter: role of PI3K/PDK-1

Kahina Toualbi-Abed, Fanny Daniel1, Meryem C. Güller, Agnès Legrand2, Jose-Luis Mauriz3, Alain Mauviel and Dominique Bernuau*

Institut National de la Santé et de la Recherche Médicale (INSERM) U697, Université Paris 7 Denis Diderot, 75010 Paris, France
1 INSERM U773, Centre de Recherche Bichat Beaujon CRB3, Université Paris 7 Denis Diderot, site Bichat, Paris, France
2 INSERM U591, Hôpital Necker, 75015 Paris, France
3 Ciberehd and Institute of Biomedicine, University of Leon, 24071 Leon, Spain

* To whom correspondence should be addressed. Unité INSERM U697, Pavillon Bazin, Hôpital Saint-Louis, 1 Avenue Claude Vellefaux, 75010 Paris, France. Tel: +33 1 53 72 20 71; Fax: +33 1 53 72 20 51; Email: bernuau{at}stlouis.inserm.fr


   Abstract
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 Abstract
Introduction
 Materials and methods
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 Discussion
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 References
 
Nuclear factor kappaB (NF-{kappa}B) and activator protein 1 are transcription factors involved in the regulation of cell proliferation that play important roles in tumorigenesis. We investigated whether these two factors cooperate for transcriptional regulation of cyclin D1 (CCND1), a gene whose deregulation is critical during carcinogenesis. We demonstrate that overexpression of JunD in human hepatocarcinoma cells strongly activates transcription mediated by the {kappa}B2 site of the CCND1 promoter in reporter assays, in a manner strictly dependent on the presence of NF-{kappa}B proteins. Serum stimulation increased the expression of p65, p50, c-Fos, c-Jun and JunD and induced the recruitment of p65, p50 and JunD to the {kappa}B2 site of the promoter in DNA pull-down assays. Chromatin immunoprecipitation (ChIP) analysis confirmed the serum-induced recruitment of JunD to the promoter in vivo and showed that the presence of JunD was dependent on the presence of p65 and p50, indicating a protein–protein-dependent mechanism of JunD recruitment. Serum-induced activation of protein binding to {kappa}B2 correlated with high levels of phosphoinositide-dependent protein kinase-1 (PDK-1) phosphorylation. Both LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), and overexpression of a dominant-negative form of PDK-1 inhibited the JunD-stimulating effect in reporter assays. LY294002 also prevented the serum-induced recruitment of JunD, but not p65 or p50 to the promoter in ChIP assay. JunD–p65 complexes, identified in vivo by co-immunoprecipitation, were decreased by LY294002 and by small interfering RNA inhibition of PDK-1. Taken together, our data demonstrate a PI3K/PDK-1-dependent functional cooperation of NF-{kappa}B and JunD in the transcriptional regulation of CCND1 by serum.

Abbreviations: ALLN, N-acetyl-leucyl-leucyl-norleucinal; AP-1, activator protein 1; CCND1, cyclin D1; ChIP, chromatin immunoprecipitation; DTT, dithiothreitol; HDAC, histone deacetylase; IHH, immortalized human hepatocyte; NF-{kappa}B, nuclear factor kappaB; PCR, polymerase chain reaction; PDK-1, phosphoinositide-dependent protein kinase-1; PI3K, phosphatidylinositol 3-kinase; RSV, rous sarcoma virus


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
The regulator of cell-cycle progression cyclin D1 (CCND1) is upregulated in a variety of cancer cells by mechanisms involving mainly transcriptional activation. Analysis of the human CCND1 promoter has revealed the presence of two {kappa}B sites located at –840 and –33, designated {kappa}B1 and {kappa}B2 (1), respectively, and mutational analysis demonstrated that p50–p65 binding to the –33 {kappa}B2 site was important for CCND1 transcriptional regulation during proliferation induced by serum or growth factors or by stimulation by the Ras/Rac pathway (1,2). Nuclear factor kappaB (NF-{kappa}B) belongs to the Rel family of transcription factors. It consists of two groups of NF-{kappa}B subunits. The first group includes the Rel proteins RelA (or p65), RelB and c-Rel, and the second group includes p105 and p100 proteins, the precursors of p50 and p52 proteins (3). These two groups form homo- or heterodimers that bind a common sequence motif known as the {kappa}B site. Among these dimers, p65-containing complexes are responsible for most of the transcriptional activity of NF-{kappa}B (3). Although this transcription factor was first identified as a regulator of immune and inflammatory responses, recent studies indicate that NF-{kappa}B also functions in promoting cell growth and tumor progression. Specifically, the importance of NF-{kappa}B in the activation of CCND1 has been outlined in several recent studies (4–6). Activator protein 1 (AP-1), a dimeric transcription factor formed of proteins of the Jun (c-Jun, JunB and JunD), Fos (c-fos, FosB, Fra-1 and Fra-2) or Acivating transcription factor/cAMP responsive element binding protein families, is also activated during cell proliferation and transformation in a variety of cells, including hepatocytes (7,8). The effects of AP-1 on proliferation have also been linked to the transcriptional regulation of CCND1 through binding of AP-1 proteins to both an AP-1 site at –950 bp and a cyclic adenosine 3',5'-monophosphate-responsive site at –66 bp (1,9–12).

Activation of CCND1 is widely recognized as a promoting or aggravating event during the course of carcinogenesis. We were therefore interested in investigating whether the NF-{kappa}B and the AP-1 pathways may synergize for transcriptional activation of CCND1. Physical association of c-Jun and c-Fos through their leucine zipper domain with the Rel homology domain of the p65 subunit of NF-{kappa}B was described previously (13), and this association has been shown to activate the transcription of {kappa}B-regulated genes in promoter studies (13,14). However, a functional cooperation between NF-{kappa}B and AP-1 proteins on the CCND1 promoter has never been investigated. In this report, we demonstrate a functional synergism between NF-{kappa}B and JunD on the {kappa}B2 site of the CCND1 promoter. We show that this mechanism of CCND1 regulation occurs during the serum-induced proliferative response of immortalized hepatocytes and is tightly reliant on both JunD activation and the stimulation of the phosphatidylinositol 3-kinase (PI3K)/phosphoinositide-dependent protein kinase-1 (PDK-1) pathway. We further demonstrate that the PI3K/PDK-1 pathway regulates both the physical association between JunD and p65 and the binding of JunD–p65 complexes on the CCND1 promoter.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Antibodies and reagents
LY294002 and LY303511 were purchased from Calbiochem (San Diego, CA). N-acetyl-leucyl-leucyl-norleucinal (ALLN) was obtained from Biomol Research Laboratories (Plymouth Meeting, PA). Antibodies to c-Jun, JunD, c-Fos, Fra-1, p50, p65, CCND1, PDK-1 and SnRNP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to phospho-PDK-1(S241), phospho-AKT(S473) and total AKT were supplied by Cell Signaling Technology (Beverley, MA).

Reporter plasmids and expression vectors
The {kappa}B2(–42, +14)-Luc reporter plasmid contains the luciferase gene under the control of a (–42, +14) fragment of the CCND1 promoter, which includes the proximal {kappa}B2 site (15). A mut{kappa}B2-Luc construct was constructed by polymerase chain reaction (PCR)-assisted mutation in the {kappa}B sequence substituting GGGG by CGCG. The Rous sarcoma virus (RSV) expression vectors for junB and junD provided by M.Yaniv (Institut Pasteur, Paris, France) include the complete murine coding sequence for junB or junD under the control of the RSV promoter. The expression vector pCMV/c-jun generously provided by D.Kardassis (Institute of Molecular Biology and Biotechnology, Heraklion, Crete, Greece) contains the human c-jun coding sequence cloned in the pcDNA1 vector. The transdominant-negative I{kappa}B{alpha} A32/A36 plasmid that contains an inactive A32/A36 mutation was supplied by R.Weil (Institut Pasteur). The PDK-1-KHS241A that encodes the human PDK-1 sequence with an inactivating mutation on serine 241 cloned in the pCDEF vector was kindly provided by A.Weiss (University of California, San Francisco, San Francisco, CA). The AKT-DN plasmid that contains a HA-tagged catalytically inactive K79M-mutated human AKT sequence cloned in the pCMV6 vector was a gift of A.Groyer (INSERM U773, Paris, France).

Cell culture, DNA transfection and luciferase assays
The immortalized human hepatocyte (IHH) cell line kindly provided by H.Moshage (University Hospital Groningen, Groningen, The Netherlands) was grown in William’s E medium supplemented with 10% (vol/vol) fetal calf serum, 1% (vol/vol) sodium pyruvate, 1% (vol/vol) penicillin and 1% (vol/vol) streptomycin. Cells were electroporated at 230 V and 960 µF, as reported (16).

Co-immunoprecipitation
Total proteins (500–700 µg) from cells lysed in NP-40 lysis buffer [50 mM Tris–HCl, pH 8.0, 150 mM NaCl and 1% (vol/vol) NP-40] were precleared with anti-rabbit IgG beads (CliniScience, Montrouge, France), incubated with 3 µg of relevant antibody for 1 h, followed with a 1-h incubation with anti-rabbit IgG beads. After washing, beads were suspended in Laemmli buffer containing 50 mM dithiothreitol (DTT), boiled and supernatants were immediately subjected to polyacrylamide gel electrophoresis. Immunoprecipitated proteins were revealed by western blotting, using rabbit TrueBlotTM Horseradish Peroxidase (CliniScience) as a secondary antibody.

Western blot analysis
Nuclear extracts were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Immunoreactive bands were visualized by enhanced chemiluminescence.

Reverse transcription–PCR analysis
Total RNA was extracted from cells using the Qiagen RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) according to the instructions by the manufacturer. For cDNA synthesis, 1 µg of total RNA was reverse transcribed using the ThermoScriptTM reverse transcription–PCR system (Invitrogen, Cergy Pontoise, France). Amplifications were performed using a Multiplex PCR kit according to the manufacturer’s protocol (Qiagen) in the presence of 200 nM of each oligonucleotide primer, 50 ng cDNA and a total of 33 cycles for CCND1 and 20 cycles for GAPDH. The primers used were as follows: CCND1 forward sequence, 5'-GGGGAGTTTTGTTGAAGTTG-3' and reverse, 5'-TTTCCACTTCGAAGCACAGG-3' and GAPDH forward sequence, 5'-CGGATTTGGTCGTATTGGGC-3' and reverse, 5'-GTCATACCAGGAAATGAGCTT-3'. PCR products were analyzed by electrophoresis in 1.5% (vol/vol) agarose gels.

Electrophoretic mobility gel shift assay
For preparation of nuclear extracts, cells were lysed in hypotonic buffer [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 20 mM, pH 7.9 containing 20 mM Na fluoride, 1 mM Na vanadate, 1 mM Na glycerophosphate, 1 mM ethylenediaminetetraacetic acid and 1 mM ethyleneglycol-bis(aminoethylether)-tetraacetic acid] supplemented with 0.2% (vol/vol) NP40, 1 mM DTT, 1x protease cocktail inhibitor (Roche Applied Science, Meylan, France) and 1x phosphatase inhibitor cocktails 1 and 2 (Sigma, L’Isle d’Abeau Chesnes, France). After centrifugation for 20 s at 16 000g at 4°C, the nuclear pellet was resuspended in a saline–glycerol buffer [hypotonic buffer containing 420 mM NaCl and 20% (vol/vol) glycerol) supplemented with 1 mM DTT, 1x protease cocktail inhibitor and 1x phosphatase inhibitor cocktails 1 and 2] and gently agitated for 30 min on a rotating wheel at 4°C. The lysates were then centrifuged for 20 min at 16 000g at 4°C and the supernatant containing nuclear proteins was aliquoted and stored at –80°C. Single-stranded oligonucleotides corresponding to the upper strand of the CCND1 {kappa}B2 site (5'-GAGTACAGGGGAGTTTTGTTGAAGTA-3'), a mutated form in the {kappa}B site (5'-GAGTACACGCGAGTTTTGTTGAAGTA-3'), the upper strand of the NF-{kappa}B site from the enhancer of the H2 gene (5'-TCGAGGGCTGGGGATTCCCCATCTC-3') (core sequences underlined) and the upper strand of a Oct-1 probe (5'-TGTCGAATGCAAATCACTAGA-3') were end labeled with T4 polynucleotide kinase in the presence of [{gamma}-32P]adenosine triphosphate (5000 Ci/mmol). The labeled oligonucleotides were annealed with their respective unlabeled lower strands. Electrophoretic mobility gel shift assay was performed as described previously (17) using 10–20 µg of protein. For supershift analyses, 4 µg antibody were incubated with nuclear extracts for 3 h at 4°C prior to incubation with the 32P-radiolabeled probe.

Chromatin immunoprecipitation assay
Preparation of soluble chromatin, chromatin immunoprecipitation (ChIP) and recovery of precipitated DNA were performed with the ChIP-IT kit (Active Motif, Rixensart, Belgium) according to the manufacturer’s instructions, using 8 µg antibodies. For the PCR, 40 cycles of amplification were carried out using two primer sets, one spanning the {kappa}B2 site of the CCND1 promoter (forward 5'-TCAGGGATGGCTTTTGGG-3' and reverse 5'-CAACTTCAAGAAAACTCCCC-3') and the other set spanning the upstream region of the promoter (forward 5'-CCAAGTCCTTTCAAGTCGCC-3' and reverse 5'-AGGGAGCGCGTTCATTCAG-3').

Biotinylated oligonucleotide precipitation assay
M-280 streptavidin Dynabeads® (Dynal Biotech, Compiègne, France) washed in 5 mM Tris–HCl, pH 7.5, 0.5 mM ethylenediaminetetraacetic acid and 1 M NaCl were coated with 4 µg biotinylated double-stranded oligonucleotides corresponding to the {kappa}B2 site or a mutated form of the {kappa}B2 site, as indicated by the manufacturer. The coated beads were subsequently incubated for 1 h at 4°C in phosphate-buffered saline containing 0.5% (wt/vol) dried non-fat milk. Nuclear extracts (200 µg) prepared as above were incubated with the coated beads for 3 h on a rotating wheel at 4°C in binding buffer [0.01 M Tris–HCl, pH 7.6, 5 mM ethylenediaminetetraacetic acid, 50 mM KCl, 5 mM MgCl2 and 10% (wt/vol) glycerol] in the presence of 50 µg poly(dI-dC) poly(dI-dC), 1 mM DTT and 5 µl protease inhibitor cocktail. After extensive washes in phosphate-buffered saline, DNA-bound proteins were eluted in Laemlli buffer by boiling for 5 min and were subsequently separated on a sodium dodecyl sulfate–polyacrylamide gel before identification by immunoblotting.

Small RNA interference experiments
IHHs were electroporated as indicated above (960 µF, 230 V) with human PDK-1-specific siRNA or control siRNA (10 µM final concentration; Qiagen GmbH, Hilden, Germany) and total cell lysates were prepared 72 h after transfection.


   Results
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 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
JunD cooperates with NF-{kappa}B on the {kappa}B proximal site of the CCND1 promoter
The main regulatory elements in the CCND1 promoter are located within the –1745 fragment relative to the initiation site. In this region, an AP-1-like sequence at –950, a cyclic adenosine 3',5'-monophosphate-responsive site at –66 and two {kappa}B sites {kappa}B1 and {kappa}B2 located at –840 and –33, respectively, have been identified (1,11) (Figure 1A). The {kappa}B2 site has been shown to participate in the induction of the CCND1 promoter by NF-{kappa}B as well as by Rac-dependent signals and growth factors. To study the function of the {kappa}B2 element, we used a luciferase reporter gene driven by the CCND1 minimal promoter (a –42/+14 fragment that contains only the {kappa}B2 site but no other identified regulatory element). As expected, luciferase activity driven by this minimal promoter fragment was lower than that detected using a –1745-luciferase construct of the promoter (data not shown), but was detectable under basal conditions. The effect of AP-1 overexpression was explored by transient transfections with c-Jun-, JunB- or JunD-expressing plasmids in the IHH line. Efficacy of the overexpression was verified by western blot analysis of nuclear proteins using specific antibodies (Figure 1B). Cotransfection of the {kappa}B2-Luc reporter with a c-Jun or JunB expression plasmid resulted, respectively, in a 12- and 4.6-fold increase in luciferase expression over cells transfected with an empty plasmid. In contrast, overexpression of JunD exerted a drastic (85-fold) stimulating effect on luciferase expression (Figure 1C). There was no effect of Jun protein overexpression on a reporter plasmid containing a mutated form of the {kappa}B2 site (mut{kappa}B2, Figure 1C). Overexpression of Fos proteins (c-Fos, Fra1 or Fra2) had no significant effect on {kappa}B2 transactivation (data not shown). To determine whether the effect of Jun proteins was dependent on the presence of NF-{kappa}B proteins, we cotransfected the cells with an expression plasmid encoding for a mutated non-phosphorylable form of I{kappa}B{alpha} (mutI{kappa}B{alpha}), hence acting as an inhibitor of NF-{kappa}B signaling. In the presence of this inhibitor, the moderate synergistic effects of c-Jun or JunB were not affected, whereas the strong stimulating effect of JunD was drastically decreased by 86% (Figure 1D). Taken together, these results indicate that JunD, but not the other Jun proteins, exerts a potent activating effect on {kappa}B2-mediated transcription. It was recently shown that AP-1 proteins activated by c-Jun N-terminal kinase could activate the NF-{kappa}B pathway by inducing NF-{kappa}B subunit expression (18). In our experimental setting, however, no change in the nuclear amounts of p50 and p65 proteins was observed in cells overexpressing JunD (Figure 1E) or the other Jun proteins (data not shown).


Figure 1
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  		Fig. 1. AP-1 proteins cooperate for the transactivation mediated by the {kappa}B2 site on the CCND1 promoter. (A) Schematic representation of the main regulatory elements located on the –1745 fragment of the human CCND1 promoter. (B) Nuclear proteins prepared from IHH 48 h after electroporation with RSV-c-Jun, RSV-JunB or RSV-JunD plasmid (+) or an empty vector (–) were immunoblotted with antibodies to c-Jun, JunB or JunD. (C) IHHs were cotransfected with the {kappa}B2-Luc reporter plasmid or the mutkB2-Luc reporter plasmid and with expression plasmids for c-Jun, JunB or JunD, as indicated. The plasmid RSV-CAT was used for normalization. Luciferase and CAT activities were measured after 48 h expression. Results are the mean ± standard error of three to four independent transfections and are expressed as the relative increase of luciferase activity over cells transfected with the control empty vector. (D) IHHs were cotransfected with the {kappa}B2-Luc reporter plasmid and with expression plasmids for c-Jun, JunB or JunD in the presence or in the absence of mutI{kappa}B{alpha}. The plasmid RSV-CAT was used for normalization. Luciferase and CAT activity were measured after 48 h expression. Results are the mean ± standard error of three independent transfections and are expressed as the relative increase of luciferase activities over cells transfected with the control empty vector. (E) Western blot analysis of NF-{kappa}B nuclear proteins in nuclear extracts from IHH prepared 48 h after electroporation with RSV-JunD (+) or an empty plasmid (–). Blotting the membranes with the snRNP antibody was used as a loading control.

 
JunD binds to the proximal {kappa}B element of the CCND1 promoter in serum-stimulated cells
To evaluate the physiological implication of JunD–NF-{kappa}B synergism, we studied the transcriptional induction of CCND1 during the serum-induced proliferative response of IHH. After 24-h serum starvation, cells were cultured in the presence of 10% fetal calf serum for increasing periods of time. Western blot analysis of nuclear proteins showed that AP-1 was stimulated by serum, with elevation of c-Jun and c-Fos levels between 1 and 4 h, and a strong increased expression of JunD 2 h following serum addition (Figure 2A). The levels of p50 and p65 also increased upon serum stimulation with a maximum at 2 h (Figure 2A). CCND1 messenger RNA levels were increased after 2 h of serum refeeding (Figure 2B) with increased levels of CCND1 protein between 2 and 8 h (Figure 2C).


Figure 2
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  		Fig. 2. Activation of AP-1, NF-{kappa}B and CCND1 proteins by serum. (A) IHHs deprived of serum for 24 h were kept unstimulated (0 h) or stimulated by 10% serum for the indicated times. Nuclear extracts (50 µg) were immunoblotted with antibodies against c-Jun, JunD, c-Fos, p65 or p50 and snRNP, as a loading control. (B) Analysis of CCND1 messenger RNA at different times following serum refeeding of starved cells by reverse-transcription analysis. Detection of Glyceraldehyde3-phosphate dehydrogenase messenger RNA served as an internal control. (C) Total extracts (50 µg) from IHH deprived of serum (0 h) or stimulated by 10% serum for the indicated times were immunoblotted with CCND1 antibody, and βactin, as a loading control.

 
A possible implication of the {kappa}B2 site of the CCND1 promoter during the serum-induced transcriptional response was first investigated by electrophoretic mobility gel shift assays, using a 32P-labeled {kappa}B2-specific probe. In serum-deprived IHH, the binding of nuclear proteins to the {kappa}B2 probe was almost undetectable, whereas serum refeeding induced a progressive increase in the intensity of two retarded bands between 1 and 6 h (Figure 3A). The specificity of these bands was verified by the absence of binding when an oligonucleotide containing a mutated {kappa}B2 site was used and by competition experiments showing decreased binding in the presence of increasing amounts of a {kappa}B2 wild-type unlabeled probe but not in the presence of a mutated {kappa}B2 unlabeled probe (Figure 3B).


Figure 3
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  		Fig. 3. Activation of the {kappa}B2 site of CCND1. (A) IHHs deprived of serum for 24 h were kept unstimulated (0 h) or stimulated by 10% serum for the indicated times. Electrophoretic mobility gel shift assay was performed using nuclear extracts (15 µg) and a 32P-labeled {kappa}B2 probe. Arrows indicate the position of the specific retarded bands; NS, non-specific bands. (B) Specificity of the binding of nuclear proteins to the {kappa}B2 probe. Electrophoretic mobility gel shift assay was performed using equal amount (15 µg) of nuclear proteins prepared from IHH stimulated with 10% serum for 2 h. The extracts were incubated with a 32P-labeled {kappa}B2 probe or a 32P-labeled mutated {kappa}B2 (mt{kappa}B2) probe. Specificity of the retarded bands was verified by performing the electrophoretic mobility gel shift assay in the presence of increasing concentrations of cold wild-type, kB2 probe or mutated kB2 probe. The arrows point to the two specific retarded bands; NS, non-specific band. (C) Electrophoretic mobility gel shift assay was performed with nuclear extracts (15 µg) from IHH cultured in serum-containing medium, in the presence or in the absence of antibody to p50 or p65 NF-{kappa}B proteins. Arrows point to the two specific retarded bands; SS, supershifted band. (D) DNA pull-down assay was performed with nuclear extracts (200 µg) from IHH stimulated by serum for 2 h. The extracts were incubated with a biotinylated {kappa}B2 probe or a mutated {kappa}B2 probe followed by purification with streptavidin-coated magnetic beads. The proteins bound to DNA were eluted from the beads and immunoblotted with antibodies specific to p65, p50, JunD, c-Jun or c-Fos. Fifty micrograms of non-precipitated proteins (NP) were blotted in parallel.

 
Supershift experiments were performed to identify the proteins bound to the {kappa}B2 probe in serum-stimulated cells. Addition of the p50 NF-{kappa}B antibody to the reaction induced the disappearance of the lowest retarded band along with the formation of a supershifted band (Figure 3C). On the other hand, no effect could be obtained with several different p65 antibodies. Similarly, antibodies to c-Jun, JunD, JunB, c-Fos or Fra1 were constantly unable to modify or shift the retarded bands (data not shown). Since some DNA bound complexes are non-accessible to antibodies in supershift assays, due to the peculiar conformation of the protein–DNA complex, we decided to use DNA pull-down assays which allow analysis of the proteins bound to DNA after detachment of their DNA target. In serum-starved IHH, no NF-{kappa}B or AP-1 proteins bound the {kappa}B2 probe (data not shown). Two hours after serum refeeding, p65, p50 and JunD were detected in the complex precipitated by the biotinylated {kappa}B2 probe but not by the biotinylated mutated {kappa}B2 probe (Figure 3D). Meanwhile, blotting of eluted proteins with c-Jun, JunB, c-Fos or Fra1 antibodies constantly yielded negative results (Figure 3D and data not shown). These data indicated that a protein complex containing p50, p65 and JunD is detectable on the {kappa}B2 element of the CCND1 promoter after serum stimulation of IHH.

JunD–NF-{kappa}B synergism on the CCND1 promoter is regulated by a PI3K/PDK-1-dependent pathway
The PI3K pathway is known to be an important regulator of both CCND1 transcription (19) and NF-{kappa}B activity (20). We therefore tested the hypothesis that PI3K activation may be responsible, at least in part, for the JunD–NF-{kappa}B synergism on the CCND1 promoter. Treatment of IHH with LY294002, a potent and specific inhibitor of PI3K, blunted the activation of CCND1 messenger RNA by serum in comparison with untreated cells (Figure 4A), in accordance with existing reports (1,19,21,22). Importantly, electrophoretic mobility gel shift assays revealed a reduced binding of nuclear proteins to the {kappa}B2 probe in response to serum in LY294002-treated cells (Figure 4B). Moreover, DNA pull-down assays revealed that JunD was no longer present in the complex bound to the {kappa}B2 site in LY294002-treated cells stimulated with serum for 2 h, contrasting with the persistent detection of p50 and p65 (Figure 4C). On the other hand, LY294002 treatment did not decrease the nuclear amounts of p50, p65 nor JunD (Figure 4C, compare lanes 1 and 4) indicating that the absence of detection of JunD in the complex binding to the {kappa}B2 element of the promoter after serum stimulation in the presence of LY294002 is not due to a decreased availability of this protein.


Figure 4
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  		Fig. 4. Activation of the {kappa}B2 site is dependent on activation of the PI3K/PDK-1 pathway. (A) RNA was extracted from starved IHH treated (LY+) or not (LY–) with LY294002 (20 µM) for 16 h, unstimulated (0 h) or stimulated by serum for the indicated times. Reverse transcription–PCR was carried out for CCND1 and GAPDH messenger RNA expression, as described in Materials and Methods. Note: the LY–data are identical to those shown in Figure 2B. (B) Electrophoretic mobility gel shift assay was performed using a 32P-labeled {kappa}B2 or Oct-1 probe and 15 µg nuclear extracts from IHH deprived of serum for 24 h and then stimulated (+) or not (–) by serum for 2 h and treated (LY+) or not (LY–) with LY294002 (20 µM) for 16 h. The 32P-labeled Oct-1 probe was used as a loading control. Arrows indicate the two specific retarded bands; NS, non-specific band. (C) DNA pull-down assay was performed with nuclear extracts (200 µg) from cells treated or not with LY294002 for 16 h and stimulated by serum for the last 2 h. The extracts were incubated with a biotinylated {kappa}B2 probe (lanes 2 and 5) or a biotinylated mutated {kappa}B2 probe (lanes 3 and 6), followed by purification with streptavidin-coated magnetic beads. The proteins bound to DNA were eluted from the beads and immunoblotted with antibodies specific to p65, p50 and JunD. Fifty micrograms of non-precipitated proteins were blotted in parallel (lanes 1 and 4). Note: the LY–data (lanes 1–3) are identical to those shown in Figure 3D. (D) IHH transiently transfected with the {kappa}B2-Luc reporter, the RSV-CAT plasmid and expression vector for JunD or an empty plasmid were treated (+) or not (–) with LY294002 (20 µM) or LY303511 (20 µM) for 16 h before measurement of luciferase and CAT activities. The results are the mean ± standard error of three independent transfections and are expressed as the relative increase of luciferase activity over cells transfected with a control empty vector. (E) Total extracts (100 µg) from cells deprived of serum (0 h) or stimulated by serum for the indicated times were subjected to immunoblotting with antibodies to phosphorylated AKT, total AKT, phosphorylated PDK-1 or total PDK-1. (F) IHHs were transiently transfected with the {kappa}B2-Luc reporter and an expression plasmid for JunD, without or with increasing amounts (5, 10 and 20 µg) of PDK-1-KDS241A or AKT-DN expression plasmid and RSV-CAT for normalization. Luciferase and CAT activities were measured after 48 h expression.

 
To further assess the effect of PI3K on {kappa}B2-dependent transactivation, we performed promoter luciferase assays in the presence of LY294002. Treatment of IHH with LY294002 following ectopic expression of JunD reduced the potentiated luciferase activity mediated by the {kappa}B2-Luc reporter by 44% whereas treatment with LY303511 which contains a single atom substitution in the molecule did not modify the activating effect of JunD, confirming the specificity of the LY29402-induced inhibition (Figure 4D). On the other hand, PD98059, an inhibitor of the extracellular-regulated kinase/mitogen-activated protein kinase-signaling cascade had no effect (data not shown).

Two main downstream targets of PI3K are PDK-1 and AKT. We tested which of this pathway might be involved in the JunD–NF-{kappa}B synergistic effect. While AKT phosphorylation was barely detectable in starved cells and its level was weakly induced after serum stimulation, basal phosphorylation of PDK-1 was strikingly high in quiescent IHH, and serum refeeding induced a still higher level at 30 min and 1 h (Figure 4E), indicating a potent activation of the PDK-1 pathway in both serum-starved and serum-stimulated IHH. We next evaluated the effect of the blockade of PDK-1 and AKT on the {kappa}B2-Luc reporter activity in the presence of overexpressed JunD. Inhibition of PDK-1 activity by expression of PDK-1-KHS241A, a mutant form of PDK-1 with a kinase-inactivating S241A mutation (23), induced a dose-dependent inhibition of the JunD–NF-{kappa}B synergism, whereas expression of increasing amounts of a dominant-negative form of AKT (AKT-DN) had no significant effect (Figure 4F). These data strongly suggest that the JunD synergistic effect on {kappa}B2-driven CCND1 transcription is modulated by a PI3K/PDK-1-dependent pathway.

Analysis of the proteins bound to the {kappa}B proximal site of the CCND1 promoter in vivo by ChIP experiments
To obtain direct in vivo evidence that serum induces JunD to cooperate with NF-{kappa}B in a PI3K-dependent pathway, we performed ChIP experiments using cross-linked chromatin from serum-deprived IHH before or after serum refeeding (2 h) and a PCR primer pair for amplification of a 171 bp fragment encompassing the {kappa}B2 site of the CCND1 promoter, which does not include other identified regulatory elements. No PCR signal was obtained when primers amplifying a far upstream region of the promoter were used (data not shown). The PCR analysis specifically produced the expected 171 bp fragment when chromatin from serum-deprived cells was immunoprecipitated with a histone deacetylase (HDAC) 1 antibody (Figure 5, lane 1). HDAC1 is a transcriptional repressor known to be present on most inactive promoters. On the other hand, no PCR signal was obtained following immunoprecipitation of chromatin with antibodies to NF-{kappa}B or AP-1 proteins (Figure 5, lane 1). Stimulation by serum induced the recruitment of p50 and p65 to the promoter, paralleled with the disappearance of HDAC1 (Figure 5, lane 2). To ascertain the specificity of these results, chromatin of cells treated by ALLN, a potent inhibitor of NF-{kappa}B activation, which inhibits the 26S proteasome thereby preventing the nuclear translocation of p50 and p65, was also analyzed. ALLN treatment completely prevented the serum-induced recruitment of p50 and p65 to the promoter (Figure 5, lane 4). Consistent with our in vitro binding studies, we detected the presence of JunD, but not that of c-Jun, JunB, c-Fos or Fra-1 on the CCND1 promoter at 2 h of serum stimulation (Figure 5, lane 2 and data not shown). Importantly, JunD binding to DNA was tightly coupled with the presence of p50 and p65 proteins since ALLN also abolished JunD recruitment on the promoter (Figure 5, lane 4), suggesting that JunD fixation to DNA is not direct. Finally, JunD was no more detected in chromatin extracts prepared from LY294002-treated cells stimulated with serum for 2 h, confirming the influence of the PI3K pathway on JunD recruitment to the {kappa}B2 site in vivo.


Figure 5
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  		Fig. 5. Recruitment of NF-{kappa}B and AP-1 proteins to the {kappa}B2 site of the CCND1 promoter in vivo. ChIP analysis was performed with chromatin prepared from cells deprived of serum (lane 1) or stimulated by serum for 2 h (lanes 2–4) and previously treated (lane 3) or not (lane 2) with LY294002 for 16 h or treated for 2 h with serum in the presence of 10 µmol ALLN (lane 4). The chromatin was precipitated with p65, p50, JunD or normal rabbit IgG. PCR was performed from these immunoprecipitates or with input chromatin using a primer covering the {kappa}B2 site of the CCND1 promoter. PCR products (171 bp) were resolved by agarose gel ethidium bromide staining. Representative of two different experiments.

 
PDK-1 increases the formation of JunD–p65 complexes
Recent studies indicated that p65 activates transcription by interacting with coactivators exhibiting HDAC activity, including p300/CBP, the steroid receptor coactivator-1 and the p300/CBP-associated factor (24–27). P65 is also able to recruit corepressor complexes, such as HDAC-1, -2 and -3 (28–30) that are believed to repress NF-{kappa}B-dependent transcription. Association of p65 with coactivators or corepressors is modulated by phosphorylations of the protein (26,28,31). Physical association between the leucine zipper domain of c-Jun and the Rel homology domain of p65 has been described previously (13). Since JunD shares the same bZIP domain as c-Jun, we hypothesized that JunD might act as a coactivator of p65 by direct binding to p65 and that this association is regulated by the PI3K/PDK-1 pathway. To test this hypothesis, we first demonstrated the presence of JunD–p65 complexes in extracts from IHH. As shown in Figure 6A, p65 was easily detected in cell lysates from IHH growing in the presence of serum immunoprecipitated with anti-JunD, and conversely JunD was revealed in cell lysates immunoprecipitated with anti-p65. We also detected endogenous c-Jun–p65 complexes in these cell extracts (Figure 6A). Notably, treatment with LY294002 dramatically reduced the detection of JunD–p65 complexes, whereas c-Jun–p65 complexes were not modified (Figure 6B) indicating that PI3K activity is specifically necessary for the assembly of JunD–p65 but not c-Jun–p65 complexes. SiRNA-mediated knockdown of PDK-1 controlled by western blotting of cell extracts prepared 48 h after transfection (Figure 6C, upper panel) reduced the amount of JunD–p65 complexes (Figure 6C, lower panel), without decreasing p65 or JunD levels, suggesting an involvement of this kinase in the formation of JunD–p65 complexes. Altogether, these data indicate that PI3K/PDK-1 influences the association of JunD with p65, thereby influencing {kappa}B-dependent CCND1 transcription.


Figure 6
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  		Fig. 6. In vivo regulation of JunD and p65 association by PI3K/PDK-1. (A) Total cell extracts (500 µg) from IHH were immunoprecipitated with p65 antibody or rabbit IgG and immunoblotted with anti-JunD antibody or were immunoprecipitated with anti-JunD antibody, anti-c-Jun antibody or rabbit IgG and immunoblotted with anti-p65 antibody. (B) Total cell extracts (500 µg) from IHH treated (LY+) or not (LY–) with LY294002 (20 µM) for 16 h were immunoprecipitated with JunD antibody, c-Jun antibody or rabbit IgG and immunoblotted with anti-p65 antibody. Fifty micrograms of non-precipitated proteins (NP) were blotted in parallel. (C) Total extracts from IHH transiently transfected for 48 h with control or PDK-1-specific siRNA were immunoblotted with anti-PDK-1 or JunD antibodies (upper panels) or immunoprecipitated with JunD antibody or rabbit IgG and immunoblotted with anti-p65 antibody. Fifty micrograms of NP were blotted in parallel (lower panel).

 

   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
The involvement of the AP-1 complex in the transcriptional regulation of CCND1 expression has been previously related to the binding of AP-1 proteins to both the AP-1 and/or the cyclic adenosine 3',5'-monophosphate-responsive sites present within its promoter region (10,11). Increasing evidence indicate that AP-1 can also exert a transactivating function independently of its fixation to AP-1 sites by protein–protein interactions with other transcriptional regulators, including SP1, β-catenin, NF-{kappa}B and Smads (16,32–34). Regarding NF-{kappa}B, a physical association of the basic leucine zipper regions of c-Jun and c-Fos with the Rel homology domain of the p65 NF-{kappa}B subunit resulting in enhanced transactivation of {kappa}B-regulated genes has been previously demonstrated (13). In the present report, we describe a similar mechanism that contributes to CCND1 gene transactivation via the proximal {kappa}B element of the promoter. Notably, consistent with our previous report on the functional importance of JunD for the {kappa}B-dependent regulation of CCND1 transcription in hepatocytes (15), we show that JunD, but not the other Jun proteins, is able to induce a strong synergistic effect on {kappa}B2 transactivation which is tightly related to NF-{kappa}B since it is almost totally abrogated by inhibition of the nuclear translocation of NF-{kappa}B proteins in promoter reporter assays. In contrast, the weak effect of c-Jun and JunB overexpression on {kappa}B2 transactivation (see Figure 1C) is not decreased by coexpression of the mutI{kappa}B{alpha} inhibitor, suggesting that c-Jun and JunB may use another mechanism of transactivation on the minimal CCND1 promoter examined. Accordingly, Jun proteins have been shown to interact with several general transcription factors, such as TBP, TFIID and TFIIB (35,36), that might potentially be active on the promoter fragment used in our experiments since it contains the major transcription initiation site (37). Consistent with our promoter studies, we show for the first time that JunD–NF-{kappa}B synergism is involved in the serum-induced transcriptional activation of CCND1 in vivo and correlates with both AP-1 stimulation and high levels of PI3K/PDK-1 activation. The combined use of DNA pull-down and ChIP assays focused on the proximal {kappa}B2 element of the CCND1 promoter indicated that in serum-deprived IHH, HDAC1 binds to this site, suggestive of a repressive state of this regulatory element (28). At this stage, no p50 was found on the promoter, indicating that repression mediated by binding of HDAC1 to p50–p50 homodimers, as in the model described by Zhong et al. (29) is not taking place on the CCND1 promoter during serum deprivation. Following serum stimulation, HDAC1 is displaced from the {kappa}B2 site of the promoter, allowing the recruitment of both p50–p65 and JunD, but not c-Jun. We recently observed that AP-1 proteins, including JunD, together with c-Jun, JunB and c-Fos bind a TCF-binding element at –539 bp of the CCND1 promoter during serum stimulation of HCT116 cells (16). The AP-1 element at –954 is also targeted in vitro by c-Jun, JunB, JunD and c-Fos during Ras transformation (9). None of these sites could interfere with the results of our ChIP analysis since they are at a large distance from the {kappa}B2 site. Altogether, these observations indicate that the role of AP-1 proteins in the transcriptional regulation of CCND1 is highly complex. At variance with c-Jun proteins, the role of JunD on cellular proliferation remains debated (38). However, most of the data were obtained from fibroblasts and not from epithelial cells. Moreover, the fixation of JunD-containing dimers on target gene promoters has been focused exclusively on the AP-1 site. The results of the present study strongly suggest that JunD may also play a contributing role on cell proliferation, when cooperating with p65 for the formation of {kappa}B-binding complexes.

Another important result herein is the identification of a new mechanism that clarifies the previously underscored influence of the PI3K pathway on CCND1 transcriptional regulation (1,19) and on PI3K–NF-{kappa}B interaction. In promoter transfection assays, we observed that the stimulatory effect of AP-1 on {kappa}B2-mediated transactivation was almost completely abrogated in the presence of the specific PI3K inhibitor LY294002. Although initially believed to operate as components of distinct signaling pathways, the PI3K and the NF-{kappa}B pathways can converge. PI3K- and PDK-1-induced phosphorylation of I{kappa}B leading to its ubiquitination and degradation with a subsequent increased p50–p65 nuclear translocation has been reported (20,39,40). Such a mechanism was clearly not involved in the present study since LY294002 treatment did not modify the binding of p50 and p65 on the CCND1 promoter neither in DNA pull-down nor in ChIP assays. In contrast, PI3K blockade prevented the binding of JunD on the {kappa}B2 site and drastically decreased the formation of JunD–p65 complexes, suggesting that the absence of JunD binding to {kappa}B2 is mainly due to the lack of available complexes. Of note, p65 association with coactivators has been reported to depend on PI3K/PDK-1-dependent phosphorylations (31,41,42). Besides, protein kinase A, a known target of PDK-1 (23,43), was also shown to favor p65 association with coactivators (23,26,29,43). Accordingly, we show herein that an important downstream effector of PI3K for JunD–NF-{kappa}B synergism is predominantly PDK-1. Knock down of PDK-1 by siRNA dose dependently inhibited the synergistic effect of JunD on {kappa}B2-mediated transactivation and decreased the abundance of JunD–p65 complexes. In contrast, inhibition of the PI3K pathway by LY294002 did not impact on the formation of c-Jun–p65 complexes. In contrast with c-Jun, little is known about phosphorylation changes modulating the activity of JunD. We postulate that PI3K/PDK-1-dependent JunD phosphorylation may also influence its association with p65. Such a PI3K-dependent mechanism remains to be characterized in details.

Activation of several cellular signaling pathways, including PI3K and NF-{kappa}B, has been described in transcriptome analyses of human hepatocarcinomas (44,45), and the concomitant activation of NF-{kappa}B and AP-1 with nuclear accumulation of JunD is frequently observed in tumor samples (46,47). Therefore, our data support a possible role for the mechanism described herein in the activation of CCND1 during the pathogenesis of human hepatocellular carcinoma.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Post-doctoral grant from Institu National de la Santé et de la Recherche Médicale, France, to J.L.M.


   Acknowledgments
 
We thank M.Yaniv, A.Weiss, R.Weil and D.Kardassis for their gift of plasmids. The IHH cell line was kindly provided by Dr H.Moshage. We also acknowledge A.Groyer, G.Courtois, S.Dennler and F.Verrecchia for critical reading of the manuscript and J.André for help in the reverse transcription–PCR and western blot experiments.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received September 24, 2007; revised December 14, 2007; accepted December 15, 2007.


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Transcriptional regulation of the cyclin D1 gene at a glance
J. Cell Sci., December 1, 2008; 121(23): 3853 - 3857.
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