InVitro CelL. Dev. Biol. 30A:761-768. November 1994 © 1994 Society forInVitro Biology 1071-2690/94 $02.50+0.00 A NOVEL PATHWAY FOR RETINOIC ACID-INDUCED DIFFERENTIATION OF F9 CELLS THAT IS DISTINCT FROM RECEPTOR-MEDIATED TRANS-ACTIVATION ISSAY KITABAYASHI,ROBERT CHIU, KAZUHIKOUMESONO, RONALD M. EVANS, GABRIEL GACHELIN, AND KAZUSHIGE YOKOYAMA ~ Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, lbaraki 305, Japan (I.K., K.Y.); Department of Surgery/Oncology, UCLA School of Medicine, and Jonsson Comprehensive Cancer Center, Los Angeles, CA 90024 (R.C.); Gene Expression Laboratory, Howard Hughes Medical Institute, The Salk Institute for Biological Studies, P. O. Box 85800, San Diego, CA 92186-5800 (K.U., R.M.E.); Department of lmmunology, Institut Pasteur, 28 Rue du Dr. Roux, 75724 Paris Cedex 15, France (G.G.) (Received 28 December 1993; accepted 22 March 1994) SUMMARY Retinoic acid (RA) has striking effects on vertebrate development and induces differentiation of several lines of ceils including embryonal carcinoma F9 cells. It is generally accepted that the actions of RA are mediated by nuclear receptors for RA. However, we now provide evidence that F9 cells can differentiate in response to RA without trans-activation by nuclear receptors. Irreversible differentiation of F9 cells was induced by 18 h of exposure to RA with subsequent incubation in the absence of RA. This induction of differentiation was not blocked after inhibition of protein synthesis and mRNA synthesis during the 18-h treatment with RA, xbutthe endogenous RA receptors failed to activate transcription from their target genes that contain the receptor-binding sequences. During the commitment to RA-induced differentiation, at least five sets of four pbosphorylated proteins underwent changes in the absence of protein synthesis de novo. These results suggest that there is a novel pathway for the action of RA that is independent of nuclear receptor-mediated trans-activation. Key words: F9 ceils, retinoic acid receptor, c-jun, transcriptional regulation, cell differentiation. INTRODUCTION Retinoids, including retinol (vitamin A) and its active metabolite retinoic acid (RA), have profound effects on cell proliferation and differentiation (31,34) and they appear to play a key role in verte- brate development (5,36). The molecular mechanisms underlying the effects of retinoids are not well understood. Important insights have been gained through the discovery of cellular binding proteins and nuclear receptors for retinoids. Cellular binding proteins for retinol (CRBPs) and retinoic acid (CRABPs) belong to a family of low molecular weight proteins that are all apparently involved in binding hydrophobic ligands (37). However, the biological roles of these binding proteins remain to be clarified (1). The nuclear recep- tors for RA (RARs), such as RAR-t~, RAR-/~, and RAR-'y, belong to a large family of ligand-activated transcriptional factors (2,13,14). An additional class of more distantly related retinoid X receptors (RXRs) has also been described (22,24). These receptors bind to specific nucleotide sequences near the promoters of respon- sive genes and activate their transcription in the presence of reti- noids. A retinoic acid response element (RARE) has been identified in the promoter region of the RAR-fl gene (11,15,35). A palin- dromic thyroid hormone response element (TREp) has also been found to serve as an efficient RARE for both RARs and RXRs (22,40). A retinoid X response element (RXRE), found in the pro- 1To whom correspondenceshould be addressed. moter region of the CRBP type II gene, is activated by RXR but not by RAR, although RA does not activate the RXRE in F9 cells without overexpression of the RXR gene (23). Recent reports show the targeted disruption of the whole RAR-a gene results in early post-lethality and testis degeneration (21) and of the whole RAR-'y gene results in the growth deficiency, early lethality, and male steril- ity (20). Embryonal carcinoma F9 ceils can be maintained as a stable population of undifferentiated stem cells in vitro (3) and can be induced to differentiate into ceils that resemble those of the primi- tive extraembryonic endoderm of the mouse embryo by treatment with RA (34). RAR-a and -7 mRNAs are constitutively expressed in F9 stem cells and the levels of both mRNAs are not affected by treatment with RA (16,25). The level of expression of the RAR-fl gene is very low in F9 cells, but the gene is rapidly and strongly induced by treatment with RA (10,25). Loss of RAR-'y function in F9 cells by gene disruption results in aberrant expression of some differentiation-marker upon RA treatment, but not all differentia- tion-specific genes are affected (4). They suggest an idea that each RAR may regulate different sets of RA-responsive genes during the development process by RA. The RA-induced differentiation of F9 cells involves induction of expression of the c-jun gene (19,43), which encodes a major compo- nent of transcription factor AP-1 (8,42). It has been shown that forced expression of the c-jun gene leads to differentiation of em- bryonal carcinoma (EC) cells, such as F9 and P19 cells, in the 761