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Home , Receptor (biochemistry), Retinoic acid receptor, Vitamin D receptor

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 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 , National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

The chimeric receptor, RAR␣/VDR, contains the DNA-binding superfamily are dimeric, -inducible- fac- ␣ domain of the (RAR ) and the ligand- tors, which activate nuclear target by binding to specific binding domain of the 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 , (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 (ER): RAR␣/ER and ER/RAR␣/ER. Both hexad repeats (for review see Refs 11 and 12). RAR␣/ER and ER/RAR␣/ER bind ␤- with high affinity, To fine tune -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 .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 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 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 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 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/ 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 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 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; 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 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. bridge, MA, USA), the mouse ␤- promoter gene was inserted into the HindIII cloning site of pUC-13 immediately 5′ of the lacZ gene. The human isoforms of RAR␣, RXR␣,ER and VDR cDNA were cloned into pSG5 expression vectors.9 COS-1 cells: nucleosol fractions and ligand-binding For RAR␣/VDR (Figure 2a), the VDR-ligand-binding domain assays (PCR-amplified (sequence verified) cDNA of amino acids 123 to 427 with engineered BamHI sites at either end) was inserted pSG5 expression vectors for RAR␣, and the chimeras were into the corresponding cloning site of pSG5-RAR␣ (cDNA for transfected into COS-1 cells by electroporation.9 Nucleosol amino acids 1 to 199) immediately 3′ of the codon for RAR fractions were prepared 72 h after transfection and stored at residue 199. For RAR␣/ER, the ligand-binding domain of ER −80°C.32,35 Receptor ligand-binding assays of nucleosol frac- (cDNA for amino acids 302 to 395) replaced that of VDR in tions have been described in detail elsewhere,9,32 and pSG5-RAR␣/VDR. The ER/RAR␣/ER construct had the C involved either FPLC size exclusion chromatography or PD10 region of RAR␣ (cDNA for amino acids 88 to 153) replacing (Pharmacia, Uppsala, Sweden) desalting columns. Data analy- the C region of ER (cDNA for amino acids 185 to 250).30 The sis of saturation kinetics followed the method of Scatchard36 plasmid, pSV2-neo, has been described previously.31 as described previously.9,37

Cell culture and generation of stable transfectants Transactivation assays in CV-1 cells

CV-1, COS-1, and F9 cells were maintained as described.32,33 The procedures for transient cotransfection of CV-1 cells, The heat-inactivated fetal calf serum was charcoal-stripped for ligand incubations (36 h), preparation and analysis of cell lys- F9 cells and for CV-1 cells when ␤-estradiol was the ligand. ates have been described previously.38 All cotransfections In the latter case, the media was also phenol-free. F9 cells contained 0.5 ␮g receptor expression plasmid, 5 ␮g reporter ␣ ␤ ␮ ␤ were cotransfected at a 9:1 ratio of pSG5-RAR /VDR or plasmid (eg (RARE)3-TK-LUC), 5 g control plasmid-, p Ac- Bluescript IIKS to pSV2-neo for a total of 20 ␮g plasmid DNA lacZ, 4–5 ␮g carrier plasmid-Bluescript IIKS, for a total of per 2 × 106 cells (100 mm culture plate) using the 15 ␮g per 100 mm plate. Ligand-mediated transcriptional phosphate precipitation method.34 G418-sulfate selection activity was expressed as either fold induction of normalized pressure (400 ␮g/ml) was applied after one cell passage. Neo- luciferase activity above that observed in the absence of mycin resistant clones were grown up in mass cultures ligand, or as a percentage of maximal normalized response. Functional properties of RAR␣/VDR SM Pemrick et al 556 Co-immunoprecipitation of RXR␣ heterodimers with (BioRad, Richmond, VA, USA). Cells transfected with pSG5- − RXR␣ antibody RAR␣/ER or pSG5-ER/RAR␣/ER were treated with 10 8 M ␤- estradiol for 1 h before harvesting. The RARE oligonucleo- RAR␣ and chimeric receptor cDNA were transcribed in vitro tide27 was labeled by filling in the 5′ overhangs with dATP, into capped mRNA using the mCAP (Stratagene) with T3 dTTP, dGTP and ␣-32P dCTP using the Klenow fragment of E. polymerase. Purified mRNA was translated in vitro using rab- coli DNA polymerase 1. Specific competitor oligonucleotides bit reticulocyte lysates (Promega) in the presence of 35S-meth- were prepared similarly, but with unlabeled dCTP. Nonspe- ionine.39,40 Construction of a baculovirus containing RXR␣ cific competitor oligonucleotides consisted of two copies of cDNA and preparation of RXR nucleosol fractions have been a consensus DNA-binding site for the .45,46 The described.41 Affinity purified rabbit antibody was directed to preincubation and incubation protocols for the whole cell a 22 amino acid sequence of the D domain of RXR␣.41 To extracts and RXR were as described in detail elsewhere.38 measure RXR heterodimer formation, nuclear extract contain- Protein-DNA complexes were resolved on a native 5% ing RXR␣ (125 ng protein) was incubated with 35S-labeled in (acrylamide-N,N′-methylenebisacrylamide 29:1) polyacryla- vitro translated RAR␣ or its chimeras, in 100 ␮l buffer A mide gel (2 h at 20 mA). Gels were dried (1 h at 80°C) and (20 mM N-2 hydroxyethylpiperazine-N′-2-ethanesulfonic acid exposed to film as described above.

HEPES, pH 7.9), 50 mM NaCl, 1 mM Na2EDTA, 5% glycerol, 0.05% Triton X-100 plus 0.5% BSA (w/v) for 1 h (4°C), fol- lowed by incubation with RXR␣-specific antibody or normal Results serum for 16 h (4°C). Samples were further incubated (2 h) ␮ ␣ with pre-washed protein A-agarose beads (15 l; Boehringer RAR /VDR binds D3 with high affinity and specificity Mannheim), and washed in buffer A plus and minus 0.1% BSA. Bound material was eluted in SDS-PAGE sample buffer Nucleosol fractions from RAR␣/VDR transfected COS-1 cells, 42 ° 3 and analyzed on 8% polyacrylamide gels. Gels were pro- were treated (4 h, 4 C) with HD3 and analyzed by size- cessed for fluorography, impregnated with Enlightening exclusion FPLC. The elution profile consists of one radioactive (Dupont-NEN), dried and exposed to film.40 peak at 25.3 min, corresponding to the predicted molecular weight of 56.4 kDa for RAR␣/VDR. VDR, however, elutes at 25.9 min because of a lower predicted molecular weight Gel mobility shift assays (48.3 kDa). In the presence of a 100-fold molar excess of unla- beled ligand, the elution profile is flat providing no evidence ␣ COS-1 cells were transfected by the DEAE-dextran procedure for nonspecific binding of D3 to either RAR /VDR or VDR with 10 ␮g each of pSG5 (Control) or receptor expression vec- (data not shown). tors. Whole cell extracts were prepared,38,43 and protein con- The principles of saturation kinetics were used to quantitate 44 ␣ centration determined using a commercial Bradford reagent the affinity of D3 for VDR- and for RAR /VDR-enriched

3 ␣ ␣ Figure 1 Saturation kinetics of the binding of H1, 25-dihydroxy-vitamin D3 (D3) to VDR (a) and RAR /VDR (b) enriched COS-1 nucleosol fractions. Specific binding (solid squares) is defined as total binding (open circles) minus nonspecific binding (solid circles). Scatchard analysis ␣ of the saturation kinetics yields a constant (Kd) of 0.3 nM for VDR (c) and 0.5 nM for RAR /VDR (d). Functional properties of RAR␣/VDR SM Pemrick et al 557 nucleosol fractions (Figure 1). This approach confirms mini- fold by 100 nM D3. The half-maximal induction value was not mal nonspecific binding of D3 and similar expression levels calculated with the RXRE because of the shallow transactiv- for both VDR and RAR␣/VDR (Figure 1a and b). Scatchard ation profile. A similar response element selectivity is ␣ analyses (Figure 1c and d) of the specific binding of D3 to VDR observed for RAR in the presence of tRA (see below and ␣ and to RAR /VDR provide approximate Kd values of 0.3 and in Table 1). 0.5 nM, respectively.

Comparison of RAR␣/VDR with RAR␣/ER and ␣ ␣ RAR /VDR induces D3-mediated transcriptional ER/RAR /ER activation Both RAR chimeras with ER ligand-binding domains bind 3H- The transactivation properties of RAR␣/VDR are analyzed with ␤-estradiol and exhibit a retention profile by size exclusion ␣ two reporter genes as a function of the D3 concentration. FPLC similar to that of ER (not shown). Neither RAR /ER nor Background levels of transactivation mediated by the reporter ER/RAR␣/ER, can induce ␤-estradiol-mediated transactivation genes in the absence of RAR␣/VDR are very low and unre- from the RARE (Table 1). This inactivity is not due to the cellu- sponsive to D3 (not shown). As shown in Figure 2b, lar milieu of CV-1 cells. Wild-type ER, when expressed in CV- ␣ ␤ ␤ RAR /VDR is a strong transcriptional activator from (RARE)3- 1 cells is capable of five-fold induction of -estradiol- TK-LUC, and a weak transcriptional activator from mediated transactivation from an ERE reporter gene (not RXRE(ApoA1)-LUC. With the RARE, induction of transactiv- shown). ␣ ation by RAR /VDR correlates with the D3 concentration. Maximal induction is 26-fold and occurs at 20 nM D3. Half- maximal induction occurs at 0.5 nM D3, in good agreement Ability to form heterodimers with RXR with the observed Kd for binding of D3 to this chimera (Figure 2). With the RXRE(ApoA1) reporter gene, maximal In a representative experiment, the ability of the three chim- induction of transactivation by RAR␣/VDR is less than four- eras to form heterodimers with RXR␣ was compared with RAR␣. Figure 3 shows RAR␣ and RAR␣/VDR form heterodi- mers with RXR␣; whereas, RAR␣/ER and ER/RAR␣/ER fail to heterodimerize with RXR␣. Noticeably, the amount of the het- erodimer complex is greater for RAR␣ than for RAR␣/VDR. Increasing the autoradiograph exposure time from 3 h (Figure 3) to 16 h (not shown) gives an additional weak signal indicating possible heterodimer formation for RAR␣/ER, but not for ER/RAR␣/ER.

Ability to bind to a RARE

The relative ability of the receptors, ER and RAR␣, vs the chimeras, to bind to a RARE was determined by the gel mobility shift assay. Figure 4 shows only RAR␣ and RAR␣/VDR bind to the RARE. The specificity of this binding

Table 1 Relative comparison of induction of transcriptional acti- vation by RAR␣ and its chimeras from direct repeat and palin- dromic HREs

Receptor Direct repeat HREs Palindromic HREs

␤(RARE)3 RXREs TREpal (TRE3)3 ApoA1 CRBPII

RAR␣ 100 4 0 100 20 RAR␣/VDR 70 3 1 33 33 RAR␣/ER 0 0 6 0 27 ER/RAR␣/ER 0 0 15 32 100 RXR␣ 7 100 100 100 100

Differential activation of a variety of synthetic RA-responsive pro- moters by RAR, RXR, and chimeric RARs. For each receptor, trans- Figure 2 The RAR␣/VDR chimeric receptor. (a) Diagrammatic rep- activation was expressed as fold induction of LUC activity by ligand resentation of the formation of the RAR␣/VDR construct from the above background (no ligand). For each HRE, a value of 100 per- DNA-binding domain of RAR␣ and the ligand-binding domain of cent was assigned the receptor exhibiting the highest fold induction VDR. (b) Comparison in CV-1 cells of the ability of RAR␣/VDR to of ligand-mediated transcriptional activation, and the ligand- induce transcriptional activation from a RARE reporter gene mediated transcriptional activity of the other receptors ranked as ␤ ( (RARE)3-TK-LUC) and from a RXRE reporter gene (RXRE(ApoA1)- percent of maximum. Data are calculated from mean of duplicate ␤ LUC) as a function of the concentration of D3. Data are expressed as experiments. The 100% values are: (RARE)3, 41-; ApoA1, 25-; means of duplicate experiments. CRBPII, six-; TREpal, two-; (TRE3)3, three-fold induction. Functional properties of RAR␣/VDR SM Pemrick et al 558

Figure 3 RAR␣ and the chimera, RAR␣/VDR form heterodimers with RXR. The apparent molecular weights of 35S-labeled in vitro translated RAR␣ and the chimeras (RAR␣/VDR, RAR␣/ER and ER/RAR␣/ER) were determined by 8% SDS-PAGE (first lane in each panel: Ab ‘−’, RXR ‘−’). RXR heterodimers were detected by the tech- nique of co-immunoprecipitation. Nuclear extracts ‘+’ and ‘−’ baculo- viral-expressed RXR␣ (RXR ‘+’ or RXR ‘−’) were incubated (1 h, 4°C) with 35S-labeled in vitro translated RAR␣ or its chimeras, followed by incubation (16 h, 4°C) with RXR␣-specific antibody (Ab ‘+’) or normal serum (Ab‘−’). The 35S-labeled RXR heterodimer-antibody complex (Ab ‘+’, RXR ‘+’), if present, bound to protein A-agarose beads and could be eluted in SDS-PAGE sample buffer for autoradiographic analysis following fluorography of 8% gels.

Figure 5 A representative Northern blot of total RNA from six stable transfectant F9 cell lines, four F9-neo lines containing the neo- mycin gene and two, F9-neo-RAR␣/VDR lines (line 1,2B (left), line 4,2B (right) containing both the neomycin and the RAR␣/VDR genes. The Northern blot was hybridized with a random-labeled probe for the ligand-binding domain of VDR. RNA size markers are indicated.

is demonstrated by the ability of a 100-fold excess of unlab- Figure 4 Gel mobility shift assay (EMSA) comparing the relative ability of ER and RAR␣ vs the chimeras, to bind to the ␤RARE. Only eled RARE oligo, but not a nonspecific oligo, to eliminate the RAR␣ (␣) and RAR␣/VDR (␣-V) bind to the 32P-labeled RARE probe; radioactive signal. This experiment was performed with whole binding is specific as determined by the ability of a 100-fold excess cell extracts from transiently transfected COS-1 cells, which of unlabeled RARE (S oligo), but not nonspecific oligo (N oligo) to also contain endogenous RXR␣. Binding to the RARE was eliminate detection of a labelled receptor-RARE complex. Binding to measured both in the presence (Figure 4) and absence (data the RARE performed in 150 mM KCl with 32P-␤RARE probe in the ␣ ␣ ␣ not shown) of exogenous RXR . In both cases, RAR and absence (C) or presence of either pSG5 (M, mock) ER, ER/RAR /ER ␣ (E-␣-E), RAR␣/ER (␣-E), ␣,or ␣-V transfected COS-1 whole cell extracts RAR /VDR form only one type of complex (heterodimer) on (0.5 ␮g + 0.5 ␮g RXR␣). Representative autoradiograph of PAGE in the RARE; ER and ER/RAR␣/ER do not bind to the RARE. How- 5% gels. ever, RAR␣/ER gives some indication of binding to the RARE Functional properties of RAR␣/VDR SM Pemrick et al 559 a b c

d e f

Figure 6 Changes in morphology of F9 wild-type cells (a, b, c) and F9-neo-RAR␣/VDR cells (d, e, f), 96 h after treatment with either 0.1% ␮ ␮ ␮ ethanol (a, d), 1 M tRA (b, e) or 100 nM D3 (c, f) in the presence of 250 M dibutyryl cAMP, and 500 M theophylline. All figures are phase contrast micrographs (green filter) × 500.

under low stringency conditions and high chimera concen- (RXREs, ApoA1 and CRBP II); palindromic (TREpal and ␣ trations (data not shown). (TRE3)3). In general, the activity profile of RAR /VDR follows most closely that of RAR␣ on the various HREs. Both RAR␣ and RAR␣/VDR are considerably active on the RARE, - Transcriptional activity from a variety of HREs tively inactive on the RXREs, and marginally active on the pal- indromic HREs. However, RAR␣/VDR is only one-third as In Table 1, RAR␣ and RXR␣ are compared with the chimeras active as RAR␣ from the TREpal reporter gene. RAR␣/ER and for the ability to induce ligand-mediated transcriptional acti- ER/RAR␣/ER, on the other hand, are relatively inactive on DR ␤ vation from the following HREs: DR-5 ( (RARE)3); DR-2 HREs, but can be active on palindromic HREs. For example, ER/RAR␣/ER is transcriptionally active from both palindromic ␣ HREs, whereas RAR /ER is active only from the (TRE3)3 reporter gene. RXR␣ appears to be maximally active, not only from RXREs, but also, from the palindromic HREs. However, the absolute value of ligand-mediated transactivation induced by RXR␣ is much less on the TREs, compared with the RXREs (three- vs 25-fold induction, respectively).

Qualitative analysis of the biological activity of RAR␣/VDR

Stable transfectant F9 cell lines were generated which contain either the neomycin gene alone (F9-neo), or together with the RAR␣/VDR gene (F9-neo-RAR␣/VDR). Figure 5 shows a Northern analysis of total RNA from the above F9 cell lines probed for the VDR-ligand-binding domain transcript. Only F9-neo-RAR␣/VDR cells exhibit a radioactive RNA species migrating at 2500 bps, as expected for RAR␣/VDR mRNA. There is no indication, even after exposure times as long as 2 weeks, that VDR mRNA is expressed in F9-Wt cells (Figure 5 Figure 7 Changes in proliferation of F9 wild-type (F9-Wt) and F9- neo-RAR␣/VDR cells from 0 to 96 h after treatment with either ethanol and data not shown).

(0), tRA (RA) or D3 (D3) in the presence of dibutyryl cAMP and theo- In the presence of tRA, F9 cells differentiate into either par- phylline. Drug concentrations are provided in Figure 6. ietal or primitive endodermal cells depending upon the pres- Functional properties of RAR␣/VDR SM Pemrick et al 560 ence or absence of dibutyryl cAMP, respectively. Spontaneous dimerization interfaces is improbable, because ER does not differentiation is negligible and not enhanced by dibutyryl heterodimerize with RXR, but instead forms ER homodimers, cAMP alone. As shown in Figure 6a and d, both F9-Wt and which selectively bind to palindromic HREs. In fact, the target F9-neo-RAR␣/VDR cells retain the compact morphology of DNA (HRE) acts as a necessary allosteric activator of recog- undifferentiated cells 96 h after treatment with 250 ␮M dibu- nition for steroid receptor homodimers.50 It was not surprising, tyryl cAMP and 500 ␮M theophylline. Four days after treat- therefore, that ER/RAR␣/ER and RAR␣/ER were transcrip- ment with 1 ␮M tRA (plus dibutyryl cAMP and theophylline), tionally inactive from DR HREs (Table 1), even though under both F9-Wt and F9-neo-RAR␣/VDR cells differentiate morpho- certain conditions RAR␣/ER (but not ER/RAR␣/ER) could bind logically into parietal endodermal cells, characterized by long to RXR␣ and to the RARE, albeit to a much lesser degree than cellular processes, rounded cell shape with distinct borders, either RAR␣ or RAR␣/VDR (see Results). and an increase in distance between cells (Figure 6b and e). Unliganded RAR, and the unliganded chimeras can inhibit Critical to the present study, when 1 ␮M tRA is replaced by ligand-mediated transcriptional activation by other RARs on ␤ 51 30–100 nM D3 in the above treatment protocol, F9-neo- (RARE)3-TK-LUC. Dominant-negative activity by unli- RAR␣/VDR cells differentiate into parietal endodermal cells ganded receptors was not an artefact due to squelching of gen- (Figure 6f); whereas, the F9-Wt cells retain the compact mor- eral transcription factors,52 because inhibition was relieved by phology of undifferentiated cells (Figure 6c). increasing the concentration of expression plasmid for the In the presence of nontoxic concentrations of tRA (ie 1– liganded receptor (SM Pemrick, unpublished observations). 500 ␮M),47 the growth rate of F9-Wt cells decreases concomi- Dominant-negative activity has been observed by mutated tant with differentiation. In Figure 7, this antiproliferative RARs,53,54 PML/RAR␣,55 and by native RARs in the presence 56–58 effect of tRA is compared with the effect of D3 for F9-Wt vs of nuclear receptor co-repressors, that shift the equilib- F9-neo-RAR␣/VDR cells. Four days following drug treatment, rium of unliganded receptors toward the repressive state.58 tRA decreases by 50–60% the growth rate of both F9-Wt and Obviously, overexpression of RAR␣/VDR in F9 cells could ␣ F9-neo-RAR /VDR cells. D3, however, has no effect upon the conceivably, in the absence of D3, suppress RAR-mediated growth rate of F9-Wt cells, but decreases by approximately gene pathways.52,56–58 For this reason, the differentiation 55% the growth rate of F9-neo-RAR␣/VDR cells, similar to experiments (Figures 6 and 7) were performed on F9-neo- that observed in the presence of tRA for both F9-Wt and F9- RAR␣/VDR cell lines with low expression levels of the chim- neo-RAR␣/VDR cells. eric receptor gene (ie line 1,2B in Figure 5). Fortunately, this concern may be unwarranted, because we were able to gener- ate healthy RAR␣/VDR mouse lines which overexpressed the Discussion RAR␣/VDR transgene.59 The goal of genetic experiments which disrupt or ‘knockout’ The present study analyzed the properties of RAR␣/VDR in both alleles of one or two RAR isoforms2,15,60,61 is to assign three cellular systems (COS-1, CV-1, F9) to conclude that both specific tRA-mediated effects and gene pathways to specific RAR␣/VDR and RAR␣ exhibit similar fold-induction of ligand- RAR isoforms. What has been achieved, however, provides mediated transcriptional activation from a variety of HREs evidence for functional redundancy or receptor recruitment (Figure 2b and Table 1), form heterodimers with RXR among the isoforms.2,60,62 In contrast, the chimeric receptor (Figure 3), bind to the RARE (Figure 4) and induce ligand- approach attempts to induce RAR-mediated effects by a ligand mediated differentiation in F9 cells (Figures 6 and 7). Besides unrelated to tRA without disturbing retinoid responsiveness the obvious heterologous ligand-binding domain, RAR␣/VDR (see Figures 6 and 7), and derepress or augment ligand- differs from RAR␣ in two minor respects: less active from the mediated gene transcription induced by RARs. Both experi- TREpal reporter gene (Table 1); weaker RXR-binding proper- mental strategies are complementary, yielding similar gen- ties under equilibrium binding conditions (Figure 3). Others48 eral interpretations. have observed that RXR heterodimers which are unstable The usefulness of RAR␣/VDR as a biological probe is most under in vitro assay conditions may be sufficiently stable in obvious for, but not restricted to, cells and tissues which do

vivo to induce gene transcription. This is obviously the case not express VDR or respond to D3. For example, U937 for RXR-RAR␣/VDR in F9 cells (Figures 6 and 7). It remains to (monocytic), HL-60 (myelocytic) and THP-1 (mature ␣ be determined whether RAR /VDR is functional in a wide var- monocytic) cells differentiate in response to either tRA or D3. iety of model systems. For example, in NB4 cells, an APL In general, D3 causes monocytic differentiation, which can ␣ ␤ 63 model system, RAR /VDR would have to successfully com- be potentiated by TGF- 1; whereas, tRA causes granulocytic pete against PML/RAR␣ for RXR. This may require relatively differentiation.64 Expression of PML/RAR␣ in mutant U937 ␣ high RAR /VDR expression levels depending upon the rela- cells blocks specifically the ability of D3 alone or combined ␣ ␣ 65 tive stability of RXR-PML/RAR vs RXR-RAR /VDR in vivo. with TGF-B1 to induce differentiation. NB4 cells express ␣ ␣ ␣ 66 66,67 The chimeras, RAR /ER and ER/RAR /ER, were analyzed PML/RAR and possibly VDR, but respond poorly to D3. to test the general applicability of swapping ligand-binding Recent evidence suggests any D3 effects in NB4 cells are inde- domains between nuclear receptors, without destroying tran- pendent of nuclear receptor binding.68 The subline NB4.306, 66 scriptional activation. We conclude neither RAR chimera with is resistant to both tRA and D3, and does not express a func- an ER ligand-binding domain is capable of substituting for tional PML/RAR␣.69 However, NB4.306 cells do express RAR␣ in cellular systems (this study, and JF Grippo, unpub- RAR␣ and appear to retain some responsiveness to tRA, such lished data in F9 cells). Even though RAR␣/ER49 and as CD18 expression.69 These findings suggest RAR␣/VDR is ER/RAR␣/ER can be active from palindromic HREs, neither is likely to be an useful experimental tool in tRA-sensitive and ␣ ␤ active from the preferred HRE of RAR , (RARE)3 (Table 1). In possibly tRA-resistant NB4 cells to probe gene pathways regu- the case of RAR (VDR or TR), the DR HRE correctly positions lated by RAR␣. dimerization interfaces within the DNA- and ligand-binding The potential usefulness of RAR␣/VDR as a gene therapy is

domains to within close proximity of specific interfaces on not limited to cells and tissues unresponsive to D3. In mam- RXR.12,23 For RAR␣/ER and ER/RAR␣/ER, correct alignment of malian cells, overexpression of targeted genes by foreign Functional properties of RAR␣/VDR SM Pemrick et al 561 nuclear receptors such as the insect ,70 and 13 Chambon P. The retinoid signaling pathway: molecular and gen- by chimeric receptors such as RAR␣/VDR, offers considerable etic analysis. Semin Cell Biol 1994; 5: 115–125. 14 Fanjul AN, Bouterfa H, Dawson M, Pfahl M. Potential role for potential as part of gene therapy strategies to control cancer. ␥ 71 retinoic acid receptor- in the inhibition of cells by Adapting gene therapy technologies to induce cell lineage- selective retinoids and interferons. Cancer Res 1996; 56: 1571– specific terminal differentiation and could represent 1577. a major advance in cancer therapy. For example, this tech- 15 Boylan JF, Lohnes D, Taneja R, Chambon P, Gudas LJ. Loss of nique may make it possible to derepress RAR-induced gene retinoic acid receptor ␥ function in F9 cells by gene disruption pathways, to trigger terminal differentiation or growth inhi- results in aberrant. Hoxa-1 expression and differentiation upon bition of a wide variety of neoplastic lesions. retinoic acid treatment. Proc Natl Acad Sci USA 1993; 90: ␣ 9601–9605. We suggest RAR /VDR be explored initially as a gene ther- 16 Green S, Chambon P. Nuclear receptors enhance our understand- apy in retinoid-responsive leukemias (eg APL or acute myelog- ing of transcription regulation. Trends Genet 1988; 4: 309–314. enous leukemia (AML)) for the following reasons. Firstly, this 17 Thompson CC, Evans RM. Trans-activation by thyroid hormone exploration is now feasible. 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