©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Separation of Transactivation and AP1 Antagonism Functions of Retinoic Acid Receptor (*)

(Received for publication, October 11, 1994 )

Sunil Nagpal (§) Jyoti Athanikar Roshantha A. S. Chandraratna (§)

From the From Retinoid Research, Departments of Biology and Chemistry, Allergan Inc., Irvine, California 92713

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Retinoic acid receptors (RARs) regulate gene expression either by directly binding to the RAR-responsive elements or by antagonizing the action of c-Jun/c-Fos (AP1). AP1 is involved in the expression of metalloproteases, cytokines and other factors which play critical roles in the turnover of extracellular matrix, inflammation and hyperproliferation in diseases such as psoriasis, rheumatoid arthritis and in tumor metastases. We demonstrate here that synthetic retinoids inhibit 12-O-tetradecanoylphorbol-14-acetate-induced transcription from the stromelysin AP1 motif through RARalpha, -beta, and -. Interestingly, these diaryl acetylenic retinoids, which are potent agonists only for RARbeta and RAR, but not for RARalpha, in transactivation assays, are able to inhibit AP1-dependent gene expression through RARalpha. Thus these analogs can differentially affect the transactivation and AP1 antagonistic functions of RARalpha. These results demonstrate that the transactivation and AP1 antagonistic functions are separable, and it should be possible to develop retinoids that are completely specific for AP1 antagonism through all RARs. Furthermore, using an RAR-selective ligand, we also demonstrate the separation of ligand binding and AP1 antagonism functions of RARs.


INTRODUCTION

All-trans-retinoic acid (RA) (^1)and its synthetic analogs elicit their biological effects by binding to and activating the RARs which belong to the steroid/thyroid receptor superfamily(1, 2) . Three different RARs (alpha, beta, and ) have been identified(1, 2) , which, upon ligand binding, regulate gene transcription either by activating the expression of genes containing RAREs in their promoter regions (1) or by inhibiting the expression of certain genes by antagonizing AP1 (c-Jun/c-Fos)-mediated gene expression(3) . RARs contain two activation functions: AF-1, a ligand-independent A/B region; and AF-2, a ligand-dependent E region transactivation function(4, 5) . The regions of the RARs contributing to the AP1 antagonism function are not identical to those involved in transcriptional activation. The C-region of RARalpha contains a major ligand-dependent AP1 antagonism function (6) . (^2)RARs are known to bind directly to RAREs(1) , and this protein-DNA interaction is obligatory for ligand induced transcriptional activation of responsive genes. However, RARs do not bind to the AP1 response element in vitro(7) . Thus, RARs may antagonize AP1 function by binding (directly or indirectly) to c-Jun/c-Fos to form an inactive complex or may interact with and sequester another nuclear accessory factor that is required for AP1-mediated transcriptional activation(3) . Given the observed differences in the regions of the RARs associated with transcriptional activation and AP1 antagonism, it is possible that the structural features of the RARs required for the protein-DNA interactions leading to transcriptional activation may be somewhat different from those required for the protein-protein interactions involved in AP1 antagonism. We report here the pharmacological separation of transactivation and AP1 antagonism functions of RARalpha using synthetic retinoid analogs. We demonstrate that analogs that selectively transactivate through RARbeta and RAR and only weakly through RARalpha are potent inhibitors of AP1 function through RARalpha. These results indicate that it should be possible to develop retinoids that are completely specific for AP1 antagonism through all RARs. Such retinoids may be of considerable clinical utility, since they should be effective in treating hyperproliferative and inflammatory diseases with fewer toxic side effects resulting from the activation of retinoid-responsive genes. Furthermore, using a stilbene synthetic retinoid analog, we demonstrate that although ligand binding is obligatory for AP1 antagonism, ligand binding, and AP1 antagonism are also independent properties of RARs.


EXPERIMENTAL PROCEDURES

Recombinant Plasmids

Expression vectors for RAR and RXR (alpha, beta, and ) as well as recombinant baculoviruses containing RARalpha, -beta, and - cDNAs have been described previously(8, 9) . ERE-tk-CAT and estrogen receptor (ER)-RAR expression vectors have been described(10, 11) . The TPA-responsive reporter containing an AP-1 sequence of the rat stromelysin-1 promoter (84S-CAT) has been reported (7) .

Transfection of Cells and CAT Assay

For chimeric receptor transactivation assays, HeLa cells grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS) were transfected using the cationic liposome-mediated transfection procedure. Cells were plated 18 h before transfection at about 40% confluence (50,000 cells/well) in a 12-well plate. Cells were transfected with ERE-tk-CAT (0.5 µg) along with one of the ER-RAR chimeric expression vectors (0.1 µg). Transfections were performed by Lipofectin (2 µg/transfection, Life Technologies, Inc.), and the cells were treated with retinoids 18 h post-transfection. The detailed transfection procedure has been described(11) . For holoreceptor transactivation assays, CV1 cells (5,000 cells/well) were transfected with an RAR reporter plasmid DeltaMTV-TREp-LUC (50 ng) along with one of the RAR expression vectors (10 ng) in an automated 96-well format by calcium phosphate procedure(8) . For RXR transactivation assays, an RXR-responsive reporter plasmid CRBP II-TK-LUC (50 ng) along with one of the RXR expression vectors (10 ng) was used(8, 9) . RXR-reporter contained DR1 elements from human CRBP II promoter(1, 8) . A beta-galactosidase (50 ng) expression vector was used as an internal control in the transfections to normalize for variations in transfection efficiency. The cells were transfected in triplicate for 6 h, followed by incubation with retinoids for 36 h, and the extracts were assayed for luciferase and beta-galactosidase activities. The detailed experimental procedure for holoreceptor transactivations has been described(8, 9) . For retinoid-mediated AP1 antagonism assays, transfections were performed in HeLa cells using 0.6 µg of AP1-CAT and 0.08 µg of human RARalpha, -beta, and - expression vectors, along with 2 µg of Lipofectamine (Life Technologies, Inc.) for each well in a total volume of 500 µl. DNA was precipitated with Lipofectamine for 30 min at room temperature before transfer to cells. Five hours post-transfection, 500 µl of Dulbecco's modified Eagle's medium containing 20% charcoal-treated FBS was added. All the transfections were performed in triplicate. Retinoids were added 18 h post-transfection, and 6 h later the cells were treated with TPA (2 nM) to induce AP-1 activity. The next day after washing twice with phosphate-buffered saline (without calcium and magnesium), the cells were harvested and lysed for 60 min with occasional agitation using a hypotonic buffer (100 µl/well) containing DNase I, Triton X-100, Tris-HCl, and EDTA(11) . CAT activity was assayed in 50 µl of the lysed cell extract using [^3H]acetyl CoA (DuPont NEN) in a 96-well plate. The CAT activity was quantified by counting the amount of ^3H-acetylated forms of chloramphenicol using a liquid scintillation counter(11) .

In Vitro RAR Binding Assays

For in vitro RAR binding experiments, baculovirus/Sf21 insect cell system was used to express human RARalpha, -beta, and - as described(9) . Suspension-grown Sf21 cells were infected with the recombinant viruses at a multiplicity of infection of 2 for 48 h, followed by disruption of the infected cells in 10 mM Tris, pH 7.6, 5 mM dithiothreitol, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 0.4 M KCl as described (8, 9) . The binding assay contained 5-20 µg of extract protein along with [^3H]all-trans-retinoic acid (5 nM) and varying concentrations (0-10 ;mz) of competing ligand in a 250-µl reaction. The binding assays were performed as described previously(8, 9) .

PCR Amplification of Human Stromelysin-1 in Cultured Keratinocytes

For the isolation of keratinocytes, fresh human foreskins were washed in ethanol (70%) for 10 s followed by two washings in keratinocyte growth medium (KGM, Clonetics). Foreskins were cut into small pieces (4 mm diameter), incubated with trypsin (0.05%, Life Technologies, Inc.) for 24 h at 4 °C, centrifuged (1,000 rpm) for 6 min, filtered through a nylon mesh membrane, and cultured in KGM containing 10% FBS. The media was replaced after 3 days with KGM without FBS, and keratinocytes were maintained in serum-free media and treated with AGN 190168 (1 µM) after three passages for 24 h. Total RNA was isolated from AGN 190168-treated and mock-treated cultures by guanidine thiocyanate (Promega) method, reverse transcribed using oligo(dT) and PCR-amplified using stromelysin-1 and glyceraldehyde-3-phosphate dehydrogenase (internal control) primers. Samples (10 µl) were removed from PCR reaction at three cycle intervals (as indicated in Fig. 3), electrophoresed on 2% gel, and analyzed by ethidium bromide staining. Stromelysin-1 primers: 5`-TGATGCTGTCAGCACTCTGAGGGG-3` and 5`-TCAACAATTAAGCCAGCTGTTACT-3` amplified a 546-bp fragment. The rightmost lane in A and B is the DNA marker lane.


Figure 3: AGN 190168 inhibits the endogenous expression of stromelysin-1 in primary human foreskin keratinocytes. Analysis of stromelysin-1 mRNA (A) and glyceraldehyde-3-phosphate dehydrogenase mRNA (B) in control (mock-treated, upperpanel in A and B) and AGN 190168-treated (lowerpanel in A and B) primary foreskin keratinocytes was performed by RT-PCR. Total RNA from mock-treated and AGN 190168-treated keratinocytes was reverse transcribed using oligo dT) and PCR-amplified using stromelysin-1 and glyceraldehyde-3-phosphate dehydrogenase primers. Samples (10 µl) were removed from PCR reaction at three cycle intervals (as indicated in the figure), electrophoresed on 2% gel, and analyzed by ethidium bromide staining. The rightmost lane in A and B is the DNA marker lane.



ODC Inhibition Assays

Ornithine decarboxylase (ODC) activity inhibition by retinoids was determined in TPA-treated female mice. Retinoids (5 mice/dose and 3 doses/experiment) were applied dorsally in 100 µl of acetone 1 h prior to TPA treatment (40 ng/mouse). ODC activity was measured in epidermal extracts obtained 4 h later and normalized to total epidermal protein content(12) . The dose of retinoid (nM/mouse) resulting in 80% inhibition of ODC activity was estimated in multi-dose assays.


RESULTS AND DISCUSSION

Synthetic Acetylenic Retinoids Are RARbeta/-selective in Transactivation Assays

The synthetic acetylenic retinoids used in this study (see Table 1) are RARbeta/-selective for transactivation since they activated the expression of an estrogen receptor element reporter construct (ERE-tk-CAT) in the presence of ER (ABC)-RARbeta(DEF) and ER(ABC)-RAR(DEF) but only weakly through ER(ABC)-RARalpha(DEF) in HeLa cells (Table 1). In these chimeric receptor assays, the DNA binding domain was provided by the ER, whereas the transactivation was retinoid-dependent by virtue of the presence of ligand binding domain of RARs. Since the A/B region of RARs co-operate with their respective AF-2s(4, 5) , we also determined the pattern of transactivation of these retinoids using RAR holoreceptors. The RAR beta/ selectivity of these analogs was maintained in the RAR (alpha, beta, and ) holoreceptor transactivation assays (Table 2). AGN 191936 exhibited significant transactivation through the RARbeta and RAR holoreceptors. All of these retinoids, except AGN 191936, were effective inhibitors of TPA-induced ODC activity in hairless mouse skin (Table 1), thereby demonstrating their anti-proliferation activity in an in vivo model. Since the promoter region of the ODC gene has a TPA and c-Fos-responsive motif (13) , the inhibition of TPA-induced ODC activity observed for these analogs probably reflects their ability to inhibit c-Fos-dependent function through the complement of retinoid receptors present in mouse skin. The binding affinities of the retinoids were determined in vitro using baculovirus expressed RARalpha, -beta, and - (Table 2). Interestingly, most of these analogs bound with reasonable affinity to all three RARs, although they were ineffective transactivators through RARalpha. Furthermore, all the retinoids examined were RAR-specific and neither bound (K(d) > 10 ;mz) to baculovirus produced RXRalpha, -beta, and - (data not shown) nor transactivated an RXR-responsive promoter construct (human CRBP II promoter DR1 elements) in the presence of RXR expression vectors (Table 2). An RAR-selective pan-agonist, TTNPB (p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-1-propenyl]benzoic acid) was used as a positive control in the holoreceptor transactivation and binding assays (Table 2).





Separation of Transactivation and AP1 Antagonism Properties of RARalpha

We used a stromelysin-1 promoter construct (84S-CAT, carrying -84 to +6 base pairs of the rat stromelysin-1 promoter) containing an AP1 motif as its sole enhancer element (7) to study the antagonism of AP1-mediated gene expression by these retinoids through each RAR subtype. RA inhibited the TPA-induced expression of 84S-CAT through RARalpha, -beta, and - in a dose-dependent manner in HeLa cells (Fig. 1A). Open bars represent inhibition of 84S-CAT expression by the addition of retinoids without any transfected receptor expression vector. Similarly, retinoid analogs that were selective for RARbeta/ in transactivation assays, ester AGN 190168, and its acid derivative AGN 190299 also inhibited 84S-CAT expression in a dose-dependent manner through RARalpha, -beta, and - (Fig. 1, B and C). These results demonstrate that even though these retinoids do not effectively activate gene expression through RARalpha (Table 1), they still can antagonize the AP1-dependent expression of 84S-CAT through RARalpha in a potent manner. In contrast, AGN 191936, which was a poor inhibitor of ODC activity (Table 1), did not significantly inhibit the TPA-induced activity of 84S-CAT through RARs (Fig. 1D). To rule out the possibility that RARalpha-mediated AP1 antagonism is unique to these three retinoids, we analyzed four more RAR beta/-selective retinoids from the same diaryl acetylenic structural class (AGN 190121, 191554, 191636, and 191639) for mediating AP1 antagonism through RARalpha. All these retinoids down-regulated the expression of 84S-CAT through RARalpha in a dose-dependent manner (Fig. 2) and with different potencies. IC values for RARalpha-mediated AP1 antagonism (50% inhibition of 84S-CAT expression) for AGN 190121 was <1 nM. AGN 191554, 191636, and 191639 were less potent with IC values of 20, 36, and 17 nM, respectively. These results demonstrate that the acetylenic analogs that selectively transactivate through RARbeta/ are still capable of antagonizing the AP1-dependent gene expression through RARalpha. All the synthetic retinoids with AP1 antagonism activity were also potent inhibitors of ODC activity in vivo (Table 1). All the synthetic retinoids, being RAR-specific, did not significantly inhibit AP1-dependent gene expression through RXRalpha, -beta, and - (Table 2).


Figure 1: Retinoids selective for transactivation through RARbeta/ inhibit the expression of 84S-CAT through all RARs. Each panel shows the percent inhibition in the expression of 84S-CAT without (open bars) or with transfected RARalpha (dark bars), RARbeta (striped bars), or RAR (gray bars) in the presence of RA (A), AGN 190168 (B), AGN 190299 (C), and 191936 (D). Inhibition values are relative to the CAT activity obtained during transfection of HeLa cells with the reporter and the receptor constructs but in the absence of ligand. Transfections were performed by Lipofectamine (2 µg/transfection, Life Technologies, Inc.) and the variations in transfection efficiency did not exceed more than 15% as adjudged by transfection with a beta-galactosidase expression vector pCH110 (Pharmacia Biotech Inc.). Retinoids were added 18 h post-transfection, and 6 h later cells were induced with TPA(2 nM). Cells were harvested after an additional 15 h of incubation, and CAT activity was measured by scintillation counting. All the experiments were performed in triplicate, and standard deviation of the mean is indicated.




Figure 2: Retinoids selective for transactivation through RARbeta/ antagonize AP1 action through RARalpha. Each panel shows the percent inhibition in CAT activity after TPA-induction of 84S-CAT by increasing concentrations of the retinoid either without (open bars) or with human RARalpha (dark bars), relative to CAT activity observed in the absence of retinoids. 84S-CAT (0.6 µg) was transfected in HeLa cells with or without human RARalpha (0.08 µg) expression vector in the presence of AGN 190121 (A), 191554 (B), 191636 (C), and 191639 (D). Transfections were performed by Lipofectamine, all the experiments were performed in triplicate, and the standard deviation of the mean at each concentration of the retinoid is indicated.



Ligand Binding to RARs Alone Is Not Sufficient for AP1 Antagonism

The stilbene retinoid analog AGN 191936 also exhibited an interesting profile of activity. It bound effectively to all three RARs in vitro (Table 2) and transactivated in an RARbeta/-selective manner through the holoreceptors (Table 2) but did not significantly inhibit AP1-dependent gene expression through any of the RARs (Fig. 1D). Thus, AGN 191936 is an example of a compound that can transactivate but not inhibit AP1 function, again demonstrating that these functions are separable. AGN 191936 is also a relatively ineffective inhibitor of ODC activity, probably reflecting its inability to inhibit AP1 function. Interestingly, AGN 191936 did not transactivate through the chimeric receptors (Table 1), suggesting that A/B region-based AF-1 is required for transactivation for this compound. Since AGN 191936 bound to all three RARs (Table 2) but did not inhibit AP1-dependent gene expression through RARs (Fig. 1D), these results indicate that although ligand binding is mandatory for AP1 antagonism, ligand binding and AP1 antagonism are also separate functions of RARs.

Biological Implications

These acetylenic retinoids were also found to be active in a variety of biological assays of anti-proliferation, cytokine production and metalloproteinase expression. Since ODC is constitutively active during cell transformation and it is a key regulator of the biosynthesis of polyamines, which are required for cell proliferation(14) , inhibition by these compounds of ODC activity in hairless mouse skin demonstrates anti-proliferative activity, which may be of relevance to therapeutic use in diseases such as psoriasis. Indeed, AGN 190168 has been shown to be effective in topical treatment of psoriasis in a clinical trial (15) . Two of these compounds (AGN 190299 and AGN 190121) were tested and shown to be effective (data not shown) in other models of anti-proliferation (B-cell myeloma and cervical carcinoma cell growth). Furthermore, AGN 190168 was a potent inhibitor of endogenous stromelysin-1 gene expression in cultured human keratinocytes (Fig. 3) and of endogenous IL-6 production in lesionally derived Kaposi's sarcoma (KS) cells (data not shown).

AP1 and RARs are effectors of the opposite pathways of cell proliferation and differentiation(3, 16, 17, 18, 19) , and they mutually antagonize each other both at the level of transactivation and DNA binding(7, 20) . The involvement of the AP1 motif in RA-mediated negative regulation of collagenase(6, 20, 21) , stromelysin(7) , TGF-beta1(22), and human papillomavirus 18 regulatory regions (23) has been documented. RA also inhibits the expression of IL-6(24) , which is TPA-inducible and contains an AP1 motif in its promoter region(25) . IL-6 is highly elevated in rheumatoid arthritis, psoriasis, and KS, and it is a potent mitogen for KS cells(24, 26, 27) . The elevation of metalloproteinases such as collagenase can contribute to the pathogenesis of chronic inflammatory diseases such as rheumatoid arthritis (3, 20) and tumor metastasis(28) . Antagonism of AP1 action also explains RA inhibition of collagenase production by fibroblasts, monocytes, and keratinocytes (29, 30, 31) and it may be the underlying mechanism for the therapeutic effect of RA on photodamaged human skin (32) . Thus, the cross-talk between the retinoid and AP1 signal transduction pathways could clearly be manipulated for therapeutic benefit in inflammatory and hyperproliferative diseases as demonstrated by the clinical utility of retinoids in treating psoriasis, acute promyelocytic leukemia, premalignant dysplasias, and KS(15, 33, 34, 35) . However, the wider use of retinoids is often hampered by the toxicity associated with retinoid therapy(36) . As to what complement of therapeutic and toxic effects are associated with the AP1 antagonism or transcriptional activation properties of retinoids remains unknown and could effectively be determined only by the development of analogs that are specific for each pathway. Our demonstration that AP1 antagonism and transactivation are separable functions of RARalpha suggests that the synthesis of retinoids with only AP1 antagonism properties is possible. Such retinoids might have greatly improved therapeutic:toxic ratios in the treatment of inflammatory and hyperproliferative disorders. A similar strategy could be applied to glucocorticoids, which also antagonize AP1 and are potent anti-inflammatory molecules with serious side effects.


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. Tel.: 714-752-4518; Fax: 714-253-5578.

(^1)
The abbreviations used are: RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; RARE, RAR-responsive element; CRBP II, cellular retinol binding protein II; AF, activation function; ER, estrogen receptor; ODC, ornithine decarboxylase; KS, Kaposi's sarcoma; IL, interleukin; TPA, 12-O-tetradecanoylphorbol-14-acetate; CAT, chloramphenicol acetyltransferase; PCR, polymerase chain reaction; FBS, fetal bovine serum; ERE, estrogen-responsive element.

(^2)
S. Nagpal, J. Athanikar, and R. A. S. Chandraratna, unpublished observation.


ACKNOWLEDGEMENTS

We thank R. Heyman for RAR and RXR expression vectors, DeltaMTV-TRE(p)-LUC and CRBP II-TK-LUC; P. Chambon for 84S-CAT; M. Pfahl for ER-RAR chimeras and ERE-tk-CAT; D. W. Gil, T. Breen, and T. Arefieg for chimeric transactivations; C. Suto for holoreceptor transactivations; D. Mais for RAR binding; S. Patel for RXR antagonism; S. Thacher and S. Friant for cultured human primary foreskin keratinocytes; and T. Arefieg for ODC assays. We also thank S. Thacher and E. Klein for critical review of the manuscript.


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