Leukemia (1998) 12, 554–562 1998 Stockton Press All rights reserved 0887-6924/98 $12.00 http://www.stockton-press.co.uk/leu Characterization of the chimeric retinoic acid receptor RAR␣/VDR ´ SM Pemrick1, P Abarzua2, C Kratzeisen2, MS Marks3, JA Medin3, K Ozato3 and JF Grippo1 Departments of 1Metabolic Diseases and 2Oncology, Hoffmann-La Roche, Inc., Nutley, NJ; and 3Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA The chimeric receptor, RAR␣/VDR, contains the DNA-binding superfamily are dimeric, ligand-inducible-transcription fac- ␣ domain of the retinoic acid receptor (RAR ) and the ligand- tors, which activate nuclear target genes by binding to specific binding domain of the vitamin D receptor (VDR). The ligand- binding properties of RAR␣/VDR are equivalent to that of VDR, DNA sequences, termed HREs, within the promoter region. ␣ with an observed Kd for 1 ,25 dihydroxy-vitamin D3 (D3)of HREs are arranged as palindromic or direct repeats (DR) of a 0.5 nM. In CV-1 cells, both RAR␣ and RAR␣/VDR induce compa- hexad consensus sequence (PuGGTCA). The steroid receptors, rable levels of ligand-mediated transcriptional activity from the such as GR and ER, bind as homodimers to palindromic HREs, ␤ retinoic acid responsive reporter gene, (RARE)3-TK-lucifer- whereas RAR, TR and VDR (the non-steroid receptors) form ase, in the presence of the ligand predicted from the receptor ligand-binding domain. Two chimeric RAR receptors were con- heterodimers with RXR and bind preferentially to DR HREs, structed which contained the ligand-binding domain of the each heterodimer selecting for a specific distance between estrogen receptor (ER): RAR␣/ER and ER/RAR␣/ER. Both hexad repeats (for review see Refs 11 and 12). RAR␣/ER and ER/RAR␣/ER bind ␤-estradiol with high affinity, To fine tune retinoid-induced gene regulation, there are and are transcriptionally active only from palindromic RAREs ␣ RAR and RXR subtypes and numerous isoforms, which are (TREpal and/or (TRE3)3). Only RAR /VDR matched in kind and 11 degree the functional characteristics of RAR␣: (1) maximally conserved more across species than within a species. One active from the ␤(RARE); (2) moderately active from the TREs; hypothesis states dimeric combinations of RAR and RXR sub- (3) inactive from the retinoic X receptor response elements types and isoforms regulate subsets of RAREs to exert the plei- (RXREs) ApoA1 and CRBP II; (4) forms heterodimers with otropic effects of retinoids.13 Approaches to explore this ␣ ␤ RXR ; and (5) binds to the RARE. F9 embryonal carcinoma hypothesis have included: rational development of receptor- cell lines were generated which express RAR␣/VDR mRNA (F9- RAR␣/VDR cells) and compared with F9 wild-type (F9-Wt) cells, specific ligands (eg see Ref. 14); targeted disruption which do not express VDR mRNA. Treatment with all-trans reti- (‘knockout’) via homologous recombination of one or both noic acid (tRA) inhibits cell growth and induces the differen- alleles for a particular receptor subtype.2,15 In addition, recep- tiation morphology in both F9-Wt and F9-RAR␣/VDR cells; tor-based gene therapies could feasibly be developed to whereas, treatment with D3 is similarly effective only for F9- manipulate the threshold concentration of specific RXR-RAR RAR␣/VDR cells. It is concluded RAR␣/VDR is an useful ‘tool’ to pinpoint, or to augment transcription from RAREs in gene heterodimers in cancers responsive to retinoids. For the chim- pathways controlled by RAR without inhibiting the retinoid era, RAR␣/VDR described here, the inducible ligand would responsiveness of endogenous RARs. be D3, eliminating acquired drug resistance due to the pharm- Keywords: retinoic acid receptors (RARs) and chimeras; all-trans acokinetic properties of retinoids. retinoic acid; vitamin D3; permanent gene transfection and Designing functional chimeric receptors can be a straight- expression; terminal differentiation; leukemia forward experiment because all hormone receptors possess a linearly arranged modular structure (regions A to E or F), con- served to varying degrees within each family.16 For example, Introduction the chimera GR/TR, replaced the n-terminal A/B region of TR for that of GR, to enhance T induction of transcriptional acti- All-trans retinoic acid (tRA) is known to be a key regulator of 3 vation.17 The most conserved region, the C or DNA-binding growth and development in both the adult, from vitamin A domain, has two zinc finger motifs, and recognizes the base deficient (VAD) animal studies, and in the embryo, from 18 analysis of the teratogenic effects resulting from administration pair sequence of the HRE. In a now classical experiment, 1,2 the glucocorticoid HRE became responsive to ␤-estradiol by of exogenous tRA. Similarly, tRA induces differentiation and 19 inhibits proliferation of certain cell types, including HL-60 replacing the C region of ER with that of GR. Region E, the leukemia3 and F9 embryonal carcinoma cells.4,5 tRA is being ligand-binding domain, is also involved in other structurally 20–24 22 explored as a differentiation therapy in cancer because it overlapping functions, including transactivation and 23,24 induces complete remission in acute promyelocytic leukemia dimerization. The receptor polarity of DR HREs, with RXR ′ (APL).6 Unfortunately, APL patients receiving only tRA therapy at the 5 HRE half-site, plus the freedom of rotation about the ′ 25 eventually experience retinoid resistance and clinical hinge region of the 3 -dimeric partner, suggests the ligand- relapse.7,8 binding domains of RAR, VDR, and TR may be functionally At the molecular level, tRA is a ligand for the retinoic acid interchangeable. Indeed, the ligand-binding domain of TR can receptor, RAR, which also binds the RA isomer 9-cis-RA substitute for that of RAR to place both synthetic25 and natural 9,10 26 (9cRA). Related receptors, RXRs, bind 9cRA. The RARs and RAREs under the control of T3. We describe in this report, RXRs belong to the steroid/nuclear receptor superfamily, the functional characteristics of the chimeric receptor which also includes the thyroid (TR), vitamin D (VDR), gluco- RAR␣/VDR, and show in F9 cells that RAR␣/VDR can mediate corticoid (GR), and estrogen (ER) receptors. Members of this the differentiation morphology in response to D3 treatment, without disturbing retinoid responsiveness. We conclude RAR␣/VDR can be a ‘tool’ to pinpoint or to modulate gene Correspondence: SM Pemrick at her present address: Merck Research pathways regulated by RARs, without inhibiting the functional Laboratories, RY32-605, Rahway, NJ 07065-0900, USA; Fax: penetrance of endogenous RARs. 518 392 6665 Received 25 April 1997; accepted 21 November 1997 Functional properties of RAR␣/VDR SM Pemrick et al 555 Materials and methods (225 cm2 flasks) and screened for VDR and RAR␣/VDR mRNA. Materials tRA and vitamin D3 were obtained from the Department of Growth and differentiation of F9 cells Medicinal Chemistry, Hoffmann-La Roche; G418 sulfate from Gibco BRL (Grand Island, NY, USA); dibutyryl cAMP and F9-Wt and F9-RAR␣/VDR cell lines were plated (1–2 × 104 theophylline from Sigma Chemical (St Louis, MO, USA); cells/well) in 1 ml culture medium in gelatinized 24-well ␣ 3 1 ,25-[26,27- H]-dihydroxy vitamin D3 (D3) specific activity Costar dishes (Cambridge, MA, USA). After 24 h, 10– 35 (sp. ac.) 155 Ci/mmol (NET-626) and S-methionine were 100 ␮l/well of filtered drug stock solution in phosphate-buff- purchased from Dupont-NEN (Boston, MA, USA); [2,4,6,7- ered saline (PBS)-Ca2+/Mg2+ free was added to a final concen- 3H]-oestradiol, sp. ac. 92 Ci/mmol (TRE.322), and deoxycytid- ␮ ␮ tration of 0.1% ethanol, 1 M tRA, 30 or 100 nM D3, 250 M ′ ␣32 ine-5 P triphosphate, sp. ac. 3000 Ci/mmol (AA005) from dibutyryl cAMP, 500 ␮M theophylline. The cultures were Amersham Life Sciences (Arlington Heights, IL, USA). HPLC incubated for 96 h, washed with PBS, photographed (through analysis placed ligand purity at greater than 97%. an inverted microscope equipped with phase contrast optics), trypsinized, and counted manually by means of a hemacyto- meter. Plasmid construction Annealed oligo nucleotides for the following HREs were RNA preparation and Northern analysis inserted into either the BglII or the BamHI site of pTK-lucifer- 27 ␤ ase (LUC); (RARE)3, gatc(gggtagGGTTCAccgaaAGTTCA Total RNA was extracted from F9 cells by the guanidine iso- ctcg)3; (TRE3)3, gatc(ttAGGTCAgggacgTGACCTaa)3aaggccta; thiocyanate procedure (RNAzol B; Biotecx Laboratories, Hou- 11 ′ TREpal, gatctcAGGTCATGACCTga; ERE, the 5 -flanking ston, TX, USA). For Northern analysis, total RNA (20–30 ␮g) − − 28 region ( 331 to 297) of the Xenopus vitellogenin A2 gene. from F9 clones was size fractionated on 1% agarose/6% for- For RXRE(CRBPII)-SV-LUC, the RXRE (gatctgCTGTCAc maldehyde gels in 20 mM MOPS (pH 7.0 HCl), 50 mM Na AGGTCAc AGGTCAcAGGTCAcAGTT),11 was inserted into 2 + EDTA, 5 mM sodium acetate and transferred to Gene Screen the BglII cloning site of Blue Script IIKS( ) (Stratagene, La Jolla, nylon membranes (DuPont-NEN). The RNA blots were CA, USA) in front of the LUC gene and SV40 promoter. The hybridized (at 42°C) with a random primer labeled cDNA RXRE(ApoA1)-LUC reporter gene had a 490 base pair HindIII probe (Boehringer Mannheim Random Primed DNA Labeling fragment of [1Xa]-41A1.CAT containing the apoA1 enhancer 29 Kit; Indianapolis, IN, USA), for the ligand-binding domain of site A oligo A inserted in the HindIII cloning site of the VDR (Figure 2a), which had been purified on G50 sephadex pGL2-basic vector (Promega, Madison, WI, USA). The oligo Quick Spin Columns (Boehringer Mannheim). Washed blots A sequence was: gatcaTGACCCctTGAACCc TGTCCTgatc. For ␤ were exposed to Kodak X-Omat film with an intensifying p Ac-lacZ (gift of G Vasios, Osteoarthritis Sciences Inc, Cam- screen at −80°C, for varying amounts of time.