Oncogene (2002) 21, 2181 ± 2190 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc AP-1 transrepressing retinoic acid does not deplete coactivators or AP-1 monomers but may target speci®c Jun or Fos containing dimers Kazumi Suzukawa1 and Nancy H Colburn*,1 1Gene Regulation Section, Basic Research Laboratory, National Cancer Institute at Frederick, Frederick, Maryland, MD 21702- 1201, USA Retinoic acid (RA) inhibits tumor promotion in many Introduction models in vivo and in vitro, among them mouse epidermal JB6 cells. RA treatment suppresses 12-O-tetradecanoyl- Carcinogenesis is a multistep process consisting of phorbol-13-acetate (TPA) induced AP-1 activity, an initiation, promotion and progression (Fearon and activity that is required for transformation of JB6 P+ Vogelstein, 1990; Klein, 1981). Regulators of responsible cells. The molecular mechanism of AP-1 transrepression signaling pathways for each stage are potential targets by retinoids is unclear, especially as related to inhibition for cancer prevention (Boutwell, 1989; Colburn et al., of transformation. Overexpression of AP-1 components 1979, 1982). A causal relationship between tumor did not rescue TPA induced AP-1 activation nor did a promoter-induced neoplastic transformation and activa- GST pull down experiment implicate direct binding, thus tion of transcription factor AP-1 has been demonstrated rendering unlikely both a Jun/Fos-RA-RAR direct in mouse and human cell progression models (Bernstein interaction and a Jun/Fos sequestration mechanism. and Colburn, 1989; Dong et al., 1994; Li et al., 1998) and Overexpression of p300, SRC-1 or pCAF did not in a dominant negative c-jun transgenic mouse (Young et abrogate AP-1 suppression by RA, thus arguing against al., 1999). In the JB6 mouse epidermal cell clonal coactivator competition. Overexpression of the corepres- variants, AP-1 transactivation is induced by 12-O- sor silencing mediator for retinoic acid and thyroid tetradecanoylphorbol-13-acetate (TPA) in transforma- hormone receptors (SMRT) suppressed AP-1 activity. tion sensitive but not in resistant cells (Bernstein and However, SMRT but not RA inhibited cJun transactiva- Colburn, 1989). TPA induced cell transformation is tion, suggesting SMRT does not mediate RA transre- blocked by AP-1 inhibitors such as retinoids, glucocorti- pression. RA treatment also did not block TPA induced coids, and the dominant negative cJun (TAM67) (Dong ERK phosphorylation, Jun/Fos family protein expression et al., 1994; Li et al., 1996, 1997). Retinoic acid (RA) is except for cFos, or DNA binding of the AP-1 complex. eective in inhibiting papilloma formation in mouse skin The transcriptional activities of full-length JunB and and transformation of mouse epidermal JB6 cells full-length Fra-1, but not the transactivation domain (Colburn et al., 1981; Li et al., 1996; Verma and fusions, were increased by TPA treatment and sup- Boutwell, 1977). RA induces dierentiation of keratino- pressed by RA. Since these full-length fusions have bzip cytes and suppresses tumor phenotype in several models domains, the results suggest that JunB and/or Fra-1- (Bollag and Holdener, 1992; Chen et al., 1995; Fuchs and containing dimers may constitute one target of RA for Green, 1980). RA can also cause a reversible phenotypic transrepression of AP-1. suppression of the TPA-transformed JB6 cell line RT101 Oncogene (2002) 21, 2181 ± 2190. DOI: 10.1038/sj/ (Dong et al., 1995). onc/1205281 The biological eects of RA are mediated by its receptors (RARs) in the nucleus. The RARs belong to Keywords: AP-1; JB6 cells; retinoic acid; transrepres- a large superfamily of ligand-inducible transcription sion; GAL4 transactivation assay factors that include vitamin D and thyroid hormone receptors. The retinoid receptors consist of two subunits, RARs (a, b, g) and RXRs (a, b, g) (Chambon, 1996). It has been well documented that liganded nuclear receptors including RAR inhibit AP-1 activation (Fisher and Voorhees, 1996; Li et al., 1996, 1997; Pfahl, 1993; Resche-Rigon and Gronemeyer, 1998; Saatcioglu et al., 1994; Schule and Evans, 1991). Transactivation of target genes through the *Correspondence: NH Colburn, Gene Regulation Section, Basic RARE sequence is functionally dissociated from Research Laboratory Building 560 Room 21 ± 89, National Cancer transrepression of AP-1 (Bollag and Holdener, 1992; Institute at Frederick, PO Box B, Frederick, Maryland, MD 21702- Chen et al., 1995; Fanjul et al., 1994; Fuchs and Green, 1201, USA; E-mail: [email protected] Received 22 August 2001; revised 14 December 2001; accepted 19 1980; Li et al., 1996). Inhibition of tumor promotion December 2001 by RA appears to be mediated by blocking AP-1 AP-1 transrepression by retinoic acid in JB6 cells K Suzukawa and NH Colburn 2182 activation, not by activating retinoic acid response element (RARE)- dependent transcription (Li et al., 1996). Mouse JB6 cells express only RAR g and a, not RAR b, and the tumor promotion inhibiting activity has been attributed to the RAR g (Rudd et al., 1993). RAR g is functionally predominant for transrepression in keratinocytes (Goyette et al., 2000). The precise mechanism of transrepression of AP-1 by RA is not clear. Three mechanisms have been suggested. The ®rst is direct interaction between Jun or Fos proteins and liganded RAR (Pfahl, 1993). The second is competition between liganded RAR and Jun or Fos proteins to bind transcriptional coactivators (Kamei et al., 1996; Lee et al., 1998). The third is inhibition of cJun N- terminal kinase (JNK) (Caelles et al., 1997; Lee et al., 1999). The silencing mediator for retinoic acid and thyroid hormone receptors (SMRT) was identi®ed as a corepressor which interacts with unliganded RAR. The interaction between SMRT and RAR is destabi- lized by ligand (Chen and Evans, 1995). SMRT mediates the repressive eect of unliganded nuclear Figure 1 Transrepression of TPA induced AP-1 activity by RA. RA represses TPA induced AP-1 activity in a dose dependent receptors through the recruitment of histone deacety- manner. JB6 P(+) cells were transfected with 0.4 mg of AP-1- lase complexes (Nagy et al., 1997). SMRT also luciferase reporter construct in a 24 well plate. The next day, the suppresses other transcription factors (e.g. AP-1 SRF, cells were exposed to DMSO or TPA (10 ng/ml)+RA (in various NFkB, and Oct-1) (Kakizawa et al., 2000; Lee et al., concentrations as indicated) in 0.2% FBS+EMEM for 18 h. All 2000). Therefore, there is the possibility that RA experiments were performed in triplicate, and standard error of the mean is indicated. The data are shown as mean+s.d. Three treatment recruits SMRT from RAR to AP-1 com- independent experiments showed similar results plexes. The results presented here render unlikely the previously suggested mechanisms as well as recruitment of corepressor SMRT. Instead, RA appears to target RA (Figure 2). When cJun, Fra-1, Fra-2, or cFos was JunB and/or Fra-1-containing dimers. expressed, the AP-1 activation level with TPA+RA was similar to the solvent control level. Furthermore, a GST pull down assay using GST-retinoic acid receptor g (the functionally predominant RAR in JB6 cells) did Results not detect ligand dependent interaction with AP-1 components of a JB6 nuclear extract, while RXR a was RA transrepresses TPA induced AP-1 activation pulled down ligand independently as expected (data The concentrations of RA that inhibit TPA induced not shown). These results suggest that direct interac- transformation of JB6 cells (Dong et al., 1994), also tion of liganded RAR with Jun or Fos proteins is inhibit TPA induced AP-1 activation (Figure 1). Dose unlikely to contribute to AP-1 transrepression. dependent suppression by RA was observed at concentrations lower than 1076 M, with higher con- Concentration of coactivator is not limiting for centrations showing no greater suppression. transrepression It has been suggested that the amount of available No rescue of AP-1 activity from transrepression by jun or p300/CBP or SRC-1 can be limiting when AP-1 and fos overexpression RAR compete for binding to the coactivator (Kamei et If RAR inhibits AP-1 by direct interaction with Jun or al., 1996; Lee et al., 1998). We asked whether any of Fos proteins to sequester them, overexpression of the these coactivators is limiting for AP-1 activity in RA sequestered AP-1 component should cancel the inhibi- treated cells. Overexpression of coactivator p300 or tion. A luciferase assay of AP-1 activation in cells SRC-1 increased both basal and TPA induced AP-1 cotransfected with Jun or Fos expression constructs activity. However, neither of them reversed transre- was performed. Overexpression of JunD or JunB raised pression by RA (Figure 3), nor did expression of ASC- the basal level and prevented further induction of AP-1 2 (not shown) reverse transrepression. by TPA. Expression of cFos substantially increased Overexpression of pCAF (Figure 3) inhibited AP-1 basal AP-1 activity and TPA treatment produced a activation, suggesting pCAF is not involved in the further increase. Overexpression of cJun, Fra-1, Fra-2 coactivator complex of AP-1 in this context. To or cFos enhanced both basal and TPA induced levels determine whether transrepression between RAR and of AP-1 but failed to abrogate AP-1 transrepression by AP-1 is mutual, endogenous RAR activity was Oncogene AP-1 transrepression by retinoic acid in JB6 cells K Suzukawa and NH Colburn 2183 Figure 2 Overexpression of Jun or Fos proteins did not rescue AP-1 activity. Each AP-1 component or pcDNA3.1 as a control DNA was cotransfected with AP-1 reporter construct. JB6 P (+) cells were exposed to 0.1% DMSO or TPA+RA for 18 h. Triplicate samples were run for each condition.