EXPERIMENTAL CELL RESEARCH 230, 50–60 (1997) ARTICLE NO. EX963408 Modulation of the Retinoic Acid and Retinoid X Receptor Signaling Pathways in P19 Embryonal Carcinoma Cells by Calreticulin MARY SHAGO,* , †GRACE FLOCK,* CHUNG-YEE LEUNG HAGESTEIJN,‡ MICHAEL WOODSIDE,§ SERGIO GRINSTEIN,§ VINCENT GIGUE ` RE,* ,Ø,1 AND SHOUKAT DEDHAR‡ ,  *Molecular Oncology Group, Royal Victoria Hospital, Montre ´al, Que ´bec, Canada H3A 1A1; Departments of †Molecular and Medical Genetics and Medical Biophysics, University of Toronto, Toronto, Ontario, Canada M5S 1A1; ‡Division of Cancer Research, Reichmann Research Building, Sunnybrook Health Centre, Toronto, Ontario, Canada M4N 3M5; §Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8; and Ø Departments of Medicine, Oncology and Biochemistry, McGill University, Montre ´al, Que ´bec, Canada H3A 1A1 ronmental cues provided by soluble mediators such as Calreticulin is a widely expressed calcium binding hormones and growth factors and solid substratum protein that can bind to an amino acid sequence motif, components of the extracellular matrix (ECM). The cell KXGFFKR, which is present in the cytoplasmic domain deciphers these diverse signals via the expression of of all integrin a-subunits. Closely related sequences, intracellular and extracellular receptor systems. These KXFFKR and KXFFRR, are encoded in the DNA-bind- regulatory proteins include nuclear receptors for ste- ing domain of all members of the steroid/thyroid/reti- roid hormones and retinoids [1], protein kinase and G noid receptor superfamily and it has recently been protein-coupled receptors for growth factors [2, 3] and demonstrated that calreticulin inhibits their activity integrins for ECM-mediated events [4]. Cellular activa- both in vitro and in vivo. Here we present novel evi- tion by these signaling molecules often results in the dence that calreticulin can interfere directly with the activation and/or repression of specific gene networks retinoic acid (RARs) and retinoid X (RXRs) receptor whose products will ultimately dictate the decision to pathways. Calreticulin exhibits the ability to inhibit proliferate or differentiate. Deregulation of these and DNA-binding activity of both heterodimeric RAR/RXR other signal transduction pathways often leads to un- and homodimeric RXR complexes in vitro. Inhibition controlled cell growth and progression toward the ma- of RXR binding to DNA is achieved with a concentra- lignant state. tion of calreticulin that is approximately fourfold Retinoids are potent regulators of cell growth and lower than that required for inhibition of RAR/RXR differentiation and exert profound effects in develop- binding to a cognate binding site. Coprecipitation ex- ment and homeostasis [5]. Signal transduction by reti- periments suggest a direct protein:protein interaction noids is mediated by two distinct families of nuclear between calreticulin and retinoid receptors. Stable receptors encoded by six different genes referred to as overexpression of calreticulin in P19 embryonal carci- retinoic acid (RARa, b, and g) and retinoid X (RXRa, noma cells significantly decreases the rapid activation b, and g) receptors (reviewed in [6]). The vitamin A of the endogenous RA-responsive RARb gene, abro- metabolite all-trans retinoic acid (atRA) specifically ac- gates the ability of endogenous RAR/RXR complexes to bind to DNA, and inhibits the emergence of the RA- tivates RARs while the 9-cis isomer (9cRA) activates induced differentiated phenotype. These data demon- both RARs and RXRs at physiological levels. RAR and strate that calreticulin can interfere with the two dis- RXR function as ligand-activated transcription factors tinct retinoid signaling pathways through a mecha- that control gene expression through binding to specific nism likely involving direct protein:protein interac- DNA sequences located in the regulatory regions of tions and that disruption of the retinoid signal alters target genes, known as RA response elements (RAREs) biological processes in vivo.  1997 Academic Press and retinoid X response elements (RXREs). While the multiple RAR isoforms bind DNA with high affinity only as heterodimers with RXR [7 – 12], in the presence INTRODUCTION of 9cRA, RXRs form stable homodimers that specifi- Control of cell proliferation and differentiation in the cally recognize RXREs [13]. For both heterodimeric and organism results from the precise integration of envi- homodimeric retinoid receptor systems, binding to DNA is an essential step for stimulation of gene expres- 1 To whom correspondence and reprint requests should be ad- sion. dressed at Molecular Oncology Group, Royal Victoria Hospital, 687 Calreticulin is a high-capacity calcium binding pro- Pine Avenue West, Montre ´al, Que ´bec, Canada H3A 1A1. Fax: (514) 843-1478. E-mail: vgiguere@dir.molonc.mcgill.ca. tein that was originally implicated to play a role in the 50 0014-4827/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.