Functional Cooperation Between Jund and NF-Κb in Rat Hepatocytes

Functional cooperation between JunD and NF-kB in rat hepatocytes

Mohamed Rahmani1, Philippe PeÂ ron1, Jonathan Weitzman2, Latifa Bakiri2, Bernard Lardeux1 and Dominique Bernuau*,1

1Laboratoire de Biologie cellulaire, INSERM U 327, FaculteÂ de MeÂdecine Xavier Bichat et UniversiteÂ Paris 7 Denis Diderot, Paris, France; 2UniteÂ des Virus OncogeÁnes, URA 1644 du CNRS, DeÂpartement des Biotechnologies, Institut Pasteur, Paris, France

AP-1 and NF-kB are rapidly activated during liver binding site (Angel and Karin, 1991). As in other cell regeneration. Whether these parallel inductions have types, a role for the AP-1 complex on hepatocyte potential functional implications is not known. Isolated proliferation has been well documented. The fos and rat hepatocytes were stimulated with two mitogens, jun genes are rapidly and transiently up-regulated epidermal growth factor or hepatocyte growth factor during the pre-replicative phase after partial hepatect- and with tumor necrosis factor a, a cytokine involved in omy in the rat which triggers a synchronized the liver regenerative response in vivo and a strong proliferative response of hepatocytes (Fausto and inducer of NF-kB. All three cytokines increased AP-1 Mead, 1989; Thompson et al., 1986; Alcorn et al., and NF-kB binding to their cognate cis-element and 1990). Increased binding of nuclear proteins to the AP- induced a 2.5-fold activation of NF-kB-dependent 1 site has also been described (Westwick et al., 1995). transcription. Inactivation of AP-1 by TAM67, a In vitro stimulation of primary cultures of hepatocytes dominant negative mutant of AP-1 drastically inhibited with growth factors induces the same program of fos basal and cytokine-induced NF-kB transactivation. and jun gene activation, increases AP-1 binding activity Overexpression of Jun D, but not of the other Jun or and stimulates the transactivation of AP-1 dependent Fos proteins increased by threefold NF-kB transactiva- genes (Nadori et al., 1997; Rahmani et al., 1999). tion. Functional cooperation between JunD and p65 was NF-kB is another transcription factor rapidly demonstrated in a simple Gal-hybrid system. Finally, a activated in the liver after partial hepatectomy (Tewari twofold decrease in NF-kB transactivation was found in et al., 1992; FitzGerald et al., 1995; Yamada et al., hepatocytes isolated from JunD7/7 mice compared with 1997). Active NF-kB is a heterodimeric complex hepatocytes from JunD+/+ mice. Altogether these data composed of two subunits of the NF-kB family, demonstrate a functional cooperation of p65 with JunD, including p50, p52, p65 (Rel-A), c-Rel and Rel B a major constituent of AP-1 in normal hepatocytes. (Lin et al., 1998; Siebenlist et al., 1994; Verma et al., Oncogene (2001) 20, 5132 ± 5142. 1995). These proteins share a conserved NH2-terminal region, termed the rel homology domain, which Keywords: NF-kB; p65; AP-1; JunD; hepatocytes mediates DNA binding and dimerization. In most tissues the preponderant NF-kB dimer is a p50/p65 heterodimer. Regulation of NF-kB activity is mediated Introduction by interaction through the rel homology domain with the IkB family of inhibitory proteins, which sequesters The transcriptional factor AP-1, a dimer formed of the transcription factor in the cytoplasm. The IkB that proteins of the Jun family (c-Jun, JunB, JunD) and Fos controls immediate-early activation of NF-kBisIkBa. family (c-Fos, FosB, Fra1, Fra2) plays a pivotal role in Following various cellular stimuli, IkBa undergoes the regulation of crucial events in cell life such as phosphorylation, ubiquitination and degradation, lead- dierentiation, proliferation, and transformation (for ing to the release of the p50/p65 complexes, their review see Angel and Karin, 1991; Karin et al., 1997). nuclear translocation, and ultimately, the transcrip- AP-1 proteins belong to the group of basic leucine tional activation of speci®c target genes. NF-kBis zipper transcriptional factors. Heterodimerization be- activated by a variety of cytokines and mitogens. tween Fos and Jun proteins, or homodimerization Besides its well recognized role as a key regulator of between Jun proteins through the leucine zipper genes involved in in¯ammation responses to infection domain is a prerequisite for binding to the speci®c and stress (Baldwin, 1996; Barnes and Karin, 1997; DNA sequence, the TPA response element or AP-1 Franzoso et al., 1998), protection from apoptosis is an important function of NF-kB. In the liver of NF-kB p65 knockout mice, extensive apoptosis of hepatocytes result in death at birth (Beg et al., 1995). During liver *Correspondence: D Bernuau, INSERM U 327, FaculteÂ de regeneration, there is a rapid and transient induction of MeÂ decine Xavier Bichat, 16 rue Henri Huchard, 75018 Paris, NF-kB DNA binding, consisting mainly of p50/p65 France; E-mail: [email protected] Received 22 March 2001; revised 29 May 2001; accepted 30 May heterodimers and p50 homodimers (Tewari et al., 1992; 2001 Cressman et al., 1994; FitzGerald et al., 1995). NF-kB and JunD co-operation in hepatocytes M Rahmani et al 5133 Although the signi®cance of NF-kB induction during liver regeneration has not been completely clari®ed, important eects would be to prevent apoptosis and to allow for normal cell cycle progression (Iimuro et al., 1998). Cross-talk between AP-1 and the nuclear receptors for glucocorticoids and retinoic acid (Jonat et al., 1990; Lucibello et al., 1990; Miner and Yamamoto, 1992; Yang-Yen et al., 1990; Kerppola et al., 1993; Maroder et al., 1993) is well-documented. More recently, interaction of AP-1 with a number of other transcription factors (C/EBP, Smads, STATS, SP1) Figure 1 TNF-a increases AP-1 DNA binding activity. Equal amounts (10 mg) of nuclear proteins from hepatocytes either (Ubeda et al., 1999; Zhang et al., 1998; Liberati et al., unstimulated (C) or stimulated with EGF (20 ng/ml), HGF 1999; Zhang et al., 1999; Kardassis et al., 1999), co- (10 ng/ml) or TNF-a (100 U/ml) for the indicated times were activators (CBP/p300) (Bannister et al., 1995; Lee et incubated with a 32P-labeled TRE probe and electrophoresed in a al., 1996), or cell cycle regulatory proteins (pRb) 6% polyacrylamide gel. For competition experiments, a 100-fold (Nishitani et al., 1999; Nead et al., 1998) has been molar excess of unlabeled AP-1 probe was added to the incubation mixture (competitor) described. These interactions take place when AP-1 proteins are bound to their cognate cis element, but they can also occur independently of the AP-1 site through protein ± protein interactions (Ubeda et al., 1999). Cooperation of Jun or Fos proteins with the This pattern is in agreement with previous studies in p65 subunit of NF-kB producing potentiated biologi- the liver and in isolated hepatocytes, showing that p65 cal function has been previously reported (Stein et al., and p50 are the main constituents of NF-kB in liver 1993). The striking parallelism of induction of AP-1 cells (Tewari et al., 1992; Kirillova et al., 1999). and NF-kB observed when hepatocytes proliferate Stimulation with TNFa induced a biphasic activation and/or dierentiate led us to investigate whether AP-1 of the binding with an early stimulation at 30 min, and NF-kB cooperate in normal rat hepatocytes followed by a decrease of the binding at 2 h, and a under basal conditions and after treatment with second more important increase at 4 h and 6 h (Figure epidermal growth factor (EGF) and hepatocyte 2b). The binding had returned to unstimulated level by growth factor (HGF), two cell mitogens, and tumor 24 h (data not shown). Activation with EGF and necrosis factor a (TNFa), a cytokine involved in the HGF induced very similar patterns of activation of liver regenerative response in vivo and a potent NF-kB binding, with an early increase at 30 min inducer of NF-kB. followed by a decrease at 2 h and a second peak by 4 h (Figure 2).

Results AP-1 cooperates with NF-kB for transactivation on the NF-kB site TNF-a increases the binding activity to the We next determined whether AP-1 proteins could AP-1 and NF-kB sites modulate the transactivating capacity of NF-kB. As previously reported (Nadori et al., 1997; Rahmani Hepatocytes were transiently transfected with the D- et al., 1999) both EGF and HGF induced a rapid 121HIV-1LTR-CAT plasmid which contains a CAT increase in the binding of nuclear proteins to a reporter gene under the control of the two NF-kB sites consensus TRE probe, within 30 min of stimulation from the human HIV LTR, or with D-76HIV-1LTR- and persisting for the ®rst 6 h (Figure 1). TNF-a CAT which lacks the two NF-kB sites (Stein et al., stimulated the TRE-binding activity more weakly, with 1993). The cells were left untreated or they were a peak at 2 h, and a persistent slight elevation of the stimulated with EGF, HGF or TNF-a. As shown on binding by 6 h (Figure 1). We next investigated Figure 3a, in cells transfected with the D-121HIV- whether EGF and HGF induced activation of the 1LTR-CAT plasmid, all three treatments induced a binding of nuclear proteins to a consensus NF-kB site, 2.5-fold activation of CAT activity. No CAT activity in the same way as TNF-a. In nuclear extracts from was detectable in hepatocytes transfected with the D- unstimulated hepatocytes, two retarded bands of equal 76HIV-1LTR-CAT (Figure 3a). To ascertain that this intensity were detected. Their speci®city was indicated stimulating eect could be observed in another by their complete disappearance in the presence of promoter context, we also transfected hepatocytes with excess unlabeled NF-kB probe, while addition of an the Igk-Cona-Luc plasmid, which contains three NF- excess of probe with a point mutation in the NF-kB kB sites from the promoter of the light chain of site did not decrease the intensity of the two retarded immunoglobulins, and the minimal conalbumin pro- bands (Figure 2a). Supershift analysis with anti-p50 moter. The same 2.5-fold stimulation was observed and anti-p65 antibodies partially disrupted the two (Figure 3b). Therefore, in hepatocytes EGF and HGF bands and induced supershifted bands (Figure 2b). signaling pathways stimulate NF-kB with the same

Oncogene NF-kB and JunD co-operation in hepatocytes M Rahmani et al 5134

Figure 2 (a) Speci®city of the detection of NF-kB binding. Nuclear extracts were incubated with a 32P-labeled HIV NF-kB probe in the absence or in the presence of increasing concentrations of wild type or mutated NF-kB probe, and electrophoresed in a 6% polyacrylamide gel. NS: non speci®c band. (b) Activation of NF-kB binding activity by EGF, HGF and TNF-a. Equal amounts (10 mg) of nuclear protein extracts from hepatocytes non stimulated (C) or stimulated with EGF (20 ng/ml), HGF (10 ng/ml), or TNF-a (100 U/ml) for the indicated times were incubated with a 32P-labeled HIV NF-kB probe and electrophoresed in a 6% polyacrylamide gel

ecacy as TNF-a. Speci®city of the CAT activity was checked by cotransfecting this construct with a TRE- checked by cotransfection with the plasmid IkBa A32/ tk Luc reporter plasmid which contains the AP-1 A36, encoding for a mutated non phosphorylable IkB consensus TRE element from the collagenase gene. protein which blocks NF-kB nuclear translocation and Under these conditions, expression of the TRE-driven activity. As expected, in the cotransfected hepatocytes gene was decreased by about 60%, compared to the there was a drastic decrease in both the basal and the level of gene expression in the presence of the CMV CAT activity induced by EGF, HGF and TNF-a empty plasmid (Figure 4a). TAM67 cotransfection (Figure 3c). To demonstrate that the stimulating eects with the D-121HIV-1LTR-CAT induced a 70% of EGF, HGF and TNF-a on NF-kB transactivation decrease of the basal CAT activity while the CMV was a response speci®c to these cytokines, we also control vector had no eect (Figure 4b). Following transfected hepatocytes with a 46SBE reporter induction by EGF, HGF and TNF-a, stimulation of plasmid, which contains four copies of the Smad CAT activity (by 2 ± , 2.8- and threefold, respectively) responsive element involved in TGF-b signalization was still observed (Figure 4b). Thus, the presence of (Zawel et al., 1998) under the control of the Luc gene. functionnally active AP-1 dimers appears crucial for Stimulation with EGF, HGF or TNF-a did not induce the basal function of NF-kB proteins on the NF-kB any signi®cant activation of the Luc gene (data not site in hepatocytes. EGF, HGF and TNF-a stimulate shown). NF-kB activity on their own, but AP-1 proteins do We next examined whether AP-1 activity in¯uences not play a synergistic eect on this induced NF-kB NF-kB transactivation. To block AP-1 activity, we transactivation. On the other hand, NF-kB inactiva- used TAM67, a c-jun dominant negative expression tion by the IkBa A32/A36 plasmid did not decrease vector which blocks the activity of all endogenous the basal activity of a 56TRE-tk-CAT reporter Jun and Fos proteins by forming non-functional plasmid (Figure 4c) indicating that there is no heterodimers (Brown et al., 1993, 1994, 1996). The reciprocal cooperation of NF-kB on a TRE site in eciency of TAM67 in inhibiting AP-1 activity was hepatocytes.

Oncogene NF-kB and JunD co-operation in hepatocytes M Rahmani et al 5135

Figure 3 EGF, HGF and TNF-a stimulate NF-kB transactivation. (a) and (b) Hepatocytes were electroporated with D-121HIV- 1LTR-CAT or D-76HIV-1LTR-CAT (a)orIgk-Cona-Luc or empty Cona-Luc (b). They were left untreated or treated with EGF (20 ng/ml), HGF (10 ng/ml) or TNF-a (100 U/ml). After 24 h, normalized CAT or Luc activity was determined. All values are expressed relative to samples obtained from unstimulated cells transfected with the D-121HIV-1LTR-CAT (arbitrarily set to 1). Bars indicate mean+s.e.m. of three independent transfections (c). Speci®city of NF-kB activation. Hepatocytes were cotransfected with D-121HIV-1LTR-CAT and the IkBa A32/A36 plasmid or the empty vector pRc/CMV. Cells were left untreated or they were stimulated with EGF (20 ng/ml), HGF (10 ng/ml) or TNF-a (100 U/ml) for 24 h. Cells were then lysed and normalized CAT activities were measured. Value of the unstimulated sample from hepatocytes transfected with the control empty vector was arbitrarily set to 1. The results are the mean+s.e.m. from three independent experiments

(Suzuki et al., 1991), the level of activation of the TRE- JunD is involved in the cooperation of AP-1 with NF-kB tk Luc reporter gene after cotransfection of the c-jun To further determine which AP-1 protein(s) is(are) and junB expression vectors was higher than after involved in the cooperation with NF-kB transactiva- cotransfection of the junD expression vector (Figure tion, we analysed the eect of the overexpression of 5b). dierent Fos and Jun proteins on the basal and the To clarify which domain of JunD is involved in NF- cytokine-induced transactivation on the NF-kB site. As kB cooperation, we used a N-terminal deletion mutant shown on Figure 5a, only JunD overexpression of JunD, DJDN1, which is devoid of transactivation signi®cantly increased (by threefold) the basal CAT activity (Hirai et al., 1990). As with RSV-JunD, expression from the D-121-1 LTR-CAT construct. The cotransfection of the DJDN1 plasmid with the Igk- threefold activation of NF-kB transactivation was Cona-Luc reporter plasmid increased about threefold maintained, but not increased, by EGF, HGF and the basal Luc activity (Figure 5c), indicating that the TNF-a in hepatocytes overexpressing JunD. Simulta- N-terminal transactivation domain of JunD is not neous overexpression of JunD with Fra1, the most necessary for its cooperation with NF-kB. To abundant Fos protein found in hepatocyte AP-1 dimers determine whether there is a physical association (Nadori et al., 1997; Rahmani et al., 1999), did not between JunD and NF-kB, EMSA were performed amplify further NF-kB transactivation (data not using the HIV NF-kB probe and nuclear extracts shown). These data indicated that only JunD increased prepared from hepatocytes transiently transfected with the basal NF-kB activity. We checked that the the JunD and the p65 expression vectors. Addition of ineciency of the other Jun proteins, c-Jun and JunB anti-JunD antibody to the binding reaction did not in stimulating NF-kB was not an artefact due to a less decrease the intensity of the retarded band (data not potent transactivating eect of RSV-c-jun and RSV- shown). Similar negative results were obtained with the junB. As expected from previous data in the literature H2 NF-kB probe (data not shown). We also failed to

Oncogene NF-kB and JunD co-operation in hepatocytes M Rahmani et al 5136

Figure 4 AP-1 inhibition decreases NF-kB transactivation. (a) Isolated hepatocytes were cotransfected with the TRE-tk-Luc plasmid and TAM67 or the empty CMV plasmid. After 24 h normalized Luc activity was measured. (b) Isolated hepatocytes were cotransfected with the D-121HIV-1LTR-CAT and TAM67 or the CMV empty plasmid. They were left untreated or treated with EGF (20 ng/ml), HGF (10 ng/ml) or TNF-a (100 U/ml). After 24 h, the cells were lysed and normalized CAT activity was measured. Value of the unstimulated sample from CMV transfected hepatocytes was arbitrarily set to 1. Bars indicate the mean of two independent experiments. (c) Isolated hepatocytes were cotransfected with 56TRE-tk-CAT or the empty plasmid, and either IkBaA32/A36 or pRc/CMV. After 24 h cells were lysed and CAT activities were measured and normalized to the Luc activity. The data, expressed as d.p.m./mg protein, are the mean of two independent experiments

detect p65/JunD complexes by coimmunoprecipitation activity were produced, indicating ecient basal experiments (data not shown). However, indirect transcriptional activity of p65 in this system. evidence for the participation of AP-1 proteins to the Stimulation of cotransfected cells with EGF or binding to the NF-kB site was provided by examining HGF induced a twofold increase in p65-mediated EMSA from hepatocytes transiently transfected with transcription, while surprisingly TNF-a did not TAM67. Whereas there was no decrease of the binding activate p65 transactivation (Figure 6), even when activity to the AP-1 site in extracts from TAM67- TNF-a doses as high as 1000 U/ml were used (data transfected compared to CMV-transfected hepatocytes, not shown). Absence of activation of p65 transacti- as previously reported by others (Brown et al., 1993, vation by TNF-a was also found in two hepatoma 1994), binding to the NF-kB probe was slightly cell lines, HepG2 and HuH7 (data not shown), diminished in extracts from TAM67-transfected hepa- raising the possibility that TNF-a-mediated signaliza- tocytes (data not shown). tion in hepatocytes depends on the activation of NF-kB proteins other than p65. To test a possible implication of p65/p50 dimers in TNF-a-induced Functional cooperation between JunD and p65 transactivation, we cotransfected the p65-Gal4 con- The main NF-kB subunits present in the liver are struct with mp50, a plasmid allowing the expression p65 and p50 (Tewari et al., 1992). Since p50 is of the full length mouse p50. Such coexpression did devoid of transactivating function, we tested whether not modify p65 transcriptional activity in unstimu- JunD cooperates with p65 by using a simple `Gal lated hepatocytes or in TNF-a-treated cells (data not hybrid' mammalian system. Hepatocytes were tran- shown). Overexpression of JunD increased 1.6-fold siently cotransfected with a Ti-56Gal4-Luc reporter the basal Luc activity (Figure 6) whereas over- construct, which contains ®ve copies of the Gal4 expression of the other Fos and Jun proteins did DNA binding site in its promoter, and a p65-Gal4 not enhance the basal transcriptional activity of p65 plasmid allowing the expression of a full-length p65- (data not shown). When hepatocytes overexpressing Gal4 fusion protein. In unstimulated cotransfected JunD were stimulated with EGF and HGF, the hepatocytes, easily detectable amounts of Luc magnitude of Luc activation (twofold) remained

Oncogene NF-kB and JunD co-operation in hepatocytes M Rahmani et al 5137

Figure 6 Functional cooperation between JunD and p65. Hepatocytes were cotransfected with Ti-56Gal4-Luc and p65- Gal4 or the empty pSG4 plasmid in the absence or presence of RSV-JunD. The cells were left untreated, or were treated with EGF (20 ng/ml), HGF (10 ng/ml) or TNFa (100 U/ml). After 24 h, cells were lysed and normalized Luc activity was measured. Value of sample from untreated cells transfected in the absence of JunD was arbitrarily set to 1. The data are the mean+s.e.m. of three independent experiments

activity of NF-kB. For this purpose, hepatocytes were Figure 5 JunD cooperates with NF-kB. (a) Hepatocytes were isolated from the liver of mice with a targeted cotransfected with D-121HIV-1LTR-CAT and various Fos and disruption of the junD gene. These animals develop Jun expression vectors. They were left untreated or treated with normally and are viable, but during adulthood male EGF (20 ng/ml), HGF (10 ng/ml) or TNFa (100 U/ml). After mice develop multiple age-dependent defects in repro- 24 h, the cells were lysed and normalized CAT activity was measured. Value of the unstimulated cells transfected with the duction, hormone imbalance and spermatogenesis empty RSV plasmid was arbitrarily set to 1. The results are the (TheÂ pot et al., 2000). We compared the level of NF- mean+s.e.m. of three independent experiments. (b) Hepatocytes kB transactivation in junD7/7 vs junD+/+ isolated were transfected with the TRE-tk-Luc plasmid and a Jun hepatocytes, using two dierent NF-kB reporter expression vector. After 24 h normalized Luc activity was measured. (c) Hepatocytes were transfected with Igk-Cona-Luc constructs. With the D-121HIV-LTR-CAT reporter alone or in association with RSV junD, a N-deleted JunD, construct, the level of basal transactivation was DJDN1, or the RSV plasmid. After 24 h, cells were lysed and decreased by 50% in JunD7/7 hepatocytes compared normalized Luc activity was measured. Value of the sample from to JunD+/+ hepatocytes (Figure 7a). To determine the cells transfected with the empty RSV plasmid was arbitrarily set activity of an endogenous NF-kB-regulated gene, we to 1. The data are the mean+s.e.m. of three independent experiments also tested the activity of the plasmid pRD1(742,+14)-Luc, which contains the Luc gene under the control of the minimal cyclin D1 promoter including the proximal NF-kB site (Hinz et al., 1999). Using this plasmid, we again detected a 50% inhibition similar to that in activated hepatocytes non-trans- of Luc expression in JunD7/7 hepatocytes compared fected with RSV-junD (Figure 6), con®rming that with JunD+/+ hepatocytes (Figure 7a). It is unlikely JunD does not synergize with cytokines for inducing that overall inhibition of the transcriptional machinery NF-kB transactivation. Expression of JunD in the occurred in JunD7/7 hepatocytes, since transactivation absence of p65 did not induce any signi®cant of other promoters, such as a SBE-driven gene did not transactivation of the Ti-5Gal-Luc construct, in the dier between JunD+/+ and JunD7/7 hepatocytes (data absence or in the presence of EGF, HGF or TNF-a not shown). To ensure that de®cient NF-kB transacti- (Figure 6), eliminating a non-speci®c interaction of vation was not a consequence of a decreased expression JunD with this promoter. of Rel/NF-kBorNF-kB regulator genes in JunD7/7 livers, we looked at the level of IkBa, p65 and p50 mRNA using a ribonuclease protection assay. As NF-kB functional activity is depressed in shown on Figure 7b, there was no signi®cant dierences junD knockout mice in the levels of IkBa, p50 and p65 mRNAs in JunD7/7 We next investigated whether the absence of JunD in vs JunD+/+ hepatocytes. Western blot analysis of hepatocytes in vivo resulted in a reduced functional nuclear proteins also indicated that comparable levels

Oncogene NF-kB and JunD co-operation in hepatocytes M Rahmani et al 5138 demonstrated by the stimulation of NF-kB transactivation upon overexpression of JunD, but not of the other Jun or Fos proteins. Finally, functional interaction implicating a protein ± protein interaction between JunD and the p65 NF-kB subunit was demonstrated. Activation of primary hepatocytes with TNF-a induced a biphasic activation of NF-kB binding, with an early increase at 30 min followed by a return to basal level, and a second later peak of binding activity by 4 and 6 h of stimulation. A similar pattern of activation has previously been described in TNF-a- treated HepG2 cells (Han and Brasier, 1997). The early accumulation has been related to nuclear translocation of a pool of constitutive NF-kB proteins due to proteolysis of IkB, and the late induction peak to synthesis of a new pool of NF-kB proteins (Han and Brasier, 1997). In EGF- and HGF-treated hepatocytes, we also observed a biphasic pattern of NF-kB binding induction. The transcriptional activity of NF-kB was stimulated by EGF and HGF to a similar extent as by TNF-a. Transcriptional activation of a NF-kB-dependent gene in EGF- and HGF-stimulated hepatocytes Figure 7 JunD7/7 hepatocytes have impaired NF-kB function. has never been described before. Several mechanisms +/+ 7/7 (a) Hepatocytes freshly isolated from JunD or JunD mice may activate the function of NF-kB. For example, were transfected with the D-HIV-LTR-CAT or the pRD1(742,+14)-Luc plasmids. Normalized NF-kB transactiva- EGF and HGF activate the Jun-N-terminal kinase, tion was measured after 24 h. The data represent the mean of two extracellular regulated kinase and p38 mitogen acti- dierent transfection experiments. (b) GuSCN lysates prepared vated protein kinase cascades, which are known to be from hepatocytes isolated from JunD+/+ or JunD7/7 mice were able to regulate NF-kB transcriptional activity inde- hybridized with 32P-labeled IkBa, p50, or p65 cRNAprobes, and a GAPDH cRNA probe as an internal standard. Protected pendently of IkB degradation and NF-kB nuclear fragments were separated by PAGE. Quantitative analysis was translocation (Schulze-Ostho et al., 1997; Finco et al., made by counting with the Instant Imager. (c) Western blot 1997; Vanden Berghe et al., 1998). Other potential analysis of IkBa, p50 and p65. Equal amounts of total cellular mechanisms of NF-kB activation by EGF include extracts (60 mg) (for IkBa) or nuclear extracts (30 mg) prepared as activation of protein kinase A, which can phosphor- indicated in Materials and methods were analysed by Western blotting using antibodies speci®c for IkBa, p50 or p65 ylate p65 at serine 276 and activate its function (Zhong et al., 1997), or induction of IkBa phosphorylation and degradation (Sun and Carpenter, 1998). Using transient transfection with TAM67, a dominant-negative mutant of c-jun which blocks the of IkBa, p50 and p65 proteins were present in JunD7/7 activity of all endogenous Jun and Fos proteins by and JunD+/+ hepatocytes (Figure 7c). forming non-functional heterodimers (Brown et al., 1993, 1994), we showed that both the basal and the cytokine-induced NF-kB transactivation were drasti- Discussion cally suppressed by inhibition of AP-1 function, implying an important role of AP-1 proteins in the Our studies were designed to determine whether functional activity of NF-kB in normal hepatocytes. cooperation between NF-kB and AP-1 occurs in non- Accordingly, down regulation of NF-kB target genes proliferating rat hepatocytes or in hepatocytes stimu- in keratinocytes transfected with TAM67 was also lated to proliferate by treatment with either EGF or recently reported (Li et al., 2000). Overexpression of HGF, two well characterized hepatocyte mitogens, or individual Fos and Jun proteins indicated that only TNF-a, a cytokine involved in the proliferative JunD exerted a stimulating eect on basal NF-kB response of liver cells after partial hepatectomy in vivo transactivation. Further analysis of the cooperation and a strong inducer of NF-kB (Menegazzi et al., 1997; between JunD and NF-kB was provided by the use of Yamada et al., 1997; FitzGerald et al., 1995; Tewari et the Gal4 `one hybrid' technique, which indicated al., 1992). cooperation between JunD and p65 through a We show that stimulation by these three cytokines protein ± protein interaction. However, since we did induced a parallel induction of AP-1 and NF-kB not succeed in coimmunoprecipitating p65 with c-Jun, binding and activated NF-kB-dependent gene tran- demonstration of the association of the two proteins scription. Inactivation of AP-1 by transient transfec- was not possible. Therefore, an indirect mechanism of tion of a dominant negative c-jun drastically inhibited cooperation between a Jun-inducible protein and p65 the constitutive NF-kB transactivation. A predominant cannot be completely excluded. The basic-leucine role of JunD in enhancing basal NF-kB activity was zipper region of the AP-1 proteins was previously

Oncogene NF-kB and JunD co-operation in hepatocytes M Rahmani et al 5139 identi®ed as the molecular basis of the in vitro Materials and methods interaction between AP-1 and NF-kB (Stein et al., 1993). Our results showing that overexpression of an Animals N-truncated form of JunD deleted of its transactivating domain did not decrease AP-1/NF-kB cooperation Hepatocytes were isolated from adult male Sprague-Dawley rats (Charles River) weighing 180 ± 200 g. Hepatocytes were ®ts well with a possible involvement of the basic also isolated from adult junD7/7 mice aged between 6 and 8 leucine-zipper domain of JunD. Synergistic transacti- weeks and age-matched junD+/+ littermates. These mice vation by cooperation of NF-kB and AP-1 dimers derived in a C57BL/6 background have been described bound to their own sites has been well documented (TheÂ pot et al., 2000). Animals were maintained on (Thomas et al., 1997; Mastronarde et al., 1998; Tsuboi commercial chow and water ad libitum. et al., 1994; Kralova et al., 1996). There is now increasing evidence that Fos and Jun proteins can also Reagents cooperate with other regulatory proteins independently of the AP-1 site. Thus, all Jun proteins may EGF, HGF and TNFa were obtained from R&D systems. play a role of superactivator by binding to the Antibodies to p65 and p50 NF-kB subunits, c-Fos, Fra1, c- Jun, JunB and JunD were purchased from Santa Cruz and ubiquitous transcription factor SP1 on the promoter used at the concentration suggested by the manufacturer. of the p21WAF1/cip1 gene (Kardassis et al., 1999). Similarly, all Jun proteins may associate in vivo with the Smad3 and Smad4 proteins (Zhang et al., 1998; Hepatocyte culture Liberati et al., 1999; PeÂ ron et al., 2001). Jun proteins Rat hepatocytes were isolated by collagenase perfusion therefore appear to participate to the formation of (Seglen, 1976), as modi®ed by Balavoine et al. (1990). The highly organized complexes of transcription factors hepatocytes were puri®ed by Percoll gradient centrifugation thus providing a potent means of amplifying gene (Wang et al., 1985), and viability was found to be 485% by responses. Previous identi®cation of AP-1 proteins trypan blue exclusion. Hepatocytes were suspended in cooperating individually with Rel proteins on the NF- William's E medium (Life Technologies) supplemented with 100 IU/ml penicillin, 100 mg/ml streptomycin and 10% fetal kB site of the promoter of speci®c cellular genes, such bovine serum (FBS), and they were plated on collagen-coated as the tenascin gene, or the HIV-1 gene have been Petri dishes. For isolation of mouse hepatocytes, the two step provided (Mettouchi et al., 1997; Yang et al., 1999). collagenase perfusion method was adaptated to the smaller In HeLa cells and F9 embryonal carcinoma cells, c- size of the liver, using reduced perfusion rates. Fos and c-Jun but not JunB or JunD were found to synergize for NF-kB cooperation (Stein et al., 1993). Electrophoretic mobility shift assays (EMSA) Our study is the ®rst to demonstrate a cooperation of JunD on a NF-kB site. Speci®c functions have been Nuclear extracts were prepared by the method of Andrews ascribed to individual Jun proteins. JunD diers from and Faller (1991) with minor modi®cations. Cells were the other jun genes, by exerting a negative rather than suspended in buer A (10 mM HEPES/KOH, pH 7.9, 1.5 mM MgCl2,10mM KCl, 1 mM dithiothreitol, 1 mM a positive eect on cell proliferation and transforma- phenylmethylsulfonyl ¯uoride, 1 mg/ml leupeptin, 1 mg/ml 7/7 tion (Pfarr et al., 1994). JunD ®broblasts isolated aprotinin, 0.5 mM spermidine), and centrifuged at 500 g at from mice de®cient in junD gene were recently shown 48C for 30 s. The nuclear pellet was suspended in 20 mM to be more sensitive to UV-induced apoptosis and HEPES/KOH, pH 7.9 containing 25% glycerol, 420 mM liver cells were more susceptible to TNF-mediated NaCl, 1.5 mM MgCl2, 0.2 mM ethylenediaminetetraacetic cytotoxicity in junD KO mice (Weitzman et al., 2000). acid (EDTA), 4 mM dithiothreitol, 1 mM phenylmethylsulfo- Since NF-kB exerts a widely demonstrated protective nyl ¯uoride, 1 mg/ml leupeptin, 1 mg/ml aprotinin, and eect on apoptosis (Liu et al., 1996), our present centrifuged at 18 000 g at 48C for 2 min. The supernatants demonstration of a NF-kB functional de®ciency in were aliquoted and stored at 7808C. Protein concentration was determined with the BCA protein assay reagent (Pierce). junD7/7 hepatocytes suggests that impaired activation Single-stranded sense and antisense oligonucleotides corre- of NF-kB-regulated anti-apoptotic genes could repre- sponding to the TRE site (5'-TAAAGCATGAGTCAGA- sent one mechanism underlying the higher sensitivity CACCTC-3'), the NF-kB site from the enhancer of the H2 of junD7/7 hepatocytes to apoptotic stimuli. This gene (5'-TCGAGGGCTGGGGATTCCCCATCTC-3') or the ®nding might be particularly relevant since we have NF-kB site from the HIV promoter (5'-GAGTGGG- shown that JunD is the major component of the AP-1 GACTTTCCAGGCTC-3') were synthesized (Genset, Paris, complex in rat hepatocytes (Nadori et al., 1998; France). Sense oligonucleotides were end-labeled with T4 Rahmani et al., 1999). Our observations underline polynucleotide kinase in the presence of [g-32P]ATP (5 000 Ci/ the functional importance of JunD as a component of mmol, Amersham). The labeled oligonucleotides were AP-1 in hepatocytes, and the speci®city of AP-1- annealed with corrresponding unlabeled antisense oligonu- mediated gene regulation in this cell type. The cleotides. Nuclear extracts (5 ± 20 mg) were incubated in binding buer (20 m HEPES, pH 7.9, 5 mM MgCl ,4mM simultaneous activation of the two transcription M 2 dithiothreitol, 20% glycerol, 0.1 mM phenylmethylsulfonyl factors AP-1 and NF-kB observed during the ¯uoride, 5 mM benzamidine, 2 mM levamisole, 0.1 mg/ml responses of hepatocytes to growth factors (our study) aprotinin, 0.1 mg/ml bestatin) containing 2 mg poly(dI-dC) and to a variety of other stimuli might constitute a and 32P-labeled double-stranded probe (36104 c.p.m.) for means for providing maximal expression of NF-kB 20 min at 48C. The reaction mixture was then loaded onto a regulated genes. 6% polyacrylamide gel in 0.09 M Tris borate, 2 mM EDTA,

Oncogene NF-kB and JunD co-operation in hepatocytes M Rahmani et al 5140 pH 8.0 buer, and electrophoresed at 11 V/cm for 2 h at MgCl2, 1% Triton X 100, 1% bovine serum albumin, 15 % 208C. The gels were dried and exposed to X-ray ®lm for glycerol, 1 mM EDTA and 1 mM DTT), centrifuged 10 min autoradiography. at 13 000 r.p.m. and stored at 7208C. Luc activity was For supershift analyses, 2 ml of antibody speci®c to the Jun determined with a luminometer (EGG), and was rapported to family (anti-c-Jun, anti-JunB, anti-JunD), or the Fos family the amount of proteins in the nuclear extracts determined members (anti c-Fos, anti-FosB, anti-Fra1, anti-Fra2), or with the Biorad reagent (Biorad). For measurement of CAT NF-kB proteins (anti-p50 or -p65) (all from Santa-Cruz) were enzyme activity, cell extracts were prepared as described incubated with the nuclear extracts for 1 h at 48C before (Nadori et al., 1997). CAT assays were performed according addition of the 32P-radiolabeled probe and electrophoresis. to Seed and Sheen (1988), after correction for protein content using the BCA protein assay reagent (Pierce). Reporter plasmids and expression vectors Ribonuclease protection assay D-121HIV-1LTR-CAT and D-76HIV-1LTR-CAT (Stein et al., 1993) were kind gifts from K Blender, Institut fuÈ r mRNA was quanti®ed with complementary RNA probes and Genetik, Karlsruhe, Germany). The 56TRE-tk-CAT plas- a ribonuclease protection assay, performed on cells directly mid was kindly provided by B Wasylyk (IGBMC, Illkirch solubilized in 5 M guanidine thiocyanate containing 0.1 M France). The transdominant negative IkBa A32/A36 plasmid EDTA, pH 7.0, as previously described (Kaabache et al., was supplied by R Weil (Institut Pasteur, Paris, France). 1995). The following cDNA fragments were used: a 286-bp TAM67 was a generous gift from DJ Birrer (Bethesda, fragment (nt153-438, Acc. No U36277) from mouse IkBa,a Maryland, MD, USA). The RSV expression vectors encoding 245-bp fragment (nt 667 ± 911, Acc. No M57999) from mouse c-fos,c-jun, junB, junD, fra-1 and fra-2 were prepared in M NF-kBp50, a 213-bp fragment (nt 535 ± 747, Acc. No 61909) Yaniv's laboratory. The mp50 plasmid constructed in RM from mouse NF-kBp65, produced by RT ± PCR, and a 851- Evans' laboratory was supplied by JW Lee (Chonnam bp XbaI/ApaI fragment from rat glyceraldehyde-3-phosphate National University, Kwangju, Korea). Ti-56Ga14-Luc dehydrogenase (GAPDH) (Fort et al., 1985). These fragments and p65Ga14 plasmids were kind gifts from X-F Wang were cloned into pBluescript II SK+(Stratagene) at HindIII/ (Duke University, Durham, NC, USA). pRc/CMV-p65 XhoI sites and 32P-labeled antisense RNA probes were originating from R Schmidt's laboratory was obtained from generated using T7 RNA polymerase (Promega). The RNAse A IsraeÈ l (Institut Pasteur, Paris, France). The Igk-Cona-Luc protection assay and polyacrylamide gel electrophoresis reporter plasmid was generously provided by G Cherqui (PAGE) were performed as previously described (Kaabache (FaculteÂ de MeÂ decine Saint-Antoine, Paris, France). The et al., 1995). Quantitative analysis was performed from gels DJDN1 deletion mutant of JunD has been described (Hirai et directly counted with the Instant Imager (Packard) and the al., 1990). the 46SBE-Luc plasmid was kindly provided by B ratio of the signals from protected fragments to GAPDH was Vogelstein (Howard Hughes Medical Institute Research calculated. Laboratories, Baltimore, MD, USA). The pRD1(742, +14)-Luc reporter plasmid was obtained by cloning a Western blot analysis XhoI/HindIII 56-bp fragment of the rat cyclin D1 promoter (742 to +14) into pGL3 (Promega). This fragment includes For total cellular extracts, cells were rinsed in PBS and lysed the cyclin D1 minimal promoter region and the proximal NF- in lysis buer containing 20 mM HEPES (pH 7.9), 20 mM kB binding site (Albanese et al., 1995; Hinz et al., 1999). The NaF, 1 mM Na3VO4,1mM glycerophosphate, 1 mM EDTA, integrity of this construct was checked by DNA sequencing 1mM EGTA, 0.2 % Nonidet P-40, 0.5 mM PMSF, 1 mM (Genome Express). DTT, 1 mg/ml leupeptine, 1 mg/ml aprotinine. Fifty mg total cellular extracts or 30 mg nuclear proteins prepared as above were fractionated by electrophoresis on a 10% SDS ± PAGE Cell transfection and electrophoretically transferred to nitrocellulose mem- Immediately after isolation, hepatocytes at a density of branes (Schleicher and Schull, CeÂ ra-Labo). Membranes were 206106 cells/0.8 ml PBS containing 5% FBS (Le Cam, 1995) treated as previously described (Rahmani et al., 1999), and were electroporated (Gene Pulser, Bio-Rad Laboratories) at incubated in rabbit polyclonal antibody to IkBa, p50 or p65 160 V and 960 mF in the presence of 30 mg reporter plasmid, (Santa-Cruz) followed by peroxidase-labeled anti-rabbit IgG 30 mg expression vector, and 30 mg RSV-CAT or RSV-Luc (Amersham) or anti-mouse IgG (Biosys). The blots were plasmid to correct for transfection eciencies. The total developed using an Enhanced Chemiluminescence kit (Amer- amount of DNA was adjusted to 150 mg with sonicated sham) and exposed to X-ray ®lm for 5 s to 3 min. salmon sperm DNA as a carrier. After electroporation, the hepatocytes were plated in the presence of 10% FBS for 2 h, and deprived of serum for 18 h. They were then incubated in serum-free medium in the presence or absence of EGF Acknowledgments (20 ng/ml), HGF (10 ng/ml) or TNFa (100 U/ml) for 24 h. We thank K Blender, B Binetruy, R Weil, J Birrer, JW Lee, For Luc assays, cells were washed in PBS, lysed for 15 min XF Wang, A IsraeÈ l and G Cherqui for providing plasmids on ice with lysis buer (25 mM Tris/HPO4, pH 7.8, 8 mM and M Yaniv for helpful discussions.

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Oncogene