Cross-Talk Between Glucocorticoid Receptor and AP-1

Home , Glucocorticoid receptor, Transrepression

Cross-talk between glucocorticoid receptor and AP-1

Peter Herrlich*,1

1Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, PO Box 3640, D-76021 Karlsruhe, Germany

Cross-talk between dierent transcription factors, massive release of TNFa and of IL1 from macro- notably between the glucocorticoid receptor and AP-1, phages, obviously overwhelming the inbuilt inhibitory has been discovered more than 10 years ago: a bona ®de factors. Most aspects of the septic shock can be traced transcription factor, without apparent need for its own to an excessive dose of TNFa synthesized and secreted, direct DNA contact, in¯uences the activity of another which is supported by the facts that injection of TNFa transcription factor. Recent experiments have added mimics LPS in inducing septic shock (Beutler et al., interesting aspects: in addition to major insights into 1985; Tracey and Cerami, 1994; Amiot et al., 1997) and the mechanism of cross-talk, it is now clear that the that both TNFa and TNFRp55 knock-out mice are cross-talk ability of glucocorticoid receptor is essential inhibited in their response to LPS (Pfeer et al., 1993; for mouse development, while the activation of target Pasparakis et al., 1996; Marino et al., 1997; Gutierrez- promoters carrying a glucocorticoid response element Ramos and Bluethmann, 1997). Less dramatic abun- (GRE), is surprisingly, dispensable for survival under dance of TNFa is also pathologic and indicates a animal house conditions. Interestingly, the cross-talk failure of counteracting force. TNFa is, for instance, function is responsible for almost all regulatory actions overproduced in joints of patients with rheumatoid of cortisol in the immune system. It is possible that the arthritis (Saxne et al., 1988), where it is responsible for two functions of the glucocorticoid receptor can be the production of other cytokines, for the destruction activated separately by speci®c ligands. Future goals will of extracellular matrix and for tissue remodeling (Tak be to de®ne whether adverse eects of long-term et al., 1996). The best proof of this role of TNFa came corticosteroid treatment, e.g. osteoporosis, joint necroses, from the therapeutic use of soluble TNF receptor metabolic eects, can be ascribed to GRE-target gene sequestering TNFa, or of neutralizing antibodies to activation and thus be dissociated from the desirable TNF, now used in the treatment of rheumatoid actions in the treatment e.g. of autoimmune disease. arthritis (Feldmann et al., 1996; Tak et al., 1996; Oncogene (2001) 20, 2465 ± 2475. Moreland et al., 1999; Maini et al., 1998). Several inbuilt restrictions are cell-autonomous. The Keywords: immune response; NF-kB; tethering; signaling components between the LPS receptor and CREB; GR D-loop mutation; genomic footprints eector molecules in the nucleus, e.g. transcription factors, are under control of negative regulators, e.g. protein phosphatases counteracting the action of Biological units, systems of checks and balances protein kinases, which limit the duration and magni- tude of the response. In addition to cell-autonomous It is obvious that a motorcar needs both an accelerator regulation, an organismic mechanism limits the and a brake. Biological processes require this principle response: the release into the circulation of cortisol in as well, but in a much more elaborate manner. Any humans (corticosterone in rodents). Removal of the organismic or cellular biological response is the result adrenal gland, the site of cortisol production, as well as of a balance between numerous `driving' and `inhibit- manipulation of the levels of the glucocorticoid ing' forces (Frame and Balmain, 2000). As an receptor (see below) sensitizes the organism for impressive example, cells in an immune response are exaggerated immune responses including septic shock stimulated to multiply and to secrete cytokines. The (e.g. Miller and Blake Tyrrel, 1995; Barnes, 1998; response is transient, in that self-limitation is built into Reichardt et al., 2000). Glucocorticoid hormone down- the response on several levels (Feldmann et al., 1996; regulates the septic shock response and counteracts Long, 1999; Baer et al., 1999). This well-adjusted in¯ammatory and immune as well as autoimmune system can, however, be driven out of balance which reactions in general which makes glucocorticoids one leads to disease or even death of the organism. For of the most frequently prescribed drugs. instance, an overwhelming bacterial infection leads to Glucocorticoid hormone acts through its speci®c septic shock. In the case of infection with a gram- receptor, the glucocorticoid receptor (GR). The GR negative bacterium, it is the lipopolysaccharide (LPS) belongs to the family of nuclear receptors (reviewed by receptor which, in association with the toll-like Mangelsdorf et al., 1995; Mangelsdorf and Evans, 1995; receptor Tlr4 (Hoshino et al., 1999), triggers the Beato et al., 1995). In the absence of ligand, the GR is cytoplasmic, bound to chaperones, e.g. Hsp 90 and Hsp56, which keep the protein in a conformation ready *Correspondence: P Herrlich to accept the lipophilic hormone ligand. Upon hormone Cross-talk between glucocorticoid receptor and AP-1 P Herrlich 2466 binding, the GR is transported into the nucleus where it mechanism exerted by cortisol, e.g. induction of acts as a transcription factor. These properties are apoptosis in CD4/CD8 double positive thymocytes re¯ected in the protein structure: a central DNA binding and of peripheral lymphocytes. Despite this multitude domain (Luisi et al., 1991), a C-terminal ligand binding of possible cortisol targets, circumstantial and some domain whose ligand interaction appears to determine ®rm evidence (e.g. knock-outs) has reduced the the accessibility of the transactivation domain AF-2 problem somewhat in that a few central transcription (Bourguet et al., 1995; Brzozowski et al., 1997; Shiau et factors and their activation pathways are responsible al., 1998; Hu and Lazar, 1999), and an N-terminal for a large part of the molecular features of the domain which carries the ligand-independent AF-1 (see immune system. The transcription factors AP-1, NF- review by Beato et al., 1995). The active transcription kB, CREB, NF-AT and C/EBPb are responsible for factor GR is a dimer which is formed on its DNA most of the transcriptions relevant for the immune element, the glucocorticoid response element GRE, in system (KoÈ ntgen et al., 1995; for references see also head-to-tail ± tail-to-head fashion. In addition to activat- Chinenov and Kerppola, this issue; and reviews by ing transcription of speci®c target genes which carry the Pfahl, 1993; De Bosscher et al., 2000; De Bosscher, palindromic GRE (Geisse et al., 1982; Hynes et al., 2000). Counteraction of the activation and activities of 1983), the GR exerts negative in¯uences on the these transcription factors could, thus, be a mechanism expression of certain genes. In principle, all nuclear by which glucocorticoids limit excessive responses of receptors seem to possess this ability (Mangelsdorf et al., the immune system. 1995; Beato et al., 1995). The expression of the nuclear receptor and the availability of ligand determines when and where receptor actions are triggered. The supply of Cortisol-induced synthesis of speci®c inhibitors GR seems indeed to be limiting, in that an increase in GR gene dose increased resistance to LPS-induced septic In view of several molecular mechanisms which cortisol shock (Reichardt et al., 2000). For cortisol actions one and the GR could establish, it is useful to classify these needs to take into account not only the abundance of according to simple criteria: are the actions immediate GR. Cortisol acts as ligand for both the GR and the or delayed? Do they require intermediary protein mineralocorticoid receptor MR (Reul and de Kloet, synthesis? Which step of interference can be traced: 1985). In the cells of the immune system, GR is the e.g. on the intracellular level a block of signal predominant mediator of cortisol action. transduction between the plasma membrane and the Cortisol controls many organismic functions, the nucleus or a block within the nucleus? level of cortisol is therefore under tight control. Delayed onset of inhibition occurs if a GR- Hypothalamic peptide hormones regulate, through the dependent inhibitor protein needs to be synthesized. synthesis and release of ACTH, the output of cortisol Among GR target genes carrying GRE elements in from the adrenal cortex (e.g. Chrousos, 1995). Cortisol their promoters, there may be some with `brake' exerts feedback control on several stages of the properties. This most straight-forward explanation for hypothalamus-to-adrenal gland pathway through the a negative action of a transcription factor, GR, is GR. Interestingly, neural tissues express both GR and exempli®ed by the induced transcription of the MR which both respond to cortisol. It would make inhibitor of NF-kB: IkB. As mentioned above, sense if only the GR requiring higher doses of cortisol cytokine gene promoters as well as those of cytokine than MR would be active in the feed-back control (de target genes are predominantly addressed by the Kloet et al., 1993). Downstream targets of cortisol and transcription factors AP-1 and NF-kB. Glucocorticoids GR are many fold, from maintaining and in¯uencing were found to activate the synthesis of IkBa (Auphan late embryonic development through metabolic stress et al., 1995; Scheinman et al., 1995) which limits the responses in the liver and stress protection in many duration of NF-kB activity. As documented by organs, to hemopoietic dierentiation and behavioral introduction of IkB mutants which cannot be de- endpoints (Karin, 1998). graded, elevated IkB could prevent NF-kB activation, I will discuss here predominantly the mechanism of a mechanism which would suce to explain cortisol the anti-in¯ammatory and immune-suppressive actions action on NF-kB dependent expression of genes. of cortisol (see also previous reviews: Pfahl, 1993; It is possible that among GR target genes there are Beato et al., 1995; GoÈ ttlicher et al., 1998; Karin, 1998; other inhibitors of transcription factors or of signal Epinat and Gilmore, 1999). The cells involved in the transduction, e.g. phosphatases. A precedence for the in¯ammatory and immune responses exhibit unique latter has been found: Dexamethasone, a non-degrad- features: upon stimulation they migrate (e.g. dendritic able cortisol analog, inhibits JNK and Erk activation by cells, T-lymphocytes) or proliferate (T and B lympho- a mechanism requiring protein synthesis (Hirasawa et cytes). They produce and release numerous in¯amma- al., 1998; Rider et al., 1996). The induction of a tory mediators, chemokines and cytokines (T cells, phosphatase would be a plausible hypothesis. Interest- macrophages, endothelial cells). Inhibition by cortisol ingly, the expression of MAP kinase phosphatase, MKP- could therefore address numerous processes and the 1, is enhanced by all-trans-retinoic acid possibly resulting synthesis of many immune-system-speci®c proteins, e.g. in inhibition of JNK activity (Lee et al., 1999). GR may adhesion molecules, cytokines, MHC components and also induce the synthesis of a phosphatase. Along these receptors. Even selective cell death could be part of the lines, in another slow action of a nuclear receptor

Oncogene Cross-talk between glucocorticoid receptor and AP-1 P Herrlich 2467 induced by trans-retinoic acid the abundance of Jun not known whether these interactions re¯ect a protein, not the transcription of c-jun, appears to be functional consequence. reduced in human skin (Fisher et al., 1998) possibly by Thus, the interference with signal transduction to activated proteasome-dependent degradation. critical transcription factors, e.g. AP-1, NF-kB, CREB etc., could represent the predominant immediate mechanism of GR action. However, it is plausible that Inhibition of signal transduction the downregulation of the immune reactions is so important for the organism that safety steps exist on The most elaborate interference by GR with pro- several levels. With respect to immediate interference in¯ammatory and immune functions is immediate and by GR with components of signal transduction the does not require protein synthesis (van de Stolpe et al., exact molecular mechanisms ± cytoplasmic or nuclear, 1993; Wissink et al., 1998), thus suggesting a direct direct or involving partner components ± are yet to be interference with a component of the signaling chain or resolved. with transcription factor function. With AP-1 and NF- kB playing key roles in the immune response, the pathways to these transcription factors and the function Tethering mechanisms of these factors caught most attention. All nuclear receptors including the GR inhibit AP-1 target gene In addition to the proper function as a DNA-binding transcription (Jonat et al., 1990; SchuÈ le et al., 1990; transcription factor, the GR has been found to Yang-Yen et al., 1990; Shemshedini et al., 1991). modulate other transcription factors, essentially with- Although in the initial studies no interference with Jun out the need for DNA contact. This was named a or Fos phosphorylation was discovered (Jonat, 1990; tethering mechanism which later turned out to occur Jonat et al., 1990), a rapid and transcription-independent also in other examples of transcription factors. GR inhibition of Jun phosphorylation and of JNK activity modulates negatively ± in most examples proven: with- has been reported (Caelles et al., 1997; GonzaÂ lez et al., out DNA binding ± the activity of CREB (Akerblom et 2000). As JNK activity was determined by immune al., 1988; see also Figure 1; Yordanov and Rahmsdorf, precipitate assay with an excess of GST-Jun as substrate, unpublished), of Jun:Fos (AP-1) (Jonat et al., 1990; it is unlikely that the substrate was shielded from enzyme SchuÈ le et al., 1990; Yang-Yen et al., 1990), of Oct-1 action by GR bound to the substrate. Rather ligand- (Kutoh et al., 1992), of Spi-1/PU-1 (Gauthier et al., induced GR or an associated factor seems to down- 1993), of GATA-1 (Chang et al., 1993) and of NF-kB modulate JNK enzyme activity. In addition, JNK (Ray and Prefontaine, 1994). Interestingly, the tether- activity was inhibited in both cytoplasm and nucleus ing interaction can also lead to synergy, e.g. with AP-1 and nuclear uptake of JNK was not aected (GonzaÂ lez et or NF-kB in certain tissues or cell types (Shemshedini al., 2000). Although it is disturbing that in these et al., 1991; Maroder et al., 1993; Wang et al., 1997), experiments hormone was added 45 min prior to with Jun homodimers (at a so-called composite element treatment with the JNK activating agent, the inhibition which requires however DNA binding of the GR: may be `immediate' which is in contrast to the delayed Diamond et al., 1990; as well as by pure tethering: eect mentioned above which requires more than 6 h of Teurich and Angel, 1995), with C/EBP (Nishio et al., hormone pretreatment. GR interferes also with the 1993) and with Stat-5 (StoÈ cklin et al., 1996). activation of Erk (but not of p38). While in endothelial Features of these tethering interactions, also called cells this inhibition was rapid (GonzaÂ lez et al., 1999), it `cross-talk', have been reviewed previously (Beato et took 18 h of hormone treatment to see the eect in mast al., 1995; Karin, 1998; GoÈ ttlicher et al., 1998). In brief, cells (Rider et al., 1996) suggesting the involvement of the cross-talk can be reciprocal, GR inhibiting Fos:Jun intermediate steps. Also in macrophages, protein synth- or NF-kB at their target genes, and elevated Fos or esis was required for MAP-kinase inhibition (Gewert et Jun or RelA (p65) interfering with GR activity at GRE al., 2000). It is puzzling that in certain tumor cells nuclear promoters. Although from in vitro experiments it receptors synergize with MAP kinases, e.g. activating seemed that GR reduced AP-1 DNA binding, genomic Erk, rather than inhibit the activation (Di Domenico et footprints and chromatin immunoprecipitations (see al., 1996; Peterziel et al., 1999). below) do not support this interpretation. Rather the There have also been reports on cytoplasmic inhibited factor remains DNA-bound. Direct interac- interference of GR-chaperone complexes with signal tions of Jun with GR have been reported (Jonat et al., transduction (see e.g. Tumlin et al., 1997; Cissel and 1990) but their signi®cance is yet unknown. Mutual Beaven, 2000). These are yet ill-understood and I will interference between AP-1 and nuclear receptors is not discuss them in further detail. It has been proposed probably not limited to the GR, but occurs with that signaling components associate with Hsp90 or retinoic acid receptors (Lafyatis et al., 1990; Nicholson Hsp70 the availability of which could depend on et al., 1990; SchuÈ le et al., 1990; Yang-Yen et al., 1991; hormone-dependent release (see also review by Pratt, Salbert et al., 1993), estrogen receptor (Doucas et al., 1997). Ligand-activated nuclear receptors, shown for 1991; Shemshedini et al., 1991), thyroid receptor ERa, can associate with and modulate cytoplasmic (Desbois et al., 1991; Zhang et al., 1991; Lopez et al., PI3-kinase (Simoncini et al., 2000). Raf-1 and 14-3-3 1993; Wondisford et al., 1993) and even the fusion coimmunopurify with GR (Widen et al., 2000), but it is receptor PML-RAR (Doucas et al., 1993).

Oncogene Cross-talk between glucocorticoid receptor and AP-1 P Herrlich 2468

Figure 1 Glucocorticoids interfere with the positive and negative functions of CREB determined by the forskolin-induced transcription of c-fos,c-jun and junB (data by Iordanov and Rahmsdorf, unpublished; Ph.D. thesis Iordanov, 1996). Quiescent NIH3T3 cells were pretreated with 1077 M dexamethasone (D) for 15 min or not treated. Then 10 mM forskolin (F) was added and speci®c RNA levels at various times thereafter (min) were measured by Northern blotting of Poly(A)+ RNA (arbitrary scale). Dexamethasone prevents the increase of c-fos and JunB abundance. Forskolin inhibits c-jun expression which is counteracted by dexamethasone after a delay of 15 min

Dissociation of transrepressing and transactivating inducible transcription of GRE promoters, but fully nuclear receptor function represses AP-1 promoters (Heck et al., 1994). One of the D-loop mutations, A458T, was inserted A major step towards understanding and de®ning the by homologous recombination using the CRE/loxP tethering (cross-talk) function was achieved by the system into the mouse germ line (Reichardt et al., detection of ligands which preferentially favor either 1998). The GR mutant allele was called dim for the transrepressing or the transactivating function of dimerization defective. GRdim/dim mice are viable under nuclear receptors (for GR: VayssieÂ re et al., 1997; animal house conditions (Reichardt et al., 1998) while Vanden Berghe et al., 1999; for other nuclear receptors: GR7/7 die postnatally because of a lung maturation Fanjul et al., 1994; Chen et al., 1995). This ®nding de®ciency which occurs for still unknown reasons (Cole suggested that the ligand binding domain can deter- et al., 1995; Tronche and Kellendonk, cited in Tronche mine which molecular property a nuclear receptor can et al., 1998). It is clear that it is the DNA binding exhibit and that probably dierent molecular surfaces independent function which is required for develop- are responsible for either function. It should thus be ment. Dexamethasone treatment of GRdim/dim mice does possible to dissociate the transrepressing and transacti- not lead to transcription of GRE target genes, e.g. of vating function by mutation. liver tyrosine aminotransferase (Reichardt et al., 1998), Indeed, this has been possible. In particular, several or of several target genes in skin (Tuckermann et al., GR mutants cannot at all or poorly transactivate, but 1999). GRE binding assays in vitro with liver extracts downmodulate AP-1 and NF-kB activity by the of GRdim/dim mice were negative in contrast to extracts tethering function. LS7, in the N-terminal zinc ®nger from wild type siblings. The action as a bona ®de GR of the DNA binding domain, is a poor transactivator transcription factor is obviously not essential during at GRE promoters (Godowski et al., 1989) but normal and protected adult life. GRdim/dim may, pro®cient in repressing AP-1 and NF-kB (Helmberg however, suer from certain stress conditions. For et al., 1995). Deletions of the AF-2 can obviously not instance, hypoxia-induced erythropoiesis is impaired induce gene expression in response to hormone (shown (Bauer et al., 1999) which is caused by de®ciency of for several nuclear receptors in Kamei et al., 1996). The erythroblast progenitor expansion (Reichardt et al., AF-2 mutation does, however, not aect the transre- 1998; Bauer et al., 1999). Thus, GRE-dependent pressing function (GoÈ ttlicher, unpublished). transcription is required for survival upon certain GR activates genes as GR dimer. The dimers form stress, but not under protected conditions. The viability at the DNA element, but require a de®ned region of of GRdim/dim mice in contrast to GR7/7 suggests that the the DNA binding domain, the D-loop, to stabilize the transcription factor modulating activities or some other dimer (Luisi et al., 1991; Dahlman-Wright et al., 1991). DNA binding independent function are essential for Mutations in the D-loop severely reduce or even life. abolish DNA binding (Umesono and Evans, 1989; The D-loop mutation in the mouse permits to Dahlman-Wright et al., 1991; Heck et al., 1994; classify GR functions: those which depend on expres- Reichardt et al., 1998). It has been argued that sion from GRE promoters or which require dimeriza- promoters carrying an array of GREs such as the tion of the GR for some other reason, and others that mouse mammary tumor virus promoter, may depend are exerted by GR monomers. As had been shown by less on stabilization by the D-loop (Karin, 1998). cell culture experiments (Heck et al., 1994), phorbol Promoters with multiple adjacent GREs are, however, ester induced expression of metalloproteases in em- the rare exception. Expression of D-loop mutants in bryonic ®broblasts and in the intact skin from GRdim/dim GR-defective CV-1 cells does not confer hormone- mice is inhibited by dexamethasone to an extent similar

Oncogene Cross-talk between glucocorticoid receptor and AP-1 P Herrlich 2469 to wild type (Reichardt et al., 1998; Tuckermann et al., accomplished by GR monomers. Further, the data 1999). The metalloprotease promoters depend on AP-1 encourage the search for agents which induce the and NF-kB. Glucocorticoid-induced genes in the skin, `monomer-property' of the GR only. encoding plasma glutathione peroxidase PGX-3 and Cortisol derivatives are most commonly used to Hsp 27, however, are not activated in GRdim/dim mice. combat in¯ammation and immune disease including Thus, the tethering reaction does not depend on GR rheumatoid arthritis and asthma. The long-term dimerization and DNA binding. application of glucocorticoid hormones goes along Interestingly, the feed-back regulation of cortisol on with severe side eects: osteoporosis, joint necroses, the hypothalamus-adrenal gland axis can in part be inhibition of skin maintenance, hyperglycemia and exerted by GR monomers: CRF levels of the other metabolic alterations (see e.g. Lukert and Raisz, hypothalamus were normal as in wild type mice 1990; Karin, 1998). It is therefore important to know indicating that the control of synthesis, possibly whether the therapeutic eect can be dissociated from through tethering reaction on CREB at the CRF these side eects. A ®rst step towards this distinction promoter, worked in GRdim/dim mice (Reichardt et al., can be achieved by the exploitation of the GRdim/dim 1998). Neural tissues express also the MR. The MR mouse. Can the dimerization and DNA binding has however no cross-talk ability as far as we know: defective GR mount the immune suppressive action see (Cato et al., 1992; Pearce and Yamamoto, 1993; in the adult organism? In a second more demanding Heck et al., 1994); interestingly, the GR abundance in step one can ask whether GRdim/dim mice develop the that the hypothalamus-to-adrenal gland axis seems unwanted side eects of long-term cortisol treatment. limiting in that the increased GR gene dosis caused Indeed phorbol ester induced skin in¯ammation as downregulation of corticosterone release: (Reichardt et measured by ear swelling could be prevented by al., 2000). The feed-back regulation of pituitary dexamethasone in GRdim/dim (Reichardt et al., 2000, enzymes such as prolactin and POMC, however, did 2001). Also the release of systemic TNFa and IL-6 was not function, as the levels of these enzymes as well as inhibited by dexamethasone. Mimicking a bacterial of ACTH were elevated in the pituitary of GRdim/dim. infection, LPS was injected systemically and the The promoters of these genes, encoding prolactin and response monitored over time by measuring serum TNFa POMC, carry speci®c DNA elements (so-called and IL-6. Both genotypes (GR+/+ or GRdim/dim) nGREs) which bind the GR and apparently in¯uence responded with massive synthesis and release of both the GR to behave as a repressing factor (Sakai et al., cytokines. These cytokines induced the enhanced 1988; Drouin et al., 1993). This action requires an release of glucocorticoids (through activating the intact GR D-loop. The hypothesis that the DNA hypothalamus-to-adrenal gland axis) which limited element or its arrangement in¯uences the structure of a eectively the cytokine production within 3 h (Reich- transcription factor and even of those relevant here, is ardt et al., 2001) ± documenting the `brake' mechanism not without precedent: AP-1, RAR-RXR and TR have mentioned in the introduction. The LPS dependent been shown to alter their conformation and function induction and repression of TNFa and IL-6 were upon binding to DNA (Patel et al., 1990; Ikeda et al., reproduced in isolated macrophages (Reichardt et al., 1996; Mangelsdorf et al., 1991; Chen and Evans, 1995). 2001). In T lymphocytes the phorbol ester and Ca- Conformational changes of the GR upon interaction ionophore dependent synthesis of other cytokines, e.g. with GRE-like elements can be concluded both, from IFNg and IL-2, was also inhibited by hormone in both the detection of mutations which arrest the GR in one genotypes, GR+/+ or GRdim/dim (Reichardt et al., 2001). conformation, and from structural analyses (Starr et In T lymphocytes from GRdim/dim mice carrying an NF- al., 1996; van Tiborg et al., 2000). kB dependent reporter transgene, the reporter behaved Unexpectedly, ACTH serum levels of GRdim/dim mice like endogenous cytokine genes indicating that one of were not elevated suggesting that there is still another the target factors of GR repression in vivo is NF-kBin level of control in the release of ACTH from the T lymphocytes. Repression occurred independently of pituitary, obviously exerted by GR monomers (Reich- the GR dimerization and DNA binding function and ardt et al., 1998). Nevertheless the corticosterone levels did not involve IkB synthesis in these cells (Reichardt in serum were higher than in wild type. Whether this is et al., 1998). It thus seems that glucocorticoids do not indicative of a GR dependent control function of induce IkB in all cell types and the induction of IkBis cortisol levels in the adrenal gland or periphery, is not not required for transrepression of NF-kB (see also yet clear. Heck et al., 1997; Dumont et al., 1998). The conclusion thus is that the immune response as determined so far, is inhibitable by cortisol in the The anti-in¯ammatory and immune suppressive function absence of a dimerization and DNA binding function requires no dimerization and DNA binding of the GR of the GR. The hope is therefore justi®ed that side eects of longterm cortisol treatment may not occur in Data that will be described below, indicate that the GRdim/dim mice, which would then trigger eorts to important physiological role of cortisol and GR in mimick the GRdim/dim situation by speci®c GR ligands limiting undue reactions, e.g. the expansion of permitting only the cross-talk function to occur. lymphocytes after antigenic stimulation or the un- Not all immune suppressive functions of the GR wanted overreaction to the presence of LPS, is could be detected in GRdim/dim mice. The killing of CD4/

Oncogene Cross-talk between glucocorticoid receptor and AP-1 P Herrlich 2470 CD8 double-positive thymocytes ± not relevant for the immune response in the adult ± in response to dexamethasone is defective (Reichardt et al., 1998; GoÈ ttlicher, unpublished, scheme in Figure 2). Periph- eral primary T and B lymphocytes were, however, sensitive entering apoptosis upon cortisol treatment of GRdim/dim mice (GoÈ ttlicher, unpublished). Also a T cell lymphoma line, Jurkat equipped with the GR mutant receptor LS7, were sensitive to dexamethasone-induced apoptosis (Helmberg et al., 1995) which may resemble the well-established treatment of human lymphomas and leukemia with glucocorticoids. It is likely that induced apoptosis in normal and transformed lymphocytes is mediated by the eective inhibition of cytokine release from either T cells, macrophages or autocrine tumor cells.

Molecular mechanism of cross-talk ± a working model

Previously favored model: `Competition for a coactivator, squelching' Interference by the limited availability of CBP? The mutual nature of cross-talk between nuclear receptors

Figure 2 Glucocorticoid dependent regulation of thymic development of T lymphocytes. GR dimer dependent loss or reduction of cell yield are indicated

Oncogene Cross-talk between glucocorticoid receptor and AP-1 P Herrlich 2471 C)

Figure 3 Genomic footprints of the IL-2 promoter in primary T- lymphocytes. (a) Inhibition of induced IL-2 transcription by glucocorticoid. Northern blot experiments. (b) The interleukin-2 (IL-2) gene promoter has binding sites for several transcription factors including AP-1, NF-kB, Octamer factors, NF-AT and a CD28 response factor. IL- 2 de®ciency in mice is, however, only generated eciently by a c-rel knock-out (KoÈ ntgen et al., 1995) suggesting that NF-kB represents the decisive transcription factor in T-cells. Nevertheless, induced occupancy of all promoter elements is detected by an alteration of guanine methylation at 1 and at 4 h after treatment with a combination of phorbol ester and ionomycin (PI). For example band 253 indicates hypersensitivity at an Octamer site whereas bands 152 and 154 represent hypersensitivity at an AP-1 site. Weaker binding by NF-kB (205 ± 207, 209) and NF-AT (proximal site, protections at positions 137 and 138, hypermethylation at position 140) can also be detected. The footprints were enhanced at 4 h. (c) Similar footprints, with additional hypomethylation at positions 284, 285 and protection at 225/3 occurred in both the coding and non-coding strands. (b and c) We note that FK506 abolished the footprints while dexamethasone did not alter the occupancy. Over the NF-kB, for instance, the G protections at positions 205, 206 and 207 remained, so did the hypermethylated G at position 209

Oncogene Cross-talk between glucocorticoid receptor and AP-1 P Herrlich 2472 and AP-1 or other factors suggested early on that GR NF-kB remains bound to the kB promoter element. and AP-1 compete for a limiting component. CBP/ GR is tethered to the promoter by the RelA subunit. p300, fairly universally requiring coactivators, seemed NF-kB remains DNA-bound while being inhibited as good candidates (Kamei et al., 1996; Aarnisalo et al., shown by genomic footprints over the kB element in 1998). A CBP knock-out has a severe embryonic-lethal the IL-2 promoter (Figure 3 and Wade, Schmitt, phenotype (Yao et al., 1998) indicating that CBP is Herrlich and Cato, unpublished) or by chromatin indeed a limiting component in some critical cell of immunoprecipitations of IL8 and ICAM-1 promoter early development. Moreover, the signi®cance of a elements (Nissen and Yamamoto, 2000). Chromatin CBP dose is demonstrated by the existance of a human immunoprecipitations also revealed that the formation syndrome, Rubinstein ± Taybi Syndrome, which is of the preinitiation complex occurred with normal caused by CBP haploid insuciency (Petrij et al., eciency, not aected by hormone treatment. This 1995) suggesting that even moderate decreases in CBP suggests that the co-activator assembly occurred abundance can aect cellular function. However, it is essentially undisturbed. However, dexamethasone unlikely that the limiting abundance accounts for the blocked the phosphorylation of one of the serines repressive action of GR: Overexpression of CBP did (serine-2) in the cytoplasmic tail of RNA polymerase II not obliterate the inhibition of IL-6 promoter activity (Nissen and Yamamoto, 2000). Thus, GR appears to nor that of a GalDBD-Jun fusion dependent reporter block an as yet unknown speci®c stimulus transfer (De Bosscher et al., 1997, 2000). Cross-talk with between transcription factor, co-activators bound to it, dissociating mutants of the GR and the eect of and preinitiation complex. dissociating ligands for RAR and GR which do not permit coactivator binding and target gene activation Working model by the respective receptors, are not easily explainable by competion for CBP (Fanjul et al., 1994; Chen et al., Many new aspects have been reported in the last few 1995; VayssieÂ re et al., 1997; Vanden Berghe et al., years which permit us to derive a plausible model of 1999). Further, one would need to assume that Jun cross-talk. The model must incorporate the following homodimers which synergize with GR (Diamond et al., observations: 1990; Teurich and Angel, 1995), and Jun:ATF2 heterodimers which are not modulated by GR . The cross-talk between GR and other transcription (unpublished data), do this by circumventing limita- factors is mutual which would be best compatible tions for CBP while squelching occurs with Jun:Fos with the formation of a complex of the interfering heterodimers. Finally, GR mutants defective in the transcription factor with the active one, a complex transactivating domains AF-1 and AF-2 as well as that will possibly involve additional partner those defective in the LBD altogether can cross-talk proteins. This direct formation of a complex (Jonat et al., 1990; and unpublished). The squelching of suggested by e.g. coprecipitation experiments (Jonat p300 or CBP would need to be achieved very indirectly et al., 1990; Wissink et al., 1997), has found support to remain a plausible hypothesis. by genomic footprints and chromatin immunoprecipitations (see above). Cross-talk by squelching unknown co-factors? The . Although it is possible that cross-talks between possibility that competition for other shared factors dierent factors follow dierent rules, it is attractive required by AP-1 and GR alike could be the to postulate common mechanisms: nuclear receptors mechanism of cross-talk, has been raised by the ®nding and possibly numerous other transcription factors of conformational GR mutants which act both as participating in cross-talk type of modulation may negative-dominant factors on GR-driven promoters share relevant properties. GR shares the cross-talk and as factors participating in cross-talk (Lefstin et al., function with other nuclear receptors. These may 1994; van Tiborg et al., 2000). Mutants S549A and work by the same tethering mechanism, although P493R in the two zinc ®ngers of the DBD assumed a one branch of nuclear receptors, the RAR, RXR, constitutively active conformation as if they were TR branch, has adopted, perhaps in addition, the bound to a GRE. It is not clear, however, whether co-repressor mode of action. The mutual nature of the presumed squelching mechanism by which the cross-talk (e.g. GR modulating AP-1 and NF-kB, mutants interfere with GR wild type, is the basis of p65 and Fos/Jun modulating GR activity) favors the cross-talk with AP-1. Data with mutants of the genes existance of a protein complex with similar func- encoding AP-1 and NF-kB which aect coactivator tional features in both directions. binding and cross-talk (SchuÈ le et al., 1990; Wissink et . NF-kB (and presumably AP-1) are inhibited by GR al., 1997), are not yet conclusive as these mutations after the assembly of the pre-initiation complex may have aected several properties. (Nissen and Yamamoto, 2000). Thus probably the assembly of co-activators and TBP-associated A lesson from the inhibition of NF-kBbyGR factors is not destroyed during inhibition of transcription. Further, cross-talk, at least in the Against this simple squelching mechanism there is also case of NF-kB/GR, does not seem to be in¯uenced recent data on the inhibition of NF-kB by GR. Very by histone deacetylase inhibitors (Cronin and similarly to AP-1, NF-kB can be inhibited by GR while Yamamoto, cited in Nissen and Yamamoto, 2000)

Oncogene Cross-talk between glucocorticoid receptor and AP-1 P Herrlich 2473 suggesting that cross-talk does not involve the recruitment of classical co-repressors such as SMRT (Kamei et al., 1996). . Conformational changes are made plausible by numerous observations where the DNA-element in¯uences the structure of the transcription factor. This appears true also for the GR in that a particular arrangement of triple binding sites as negative GRE seem to convert the GR into a repressing factor (Drouin et al., 1993). Structural analyses support that DNA binding elements in¯uence factor structure (Lefstin and Yamamoto, 1998; van Tiborg et al., 2000). It is plausible that protein-protein interactions also bring out conformation changes. . The composition of AP-1 (e.g. Jun : Fos vs Jun : Jun or Jun : ATF2) determines the positive or negative cross-talk with GR (Diamond et al., 1990; Teurich and Angel, 1995; and unpublished data). This speci®cation of action does not depend on the DNA binding function of either transcription factor as GR shows this speci®cally also with chimeric AP-1 proteins carrying the transactivating domain fused to the DBD of Gal4 (Teurich and Angel, 1995). . Although GR mutants dierently acting on the composite element of the proliferin gene have been screened for in yeast (Starr et al., 1996); the tethering cross-talk could not be reconstructed in yeast (our unpublished data) suggesting the lack of a co-factor.

Figure 4 A model of cross-talk A requirement for additional yet unknown factors The co-immunoprecipitation data of kB promoters, but also the other features which need to be incorporated modulation of AP-1 and enhancing transcription from into a working model ± including cell-type speci®c GRE promoters. Further that factors X will be tissue- responses and the absence of cross-talk in yeast ± speci®c and exert novel molecular activities. highlight the need for novel factors participating in cross-talk. We envisage that the modulating factor is Concluding remarks associated with one or several factors X which exert their yet unknown molecular function only when The cross-talk ability of nuclear receptors and their tethered to an active transcription complex (Figure partner transcription factors including AP-1 which is 4). The modulating tethering factor has presumably not essential for development, was discovered 10 years ago. yet been switched into an `active conformation' and Much has been achieved by learning from dierent thus may not assemble co-activators on its own. systems, particularly from the cross-talk of GR with Speci®city will be achieved by both the modulating Jun:Jun, Jun:Fos and NF-kB. It will be exciting to see factor and the associated X. The tethered complex how this knowledge will be made use of for would be defective in one or several last steps prior to glucocorticoid therapy of immune diseases. polymerase take-o. The model implies that the factors X can be squelched/sequestered, given the binding motif has been de®ned. One may also predict that Acknowledgments depriving cells of X e.g. by knock-out will aect both ThehelpofDrMartinGoÈ ttlicher and Ingrid Kammerer is sides of the reciprocal cross-talk: e.g. releaving down- gratefully acknowledged.

References

Aarnisalo P, Palvimo JJ and Janne OA. (1998). Proc. Natl. Amiot F, Fitting C, Tracey KJ, Cavaillon JM and Dautry F. Acad. Sci. USA, 95, 2122 ± 2127. (1997). Mol. Med., 3, 864 ± 875. Akerblom IE, Slater EP, Beato M, Baxter JD and Mellon Auphan N, Di Donato JA, Rosette C, Helmberg A and PL. (1988). Science, 241, 350 ± 353. Karin M. (1995). Science, 270, 286 ± 290.

Oncogene Cross-talk between glucocorticoid receptor and AP-1 P Herrlich 2474 Baer M, Nedospasov S and Johnson PF. (1999). Cold Spring Frame S and Balmain A. (2000). Curr. Opin. Genet. Dev., 10, Har. Symp. Quant. Biol., 64, 437 ± 444. 106 ± 113. Barnes PJ. (1998). Clin. Sci., 94, 557 ± 572. Gauthier JM, Bourachot B, Doucas V, Yaniv M and Bauer A, Tronche F, Wessely O, Kellendonk C, Reichardt Moreau-Gachelin F. (1993). EMBO J., 12, 5089 ± 5096. HM, Steinlein P, Schutz G and Beug H. (1999). Genes Geisse S, Scheidereit C, Westphal HM, Hynes NE, Groner B Dev., 13, 2996 ± 3002. and Beato M. (1982). EMBO J., 1, 1613 ± 1619. BeatoM,HerrlichPandSchuÈ tz G. (1995). Cell, 83, 851 ± 857. Gewert K, Hiller G and Sundler R. (2000). Biochem. Pharm., Beutler B, Milsark IW and Cerami AC. (1985). Science, 229, 60, 545 ± 551. 869 ± 871. Godowski PJ, Sakai DD and Yamamoto KR. (1989). DNA- Bourguet W, Ru M, Chambon P, Gronemeyer H and Protein Interactions in Transcription. UCLA Symposia on Moras D. (1995). Nature, 375, 377 ± 382. Molecular and Cellular Biology, Vol. 95, Gralla JD (ed.). Brzozowski AM, Pike ACW, Dauter Z, Hubbard RE, Bonn Alan R. Liss: New York, pp. 197 ± 210. T, EngstroÈ mO,OÈ hman L, Greene GL, Gustafsson J-A GonzaÂ lez MV, GonzaÂ lez-Sancho JM, Caelles C, Munoz A and Carlquist M. (1997). Nature, 389, 753 ± 758. and Jimenez B. (1999). FEBS letts, 459, 272 ± 276. Caelles C, Gonzalez-Sancho JM and Munoz A. (1997). Genes GonzaÂ lez MV, Jimenez B, Berciano MT, Gonzalez-Sancho Dev., 11, 3351 ± 3364. JM, Caelles C, Lafarga M and Munoz A. (2000). J. Cell. Cato AC, Konig H, Ponta H and Herrlich P. (1992). J. Biol., 150, 1199 ± 1208. Steroid Biochem. Mol. Biol., 43, 63 ± 68. GoÈ ttlicher M, Heck S and Herrlich P. (1998). J. Mol. Med., Chang TJ, Scher BM, Waxman S and Scher W. (1993). Mol. 76, 480 ± 489. Endocrinol., 7, 528 ± 542. Gutierrez-Ramos JC and Bluethmann H. (1997). Immunol- Chen JD and Evans RM. (1995). Nature, 377, 454 ± 457. ogy Today, 18, 329 ± 334. Chen JY, Penco S, Ostrowski J, Balaguer P, Pons M, Starrett Heck S, Bender K, Kullmann M, Gottlicher M, Herrlich P JE, Reczek P, Chambon P and Gronemeyer H. (1995). and Cato ACB. (1997). EMBO J., 16, 4698 ± 4707. EMBO J., 14, 1187 ± 1197. Heck S, Kullmann M, Gast A, Ponta H, Rahmsdorf HJ, Chrousos GP. (1995). N. Engl. J. Med., 332, 1351 ± 1362. Herrlich P and Cato AC. (1994). EMBO J., 13, 4087 ± Cissel DS and Beaven MA. (2000). J. Biol. Chem., 275, 4095. 7066 ± 7070. Helmberg A, Auphan N, Caelles C and Karin M. (1995). Cole TJ, Blendy JA, Monaghan AP, Krieglstein K, Schmid EMBO J., 14, 452 ± 460. W, Aguzzi A, Fantuzzi G, Hummler E, Unsicker K and Hirasawa N, Sato Y, Fujita Y, Mue S and Ohuchi K. (1998). Schutz G. (1995). Genes Dev., 9, 1608 ± 1621. J. Immunol., 161, 4939 ± 4943. Dahlman-Wright K, Wright A, Gustafsson JA and Carl- HoshinoK,TakeuchiO,KawaiT,SanjoH,OgawaT, stedt-Duke J. (1991). J. Biol. Chem., 266, 3107 ± 3112. Takeda Y, Takeda K and Akira S. (1999). J. Immunol., De Bosscher K, Schmitz ML, Vanden Berghe W, Plaisance S, 162, 3749 ± 3752. Fiers W and Haegeman G. (1997). Proc. Natl. Acad. Sci. Hu X and Lazar MA. (1999). Nature, 402, 93 ± 96. USA, 94, 13504 ± 13509. Hynes N, Van Ooyen AJJ, Kennedy N, Herrlich P, Ponta H De Bosscher K, Vanden Berghe W, Vermeulen L, Plaisance and Groner B. (1983). Proc. Natl. Acad. Sci. USA, 80, S, Boone E and Haegeman G. (2000). Proc. Natl. Acad. 3637 ± 3641. Sci. USA, 97, 3919 ± 3924. Ikeda M, Wilcox EC and Chin WW. (1996). J. Biol. Chem., De Bosscher K. (2000). Thesis, University of Ghent. 271, 23096 ± 23104. de Kloet ER, Oitzl MS and Joels M. (1993). Cell. Mol. Jonat C. (1990). Thesis, University of Karlsruhe. Neurobiol., 13, 433 ± 455. JonatC,RahmsdorfHJ,ParkKK,CatoAC,GebelS,Ponta Desbois C, Aubert D, Legrand C, Pain B and Samarut J. H and Herrlich P. (1990). Cell, 62, 1189 ± 2104. (1991). Cell, 67, 731 ± 740. Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Di Domenico M, Castoria G, Bilancio A, Migliaccio A and Lin SC, Heyman RA, Rose DW, Glass CK and Rosenfeld Auricchio F. (1996). Cancer Res., 56, 4516 ± 4521. MG. (1996). Cell, 85, 403 ± 414. Diamond MI, Miner JN, Yoshinaga SK and Yamamoto KR. Karin M. (1998). Cell, 93, 487 ± 490. (1990). Science, 249, 1266 ± 1272. KoÈ ntgenF,GrumontRJ,StrasserA,MetcalfD,LiR, Doucas V, Brockes JP, Yaniv M, de The H and Dejean A. Tarlinton D and Gerondakis S. (1995). Genes Dev., 9, (1993). Proc. Natl. Acad. Sci. USA, 90, 9345 ± 9349. 1965 ± 1977. Doucas V, Spyrou G and Yaniv M. (1991). EMBO J., 10, Kutoh E, Stromstedt PE and Poellinger L. (1992). Mol. Cell. 2237 ± 2245. Biol., 12, 4960 ± 4969. Drouin J, Sun YL, Chamberland M, Gauthier Y, De Lean A, LafyatisR,KimSJ,AngelP,RobertsAB,SpornMB,Karin Nemer M and Schmidt TJ. (1993). EMBO J., 12, 145 ± 156. M and Wilder RL. (1990). Mol. Endocrinol., 4, 973 ± 980. Dumont A, Hehner SP, Schmitz ML, Gustafsson JA, Liden Lee HY, Sueoka N, Hong WK, Mangelsdorf DJ, Claret FX J, Okret S, van der Saag PT, Wissink S, van der Burg B, and Kurie JM. (1999). Mol. Cell. Biol., 19, 1973 ± 1980. Herrlich P, Haegeman G, De Bosscher K and Fiers W. Lefstin JA, Thomas JR and Yamamoto KR. (1994). Genes (1998). Trends Biochem. Sci., 23, 233 ± 235. Dev., 8, 2842 ± 2856. Epinat JC and Gilmore TD. (1999). Oncogene, 18, 6896 ± Lefstin JA and Yamamoto KR. (1998). Nature, 392, 885 ± 6909. 888. Fanjul A, Dawson MI, Hobbs PD, Jong L, Cameron JF, Long EO. (1999). Annu. Rev. Immunol., 17, 875 ± 904. Harlev E, Graupner G, Lu XP and Pfahl M. (1994). Lopez G, Schaufele F, Webb P, Holloway JM, Baxter JD and Nature, 372, 107 ± 111. Kushner PJ. (1993). Mol. Cell. Biol., 13, 3042 ± 3049. Feldmann M, Brennan FM and Maini RN. (1996). Annu. Luisi BF, Xu WX, Otwinowski Z, Freedman LP, Yamamoto Rev. Immunol., 14, 397 ± 440. KR and Sigler PB. (1991). Nature, 352, 497 ± 505. Fisher GJ, Talwar HS, Lin J, Lin P, McPhillips F, Wang Z, Lukert BP and Raisz LG. (1990). Ann. Intern. Med., 112, Li X, Wan Y, Kang S and Voorhees JJ. (1998). J. Clin. 352 ± 364. Invest., 101, 1432 ± 1440.

Oncogene Cross-talk between glucocorticoid receptor and AP-1 P Herrlich 2475 Maini RN, Breedveld FC, Kalden JR, Smolen JS, Davis D, SalbertG,FanjulA,Piedra®taFJ,LuXP,KimSJ,TranP Macfarlane JD, Antoni C, Leeb B, Elliott MJ, Woody JN, and Pfahl M. (1993). Mol. Endocrinol., 7, 1347 ± 1356. Schaible TF and Feldmann M. (1998). Arthritis Rheum., Saxne T, Palladino MAJ, Heinegard D, Talal N and 41, 1552 ± 1563. Wollheim FA. (1988). Arthritis Rheum., 31, 1041 ± 1045. Mangelsdorf DJ and Evans RM. (1995). Cell, 83, 841 ± 850. Scheinman RI, Cogswell PC, Lofquist AK and Baldwin Jr. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz AS. (1995). Science, 270, 283 ± 286. G, Umesono K, Blumberg B, Kastner P, Mark M, SchuÈ le R, Rangarajan P, Kliewer S, Ransone LJ, Bolado J, Chambon P. et al. (1995). Cell, 83, 835 ± 839. Yang N, Verma IM and Evans RM. (1990). Cell, 62, Mangelsdorf DJ, Umesono K, Kliewer SA, Borgmeyer U, 1217 ± 1226. Ong ES and Evans RM. (1991). Cell, 66, 555 ± 561. Shemshedini L, Knauthe R, Sassone-Corsi P, Pornon A and Marino MW, Dunn A, Grail D, Inglese M, Noguchi Y, Gronemeyer H. (1991). EMBO J., 10, 3839 ± 3849. Richards E, Jungbluth A, Wada H, Moore M, Williamson Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, B, Basu S and Old LJ. (1997). Proc.Natl.Acad.Sci.USA, Agard DA and Greene GL. (1998). Cell, 95, 927 ± 937. 94, 8093 ± 8098. Simoncini T, Hafezi-Moghadam A, Brazil DP, Ley K, Chin MaroderM,FarinaAR,VaccaA,FelliMP,MecoD, WW and Liao JK. (2000). Nature, 407, 538 ± 541. Screpanti I, Frati L and Gulino A. (1993). Mol. Starr DB, Matsui W, Thomas JR and Yamamoto KR. Endocrinol., 7, 570 ± 584. (1996). Genes Dev., 10, 1271 ± 1283. Miller WL and Blake Tyrrel J. (1995). The Adrenal Cortex. StoÈ cklin E, Wissler M, Gouilleux F and Groner B. (1996). McGraw-Hill: New York. pp 555 ± 711. Nature, 383, 726 ± 728. MorelandLW,SchiMH,BaumgartnerSW,TindallEA, Tak PP, Taylor PC, Breedveld FC, Smeets TJ, Daha MR, Fleischmann RM, Bulpitt KJ, Weaver AL, Keystone EC, Kluin PM, Meinders AE and Maini RN. (1996). Arthritis Furst DE, Mease PJ, Ruderman EM, Horwitz DA, Rheum., 39, 1077 ± 1081. Arkfeld DG, Garrison L, Burge DJ, Blosch CM, Lange Teurich S and Angel P. (1995). Chem. Senses, 20, 251 ± 255. ML, McDonnell ND and Weinblatt ME. (1999). Ann Tracey KJ and Cerami A. (1994). Annu.Rev.Med.,45, 491. Intern. Med., 130, 478 ± 486. Tronche F, Kellendonk C, Reichardt HM and Schutz G. NicholsonRC,MaderS,NagpalS,LeidM,Rochette-EglyC (1998). Curr.Opin.Genet.Dev.,8, 532 ± 538. and Chambon P. (1990). EMBO J., 9, 4443 ± 4454. Tuckermann JP, Reichardt HM, Arribas R, Richter KH, Nishio Y, Isshiki H, Kishimoto T and Akira S. (1993). Mol. Schutz G and Angel P. (1999). J. Cell. Biol., 147, 1365 ± Cell. Biol., 13, 1854 ± 1862. 1370. Nissen RM and Yamamoto KR. (2000). Genes Dev., 14, TumlinJA,LeaJP,SwansonCE,SmithCL,EdgeSSand 2314 ± 2329. Someren JS. (1997). J. Clin. Invest., 99, 1217 ± 1223. Pasparakis M, Alexopoulou L, Episkopou V and Kollias G. Umesono K and Evans RM. (1989). Cell, 57, 1139 ± 1146. (1996). J. Exp. Med., 184, 1397 ± 1411. van de Stolpe A, Caldenhoven E, Raaijmakers JA, van der Patel L, Abate C and Curran T. (1990). Nature, 347, 572 ± Saag PT and Koenderman L. (1993). Am.J.Respir.Cell. 575. Mol. Biol., 8, 340 ± 347. Pearce D and Yamamoto KR. (1993). Science, 259, 1161 ± vanTiborgMAA,LefstinJA,KruiskampM,TeubenJ-M, 1165. Boelens R, Yamamoto KR and Kaptein R. (2000). J. Mol. Peterziel H, Mink S, Schonert A, Becker M, Klocker H and Biol., 301, 947 ± 958. Cato AC. (1999). Oncogene, 18, 6322 ± 6329. Vanden Berghe W, Francesconi E, De Bosscher K, Resche- Petrij F, Giles RH, Dauwerse HG, Saris JJ, Hennekam RC, Rigon M and Haegeman G. (1999). Mol. Pharmacol., 56, Masuno M, Tommerup N, van Ommen GJ, Goodman 797 ± 806. RH, Peters DJ et al. (1995). Nature, 376, 348 ± 351. VayssieÂ re BM, Dupont S, Choquart A, Petit F, Garcia T, Pfahl M. (1993). Endocr. Rev., 14, 651 ± 658. MarchandeauC,GronemeyerHandResche-RigonM. Pfeer K, Matsuyama T, Kundig TM, Wakeham A, (1997). Mol. Endocrinol., 11, 1245 ± 1255. Kishihara K, Shahinian A, Wiegmann K, Ohashi PS, Wang Y, Zhang JJ, Dai W, Lei KY and Pike JW. (1997). J. Kronke M and Mak TW. (1993). Cell, 73, 457 ± 467. Immunol., 159, 534 ± 537. Pratt WB. (1997). Annu. Rev. Pharmacol. Toxicol., 37, 297 ± Widen C, Zilliacus J, Gustafsson JA and Wikstrom AC. 326. (2000). J. Biol. Chem., 275, 39296 ± 39301. Ray A and Prefontaine KE. (1994). Proc. Natl. Acad. Sci. Wissink S, van Heerde EC, Schmitz ML, Kalkhoven E, van USA, 91, 752 ± 756. der Burg B, Baeuerle PA and van der Saag PT. (1997). J. Reichardt HM, Kaestner KH, Tuckermann J, Kretz O, Biol. Chem., 272, 22278 ± 22284. Wessely O, Bock R, Gass P, Schmid W, Herrlich P, Angel Wissink S, van Heerde EC, van der Burg B and van der Saag P and Schutz G. (1998). Cell, 93, 531 ± 541. PT. (1998). Mol. Endocrinol., 12, 355 ± 363. Reichardt HM, Umland T, Bauer A, Kretz O and SchuÈ tz G. Wondisford FE, Steinfelder HJ, Nations M and Radovick S. (2000). Mol. Cell. Biol., 20, 9008 ± 9017. (1993). J. Biol. Chem., 268, 2749 ± 2754. Reichardt HM, Tuckermann JP, JoÈ ttlicher M, Vujic M, Yang-Yen HF, Chambard JC, Sun YL, Smeal T, Schmidt TJ, WeihF,AngelP,HerrlichPandSchuÈ tz G. (2001). Proc. Drouin J and Karin M. (1990). Cell, 62, 1205 ± 1215. Natl. Acad. Sci. USA, (in press). Yang-Yen HF, Zhang XK, Graupner G, Tzukerman M, Reul JM and de Kloet ER. (1985). Endocrinology, 117, Sakamoto B, Karin M and Pfahl M. (1991). New Biol., 3, 2505 ± 2511. 1206 ± 1219. Rider LG, Hirasawa N, Santini F and Beaven MA. (1996). J. Yao TP, Oh SP, Fuchs M, Zhou ND, Ch'ng LE, Newsome Immunol., 157, 2374 ± 2380. D, Bronson RT, Li E, Livingston DM and Eckner R. Sakai DD, Helms S, Carlstedt-Duke J, Gustafsson JA, (1998). Cell, 93, 361 ± 372. Rottman FM and Yamamoto KR. (1988). Genes Dev., 2, Zhang XK, Wills KN, Husmann M, Hermann T and Pfahl 1144 ± 1154. M. (1991). Mol. Cell. Biol., 11, 6016 ± 6025.

Oncogene