Redundancy and Specificity F Mechta-Grigoriou Et Al 2379

The mammalian Jun proteins: redundancy and speci®city

Fatima Mechta-Grigoriou1, Damien Gerald1 and Moshe Yaniv*,1

1UniteÂ des virus oncogenes, CNRS URA 1644, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France

The AP-1 transcription factor is composed of a mixture transcription factors. These proteins are characterized of homo- and hetero-dimers formed between Jun and Fos by a highly charged, basic DNA binding domain, proteins. The dierent Jun and Fos family members vary immediately adjacent to an amphipathic dimerization signi®cantly in their relative abundance and their domain, referred as the `Leucine zipper' (Kouzarides interactions with additional proteins generating a and Zi, 1988; Landschulz et al., 1988). Dimerization complex network of transcriptional regulators. Thus, is required for speci®c and high anity binding to the the functional activity of AP-1 in any given cell depends palindromic DNA sequence, TGAC/GTCA (Rauscher on the relative amount of speci®c Jun/Fos proteins which et al., 1988; Gentz et al., 1989; Hirai and Yaniv, 1989; are expressed, as well as other potential interacting Ransone et al., 1989; Schuermann et al., 1989; Turner proteins. This diversity of AP-1 components has and Tjian, 1989). The dierent AP-1 dimers exhibit complicated our understanding of AP-1 function and similar DNA binding speci®cities but dier in their resulted in a paucity of information about the precise transactivation eciencies (Chiu et al., 1989; Hirai et role of individual AP-1 members in distinct cellular al., 1990; Kerppola and Curran, 1991b; Suzuki et al., processes. We shall discuss recent studies which suggest 1991). The N-terminal transactivation domain is more that dierent Jun and Fos family members may have divergent and may account for the distinct transactiva- both opposite and overlapping functions in cellular tion properties of the Jun proteins. c-Jun contains a proliferation and cell fate. Oncogene (2001) 20, docking site for the Jun N-terminal kinases (JNKs) 2378 ± 2389. which phosphorylate Serines 63 and 73 thereby enhancing its transcriptional potential and stability Keywords: cell cycle; transcription; ras signaling; (Smeal et al., 1991). The JunB protein contains a JNK apoptosis docking site but lacks N-terminal acceptor serine residues (Chiu et al., 1989) still a recent study claims that JunB can be phosphorylated by these kinases (Li Introduction et al., 1999). JunD lacks a functional JNK docking site (Kallunki et al., 1996) and is not phosphorylated by The AP-1 transcription factor participates in the these kinases. Nevertheless, JunD contains an acceptor control of cellular responses to stimuli that regulate Serine and can be phosphorylated by JNK in c-Jun/ proliferation, dierentiation, immune response, cell JunD heterodimers (Kallunki et al., 1996). death and the response to genotoxic agents or stress Comparison of c-Jun sequences among species (Angel and Karin, 1991). AP-1 is composed of Jun reveals high degree of homology. The sequence of the family members (c-Jun, JunB, and JunD) that can form mouse c-Jun protein shares 96, 81 and 72% identity either homo- or hetero-dimers among themselves. Jun with its human, chick and Xenopus counterparts, proteins also dimerize with Fos family members (c-Fos, respectively (Figure 1). The C-terminal part of the c- FosB, Fra1 and Fra2) (Angel et al., 1987, 1988a; Jun protein containing the basic region and the Curran and Franza, 1988; Halazonetis et al., 1988; Leucine-zipper is totally conserved amongst these Sassone-Corsi et al., 1988; Hirai and Yaniv, 1989). Jun species while divergences are observed in the N-terminal and Fos proteins also dimerize eciently with other transactivation domain. When compared with the transcription factors such as members of the ATF/ Drosophila Melanogaster Jun protein, sequence identity CREB (Hai and Curran, 1991) or Maf/Nrl (Kataoka et falls to 25% but remains highly similar (60%) in the al., 1994; Kerppola and Curran, 1994) families of bZip region. This observation prompted us to search proteins. for an homologous sequence in Caenorhabditis Elegans. The diferent members within the Jun or Fos We found a bZip-containing Blast sequence which has families of proteins share a high degree of sequence 34% identity and 54% similarity with the mouse bZip homology. Experiments based on the use of deletion domain. Moreover, as previously shown, the bZip and chimeric constructs of Jun and Fos proteins have domain found in the Pap1 protein, an AP-1 member revealed the modular nature of their structure. Both of the ®ssion yeast S. Pombe, has 27% identity and families belong to the bZIP group of DNA binding 46% similarity with its murine counterpart (Figure 1). The relative binding anities of distinct dimer combinations depend on the speci®c DNA sequence *Correspondence: M Yaniv and on the promoter context (Halazonetis et al., 1988; The mammalian Jun proteins: redundancy and specificity F Mechta-Grigoriou et al 2379

Figure 1 Sequence alignment of c-Jun proteins among dierent species. Mouse c-Jun protein is aligned with its human, chicken Xenopus and Drosophila counterparts. We have also added the sequence of Pap1, referred as the AP-1 member in the ®ssion yeast S. Pombe as well as the sequence of abZip-containing Blast sequence found in C. Elegans (accession number in GenBank: CAA90948.1). Each amino acid is de®ned by its one letter-code. A colour code is also used to better illustrate the similarities between c-Jun proteins. It is described as follows: short side chains amino acids (A, G) in dark blue, hydrophobic residues (V, L, P, I, M) and aromatic amino acid (F, W, Y) in green, charged amino acids (including basic residues ± R, K, H ± and acidic residues ± D, E ± in red) and nucleophilic (S, T), amide (N, Q) and cystein residues in purple. Phosphorylated Serines 63 and 73 are pointed by stars and DNA binding domain (DNA BD), nuclear localization signal (NLS) and dimerization domain are also indicated

Kerppola and Curran, 1991a; Ryseck and Bravo, and S phases of the cell cycle (Lau and Nathans, 1987; 1991). Such binding sites were identi®ed in viral Almendral et al., 1988). In the following sections, we enhancers and promoters, including the polyomavirus will review the function of the three members of the (Piette et al., 1987) and papillomavirus (Thierry et al., Jun family in dierent biological processes. 1992; Bouallaga et al., 2000). Many other AP-1 binding sites have been characterized in promoters or enhancers of cellular genes encoding enzymes involved in tissue I-mitotic response remodelling, like collagenase and stromelysin, in cell cycle controlling genes such as cyclin D1 or c-jun itself, From G0 to G1 in cytokine encoding genes like IL2 (for reviews see Vogt and Bos, 1990; Angel and Karin, 1991). Proto-oncogenes frequently participate in the regula- The role of AP1 in the control of cell proliferation tion of cell proliferation through signal transduction was extensively investigated, mostly at the cellular pathways which convert extra-cellular stimuli into gene level. Several lines of evidence suggest that the Jun and expression programs. Many of these genes are also Fos proteins are important targets for mitogenic signal activated during the G0 to G1 transition when transduction pathways. First, the AP-1 binding quiescent cells are stimulated by mitogens to re-enter sequence was originally identi®ed as a TPA responsive the cell cycle. To follow carefully the dierent element (TRE) and was shown to respond to the components of AP-1 during cell growth, our laboratory tumour promoter, TPA. Treatment of cultured cells has produced polyclonal and monoclonal antibodies with mitogenic agents resulted in a strong increase in speci®c for each member of the Jun and Fos families of AP-1 binding to a TRE sequence (Piette et al., 1988; proteins. These antibodies provided valuable tools to Angel and Karin, 1991). Upon serum stimulation, determine the relative concentration and the potential many quiescent cells displayed an increase in c-jun, dimer combinations under speci®c cellular growth junB and fos mRNAs (Herschman, 1991). Based on conditions. We have ®rst analysed the variations of their rapid, high and transient transcriptional up- the Jun and Fos protein levels and AP-1 activity in regulation in response to mitogenic agents, these agents normally cycling, resting and stimulated cells (Lalle- were de®ned as `immediate early genes' or `competence mand et al., 1997). This study allowed us to classify genes' and encode products necessary for entry into G1 these proteins into three sub-groups, according to their

Oncogene The mammalian Jun proteins: redundancy and specificity F Mechta-Grigoriou et al 2380 expression patterns c-Jun, JunD and Fra-2 are polymerase a and g subunits, thymidine kinase etc. AP- expressed in cycling mouse ®broblasts and are only 1 binding sites are also found in regulatory sequences mildly induced upon serum addition to the growing of cell cycle speci®c genes. For instance, the cyclin D1 cells. When cells become quiescent upon serum gene is directly regulated by AP-1 and links it to the starvation, c-Jun level decreases and JunD level cell cycle progression. Thus, the AP-1 transcription increases. JunD is partially degraded upon serum- factor does not only mediate the transduction of extra- induced re-entry into the cycle while c-Jun is strongly cellular stimuli in the G0 to G1 transition but may also stimulated under these conditions. In contrast to the provide a speci®c function in normally cycling cells. ®rst group, the basal level of JunB, c-Fos, FosB and DfosB proteins is low in cycling mouse ®broblasts but c-Jun: positive regulator of cell cycle The involvement strongly and rapidly increases after serum stimulation. of c-Jun in promoting normal cell growth has been ®rst Fra1 is the only member of the third group. documented by the use of neutralizing antibodies or Undetectable in cycling cells, Fra-1 stimulation follow- anti-sense RNA which block entry into S phase ing serum addition is delayed to later time points and it (Kovary and Bravo, 1991b; Riabowol et al., 1992; was shown to be transcriptionally activated by c-Jun/c- Smith and Prochownik, 1992). Moreover, overexpres- Fos dimers. The AP-1 DNA binding activity follows sion of c-Jun alters cell cycle parameters and increases the kinetic of Jun and Fos protein accumulation after the proportion of cells in S, G2 and M relative to G1 serum stimulation and the composition of AP-1 dimers phases of the cell cycle (Pfarr et al., 1994). The role of involved in DNA binding re¯ects their respective c-Jun in promoting cell growth has been further abundance. These experiments completed data from highlighted from studies of c-Jun de®cient mouse earlier studies on the G0 to G1 transition in SWISS- embryonic ®broblasts (Hilberg et al., 1993; Johnson 3T3 ®broblasts (Kovary and Bravo, 1991a, 1992). et al., 1993). Fibroblasts lacking c-Jun exhibit a severe These authors also showed that the amount of each proliferation defect. Cyclin D1 is only poorly activated Jun and Fos protein governs the relative proportion of in these cells leading to an inecient progression from the dierent dimers at any time. Finally, serum or G1 to S phase and cell cycle block (Wisdom et al., mitogen stimulation activates also pre-existing AP-1 1999). Moreover, c-Jun de®cient ®broblasts accumulate complexes through post-translational modi®cations. All the tumour suppressor p53 and its downstream target, Jun and Fos proteins are phosphorylated following the cyclin-dependent kinase inhibitor p21, suggesting their synthesis or after mitogenic stimulation suggesting that one particular function of c-Jun is the negative that both transcriptional and post-transcriptional regulation of p53 transcription in ®broblasts (Schreiber mechanisms are involved in the regulation of Jun and et al., 1999). These observations link directly c-Jun Fos proteins. dependent signalling to the cell cycle machinery and In summary, AP-1 composition and activity are demonstrate that c-Jun transduces a mitogenic response strikingly controlled at the G0 to G1 transition through two dual signalling pathways. On one hand, it involving both protein synthesis and post-translational activates cyclin D1 transcription and, on the other modi®cations. The dierent combinations of the AP-1 hand, it inhibits p21 accumulation through repression complex and the surrounding DNA sequences deter- of p53 expression. In this context, it is important to mine the binding anity for a given TRE. As discussed mention a recent study on the role of c-Jun in the further, AP-1 target genes are dierently regulated by mammalian UV-response. This study shows that cells distinct AP-1 dimers. As a consequence, AP-1 lacking c-Jun undergo a cell cycle arrest in response to composition would determine the speci®c pattern of UV irradiation whereas cells expressing c-Jun consti- target gene expression and allow prediction on the tutively exhibit an increased clonogenic potential as nature of the dimeric combinations which are well as an increased apoptotic response. These results physiologically relevant under various conditions. Very demonstrate that c-Jun is necessary for UV-irradiated few of the AP-1 target genes activated during the G0 to cells to escape from p53-dependent growth arrest and G1 transition were identi®ed. One of them is cyclin D1 to re-enter the cell cycle (Shaulian et al., 2000). that will be discussed below. JunB and JunD: negative regulators of the cell cycle The protein level of the dierent AP-1 members Cell cycle is dierently modulated during the cell cycle. The Progression through the cell cycle is tightly controlled analysis of c-Jun and JunB proteins in exponentially by the sequential activation of a genetic program growing Hela or 3T3 cells showed that the level of c- including the synthesis of the cyclins and the activation Jun remains constant during the cycle whereas JunB of the cyclin-dependent kinases (cdk). Few examples of protein level decreases during mitosis. The reduction of cross talk between transcription factors and cell cycle- JunB levels during the G2/M transition is the regulated kinases have already been described. Among consequence of its phosphorylation by the p34cdc2-cyclin them the E2F factor is activated by the phosphoryla- B kinase complex followed by its proteolysis. In tion and release of its negative regulator pRb by Cdk- contrast, c-Jun undergoes N-terminal phosphorylation Cyclins. Free E2F promotes cell cycle progression by during this transition increasing its transcriptional activating the synthesis of cyclins and of proteins activity (Bakiri et al., 2000). JunB exhibits a decreased essential for DNA replication including histones, DNA dimerization propensity and a weaker DNA binding

Oncogene The mammalian Jun proteins: redundancy and specificity F Mechta-Grigoriou et al 2381 activity than c-Jun. Thus, JunB is a less potent cell cycle dependent variations in Jun protein levels transcriptional activator than c-Jun, and it was shown constitute a novel reciprocal link between the cell cycle to repress c-Jun-mediated transactivation, most likely machinery and a transcription factor (Bakiri et al., by formation of inactive c-Jun/JunB dimers (Deng and 2000). Karin, 1993). In fact, c-Jun and even more phospho-c- Jun are strong activators of the cyclin D1 promoter. Cellular transformation On the contrary, JunB inhibits its transcription in co- transfection experiments. The c-Jun/JunB protein ratio Constitutive activation of growth-promoting signalling is thus an important parameter for the control of AP-1 pathway leads to partial transformation of cells. The target gene expression, including cyclin D1. The Ras proteins play a key role in signal transduction and decrease in the abundance in JunB at the beginning regulation of cell proliferation (Mulcahy et al., 1985; of G1 and the transient phosphorylation of c-Jun Feig and Cooper, 1988; Smith et al., 1989). Their during this time will generate a wave of cyclin D1 mutation leads to abnormal proliferation and is transcription (Bakiri et al., 2000). frequently associated with human tumours. Mitogenic In another recent study, p16INK4a was identi®ed as a signal transduction initiated by activation of growth direct transcriptional target of JunB. Induction of factor receptor leads to conversion of Ras into its p16INK4a expression leads to inhibition of cyclin D- GTP-bound active form. The best characterized target dependent kinase activity and reduced pRb phosphor- for the membrane-tethered Ras proteins is the Raf ylation state which in turn extends G1 phase length. protein kinase, involved in the control of MAPKK Stimulation of p16INK4a transcription de®nes JunB as a (Mitogen-activated protein kinase kinase) cascade, direct negative regulator of cell proliferation (PasseÂ gue which, in turn, activate the Erk (Extracellular regulated and Wagner, 2000). In essence, JunB negatively kinase) members of the MAP kinase family. Ras regulates G1/S transition by two distinct mechanisms, partially activates also the JNKs. Several lines of inhibition of cyclin D1 transcription by competing with evidence suggest that AP-1 transmits signals from c-Jun and activation of p16INK4a. In this context, it is activated Ras to the nucleus in transformed cells. important to mention that, in dermal ®broblasts, c-Jun and JunB display antagonistic functions on keratino- c-Jun: Positive regulator of Ras-mediated transforma- cyte proliferation and dierentiation. The relative tion Ras-transformation is correlated with increased amounts of c-Jun and JunB regulate the regeneration c-Jun transcriptional activity and an increase in AP-1- and maintenance of the epidermal tissue by acting as mediated gene expression (Alani et al., 1991; Westwick positive or negative mediators of IL1-regulated et al., 1994). Blocking AP-1 activity using dominant cytokine expression, such as KGF or GM-CSF. In negative forms of c-Jun or c-Fos proteins partially ®broblasts, JunB suppresses directly or indirectly the reverts the transformed phenotype of Ras-overexpres- expression of these genes whereas c-Jun is required for sing ®broblasts (Lloyd et al., 1991; Suzuki et al., 1994). the expression of both secreted cytokines which control The induction of AP-1-responsive genes by Ras is keratinocyte proliferation and dierentiation (Szabow- severely impaired in c-jun7/7 ®broblasts. Ras-expres- ski et al., 2000). sing c-jun7/7 cells lack transformed traits, including Similarly to JunB, overexpression of JunD in loss of contact inhibition, anchorage independence and immortalized ®broblasts slows down the proliferation tumorigenicity in mice showing that c-Jun is required of the cells and induces their accumulation in G1 (Pfarr for Ras-driven malignant conversion (Johnson et al., et al., 1994). Moreover, JunD7/7 immortalized ®bro- 1996). Deregulated expression of c-Jun or its viral blasts display higher proliferation rates than their wild counterpart v-Jun triggers transformation in avian type counterparts. However, as discussed below, JunD primary embryo ®broblasts (Vogt et al., 1989; Bos et is still essential for the survival of primary mouse al., 1990; Castellazzi et al., 1990). Although over- embryonic ®broblasts (Weitzman et al., 2000). JunD expression of c-Jun alone leads to partial transforma- may act as a negative regulator of cell growth by tion of mouse ®broblasts (SchuÈ tte et al., 1989), the co- establishing or maintaining the cells in a quiescent expression of both c-Jun and Fra1 strongly increases state. In ovarian follicles, JunD expression correlates the transformed traits. Hence, ectopic co-expression of with the transition from proliferating granulosa cells to c-Jun and Fra-1 in ®broblasts leads to a transformed dierentiated growth arrested luteal cells and may play phenotype with properties reminiscent of that of Ras- a critical role in terminating cell proliferation (Sharma transformed cell lines (Mechta et al., 1997). The and Richards, 2000). intrinsic transforming capacity of c-Jun and Fra-1 In conclusion, c-Jun and JunB or JunD display suggests that these two AP-1 members are crucial in opposite eects on cell cycle progression. While c-Jun establishing Ras-mediated transformation of NIH3T3 is a strong transcriptional activator of cyclin D1, JunD ®broblasts. An elegant study has addressed the is a weak positive activator and JunB even an inhibitor contribution of the dimer partner into the c-Jun- of this promoter. Increasing the abundance of JunB or dependent transformation and showed that c-Jun JunD relative to c-Jun decreases cyclin D1 transcrip- mediated transformation acts by two distinct pathways tion. The temporal changes in AP-1 composition and controlling either anchorage independence or autocrine activity during the M-G1 transition may constitute an growth, depending on its associated partner. In the ®rst interesting way to control cell cycle progression. The case, ATF2 is the dimerization partner while for the

Oncogene The mammalian Jun proteins: redundancy and specificity F Mechta-Grigoriou et al 2382 second it involves Fos or Fra proteins (van Dam et al., vation of Menin may aect the capacity of JunD to 1998). control p19Arf (see below). Simultaneous overexpression of c-Jun and c-Fos in JunB is another Jun member that antagonizes the bone enhances the rate of osteosarcoma formation, Ras-signalling pathway. Avian cells overexpressing suggesting that c-Jun and c-Fos proteins cooperate in JunB develop colonies in agar with a reduced eciency vivo in bone tumour development (Wang et al., 1995). than c-Jun and do not grow in low-serum containing These data illustrate that the c-Jun containing AP-1 medium (Castellazzi et al., 1990). Moreover, in mice, dimers are essential downstream eectors for the Ras JunB inhibits Ras- and Src-mediated neoplastic signalling pathway in cellular transformation, as well transformation and tumour growth by increasing as important players in multi-step carcinogenesis. p16INK4a gene transcription (PasseÂ gue and Wagner, 2000). An additional link between JunB and regulation JunB and JunD: negative regulators of Ras-mediated of cell proliferation was recently established in vivo by transformation Members of the AP-1 family are showing that the lack of JunB in the myeloid lineage dierently regulated by constitutive oncogenic Ras leads to increased cell proliferation and development of expression. While stable Ras-transformed ®broblasts myeloid leukaemia (PasseÂ gue et al., 2001). accumulate higher levels of c-Jun, junB, Fra-1 and Fra- All these observations suggest that temporal and 2 proteins relative to their normal counterparts qualitative modulation of AP-1 composition provides a (Mechta et al., 1997), JunD protein level decreases paradigm of speci®city in target gene regulation in strongly in Ras-transformed ®broblasts (Pfarr et al., dierent cell-transformation processes (Figure 2). Ras 1994). Moreover, in these cells, serum inducibility of transformation increases the cyclin D1 protein level, a JunB and Fra2 is markedly attenuated whereas the major regulator of the G1/S transition previously elevated levels of c-Jun and Fra-1 remain constant identi®ed as an AP-1 target gene. Increased cyclin D1 under these conditions. Furthermore Ras signalling expression in Ras-transformed cells shortens their G1 leads to a complete loss of serum inducibility of c-Fos phase (Liu et al., 1995; Robles et al., 1998). Moreover, and FosB (Mechta et al., 1997). Selective and sustained overexpression of cyclin D1 in immortalized ®broblasts MAPK activation induces also speci®c AP-1 members accelerates G1 progression and decreases serum like c-Jun whereas persistent activation of MAPK is requirement for growth. Thus, transformation by Ras not sucient to induce c-fos (Cook et al., 1999). leads to accumulation and activation of AP-1 tran- Besides their antagonistic roles on normal cell scription factor that in turn increases cyclin D1 protein proliferation, a striking dierence in response to Ras level and accelerates G1 progression. AP-1-mediated signalling between c-Jun and JunD was observed in induction of cyclin D1 explains how changes in AP-1 ras-transformed ®broblasts. The ratio of c-Jun to JunD composition mimic in part the proliferative eects of proteins in such cells correlates with the severity of the activated Ras. Dierences in the transcriptional transformed phenotype. While c-Jun co-operates with activities of JunB and JunD in comparison with c- Ras, excess JunD partially suppresses the transformed Jun explain how the two ®rst antagonize proliferation phenotype (Pfarr et al., 1994). Moreover, in cell lines and Ras-mediated transformation by dierently reg- derived from bovine-papillomavirus-induced tumours ulating cell cycle regulators like cyclin D1 (Pfarr et al., in mice, the c-Jun to JunD ratio increases as cells 1994; Mechta et al., 1997; Bakiri et al., 2000). In progress from aggressive ®bromatosis to malignant agreement with this hypothesis, JunB was shown to ®brosarcoma (Bossy-Wetzel et al., 1992). The down inhibit the transforming activity of a c-Jun (Schutte et regulation of JunD protein in dierent transformed cell al., 1989). In conclusion, junB and JunD proteins systems is consistent with the hypothesis that JunD function as a buer of Ras oncogene and modulate functions as an inhibitor of cell proliferation. For their potent c-Jun partner hence decreasing its harmful instance, in human ovarian tumours, the expression eects. level of junD is down-regulated when compared with normal ovarian epithelial cells (Neyns et al., 1996). Moreover, c-Jun protein is barely undetectable in normal mucosa whereas JunD protein is expressed at high level. Finally, while c-jun expression is signi®- cantly increased in human colorectal adenocarcinoma, junD expression remains steady (Wang et al., 2000) suggesting antagonistic roles of c-Jun and JunD in epithelial cell growth and colorectal tumorigenesis. In this context it is important to recall that JunD interacts with Menin encoded by the Men1 (Multiple Endocrine Neoplasia type 1) tumour suppressor gene. Menin inhibits the transcriptional activity of JunD. This provides an intriguing link between junD and growth control in vivo (Agarwal et al., 1999). It is possible that in cell types giving rise to this tumour, JunD acts as a Figure 2 Schematic representation of cell cycle gene regulation positive regulator of growth. Alternatively, the inacti- by Jun proteins

Oncogene The mammalian Jun proteins: redundancy and specificity F Mechta-Grigoriou et al 2383 Apoptosis and senescence induction and JNK activation (Lenczowski et al., 1997). Furthermore, AP-1 regulates directly the murine Apoptosis or programme cell death is commonly used anti-apoptotic Bcl-3 gene, which acts as a survival to eliminate damaged or non essential cells during factor for T cells in the absence of growth factors development, to maintain tissue homeostasis or to (Rebollo et al., 2000). In TNFa-treated HeLa cells, eliminate oncogene-expressing cells in adult organisms. blocking MAPK cascade prevents JNK activation Numerous studies have shown that AP-1 is involved in without aecting apoptosis (Liu et al., 1996) claiming the onset or control of apoptosis. AP-1 can trigger that JNK activation is not associated with TNFa- both pro- or anti-apoptotic signals depending on the mediated apoptosis. Finally, analysis of mice lacking dimer composition, the cell types or the death-inducing both jnk1 and jnk2 assign both pro- and anti-apoptotic treatments. functions to these kinases in early brain development (Kuan et al., 1999; Sabapathy et al., 1999). c-Jun: pro-apoptotic gene c-jun expression is induced The role of c-Jun in apoptosis is thus complex, in response to treatments which frequently triggers mostly cell-type speci®c and depends on the nature of apoptosis, among them UV, ionizing radiation, hydro- the death-inducing signal. One current hypothesis to gen peroxide and Tumour Necrosis Factor a (TNFa) explain these apparent contradictory results links c-Jun (Brenner et al., 1989; Devary et al., 1991; Manome et containing AP-1 dimers to de novo protein synthesis. al., 1993). c-jun and c-fos expression is also up- Hence, it has been shown that an active pro-apoptotic regulated in lymphocytes that undergo apoptosis in machinery pre-exists in the cells. AP-1 activity would response to IL6 depletion and c-jun or c-fos antisense be required for apoptosis in cells in which the death oligonucleotides protect these cells from death response eectors are present in limiting amounts and in which (Colotta et al., 1992; Preston et al., 1996). Other data protein synthesis is required to initiate the apoptotic strongly suggest that increased c-Jun activity (phos- suicide program (Karin et al., 1997). phorylation and neosynthesis) mediates apoptosis in neurons, after removal of survival factors (Watson et JunB and JunD: apoptosis and senescence pro- al., 1998). c-Jun mediates the neuronal dierentiation grams The study of JunD7/7 primary embryonic eects of NGF on PC12 cells (Xia et al., 1995). In ®broblasts showed that these cells undergo premature dierentiated PC12 cells, removal of NGF triggers the replicative growth arrest, also named senescence, and JNK signalling pathways and results in apoptosis of displayed increased sensitivity to death-promoting the cells (Leppa et al., 1998). Overexpression of c-Jun agents, including UV irradiation or TNFa treatment in sympathetic neurons induces apoptosis while a (Weitzman et al., 2000). JunD protects normal cells dominant-negative mutant of c-Jun blocks Nerve from senescence or from apoptosis induced by stress Growth Factor (NGF) withdrawal-induced pro- stimuli by modulating a p53-dependent pathway. grammed cell death (Estus et al., 1994; Ham et al., JunD7/7 ®broblasts exhibit increased levels of p19Arf. 1995). Moreover, c-Jun accumulates in neurons under- p19Arf sequesters Mdm2 blocking its capacity to going apoptosis by hypoxia in vivo (Dragunow et al., promote p53 degradation. Thus the increased level of 1993). Amino-terminal phosphorylation of c-Jun is also p19Arf in JunD7/7 cells leads to accumulation of p53 in required for ceramide-induced apoptosis of myeloid or the nucleus and to increased sensitivity to pro- lymphoid tumour cell lines (Verheij et al., 1996) and apoptotic signals. Moreover, the anti-apoptotic eect neuronal stress-induced apoptosis in vivo (Behrens et of JunD was con®rmed in vivo in TNFa-induced al., 1999). Finally, inducible c-Jun protein transmits an hepatic cell death. JunB overexpressing primary apoptotic death signal to immortalized ®broblasts that ®broblasts exhibit an increased p16INK4a protein level, can be antagonized by Bcl-2. This pro-apoptotic eect block cell cycle and also undergo premature senescence is speci®c of c-Jun and is not observed with JunB (PasseÂ gue and Wagner, 2000). JunB decreases the inducible constructions (Bossy-Wetzel et al., 1997). All proliferation rate of primary ®broblasts by bringing these results provide evidence that c-Jun is a potent up the p16INK4a known to favour the premature onset of inducer of programmed cell death in various cell types. senescence (Serrano et al., 1997). In conclusion, these data suggest that the dynamic c-Jun: anti-apoptotic gene Although a number of changes in Jun and Fos composition after stress-stimuli reports showed that AP-1 and JNK are involved in balance between distinct signals, including pro- or anti- apoptosis, other data argue that they are not essential apoptotic pathways, that play a key role in de®ning for cell death c-Fos appears to be anti-apoptotic in UV whether cells undergo senescence, apoptosis or survival. irradiated immortalized ®broblasts (Schreiber et al., 1999) whereas c-fos de®cient mice undergo normal Regulation of Juns by signalling pathways developmental apoptosis (Gajate et al., 1996). c-Jun7/7 embryos exhibit higher rates of apoptosis in liver than The biological function of AP-1 was initially restricted control animals (Eferl et al., 1999) and c-Jun is not to the transmission of growth promoting signals. essential for programmed cell death and axon growth Stimulation of dierent signal transduction pathways in the retina (Herzog et al., 1999). Moreover, in Jurkat led to the onset of distinct transcription programs and cells, AP-1 is not increased during Fas-mediated cell genes encoding Jun and Fos protein underwent similar, death although Fas cross linking leads to Ras but not identical, regulation in response to various

Oncogene The mammalian Jun proteins: redundancy and specificity F Mechta-Grigoriou et al 2384 stimuli. The expression of jun and fos gene products is and Erk2 MAPKs (mostly stimulated by growth temporally modulated suggesting that the AP-1 factors or phorbol esters) are inecient in phosphor- composition vary in the cell as a function of time ylating the N-terminal part of c-Jun although they and stimulus but also according to the cellular have been shown to phosphorylate a cluster of dierentiation state and its environment. De®ning inhibitory residues located next to the basic domain. how the dynamic transcription factor AP-1 responds to dierent environmental cues is an important issue in Regulation of JunB Sequence analysis of the junB an eort to understand their respective functions. promoter reveals several potential regulatory elements, well conserved amongst rat, mouse and human Regulation of c-Jun The c-jun gene is expressed in sequences (Kawakami et al., 1992). These include Stat3 many dierent cell types at low levels and its (Coer et al., 1995), CRE, SRE (Kitabayashi et al., expression is enhanced in response to many stimuli 1993) and CAAT enhancer binding domains. An IL6 including TPA (in a protein-kinase C-dependent (Interleukine-6) regulatory region containing an in- manner), growth factors (EGF, NGF, FGF), UV verted repeat (IR) was ®rst identi®ed as conferring irradiation or cytokines (Hill and Treisman, 1995; phorbol-ester and protein kinase A inducibility of the Karin and Hunter, 1995). The c-jun promoter region is junB gene (de Groot et al., 1991a). A GC-rich area highly conserved between mouse, rat and human and provides potential binding sites of several transcription the 200 nucleotides upstream of the murine and human factors including SP1 or Krox-24 (Egr1) but its transcription initiation site share 94% identity (Mechta function is not yet completely established. A SRE1 and Yaniv, 1994). The c-jun promoter contains sequence (Serum Responsive Element) confers junB- potential binding sites for several transcription factors, inducibility to growth factors and recruits ternary including SP1 (Rozek and Pfeifer, 1993), NF-Jun complex factor (TCFs). Another SRE site (SRE2) (Nuclear factor-jun) (Brach et al., 1992), CTF (CCAAT located distantly and functionally distinct from SRE1, Transcription Factor) and AP1 itself (Angel et al., as it does not respond to growth factor, suggests that 1988b). Induction of c-jun expression by serum, junB regulation proceeds through a complex interplay phorbolester, or TGFb is mediated through a TRE- between proximal and distal regulatory regions like site (jun1) located in the proximal region of the (Phinney et al., 1994). murine c-jun regulatory sequences. This TRE sequence JunB diers in biological properties from its diers from the consensus TRE by one base pair homologue c-Jun and was de®ned as a negative insertion and is preferentially recognized by a c-Jun/ regulator of cell growth. This functional dierence is ATF2 heterodimer rather than a conventional cJun/c- due to changes in amino-acid sequences which Fos AP-1 dimer (van Dam et al., 1993). In the distal decrease its dimerization potential (see above) and part of the c-jun promoter, a second AP-1 like site eliminate the JNK phosphorylation sites, and there- (jun2) also mediates the c-jun responsiveness to TPA or fore decrease its transactivation potency. First, insulin treatment and growth factor stimulation (Stein elevation of cAMP concentration arrests 3T3 ®bro- et al., 1992; Hagmeyer et al., 1993). The jun2 site also blasts in mid-G1 of the cell cycle and regulates binds c-Jun/ATF2 heterodimers. As ATF2 alone dierently c-Jun and JunB. While c-Jun expression is cannot confer TPA-inducibility of c-jun, the c-jun gene completely inhibited, JunB expression is induced in is thus up-regulated by its own product. Despite its response to PKA (cAMP-dependent Protein Kinase) inducible expression, most cell types contain a certain activation (Mechta et al., 1989). The negative eect basal level of c-Jun protein prior to stimulation and the of JunB on cell proliferation is also consistent with TRE site in its promoter is constitutively occupied its induction by inhibitors of cell growth such as (Angel et al., 1988b; Rozek and Pfeifer, 1993). TGFb (Transforming Growth Factor b) or inhibitors Following exposure to stimuli, the N-terminal Jun of myogenic dierentiation, like BMP2 (bone Mor- Kinases (JNK), members of the MAPK family, are phogenetic Protein), which alters cell lineage from the activated leading to the rapid phosphorylation of pre- myogenic to the osteogenic lineage (Chalaux et al., existing c-Jun and ATF2 proteins (Devary et al., 1992; 1998). Smad proteins were identi®ed as downstream Gupta et al., 1995). Phosphorylation of c-Jun on eectors of the TGFb-dependent signal transduction residues Ser63 and Ser73, located within its transacti- pathway. Smad binding elements (CAGACA) that vation domain, potentiates its transactivation proper- mediate responsiveness to several members of the ties by recruiting the coactivator protein, CBP, a TGFb family were identi®ed within the junB histone acetylase (Arias et al., 1994), thereby enhancing promoter (Jonk et al., 1998; Lopez-Rovira et al., c-jun transcription. Thus, stimulation of AP-1 activity 2000). Multimerization of this element leads to a in response to JNK-mediated stimuli (such as UV strong TGFb inducible enhancer that is also irradiation, ras activation, NGF removal or TNFa responsive to BMPs suggesting that this speci®c site treatment) proceeds through two distinct steps: en- mediates junB induction in response to TGFb and dogenous basal c-Jun protein is ®rst activated by post- BMP treatments. translational modi®cations and the phosphorylated The junB gene belongs to the immediate early genetic form of c-Jun induces subsequently its own transcrip- response of leukaemic myeloid (M1) cells to IL6, a tion by a positive auto-regulatory loop. The JNKs are cytokine which induces dierentiation and growth the only kinases which activate speci®cally c-Jun; Erk1 arrest in these cells. The analysis of junB induction in

Oncogene The mammalian Jun proteins: redundancy and specificity F Mechta-Grigoriou et al 2385 response to IL6 revealed that the cis-acting elements malformation in the heart out¯ow tract (Eferl et al., involved may be cell-type speci®c. The Stat3 and CRE- 1999). Embryos lacking junB or fra1 die earlier than c- like binding sites in the junB promoter are required for jun with multiple defects in extra-embryonic tissues. IL6 responsiveness in HepG2 liver cells (Nakajima et The JunB7/7 lethality results from abnormal interac- al., 1993). Conversely, the CAAT box and the IR were tion between the embryonic vascular system and the identi®ed as IL6-response element in M1 cells. These maternal circulation (Schorpp-Kistner et al., 1999). sites are constitutively occupied suggesting that IL6- Similarly, the placental labyrinth layer of Fra1 mutant mediated induction passes through post-translational embryos is largely a-vascular with decreased number of modi®cation of pre-existing complexes (Sjin et al., vascular endothelial cells (Schreiber et al., 2000). 1999). Injections of junB7/7 or fra17/7 ES cells into tetraploõÈ d wild type blastocysts rescued the placental defects and Regulation of junD In contrast with c-jun and junB, restored normal growth suggesting that de®ciencies in junD is relatively refractory to serum stimulation (Hirai extra-embryonic tissues are the prime cause of et al., 1989) and its basal expression remains high in embryonic lethality of junB7/7 or fra17/7 mutants. many cell types. The murine junD promoter contains In contrast to c-jun and junB genes, inactivation of several identi®ed binding sites: a CRE sequence junD does not lead to death in utero suggesting that recognized by CREB/ATF family members, a Sp1 site, JunD is not essential for embryonic development. an octamer motif, a CAAT box, a GC-rich region Homozygotes junD7/7 mice appear quite healthy, recognized by Krox-24 and an AP-1 consensus although they exhibit postnatal growth retardation sequence. The octamer motif is the major regulator and males display an age-dependent defect in repro- of junD expression and is speci®cally recognized by the duction (Thepot et al., 2000). ubiquitous Oct1 protein (de Groot et al., 1991b). Oct1 The speci®city and dierences of the biological confers the high basal level of junD transcription and functions of each jun gene is also re¯ected by their explains the wide distribution of its expression pattern. dierential expression pattern during embryogenesis. The AP-1 motif does not respond to TPA in the While JunD is widely expressed (Thepot et al., 2000), c- presence of the octamer motif, suggesting a negative Jun and JunB are expressed in non-overlapping sites interference between both factors. This interference (Wilkinson et al., 1989). c-Jun mRNA is detected may explain the low inducibility of junD in response to throughout organogenesis in restricted cell populations various stimuli. However, the TRE site is activated by within developing cartilage, gut and the central nervous JunD in the brain suggesting speci®c positive auto- system. JunB expression is observed at early stages of regulation of JunD in this tissue. embryogenesis in the primitive streak region, in the In conclusion, the transcriptional regulation of c-jun, neural tube and in the pre-chordal mesoderm as well as junB and junD diers markedly in accordance with the in extra-embryonic tissues (Schorpp-Kistner et al., existence of distinct regulatory elements (Figure 3). 1999). Later on, JunB is restricted to dierentiating High basal expression level of the junD gene is epidermal cells and endodermal gut epithelium (Wilk- mediated by an octamer motif, absent in c-jun-or inson et al., 1989). Finally, JunD has a broad junB-promoters. Moreover, the basal level of c-jun expression pattern (Thepot et al., 2000). A genetic expression is generally higher than that of junB in redundancy amongst this multi-gene family of tran- ®broblasts due to its positive autoregulatory loop. scription factor may account for the absence of Conversely, the junB gene is the most inducible phenotype in the JunD7/7 animals. Preliminary results member of the jun gene family. obtained from the intercross of c-Jun and JunD double heterozygote mice demonstrate that c-Jun7/7 JunD7/7 double mutants die earlier than the c-Jun7/7 embryos In vivo functions suggesting a functional redundancy between c-Jun and Studies of AP-1 functions in cell culture reveal distinct JunD mutations during embryogenesis (F Mechta, functions for each member. Several groups have unpublished results). Reciprocally, the low expression undertaken gene targeting strategy to investigate the of junD in the liver and the extra-embryonic tissues function of individual AP-1 members in vivo. Inactiva- may explain the hepatogenetic defect in c-Jun7/7 tion of individual AP-1 members results in distinct embryos and the placental de®ciency in JunB7/7 phenotypes, suggesting distinct functions of each gene embryos. The viability of mice lacking c-Fos or FosB in vivo. Inactivation of Jun and Fos family members suggests that the Fos-related genes may have also generates various phenotypes spanning from embryonic overlapping roles. lethality to post-natal survival associated with growth In conclusion, determination of the phenotypic retardation or behavioural defects. Disruption of c-jun, consequences of gene disruption in mice identi®es Jun junB or fra1 results in embryonic death whereas and Fos speci®c functions. AP-1 members are involved embryos lacking either junD,c-fos or fosB genes in various processes including hepatogenesis, sperma- develop normally to birth. togenesis, and vascularization. The lack of major c-jun de®cient embryos die at 13.5 days from defects in three AP-1-knock-out mice suggests that a impaired hepatogenesis (Hilberg et al., 1993; Johnson genetic redundancy may exist among some members of et al., 1993). These embryos exhibit massive apoptosis the AP-1 multi-gene family (see also contribution by in hepatoblasts and erythroblasts lineages and present Jochum et al.).

Oncogene The mammalian Jun proteins: redundancy and specificity F Mechta-Grigoriou et al 2386

Figure 3 Comparison of the three mouse Jun promoters. The organization of the most important regulatory cis-acting elements is depicted. The sequences of the regulatory elements and their relative position with respect to the transcription start site (+1) are also indicated

Conclusions 2000). Multi-gene family may be involved in ®ne modulation of complex processes in higher vertebrates. There has been rapid progress over the past few years However, the sequence of the c-Jun protein is highly towards understanding the speci®c functions of each conserved among these dierent species. Moreover, the AP-1 member. In cultured cells, AP-1 has been shown isolation of a bZip containing protein domain in C. to play an important role in several processes including elegans suggests the potential existence of a Jun protein cell cycle progression, oncogenic transformation or in this organism. In this context, it is important to apoptosis by responding to stimuli or stress. AP-1 recall that recent genetic studies have demonstrated participates in the control of these processes by ®nely that the JNK pathway is conserved among species and regulating the expression of a set of target genes required for morphogenesis and dorsal closure in (Figure 2). It was clearly established that the anity of Drosophila (Noselli, 1998) and coordinated locomotion AP-1 for a given TRE binding site is determined by its of motor neurons in Caenorhabditis elegans (Kawasaki surrounding DNA sequences and the presence of et al., 1999). neighbouring interacting proteins. It appears that the Careful examination of all the mutants animals and composition of the AP-1 dimers de®nes the nature of derived cell lines clearly demonstrated that each family genes that are expressed. The antagonistic roles of the member has speci®c functions. Some of these functions dierent Jun proteins is now clearly established in were better studied with primary or established cells in various cell systems. Their relative abundance mod- culture but they relate to defects occurring in vivo ulates speci®c gene transcription and engages cells in a under stress conditions. By applying our current precise destiny. The existence of dierent Jun proteins knowledge on in vivo concepts and through the analysis in vertebrates contrasts with the unique Jun partner yet of double mutants, we will be able in the future to identi®ed in Drosophila (Zhang et al., 1990; Riesgo- determine potential redundant, overlapping or antag- Escovar and Hafen, 1997) or Xenopus (Knochel et al., onistic roles among jun and fos genes in vivo.

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Oncogene