©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Enhancement of HL-60 Differentiation by a New Class of Retinoids with Selective Activity on Retinoid X Receptor (*)

(Received for publication, June 5, 1995; and in revised form, October 11, 1995)

Christian M. Apfel (§) Markus Kamber Michael Klaus Peter Mohr Siegfried Keidel (¶) Peter K. LeMotte (**)

From the From Preclinical Research, Department of Dermatology, F. Hoffmann-LaRoche, CH-4002 Basel, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cellular responsiveness to retinoic acid and its metabolites is conferred through two distinct families of receptors: the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Herein, we report on the identification and characterization of several conformationally restricted retinoids, which selectively bind and activate RX receptors. Under the influence of retinoids, HL-60 myelocytic leukemia cells differentiate into granulocytes. This effect is mediated by RARalpha, as has been demonstrated through the use of a selective RARalpha antagonist (Apfel, C., Bauer, F., Crettaz, M., Forni, L., Kamber, M., Kaufmann, F., LeMotte, P., Pirson, W., and Klaus, M.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7129-7133). Here, we show that conformationally restricted RXR-specific retinoids, at doses that are per se inactive, are able to potentiate by up to one order of magnitude the pro-differentiating effects of all-trans retinoic acid and an RARalpha-selective synthetic retinoid. We also present evidence that these RXR-selective ligands are able to bind to a DNA RXRbulletRAR heterodimer complex. This finding demonstrates that agonists for RARs and RXRs can synergistically promote HL-60 differentiation, which could be mediated through a heterodimer of these receptors.


INTRODUCTION

A subclass of nuclear hormone receptors have been described, termed retinoid X receptors (RXR)(^1)(2, 3, 4) , which are ligand-inducible transcription factors responsive to the 9-cis isomer of retinoic acid(5, 6) . A related subclass of receptors, the retinoic acid receptors (RAR)(7, 8, 9, 10, 11, 12) , also responds to 9-cis retinoic acid (9-cis RA), as well as to the all-trans isomer of retinoic acid (t-RA). A striking feature of RXRs is their ability to heterodimerize with several members of the steroid receptor superfamily, including the RA receptors, thyroid receptors, peroxisome proliferator-activated receptors, vitamin D receptor, and chicken ovalbumin upstream promoter-transcription factor (13, 14, 15, 16, 17, 18, 19, 20, 21) . Growing evidence suggests that heterodimers are the active species for binding to promoter response elements.

In view of the involvement of RXRs in multiple signaling pathways, RXR-selective ligands can possibly be expected to show medical usefulness through their modulation of the above mentioned and perhaps also other hormone-regulated processes. The pharmacological effects of RXR ligands, which are attributable solely to activity on RXRs, are difficult to assess using 9-cis RA as an activating ligand since 9-cis RA also functions as an RAR ligand and consequently elicits the full range of classical retinoid effects. Therefore, to gain deeper insight into RXR function as well as to evaluate their pharmacological usefulness, we have designed and characterized a number of RXR-selective compounds and report on some intriguing pharmacological properties. These compounds are sterically restricted analogues of 9-cis RA, which cannot isomerize. They exhibit high potency and high selectivity for RXRs versus RARs and belong to different structural classes than other RXR-selective compounds recently reported(22, 23, 24) . We have characterized these compounds with regard to their binding affinity to RXRs and RARs, to their transactivation potency of these receptors in transfected cells, and to their ability to induce DNA binding of RXR receptors to RXR response elements.

HL-60 is a human myeloid leukemia cell line, which is exquisitely sensitive to retinoid-induced differentiation(25) . The predominant expression of RARalpha in these cells suggests that retinoids act through this receptor(26, 27) . We have recently found additional evidence supporting this assumption by the use of an RARalpha-selective antagonist, which was able to suppress retinoid-induced differentiation (1) . Therefore, we have used HL-60 as a model system to evaluate the role of RXR in RARalpha-mediated biological responses. We now show that these new RXR-selective agonists synergistically enhance the differentiation of HL-60 cells induced by all-trans RA or an RARalpha-selective retinoid.


EXPERIMENTAL PROCEDURES

Materials

t-RA and analogues were synthesized at F. Hoffmann-La Roche Ltd. (Basel). ^3H-t-RA (50 Ci/mmol) was obtained from DuPont NEN, and ^3H-9-cis RA (22 Ci/mmol) was synthesized at F. Hoffmann-La Roche Ltd. (Basel). The structures of the retinoids presented in this paper are given in Table 1. Retinoids were dissolved in Me(2)SO as 10 mM stock solutions and kept at -80 °C. Some retinoids were unstable upon storage, and new solutions were prepared for each experiment. All retinoids were handled under dimmed light or in the dark.



Retinoid Binding Assays

For competition binding, we used Escherichia coli expressed RARs (DEF domains) or RARalpha (full-length). The binding experiments were performed as described in detail previously(1, 28) . Briefly, aliquots of receptors (crude extract) were incubated at room temperature with binding buffer containing ^3H-t-RA and increasing concentrations of unlabeled competing ligand. Bound t-RA was then separated from free ligand by charcoal/dextran extraction. Human RXRalpha in pSG5 (5) was provided by L. Sturzenbecker (Hoffmann-La Roche Inc., Nutley, NJ). The cDNA was subcloned in the E. coli expression vector pET3a(29) . Receptors were expressed and solubilized as described earlier(1) . Aliquots thereof (0.2-0.4 pmol receptor) were used to measure competitive binding. The crude extracts were incubated in glass microtubes in 0.2 ml of binding buffer (10% glycerol, 0.8 M KCl, 10 mM Tris-HCl, pH 8.0, 1.5 mM EDTA, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, trypsin inhibitor (1 µg/ml), aprotinin (100 trypsin inhibitor units/ml)), 20 nM^3H-9-cis RA, and increasing concentrations of unlabeled competing ligand. After a 3-h incubation period at 4 °C, the bound radioligand was separated from free radioligand by using NAP-5 desalting columns (Pharmacia), and the radioactivity in the eluates was quantified by liquid scintillation counting. Retinoid binding assays were performed under equilibrium conditions. Two to four competition experiments were performed with each compound. Results from different experiments varied by a factor of less than 2. Binding parameters were calculated from competition curves using the Equilibrium Binding Data Analysis computer program from Biosoft.

DNA-dependent Ligand Binding Assay

RARalpha and RXRalpha were expressed in E. coli (see binding assay). Biotinylated oligonucleotides containing the betaRARE were synthesized (see retardation assay). 15 ng of receptor crude lysates (RAR and RXR) and 20 ng of biotinylated betaRARE were incubated in RXR binding buffer for 30 min on ice. ^3H-t-RA, (20 nM) ^3H-9-cis RA (20 nM), or ^3H-9-cis RA (20 nM) plus Ro 13-7410 (1000 nM) were added and incubated for 30 min on ice. Competition was measured by incubation with a 100-fold molar excess of the unlabeled ligands. After a 3-h incubation period on ice, bound radioligand was separated from free radioligand by gel filtration (see binding assay). The DNA complex of half of the eluate was precipitated with 30 µl of streptavidin-agarose (Sigma, S-1638) and washed two times with column buffer; the bound ligand was then quantitated. For the second half of the eluate, the bound ligand was also quantitated. We found no difference in % competition in solution or upon DNA precipitation (data not shown). The results represent mean values of duplicates and are representative of more than two independent experiments. The data are presented as % competition, where 9-cis competition is defined as 100% and the control as 0%. The latter values correspond to 4-20 fmol of bound ligand, while after competition these values were at least 10-fold lower.

Transient Transfection and Enzyme Assays

Chimeric RAR cDNAs (RARalpha-ER.CAS or RARbeta-ER.CAS) were generously provided by Prof. Dr. P. Chambon (Faculté de Médecine, Strasbourg, France)(7, 30) . For the construction of the corresponding RAR-ER chimeric receptor see Apfel et al.(1) . Through the use of chimeric receptors (the DNA binding region of the estrogen receptor replacing the DNA binding region of the RARs), which recognize an estrogen response element in transcription activation assays, any background induction due to endogenous retinoic acid receptors is avoided, and so it is possible to determine the activating capacity of a retinoid on a single RAR receptor. As a reporter system, we used the SeAP (secreted alkaline phosphatase) gene (31) under the control of the vitellogenin estrogen response element fused to the herpes simplex thymidine kinase promoter (vit-TK-SeAP). COS 1 cells were transiently transfected by the DEAE-dextran method(32) , using 4-20 µg of total plasmid DNA (RAR-ER expression vector, vit-TK-SeAP reporter = 1:5) per 10-cm dish(1) . After 18 h, the cells were trypsinized and replated on 96-well plates. 4 h later, various concentrations of retinoids were added to duplicate wells. At the end of incubation (36-48 h), the cell culture supernatants were assayed for SeAP activity.

An RAR/RXR reporter gene was constructed using a synthetic oligonucleotide containing three copies of the RAR response element from the RARbeta promoter (33) in front of the basal promoter of TK and the luciferase coding region in the plasmid pGL2-basic (Promega) betaRARE-TK-luc. COS-1 cells were transiently transfected as described above, and at the end of incubation the cells were assayed for luciferase activity.

For transfection experiments in Drosophila cells (Schneider SL3), human RXRalpha was expressed using the Drosophila beta-actin promoter(34) . Two RXR reporter systems were constructed using synthetic oligonucleotides, one containing three copies of the RXR response element from the rat CRBP II gene (35) or the other containing three copies of the RARE response element from the betaRAR gene, each with the alcohol dehydrogenase gene promoter (2) cloned in front of the luciferase coding region in the plasmid pGL2-basic (Promega). The transfection was performed by the calcium phosphate-DNA coprecipitation method(32) , using 5 µg of total plasmid DNA (RXRE reporter, RXRalpha expression vector = 1:10) per well of a 6-well dish in Dulbecco's modified Eagle's medium plus 15% FCS. 18 h later, the cells were replated on 96-well plates, and 3-4 h later, various concentrations of retinoids were added to duplicate wells. At the end of incubation (36-48 h), the cells were assayed for luciferase activity.

The SeAP assays were performed as described in detail previously(1) . For the luciferase assay the medium was aspirated, and 40 µl of lysis buffer was added (25 mM Tris-phosphate, pH 7.8, 2 mM dithiothreitol, 2 mM CDTA, 10% glycerol, and 1% Triton X-100). After 15 min incubation at room temperature, 20 µl were transferred into a 96-well plate, and the reaction was triggered by adding 100 µl of luciferin buffer (270 µM CoA, 470 µMD-luciferin, 530 µM ATP, 20 mM tricine, 1.07 mM Mg(CO(3)), 2.67 mM MgSO(4), 0.1 mM EDTA, 33.3 mM dithiothreitol, adjusted pH 7.8).

The luminescent reaction products were measured in a 96-well luminometer (Luminoscan, Flow). Transcription activation is expressed as percent receptor activation, with 100% representing the activity observed with RARs in the presence of 10M t-RA (3-8-fold induction) and 100% defining the RXRalpha activity observed in the presence of 10M 9-cis RA respectively (CRBP II, 10-15-fold induction: betaRARE, 100-500-fold induction). Duplicate values varied by 5-10%. Two to four dose-response experiments were performed. The retinoid concentration giving half-maximal effect in transactivation (EC) was determined from the plots. Reproducibility was found to be within a factor of 2.

Gel Retardation Assay

Oligonucleotides corresponding to the rat cytoplasmic retinol binding protein RARE (CRBP II, 5`-gatcCAGGTCACAGGTCACAGGTCACAGTTCAA-3`), to the RARbeta2 gene promoter RARE (betaRARE, 5`-gatcGTAGGGTTCACCGAAAGTTCACTC-3`) as well as to the vitellogenin gene estrogen response element (5`-gatcTCAGGTCACAGTGACCTGA-3`) were obtained synthetically. Human RXRalpha in pSG5 was linearized using BglII, and the template was transcribed with T7 RNA polymerase (MEGAscript kit, Ambion). Purified mRNA was in vitro translated using rabbit reticulocyte lysates according to Promega's instructions using a complete amino acid mixture. The gel retardation assay was performed as described previously in detail(36) .

HL-60 Differentiation

Retinoid-induced differentiation of HL-60 cells was assayed by measuring their oxidative burst potential via the reduction of nitro blue tetrazolium (NBT)(37) . HL-60 cells were maintained in RPMI 1640 medium (Seromed, Munich) supplemented with 10% FCS, 2 mML-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acids, 50 units/ml penicillin, and 50 µg/ml streptomycin (RPMI/FCS). 30,000 cells/100 µl of RPMI/FCS were seeded into flat-bottomed microtiter wells. 10 µl of retinoids diluted in complete medium were added at the same time to yield final concentrations. After 72 h, the medium was removed and replaced with 100 µl of NBT solution (1 mg/ml in phosphate-buffered saline with 200 nM phorbol myristate acetate). Following an additional 1-h incubation at 37 °C, the NBT solution was removed, and 100 µl of 10% SDS in 0.01 N HCl was added. The amount of the reduced NBT was quantified photometrically at 540 nm using an automated plate reader. The mean of 3 wells was calculated. S.E. were between 5 and 10%.


RESULTS

Characterization of the RXR-selective Retinoids

The chemical structures of the retinoids used in this study are listed in Table 1, including 9-cis RA, t-RA, and three synthetic compounds exhibiting high RXR selectivity. As can be seen from the structures, our strategy in designing RXR-selective ligands focused on the synthesis of retinoids with restricted rotational freedom by fixing the polyene side chain into a 9-cis-like configuration. This goal has successfully been achieved by embedding the C(9)-C(10) double bond into a ring. In addition, this prevents an easy thermal or light-induced isomerization.

To determine the activity of these compounds toward RXRs and RARs we have measured their binding to the receptors in competition binding assays. Fig. 1A shows competitive binding on RXRalpha using ^3H-9-cis RA as tracer. Ro 48-2250 and Ro 47-8652 compete nearly as well as unlabeled 9-cis RA for binding to RXRalpha, whereas Ro 47-5944 is severalfold less potent. In contrast, t-RA and Ro 40-6055 (Table 1) exhibit virtually no binding to RXRalpha. On RARalpha, the three synthetic compounds show only marginally detectable binding in a competition assay with ^3H-t-RA as tracer, while t-RA and 9-cis RA exhibit potent binding (Fig. 1B).


Figure 1: Competition binding of t-RA, 9-cis RA, and synthetic retinoids to RXRalpha (A) and RARalpha (B). The radioligand was ^3H-9-cis RA for RXRalpha and ^3H-t-RA for RARalpha. Percent bound radioligand is plotted against the log of the concentration of the retinoid, t-RA (circle), 9-cis RA (box), Ro 47-5944 (), Ro 47-8652 (bullet), and Ro 48-2250 (). Results of one representative experiment are shown.



To test the ability of these compounds to transactivate receptor-responsive promoters, we used Drosophila SL3 cells as a cellular system. It has been shown in the literature that the RARE of the rat cytoplasmic retinol binding protein gene (CRBP II) is only activated by RXR homodimers while the RARE of the betaRAR gene is activated by RAR-RXR heterodimers as well as by RXR homodimers(20) . Only after cotransfection of the RXRalpha expression vector and different reporter constructs (CRBP II-adh-luciferase or betaRARE-adh-luciferase) does 9-cis RA show a dose-dependent activation of the reporter gene (Fig. 2, A and B). All three compounds showed good activity, being nearly as active as 9-cis RA on both response elements. Using a palindromic thyroid hormone response element-luciferase reporter gene, which is activated by either RARbulletRXR heterodimers or RXR homodimers (20) , we found the same results (data not shown). For an RAR-selective retinoid (Ro 13-7410, TTNPB(38) ), no activation was detectable on all three tested response elements (data not shown).


Figure 2: Transactivation dose-response curves for RXRalpha and RARalpha-ER. A, activation of the betaRARE-adh-luciferase reporter gene. B, activation of the CRBP II-adh-luciferase reporter gene. Schneider SL-3 cells cotransfected with the corresponding reporter gene and an RXRalpha expression plasmid were treated with various concentrations of retinoids; 2 days later, they were assayed for luciferase activity. C, activation of the vit-TK-SeAP reporter gene. COS-1 cells cotransfected with a vit-TK-SeAP reporter gene and the chimeric RARalpha-ER expression plasmid were treated with various concentrations of retinoids; 2 days later, they were assayed for SeAP activity. t-RA, circle; 9-cis RA, box; Ro 47-5944, ; Ro 47-8652, bullet; Ro 48-2250, . Results of a representative experiment are shown.



Transactivation measured using chimeric RAR-ERs in COS cells showed that these three compounds are roughly three orders of magnitude less active on RARs than t-RA or 9-cis RA. Fig. 2C shows as an example dose-response curves of 9-cis RA, t-RA, and the three compounds on RARalpha-ER transactivation. Table 1summarizes activation and binding data of these compounds to RARalpha, -beta, and - and to RXRalpha. As can be seen from these values, the three RXR-selective compounds show approximately a 100-fold preference for RXR versus RAR in transactivation.

DNA-bound RARbulletRXR Heterodimer Can Bind Both Receptor Ligands

To determine whether RARbulletRXR heterodimers can bind both ligands when bound to DNA, DNA-dependent ligand binding assays were performed. ^3H-t-RA bound with high affinity to RARbulletRXR heterodimer on a betaRARE (DR5) (Fig. 3A) and could be competed by unlabeled t-RA, 9-cis RA, and Ro 13-7410 but not by the three RXR-selective ligands. ^3H-9-cis RA bound also with high affinity to RARbulletRXR heterodimers and could be competed 100% by unlabeled 9-cis RA. Unlabeled t-RA or Ro 13-7410 was able to compete approximately half of the counts, which corresponds to the proportion of 9-cis RA that may be bound by the RAR receptor in the RARbulletRXR heterodimer. The RXR-selective retinoids showed also about half the competition, which would correspond to the proportion of 9-cis RA that is bound to the RXR receptor in a heterodimer. After coincubation of ^3H-9-cis RA and a 50-fold excess of unlabeled Ro 13-7410, only the RXR receptor in the DNA heterodimer should be bound with ^3H-9-cis RA. The addition of 9-cis RA and an RXR-selective ligand results in a nearly 100% competition, while t-RA and Ro 13-7410 show no competition. This result indicates that binding of 9-cis RA and RXR-selective ligands to the RXR receptors in an RARbulletRXR heterodimer, bound to DNA, does occur.


Figure 3: DNA-dependent ligand binding assay. Same molar ratios of RARalpha and RXRalpha (A) or molar ration of 0.1/1 (B) were incubated with biotinylated oligonucleotides containing the betaRARE (DR5). Incubations were performed in the presence of ^3H-t-RA (20 nM) (black bars), ^3H-9-cis RA (20 nM) (white bars), or ^3H-9-cis RA (20 nM) plus Ro 13-7410 (1000 nM) (striped bars) with the addition of the indicated unlabeled ligands (2000 nM).



In a second experiment, we dropped the ratio of RAR to RXR from 1 to 0.1. Under these conditions, the binding of ^3H-t-RA fell to near background levels (data not shown). This result suggests that under these conditions the RXR homodimer and not the heterodimer is preferentially, if not exclusively, bound to the DNA. This interpretation was supported by binding studies using ^3H-9-cis RA with and without Ro 13-7410 (Fig. 3B). As expected for an RXR homodimer complex, we see nearly complete competition with RXR-selective ligands.

RXR-selective Retinoids Potentiate RARbulletRXR Heterodimer-mediated Transactivation by an RAR-selective Retinoid

To determine the activity of these compounds in activating an RARbulletRXR heterodimer, we cotransfected CV-1 cells with a betaRARE-TK-luc reporter and the indicated receptors in the presence of 100 nM 9-cis RA, Ro 13-7410 (TTNPB, an RAR-selective retinoid(38) ), Ro 48-2250, Ro 47-5944, or a combination of the last two with 10 nM Ro 13-7410 (Fig. 4). No activation or at most a weak activation was seen in the presence of the RXR- or RAR-selective retinoids, while the betaRARE-TK-luc expression was well induced by 9-cis RA (Fig. 4, lanes 2-5). However, the simultaneous addition of an RXR-selective ligand and 10 nM RAR-selective ligand (Ro 13-7410) resulted in stronger activation (Fig. 4, lanes 6 and 7), similar to that achieved with 9-cis RA. This enhanced or sometimes synergistic effect was most pronounced by cotransfecting RXRalpha. We found the same effect using other RAR-selective ligands in combination with these RXR-selective retinoids (data not shown). t-RA at 100 nM showed the same response as 9-cis RA (data not shown).


Figure 4: Transactivation of RARalphabulletRXRalpha heterodimer using the betaRARE-TK-luc reporter gene. CV-1 cells were cotransfected with a betaRARE-TK-luc reporter gene and the empty expression vector pSG5 or the indicated receptor expression plasmid for RARalpha and RXRalpha, treated with constant concentrations of retinoids; 2 days later, they were assayed for SeAP activity. Lane 1, Me(2)SO; lane 2, 9-cis RA (100 nM); lane 3, Ro 13-7410 (100 nM); lane 4, Ro 48-2250 (100 nM); lane 5, Ro 47-5944 (100 nM); lane 6, Ro 48-2250 (100 nM) + Ro 13-4710 (10 nM); lane 7, Ro 47-5944 (100 nM) + Ro 13-4710 (10 nM). Results of one representative experiment are shown.



RXR-selective Compounds Induce Homodimerization of RXR on CRBP II and betaRARE

To further characterize the interaction of the synthetic compounds with RXRalpha, we have tested whether they are able to induce binding of RXRalpha to an RXR response element in vitro (CRBP II and betaRARE). We have visualized this binding using a gel retardation assay, in which RXR binding to the radiolabeled CRBP II or betaRARE causes slower migration of the response element in a non-denaturing polyacrylamide gel. The complexes being detected under these conditions have been shown to correspond to RXR homodimer bound to the DNA fragment(20) . As can be seen from Fig. 5A (CRBP II) and 5B (betaRARE), 9-cis RA, at a concentration of 1 µM, induces the formation of a more slowly migrating band. A band of the same size is induced with each of the synthetic RXR ligands at the same concentration on both response elements. Fig. 3B shows that this binding of the RXR homodimer on betaRARE is dose dependent. We found the same dose dependence for CRBP II (data not shown). t-RA is unable to induce band formation. The specificity of this binding is demonstrated by its competition with unlabeled response element (Fig. 5B, lane 12) and also by lack of competition using an estrogen response element (Fig. 5B, lane 13). This result implies that the synthetic RXR ligands are able to induce dose-dependent conformational changes of RXRalpha allowing DNA sequence-specific binding as detected by gel retardation.


Figure 5: Ligand effects on the binding of RXRalpha to the RXR response elements (CRBP II and betaRARE). In vitro translated RXRalpha (first column (=) contains no RXRalpha) was preincubated with the indicated retinoids or Me(2)SO alone(-) and then tested for DNA binding activity in the gel retardation assay. A double-stranded oligonucleotide containing (A) the CRBP II sequence or (B) the betaRARE sequence was used as probe. Where indicated, a 100-fold molar excess of unlabeled CRBP II or betaRARE or estrogen response element (ERE) was added. indicates the specific RXR homodimer complex, marks nonspecific band observed also with unprogrammed reticulocyte lysate.



Effect of RXR-selective Retinoids on HL-60 Differentiation

Having identified compounds with high selectivity for RXR versus RAR, we examined the effects of these compounds on a biological function, specifically the induction of differentiation of the myelocytic leukemia cell line, HL-60. Fig. 6shows the degree of HL-60 differentiation, using NBT reduction as a parameter for differentiation-induced oxidative burst potential. t-RA and Ro 40-6055 (an RARalpha-selective retinoid) are able to induce HL-60 differentiation at ED concentrations of 0.3-0.6 nM, while the RXR-selective compounds show only marginal inducing activity, with ED values of 100 nM for Ro 47-8652, 220 nM for Ro 48-2250, and 600 nM for Ro 47-5944, respectively. This is in agreement with their weak potency as activators of RARs. HL-60 cells appear to be about 10-fold more sensitive to retinoids than the transactivation reporter system.


Figure 6: Induction of differentiation of HL-60 cells by retinoids. OD is proportional to the number of differentiated HL-60 cells. t-RA, circle; RARalpha-selective retinoid, Ro 40-6055; box, Ro 47-5944; , Ro 47-8652 (bullet); Ro 48-2250, . Results of one representative experiment are shown.



RXR-selective Retinoids Potentiate RARalpha-mediated Differentiation of HL-60 Cells

We raised the question whether RXR-selective retinoids could augment the differentiation-inducing potency of t-RA or of Ro 40-6055. We have tested the combined effect of RXR ligands with t-RA at concentrations of t-RA per se not sufficient to give full levels of HL-60 differentiation and at concentrations of RXR ligands inducing no HL-60 differentiation at all. As can be seen from Fig. 7, all three RXR-selective retinoids were able to potentiate the effects of t-RA (Fig. 7, A-C) and Ro 40-6055 (Fig. 7, D-F) at concentrations at which they were not active or only minimally active by themselves. The amplification was in the range of 3-10-fold, i.e. 3-10-fold higher concentrations of t-RA or Ro 40-6055 alone are required to obtain comparable levels of HL-60 differentiation.


Figure 7: Induction of differentiation of HL-60 cells by combination of retinoids. OD is proportional to the number of differentiated HL-60 cells. Panels A, B, and C show dose-response curves with t-RA in combination with a constant concentration, as indicated, of RXR-selective retinoids; panels D, E, and F show dose-response curves with the RARalpha-selective retinoid Ro 40-6055 in combination with a constant concentration, as indicated, of RXR-selective retinoids. Results of one representative experiment are shown.




DISCUSSION

In this report, we have presented three synthetic retinoids that function as RXR-selective ligands. All three compounds contain the polyene side chain in a 9-cis-like fixed configuration through the introduction of a ring system. Since 9-cis RA is the natural RXR ligand, these analogs were planned to mimic structural features of 9-cis RA, and indeed they do show nearly equal potency on RXRalpha as compared to 9-cis RA. Gratifyingly, even though 9-cis RA is also a ligand for the RARs, these synthetic compounds exhibit virtually no activity on RARs in binding or transactivation assays. This could be indicative of a distinctly different retinoid binding pocket in RARs versus RXRs. It could, on the one hand, imply that the binding pocket in RARs cannot accept the additional size of a ring system at this position of the side chain, although with the introduction of a three-membered ring in Ro 48-2250, the size of the molecule is only minimally increased. Alternatively, 9-cis RA may adopt different conformations in binding to RARs and RXRs, and the RAR-required conformation may not be accessible to these ring-constrained synthetic derivatives. As a third possibility, mutual isomerization of the natural ligands t-RA and 9-cis RA under the assay conditions cannot be rigorously excluded.

The activity of these RXR-selective compounds is evidenced first by their binding affinity for RXRalpha, which is nearly as high as that of 9-cis RA (Fig. 1, Table 1). Second, these compounds can induce the formation of RXR homodimers on RXR response elements (Fig. 5). Third, these derivatives show potent transactivation of RXR reporter genes (Fig. 2). Fourth, these retinoids are able to potentiate the RARbulletRXR heterodimer-mediated transactivation by the RAR-selective retinoid 13-7410 (Fig. 4). Taken together, these results suggest appropriate ligand-induced conformational changes of the receptor, leading to RXR transcriptional activity.

Furthermore, these selective ligands can be used for exploring the functions of RXR. Since RXR is known to heterodimerize with RAR, we have tested the influence of these compounds on a retinoic acid-mediated process, HL-60 differentiation. Published reports suggest that RARalpha is critical in mediating this differentiation(1) . The RXR ligands per se exhibit EC concentrations higher than 100 nM in inducing HL-60 differentiation (Fig. 6). This implies that liganded RXR receptors are not sufficient to promote induction of HL-60 cells and suggests that liganded RARs are necessary for this process. The weak effect of the RXR-selective retinoids can be explained by their residual RAR-activating activity (Fig. 2B). t-RA, a potent ligand for RARs, induces HL-60 differentiation with an EC of about 0.6 nM. When t-RA is added to cultures at a concentration of 0.1 nM, essentially no differentiation is induced. When cells are treated with 0.1 nM t-RA plus 100 nM of an RXR-selective ligand, substantial induction of differentiation is obtained (Fig. 7, A-C). These effects are somewhat more pronounced if a preferential RARalpha ligand (Ro 40-6055) rather than the unselective t-RA is used (Fig. 7, D-F).

From the results obtained in these combination experiments, it is obvious that the effects of RXR-selective ligands on HL-60 differentiation are more than additive and therefore cannot be explained solely by their residual RAR-activating effects. This indicates that RX receptors, in the presence of their ligand, are able to enhance an RAR-mediated process. This could well be through the activity of an RARbulletRXR heterodimer in the presence of both receptor ligands. In support of this hypothesis, we have presented evidence (Fig. 3) that both ligands do indeed bind to a heterodimer complex on a DNA response element (betaRARE) and show together enhanced transactivation of this element in transient transfections of a reporter gene (Fig. 4). A recent study has demonstrated by direct ligand binding experiments that within an RXRbulletRAR heterodimer bound to a DR5 or DR1 response element, RXR is excluded from binding its ligand(39) . These data appear to contradict our conclusion that both ligands are able to bind such a complex and would support alternative hypotheses, for example, that two independent RAR and RXR pathways exist, and only the activation of both pathways leads to a maximal response. There may be several reasons why Kurokawa et al.(39) did not see binding of an RXR ligand in the heterodimer. One reason may be that the synthetic compound they used is unable to bind in the heterodimer, whereas the new ligands we describe here are accessible to the heterodimer. In addition, it is not clear exactly which response element they use, and it is well possible that on one response element the RXR ligand binding is blocked but not on another.

The fact that the addition of both an RAR and RXR ligand is apparently able to activate better than either alone has also been reported by two other groups. Durand et al.(40) could show that activation on an RARbulletRXR heterodimer response element (betaRARE, DR5T-TK/CAT) in HeLa cells is more than additive. They found a poor activation by an RXR-selective ligand, a moderate activation by t-RA (not a very RAR-selective retinoid) and a synergistic activation by both ligands, to the same level as 9-cis RA alone. Lotan et al.(41) describe that a combination of suboptimal concentrations of RAR-selective retinoids with RXR-selective retinoids shows more than additive effects on the inhibition of cervical carcinoma cell proliferation.

The fact that these RXR ligands can enhance a biological response mediated by a known heterodimer partner, RAR, may serve as an indication that they could also be useful for enhancing processes controlled by other receptor partners, such as thyroid receptors, vitamin D receptor, or peroxisome proliferator-activated receptors, as has also been suggested by combination experiments with 9-cis RA(18, 42) . With a view to clinical usefulness, the RXR-selective ligands could, by increasing the potency of partner receptor ligands, allow lower doses to be administered and thus, by reducing the side effects, provide superior treatment regimens for the various indications where such compounds are commonly used.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: PRPD, 60/409, F. Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland. Tel.: 41-61-688-5878; Fax: 41-61-688-8616.

Present address: Bayer AG, 42096 Wuppertal, Germany.

**
Present address: Pfizer Central Research, Groton, CT 06340.

(^1)
The abbreviations used are: RXR, retinoid X receptor; RAR, retinoic acid receptor; t-RA, all-trans retinoic acid; 9-cis RA, 9-cis retinoic acid; RARalpha and RXRalpha, retinoic acid receptors; RARE, RAR response element; RXRE, RXR response element; CRBP II, cytoplasmic retinol binding protein II; SeAP, secreted alkaline phosphatase; TK, thymidine kinase; luc, luciferase; CDTA, 1,2-diaminocyclohexan-N,N,N`,N`-tetraacetic acid; NBT, nitro blue tetrazolium; FCS, fetal calf serum.


ACKNOWLEDGEMENTS

We thank Prof Dr. P. Chambon (Faculté de Médecine, Strasbourg, France) for providing RARalpha-ER-CAS and RARbeta-ER-CAS plasmids and L. Sturzenbecker (Hoffmann-La Roche Inc., Nutley, NJ) for human RXRalpha in pSG5. We are very grateful to Anne-Cathrine Boscato, Estelle Spieser, Christian Lacoste, Olivier Partouche, and Bernard Rutten for excellent technical assistance.


REFERENCES

  1. Apfel, C., Bauer, F., Crettaz, M., Forni, L., Kamber, M., Kaufmann, F., LeMotte, P., Pirson, W., and Klaus, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7129-7133 [Medline]
  2. Mangelsdorf, D. J., Ong, E. S., Dyck, J. A., and Evans, R. M. (1990) Nature 345, 224-229 [Medline]
  3. Mangelsdorf, D. J., Borgmeyer, U., Heyman, R. A., Zhou, J. Y., Ong, E. S., Oro, A. E., Kakizuka, A., and Evans, R. M. (1992) Genes & Dev. 6, 329-344 [Medline]
  4. Leid, M., Kastner, P., Lyons, R., Nakshatri, H., Saunders, M., Zacharewski, T., Chen, J. Y., Staub, A., Garnier, J. M., Mader, S., and Chambon, P. (1992) Cell 68, 377-395 [Medline]
  5. Levin, A. A., Sturzenbecker, L. J., Kazmer, S., Bosakowski, T., Huselton, C., Allenby, G., Speck, J., Kratzeisen, C., Rosenberger, M., Lovey, A., and Grippo, J. F. (1992) Nature 355, 359-361 [Medline]
  6. Heyman, R. A., Mangelsdorf, D. J., Dyck, J. A., Stein, R. B., Eichele, G., Evans, R. M., and Thaller, C. (1992) Cell 68, 397-406 [Medline]
  7. Petkovich, M., Brand, N. J., Krust, A., and Chambon, P. (1987) Nature 330, 444-450 [Medline]
  8. Giguére, V., Ong, E. S., Segui, P., and Evans, R. M. (1987) Nature 330, 624-629 [Medline]
  9. Brand, N., Petkovich, M., Krust, A., Chambon, P., de Thé, H., Marchio, A., Tiollais, P., and Dejaen, A. (1988) Nature 332, 850-853 [Medline]
  10. Krust, A., Kastner, P., Petkovich, M., Zelent, A., and Chambon, P. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5310-5314 [Medline]
  11. Zelent, A., Krust, A., Petkovich, M., Kastner, P., and Chambon, P. (1989) Nature 339, 714-717 [Medline]
  12. Leroy, P., Krust, A., Zelent, A., Mendelsohn, C., Garnier, J. M., Kastner, P., Dierich, A., and Chambon, P. (1991) EMBO J. 10, 59-69 [Medline]
  13. Yu, V. C., Delsert, C., Andersen, B., Holloway, J. M., Devary, O. V., Näär, A. M., Kim, S. Y., Boutin, J. M., Glass, C. K., and Rosenfeld, M. G. (1991) Cell 67, 1251-1266 [Medline]
  14. Leid, M., Kastner, P., and Chambon, P. (1992) Trends Biochem. Sci. 17, 427-433 [Medline]
  15. Marks, M. S., Hallenbeck, P. L., Nagata, T., Segars, J. H., Appella, E., Nikodem, V. M., and Ozato, K. (1992) EMBO J. 11, 1419-1435 [Medline]
  16. Kliewer, S. A., Umesono, K., Mangelsdorf, D. J., and Evans, R. M. (1992) Nature 355, 446-449 [Medline]
  17. Kliewer, S. A., Umesono, K., Heyman, R. A., Mangelsdorf, D. J., Dyck, J. A., and Evans, R. M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1448-1452 [Medline]
  18. Kliewer, S. A., Umesono, K., Noonan, D. J., Heyman, R. A., and Evans, R. M. (1992) Nature 358, 771-774 [Medline]
  19. Zhang, X. K., Hoffmann, B., Tran, P. B. V., Graupner, G., and Pfahl, M. (1992) Nature 355, 441-446 [Medline]
  20. Zhang, X. K., Lehmann, J., Hoffmann, B., Dawson, M. I., Cameron, J., Graupner, G., Hermann, T., Tran, P., and Pfahl, M. (1992) Nature 358, 587-591 [Medline]
  21. Keller, H., Dreyer, C., Medin, J., Mahfoudi, A., Ozato, K., and Wahli, W. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2160-2164 [Medline]
  22. Lehmann, J. M., Jong, L., Fanjul, A., Cameron, J. F., Lu, X. P., Haefner, P., Dawson, M. I., and Pfahl, M. (1992) Science 258, 1944-1946 [Medline]
  23. Boehm, M. F., McClurg, M. R., Pathirana, C., Mangelsdorf, D., White, S. K., Hebert, J., Winn, D., Goldman, M. E., and Heyman, R. A. (1994) J. Med. Chem. 37, 408-414 [Medline]
  24. Boehm, M. F., Zhang, L., Badea, B. A., White, S. K., Mais, D. E., Berger, E., Suto, C. M., Goldman, M. E., and Heyman, R. A. (1994) J. Med. Chem. 37, 2930-2941 [Medline]
  25. Breitman, T. R., Selonick, S. E., and Collins, S. J. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2936-2940 [Medline]
  26. Largman, C., Detmer, K., Corral, J. C., Hack, F. M., and Lawrence, H. J. (1989) Blood 74, 99-102 [Medline]
  27. Nervi, C., Grippo, J. F., Sherman, M. I., George, M. D., and Jetten, A. M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5854-5858 [Medline]
  28. Apfel, C., Crettaz, M., and LeMotte, P. (1992) in Retinoids in Normal Development and Teratogenesis (Morriss-Kay, G., ed) vol pp. 65-74, Oxford University Press, United Kingdom
  29. Studier, F. W., and Moffatt, B. A. (1986) J. Mol. Biol. 189, 113-130 [Medline]
  30. de Thé, H., Marchio, A., Tiollais, P., and Dejean, A. (1987) Nature 330, 667-670 [Medline]
  31. Berger, J., Hauber, J., Hauber, R., Geiger, R., and Cullen, B. R. (1988) Gene (Amst.) 66, 1-10 [Medline]
  32. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , vol, pp Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  33. de Thé, H., Vivanco-Ruiz, M. D. M., Tiollais, P., Stunnenberg, H., and Dejaen, A. (1990) Nature 343, 111-180
  34. Bond, B. J., and Davidson, N. (1986) Mol. Cell. Biol. 6, 2080-2088 [Medline]
  35. Mangelsdorf, D. J., Umesono, K., Kliewer, S. A., Borgmeyer, U., Ong, E. S., and Evans, R. M. (1991) Cell 66, 555-561 [Medline]
  36. Keidel, S., LeMotte, P., and Apfel, C. (1994) Mol. Cell. Biol. 14, 287-298 [Medline]
  37. Pick, E., Charon, J., and Mizel, D. (1981) J. Reticuloendothel. Soc. 30, 581-593
  38. Beard, R. L., Gil, D. W., Marler, D. K., Henry, E., Colon, D. F., Gillett, S. J., Arefieg, T., Breen, T. S., Krauss, H., Davies, P. J. A., and Chandraratna, R. A. S. (1994) Bioorg. Med. Chem. Lett. 4, 1447-1452
  39. Kurokawa, R., DiRenzo, J., Boehm, M., Sugarman, J., Gloss, B., Rosenfeld, M. G., Heyman, R. A., and Glass, C. K. (1994) Nature 371, 528-531 [Medline]
  40. Durand, B., Saunders, M., Gaudon, C., Roy, B., Losson, R., and Cahmbon, P. (1994) EMBO J. 12, 5370-5382
  41. Lotan, R., Dawson, M. I., Zou, C., Jong, L., Lotan, D., and Zou, C. (1995) Cancer Res. 55, 232-236 [Medline]
  42. Carlberg, C., Bendik, I., Wyss, A., Meier, E, Sturzenbecker, L. J., Grippo, J. F., and Hunziker, W. (1993) Nature 361, 657-660 [Medline]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.