Oncogene (2003) 22, 8212–8220 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc Retinoic acid receptors interfere with the TGF-b/Smad signaling pathway in a ligand-specific manner Vale´ rie Pendaries1, Franck Verrecchia1, Serge Michel2w and Alain Mauviel*,1 1INSERM U532, Universite´ Paris VII, Institut de Recherche sur la Peau, Pavillon Bazin, Hoˆpital Saint-Louis, 1, avenue Claude Vellefaux, F-75475 Paris Cedex 10, France; 2Galderma R&D, F-06902 Sophia-Antipolis, France Transforming growth factor-b (TGF-b) and retinoic acid steroid, vitamin D, and thyroid hormone receptors, the (RA) are important regulators of cell growth and peroxisome proliferator-activated receptor, the insect differentiation. The TGF-b receptors utilize Smad pro- edysteroid receptor, and a number of orphan receptors teins to transduce signals intracellularly and regulate whose ligands are unknown (Chambon, 1996; Jetten transcription of target genes, either directly or in et al., 2001). All these nuclear receptors share several combination with other sequence-specific transcription well-characterized structural domains, including a con- factors. Two classes of nuclear receptors, the retinoic acid served DNA- binding domain, and a ligand-binding receptors (RARs) and the retinoic X receptors, are domain at the carboxyl terminus of the receptor involved in mediating transcriptional responses to RA. (Pemrick et al., 1994; Kumar and Thompson, 1999). Given the known interactions between the TGF-b and Nuclear retinoic acid receptors, the retinoic acid RAR pathways, we have investigated the role played by receptor (RAR), and retinoic X receptor (RXR) classes, RAR ligands in modulating functional interactions are each composed of a, b, and g subtypes with more between Smad3 and RARs. Using transient cell transfec- than one isoform for each receptor subtype (Chambon, tion experiments with an artificial Smad3/Smad4-depen- 1996).RARs associate with RXRs to form RAR–RXR dent reporter construct, we demonstrate that RAR heterodimers which bind target DNA sequences, overexpression enhances Smad-driven transactivation, an called retinoic acid response elements (RAREs), effect that requires both Smad3 and Smad4. We provide consisting of directly repeated hexameric half-sites with evidence that RAR effect on Smad3/Smad4-driven the consensus sequence 50-PuG(G/A)(T/A)CA-30, sepa- transcription is prevented by natural and synthetic RAR rated by one to five nucleotides depending on the target agonists, and potentiated by synthetic RAR antagonists. gene.Members of the RAR family recognize two The activity of two TGF-b-responsive human gene natural stereoisomers of RA, all-trans RA (ATRA) promoter constructs was regulated in a parallel fashion. and 9-cis RA, whereas the RXR family exclusively Using both mammalian two-hybrid and immunoprecipita- recognizes 9-cis RA.It has been suggested that tion/Western methods, we demonstrate a direct interac- individual receptor subtypes may control distinct gene tion between the region DEF of RARc and the MH2 expression patterns important for cell growth and domain of Smad3, inhibited by RAR agonists and differentiation (Chambon, 1996). enhanced by their antagonists. We propose that RARs Cellular signaling from the TGF-b family of growth may function as coactivators of the Smad pathway in the factors (e.g. activins, bone morphogenic proteins, and absence of RAR agonists or in the presence of their the TGF-bs) is initiated by binding of the ligand to antagonists, a phenomenon that contrasts with their transmembrane receptor serine/threonine kinases, TbRI known role as agonist-activated transcriptional regulators and TbRII (Derynck and Feng, 1997).Following ligand of RA-dependent genes. activation, signaling from the receptors to the nucleus Oncogene (2003) 22, 8212–8220. doi:10.1038/sj.onc.1206913 occurs predominantly by phosphorylation of cytoplas- mic mediators belonging to the Smad family (Datto and Keywords: transforming growth factor-b; nuclear recep- Wang, 2000; Attisano and Wrana, 2002).The receptor- tors; RAR; Smad; promoter region; gene expression associated Smads (R-Smads), such as Smad1, Smad2, Smad3, and Smad5, interact directly with, and are phosphorylated by, activated type I receptors of the TGF-b superfamily.Activation of the R-Smads is Introduction ligand-specific, but each then forms, upon phosphoryla- tion, heteromeric complexes with Smad4, a common The retinoid receptors belong to a large superfamily of mediator for all receptor-activated Smad pathways. ligand-inducible transcription factors that include the R-Smad/Smad4 complexes are then translocated into the nucleus where they function as transcription *Correspondence: A Mauviel; E-mail: [email protected] factors, either directly or in association with wDeceased other DNA-binding factors (Massague´ and Wotton, Received 31 March 2003; revised 20 June 2003; accepted 30 June 2003 2000).Activated T bRI may phosphorylate Smad2, Modulation of RAR/Smad interactions V Pendaries et al 8213 which drives activin-like transcriptional responses Results through association with transcription factors of the FAST family, or Smad3, which drives TGF-b-specific RAR ligands alter TGF-b-induced Smad3/4-specific transcriptional responses, directly binding specific cis- transcription elements which most often contain the nucleotidic As a first approach to examine the possible interference sequence CAGACA.Another group of Smad proteins, of RAR signaling with the TGF-b/Smad pathway, we the inhibitory Smads, including Smad6 or Smad7, determined the effect of RAR agonists and antagonists prevent phosphorylation and/or nuclear translocation on TGF-b-induced, Smad-driven, gene transactivation of R-Smad/Smad4 heterocomplexes (Datto and Wang, in WI-26 lung fibroblast cultures transfected with the 2000; Massague´ and Wotton, 2000; Attisano and Wrana, 2002). artificial (CAGA)9-lux reporter construct.The latter, which consists of nine copies of the nucleotidic motif Several recent reports have provided novel insights CAGA known to bind Smad3 and Smad4 with high regarding the interactions of nuclear receptors with the Smad signaling pathway.In the case of vitamin D, affinity, cloned upstream of the adenovirus major late promoter, to control the expression of the luciferase which controls transcription of target genes through reporter gene (Dennler et al., 1998), allows to monitor the vitamin D receptor (VDR), Smad3 acts as a Smad3/Smad4-specific transcription in response to coactivator specific for ligand-induced VDR-dependent TGF-b.As expected, TGF- b alone dramatically ele- transactivation, by forming a complex with a member of vated (CAGA) -lux activity (Figure 1a).ATRA and the steroid receptor coactivator-1 protein family 9 CD2043, a synthetic RAR agonist, had a dose- (SRC-1) in the nucleus (Yanagisawa et al., 1999). dependent inhibitory effect on TGF-b-induced transac- The estrogen receptor (ER) suppresses TGF-b-induced tivation, with maximal repressions of 35 and 55%, Smad3 activity when, at the mean time, ER-mediated respectively, at a concentration of 10À6 m.At each transcription is enhanced by TGF-b signaling À10 À8 À6 m (Matsuda et al., 2001). Direct association of Smad3 concentration tested (10 ,10 , and 10 ), CD2043 was consistently more potent than ATRA to repress with the androgen receptor has also been described, Smad-dependent transcription.Inversely, CD3106, a leading to suppression of Smad3/DNA association and synthetic RAR antagonist, dose-dependently poten- resulting in Smad3-dependent gene transactivation tiated TGF-b-induced, Smad3/Smad4-specific, tran- downstream of TGF-b (Chipuk et al., 2001). Peroxi- scriptional response (Figure 1b), with a maximal some proliferator-activated receptor g has been shown effect, three-fold above levels achieved with TGF-b to inhibit TGF-b-induced connective tissue growth alone, at a concentration of 10À6 m.RAR agonists factor expression by interfering with Smad3 (Fu et al., CD2043 and ATRA (not shown), added simultaneously 2001). with CD3106 at a 1/10 ratio (10À7 m CD2043 or ATRA, Cooperation of TGF-b with RA occurs both in À6 m physiological and pathological situations (Roberts and 10 CD3106), neutralized each other in the Smad transactivation assay (Figure 1c), consistent with RAR Sporn, 1992).An explanation may be that RA increases agonist/antagonist effects exerted at the level of the production of active TGF-b and its receptors by a variety of cell types, a mechanism responsible for the endogenous RARs. growth inhibition of human keratinocytes and U937 et al leukemia cells, for example (Nunes ., 1996; RAR and RXR overexpression enhances TGF-b-induced Defacque et al., 1997; Borger et al., 2000). Most Smad3/Smad4-specific transcription recently, it was shown that ATRA is able to decrease the levels of phosphorylated Smad2/Smad3 in response We next tried to determine whether overexpression of to TGF-b, possibly via a RARa-mediated activation of RARs and RXR would affect TGF-b response.As a nuclear serine–threonine phosphatase that remains to shown in Figure 2a, transfection of expression vectors be identified (Cao et al., 2003). Another report demon- for RARa,RARb, and RARg resulted in the upregula- strated intracellular interactions between the TGF-b and tion of Smad3/Smad4-dependent transactivation of RA pathways, whereby RAR stimulates, and fusion (CAGA)9-lux in response to TGF-b.Reproducibly, proteins of retinoid receptors antagonize, TGF-b- RARg was the most potent among RARs to enhance induced growth inhibition