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Unique Anti-Activator Protein-1 Activity of Retinoic Acid Receptor ß¹

The Burnham Institute Cancer Center, La Jolla, California 92037

	ABSTRACT

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The anticancer effects of retinoids are mainly mediated by two classes of nuclear receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which are encoded by three distinct genes (, ß, and ). Recent studies have demonstrated that RARß plays a critical role in mediating anticancer effects of retinoids. However, how RARß exerts its potent anticancer effects remains largely unknown. In this study, we investigated anti-Activator Protein-1 (AP-1) activity of RARß. In a transient transfection assay, all three RAR subtypes, RAR, RARß, and RAR, could effectively inhibit phorbol ester 12-O-tetradecanoylphorbol-13-acetate-induced AP-1 activity and the activity of oncogenes c-Jun and c-Fos on AP-1 containing reporter genes in the presence of retinoic acid (RA). However, RARß showed a strong RA-independent inhibition of AP-1 activity, whereas inhibition of AP-1 activity by RAR and RAR was RA dependent. By using several hybrid receptors that contain either the COOH-terminal portion or the NH₂-terminal portion of RARß, we demonstrated that the NH₂-terminal portion of RARß, the A/B domain, was mainly responsible for the RA-independent inhibition of AP-1 activity. This activity was not attributable to constitutive AF-1 activity of RARß, because it did not activate several RA response element-containing reporter genes. In addition, inhibition of histone deacetylase activity by trichostatin A did not overcome the inhibitory effect of RARß. In cancer cells, stable transfection of RARß exhibited strong inhibition of AP-1 activity, even in the absence of RA. Moreover, expression of endogenous AP-1-responsive gene collagenase I was strongly repressed in cancer cells stably transfected with RARß. In studying the antitransforming activity of RARß, we observed that the growth of breast cancer MDA-MB231 cells in soft agar was significantly repressed in a RA-independent manner when cells were stably transfected with RARß but not RAR. Together, our results demonstrate that RARß may exert its potent anticancer effect in part through its unique anti-AP-1 activity.

	INTRODUCTION

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Retinoids, a class of natural and synthetic vitamin A analogues, exert profound effects on many biological processes, including cell proliferation and differentiation, vision, reproduction, morphogenesis, and pattern formation, both in normal and transformed cells (1 , 2) . They have been well recognized as promising anticancer agents (3) . The effects of retinoids are mainly mediated by two classes of nuclear receptors, the RARs³ and RXRs (4, 5, 6) . 9-cis-RA is a high affinity ligand for both RARs and RXRs, whereas all-trans-RA is a ligand for only RARs. RARs and RXRs are encoded by three distinct genes (, ß, and ) and are members of the steroid/thyroid hormone receptor superfamily that function as ligand-activated transcription factors (4, 5, 6) . RARs and RXRs primarily function as RXR/RAR heterodimers that bind to a variety of RAREs and regulate their transactivation activities (4, 5, 6) . Regulation of gene expression by retinoid receptors requires interaction with additional cofactors that appears to provide a direct link to the core transcriptional machinery and to modulate chromatin structure (7) .

In addition to their positive regulation of gene transcription, retinoid receptors also function as negative transcriptional factors (8) . One of the well-known transcriptional repressive effects of retinoid receptors is their inhibition of AP-1 activity (8) . Retinoid receptors, in response to their ligands, can inhibit the effect of tumor promoter TPA by repressing the transcriptional activity of AP-1 (9 , 10) . AP-1 is composed of proto-oncogenes c-Jun and c-Fos, the activity of which is often associated with cell proliferation and tumor progression (11) . AP-1 activity is regulated by growth factors, cytokines, oncogenes, and tumor promoters that activate protein kinase C. It induces transcriptional activation by binding to TRE (11) . The mechanism by which ligand-activated retinoid receptors repress AP-1 activity remains largely unknown, although a direct protein-protein interaction between retinoid receptors and AP-1 (9 , 10) and a competition for a common coactivator (12) have been proposed. Nevertheless, the interaction between membrane and retinoid receptor signaling pathways may represent an important mechanism by which retinoids exert their potent antineoplastic effect (8) . RA could prevent transformation of JB6 mouse epidermal cells promoted by TPA (13) and counteract the effect of TPA on expression of collagenase and stromelysin, presumably through inhibition of AP-1 activity (14) . In addition, synthetic retinoids that selectively inhibit AP-1 activity and cannot induce transactivation of RA-responsive genes were able to inhibit the growth of lung and breast cancer cells (15 , 16) .

Recent studies have demonstrated that RARß plays a critical role in mediating the anticancer effect of retinoids in many different types of cancer cells (17 , 18) , including breast cancer (19, 20, 21) , lung cancer (22, 23, 24, 25, 26, 27) , ovarian cancer (28) , cervical cancer (29) , prostate cancer (30) , neuroblastoma (31) , renal cell carcinoma (32) , pancreatic cancer (33) , liver cancer (34) , and head and neck cancer (35) . Expression of RARß in RARß-negative cancer cells restored RA-induced growth inhibition, whereas inhibition of RARß expression in RARß-positive cancer cells abolished RA effects (19 , 20) . In addition, transgenic mice expressing RARß antisense sequences showed increased incidence of lung tumor (25) , whereas suppression of RARß expression was responsible for diminished anticancer activities of retinoids in animal (36) , and up-regulation of RARß is associated with a positive clinical response to retinoids in patients with premalignant oral lesions (37) . Furthermore, loss of RARß was suggested to be an early event in carcinogenesis (26 , 38, 39, 40) and may be involved in liver cancer development (41) .

How RARß exerts its anticancer effects remains largely unknown. We have demonstrated previously that expression of RARß in certain breast cancer and lung cancer cells could induce apoptosis (20 , 22) , suggesting that apoptosis induction could contribute to the anticancer activities of RARß. However, expression of RARß does not always induce apoptosis of cancer cells (20 , 22) , indicating that mechanism other than apoptosis induction mediates anticancer effects of RARß. In this study, we evaluated anti-AP-1 activity of RARß. Unlike RAR and RAR which repressed AP-1 activity in a RA-dependent manner, RARß could repress TPA-induced AP-1 activity and activity of oncogenes c-Jun and c-Fos in a RA-independent manner. By using various hybrid receptors and RARß deletion mutants, we demonstrated that the NH₂-terminal portion (A/B domain) of RARß is mainly responsible for the RA-independent anti-AP-1 activity of RARß. On several RAREs, RARß did not show any constitutive AF-1 activity associated with the A/B domain. In addition, inhibition of histone deacetylase activity by TSA did not relieve the inhibitory effect of RARß. These observations suggest that competition for common coactivator or the recruitment of receptor corepressor by RARß is unlikely the mechanism for its effect. Furthermore, we found that cancer cells constitutively expressing RARß, but not RAR, exhibited reduced AP-1 activity, even in the absence of RA treatment. Moreover, expression of endogenous AP-1 responsive gene collagenase I and the growth of cancer cells in soft agar was significantly inhibited in a RA-independent manner in cancer cells stably expressing RARß. Together, our results demonstrate that RARß has a unique anti-AP-1 activity and that this unique RA-independent inhibition of AP-1 activity may represent one of the mechanisms by which RARß exerts its potent anticancer activities.

	MATERIALS AND METHODS

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Cell Culture.
HeLa and MDA-MB231 cells were grown in DMEM supplemented with 10% FCS. SK-MES-1 cells were maintained in MEM supplemented with 10% FCS. H292 cells were grown in RPMI 1640 supplemented with 10% FCS.

Plasmid Constructions.
The CAT reporter constructs, -73Col-CAT, TRE-tk-CAT, and TREpal-tk-CAT, have been described previously (9 , 42) . Constructions of expression vectors for RAR, RARß, RAR, c-Jun, and c-Fos and of RAR hybrid receptors were described previously (9 , 42, 43, 44) . RARß/AB was constructed by cloning BamHI-flanked PCR product, using forward primer 5'-CGG GAT CCC GAA TGT ACA AAC CCT GCT TCG TC-3' and reverse T₃ primer, into pcDNA3 vector. RARß/E was generated by deleting the EcoRI fragment from the receptor.

Transient and Stable Transfection Assay.
For HeLa cells, they were plated at 1 x 10⁵ cells/well in a 24-well plate 16–24 h before transfection as described previously (43) . For cancer cells, 5 x 10⁵ cells were seeded in a six-well plate. A modified calcium phosphate precipitation procedure was used for transient transfection and is described elsewhere (43) . Briefly, 250 ng of reporter plasmid, 100 ng of ß-galactosidase expression vector (pCH 110; Pharmacia), and various amounts of expression vector were mixed with carrier DNA (pBluescript) to 1000 ng of total DNA/well. CAT activity was normalized for transfection efficiency to the corresponding ß-gal activity. For stable transfection, the pRc/CMV-RARß recombinant plasmid was stably transfected into SK-MES-1, H292, and MDA-MB231 cells using the calcium phosphate precipitation method and screened using G418 (Life Technologies, Inc., Grand Island, NY) as described (20 , 22) . The integration and expression of transfected cDNA were determined by Southern blotting and Northern blotting, respectively. The stable clones obtained have been described (20 , 22) .

Multi-RT-PCR Assay.
For multi-RT-PCR analysis, total RNAs were isolated and purified by Qiagen RNeasy Mini kit. Expression of AP-1 responsive gene collagenase I in cells was determined by the multi-RT-PCR, modified according to the method described (45) . Briefly, 1.5 µg total RNA were used for reverse transcription in 20 µl of reaction mixture containing of 500 ng of oligo (dT)_12–18, 250 µM deoxynucleotide triphosphates, first strand buffer, and 200 units of superscript II (Life Technologies, Inc.). PCR was carried out with collagenase I primers and GAPDH primers in one reaction system under optimized running conditions. PCR products were analyzed on 2.5% agarose gel and visualized by ethidium bromide staining. The primers used were as follows: collagenase I, 5'-ATTGGAGCAGCAAGAGGCTGGG-3' (sense); 5'-TTCCAGGTATTTCTGGACTAAGTCC-3'; GAPDH, 5'-CCATCACCATCTTCCAGGAG-3' (sense), 5'-CCTGCTTCACCACCTTCTTG-3'.

Soft-Agar Assay.
About 20,000 cells in culture medium containing 10% FCS, 0.3% agar (Difco, Detroit, Michigan), and 10^-7 M all-trans RA in a six-well plate were plated onto an already hardened 0.6% agar underlayer in medium supplemented with 10% FCS. The plates were incubated for 21 days with 6% CO₂. A colony was defined as >40 cells, and colonies with >40 cells were counted with a microscope.

	RESULTS

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RA-independent Inhibition of AP-1 Activity by RARß.
Inhibition of AP-1 activity by retinoid receptors is known to represent one of the mechanisms by which retinoid receptors exert their anticancer activity (8) . Previous studies have demonstrated that RARß exhibits tumor-suppressive effect and is a key retinoid receptor mediating anticancer activity of retinoids (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35) . To study the potent anticancer effect of RARß, we evaluated its anti-AP-1 activity by transient transfection assay using a CAT reporter containing the collagenase promoter (-73Col-CAT), which is known to be activated by AP-1 activity (9) . For comparison, the anti-AP-1 activity of RAR and RAR was studied. The -73Col-CAT was transiently transfected into HeLa cells together with each of RARs. Cells were then treated with or without 100 ng/ml TPA in the presence or absence of 10^-6 M RA. As shown in Fig. 1A , treatment of HeLa cells with TPA strongly induced the reporter transcription with about a 5-fold induction. Induction of the collagenase promoter activity by TPA was mainly attributable to its induction of the endogenous AP-1 activity (9) . In the absence of cotransfected RAR expression vector, RA treatment slightly inhibited the TPA-induced reporter activity, with 27% inhibition, most likely attributable to activation of endogenous retinoid receptor. When we analyzed the effect of cotransfected RARs in the absence of RA, we observed a strong inhibition of the TPA-induced reporter activity when RARß expression vector was cotransfected. The RA-independent inhibition of TPA activity by RARß was concentration dependent. About 60% inhibition was observed when 50 ng of RARß was cotransfected. RA treatment further enhanced the effect of RARß. In contrast, inhibition of the TPA-induced promoter activity by RAR or RAR required RA treatment. In the absence of RA, we did not observe any inhibition of the reporter activity when various concentrations of RAR or RAR were transfected. These data demonstrated that RARß could inhibit TPA activity in a RA-independent manner, whereas inhibition of TPA activity by RAR and RAR requires their ligand binding. To determine whether the AP-1 binding site in the collagenase promoter was responsible for the observed effects, we used the TRE-tk-CAT reporter, in which the consensus AP-1 binding site (TRE) was linked with the tk promoter (42) . About 6-fold induction of reporter transcription was observed when HeLa cells were treated with 100 ng/ml TPA (Fig. 1B ). The TPA-induced TRE-tk-CAT reporter activity was significantly repressed when RARß expression vector was cotransfected. In the absence of RA treatment, cotransfection of 50 ng of RARß expression vector led to 50% inhibition of TPA-induced TRE-tk-CAT activity. In contrast, repression of the reporter transcription by cotransfected RAR or RAR occurred only when cells were treated with 10^-6 M RA. Together, these results demonstrate that RARß could inhibit TPA activity in a RA-independent manner and that the inhibition is mediated by the AP-1 binding site.

View larger version (41K): [in this window] [in a new window]   Fig. 1. Inhibitory effect of different RARs on AP-1 activity. A, inhibition of TPA-induced collagenase promoter activity by RAR in HeLa cells. The -73Col-CAT reporter (250 ng) was cotransfected without or with the indicated RAR expression vector (100 ng) into HeLa cells. After transfection, the cells were incubated in DMEM medium containing charcoal-treated 0.5% FCS for 24 h and treated with either TPA (100 ng/ml) alone () or with 10-6 M of all-trans RA (). After 12 h, the cells were harvested, and CAT activity was determined. The activities of cotransfected ß-gal were used as reference values. B, inhibition of TPA-induced TRE-tk-CAT activity by RAR. The TRE-tk-CAT reporter (250 ng) was cotransfected with or without the indicated RAR expression vector (100 ng) into HeLa cells. After transfection, cells were incubated in DMEM medium containing 0.5% charcoal-treated FCS for 24 h and treated with either TPA (100 ng/ml) alone () or with all-trans RA (10-6 M; ). After 12 h, the cells were harvested, and CAT activity was determined. The activities of cotransfected ß-gal were used as reference values. C, inhibition of c-Jun activity by RAR in HeLa cells. D, inhibition of c-Jun and c-Fos activity by RAR in HeLa cells. The TRE-tk-CAT reporter (250 ng) was cotransfected with or without c-Jun (100 ng; C) and/or cFos (100 ng; D) expression vectors either in the presence or absence of the indicated RAR expression vector (100 ng) into HeLa Cells. After transfection, the cells were treated with () or without () all-trans RA (10-6 M) for 24 h. The cells were then harvested, and CAT activity was determined. The activities of cotransfected ß-gal were determined and used as reference values. Bars: A–D, SD. E, RXR potentiates effect of RARß. The -73Col-CAT reporter plasmid was cotransfected with the indicated retinoid receptor expression vectors into HeLa cells. After transfection, cells were incubated in DMEM containing charcoal-treated 0.5% FCS and treated with TPA (100 ng/ml), and CAT activity was determined as described in A. F, activation of RARE by RARß and RAR in HeLa cells. The TREpal-tk-CAT reporter plasmid was cotransfected with or without the indicated RAR expression vectors (100 ng) into HeLa cells. After transfection, the cells were incubated in DMEM containing the indicated concentration of RA for 24 h. Cells were harvested, and CAT activity was determined. The activities of cotransfected ß-gal were used as reference values.

RARs can heterodimerize with RXR and may function as a RAR/RXR heterodimer in cells (43) . We therefore investigated whether expression of RXR had any effect on RA-independent inhibition of AP-1 activity. As shown in Fig. 1E , cotransfection of RXR expression vector did not show any effect on TPA-induced -73Col-CAT reporter activity. However, when RXR was cotransfected with RARß, RA-independent inhibitory effect of RARß was enhanced. The enhancing effect of RXR on RARß activity was observed when three different concentrations of RARß were used. The results therefore demonstrate that RARß/RXR heterodimer could act as an effective inhibitor of AP-1 activity in the absence of RA.

To study whether the observed inhibition of AP-1 activity by RARß in the absence of RA treatment was attributable to a trace amount of endogenous retinoids, we evaluated the activation function of RARß in response to various concentrations of RA by using the TREpal-tk-CAT reporter, which is known to be activated by RAR (43) . For comparison, activation by RAR was analyzed. As shown in Fig. 1F , both RAR and RARß showed a very similar response to various concentrations of RA in activating the TREpal-tk-CAT reporter. The maximum activation of the reporter by both receptors was observed when cells were treated with 10^-6 M RA. These results demonstrate that activation of gene transcription by RARß is RA dependent and suggest that repression of AP-1 activity by RARß in the absence of exogenous RA is unlikely because of the presence of trace amounts of RA in the cells.

The A/B Domain of RARß Is Responsible for RA-independent Repression of AP-1 Activity.
To determine which domain of RARß is responsible for its RA-independent anti-AP-1 activity, we analyzed the activity of several RARß deletion mutants (Fig. 2A ). Cotransfection of the -73Col-CAT reporter with a RARß mutant deleted with the E domain (RARß/E) significantly repressed the c-Jun-induced reporter activity in the absence of RA treatment (Fig. 2B ). The observation is similar to that observed with the parental RARß receptor (Fig. 1A ), although the degree of inhibition by RARß/E was reduced. Addition of RA treatment showed slight enhancement of the inhibitory effect by RARß/E, which is likely attributable to the effect by endogenous RAR activity, because a similar degree of inhibition by RA was observed in the absence of cotransfected receptor. In contrast, cotransfection of a mutant lacking the A/B domain RARß/AB did not show any inhibitory effect on c-Jun-induced reporter activity in the absence of RA (Fig. 2B ), indicating that the A/B domain is crucial for RA-independent inhibition of AP-1 activity. However, the mutant strongly inhibited the reporter activity when cells were treated with RA, demonstrating that the A/B domain is not required for RA-dependent inhibition of AP-1 activity. When a mutant deleted with the ABC domain (RARß/ABC) was analyzed, we found that both RA-dependent and -independent activities were abolished, indicating that the ligand-binding domain (E/F domain) alone is not sufficient to confer RA-dependent inhibition of AP-1 activity and that the C domain is also required. Together, these data demonstrate that the RA-independent anti-AP-1 activity of RARß is likely mediated through the A/B domain of the receptor, whereas the RA-dependent activity requires both the DNA-binding domain and ligand-binding domain.

View larger version (42K): [in this window] [in a new window]   Fig. 2. Inhibition of AP-1 activity by RARß deletion mutants. A, schematic representation of RARß deletion mutants. B, inhibition of c-Jun-induced collagenase promoter activity by RARß mutants in HeLa cells. The -73Col-CAT reporter (250 ng) was cotransfected without or with c-Jun expression vector (100 ng) and the indicated amounts of RARß mutant expression vector into HeLa cells. After transfection, the cells were incubated in DMEM containing charcoal-treated 0.5% FCS for 24 h and treated with () or without () 10-6 M of all-trans RA. After 12 h, the cells were harvested, and CAT activity was determined. The activities of cotransfected ß-gal were used as reference values. Bars, SD.

View larger version (44K): [in this window] [in a new window]   Fig. 3. Inhibition of AP-1 activity by RAR hybrid receptors. A, schematic representation of hybrid receptors. The DNA binding domain (DBD) and the ligand binding domain (LBD) of RAR are indicated. B, inhibition of TPA-induced collagenase promoter activity by RAR hybrid receptors in HeLa cells. The -73Col-CAT reporter (250 ng) was cotransfected without or with the indicated RAR hybrid receptor expression vectors (100 ng) into HeLa cells. After transfection, the cells were incubated in DMEM containing charcoal-treated 0.5% FCS for 24 h and treated with either TPA (100 ng/ml) alone or with 10-6 M of all-trans RA. After 12 h, the cells were harvested, and CAT activity was determined. The activities of cotransfected ß-gal were used as reference values. Bars, SD.

View larger version (21K): [in this window] [in a new window]   Fig. 4. RA-independent activation of RARE by RARs. A reporter plasmid containing TREpal (A), ßRARE (B), or CRBPI-RARE (C) was transiently transfected together with the indicated amount of RAR, RARß, or RAR expression vector into HeLa cells. CAT activity was determined as described in Fig. 1 .

View larger version (35K): [in this window] [in a new window]   Fig. 5. Histone deacetylase is not required for repression of AP-1 activity by RARß. The -73Col-CAT reporter plasmid (250 ng) was cotransfected with or without the indicated concentrations of RARß expression vector into HeLa cells. After transfection, the cells were incubated in DMEM medium containing charcoal-treated 0.5% FCS and treated with either TPA (100 ng/ml) and TSA (30 ng/ml) alone or their combination. CAT activity was determined as described in Fig. 1 .

View larger version (30K): [in this window] [in a new window]   Fig. 6. Inhibition of TPA-induced AP-1 activity by RARß in human cancer cells. The TRE-tk-CAT reporter (250 ng) was cotransfected with or without the indicated RAR expression vectors (100 ng) into SK-MES-1 lung cancer cells. After transfection, the cells were incubated in DMEM containing charcoal-treated 0.5% FCS for 24 h and treated with either TPA (100 ng/ml) alone () or with all-trans RA (10-6 M; ). After 12 h, the cells were harvested, and CAT activity was determined. The activities of cotransfected ß-gal were used as reference values. Bars, SD.

View larger version (46K): [in this window] [in a new window]   Fig. 7. RA-independent inhibition of AP-1 activity by RARß in RARß-negative cancer cells stably transfected with RARß. A, stable expression of RARß in SK-MES-1 cells inhibits transactivation function of AP-1 in a RA-independent manner. WT, wild type; Vector, SK-MES-1 cells stably transfected with the empty vector. RARß#6 and RARß#7, SK-MES-1 cells stably transfected with RARß. Bars, SD. B, stable expression of RARß but not RAR in MDA-MB231 cells inhibits transactivation function of AP-1 in a RA-independent manner. WT, wild type MDA-MB231 cells; Vector, MDA-MB231 cells stably transfected with the empty vector; RAR#2 and RAR#21, MDA-MB231 cells stably transfected with RAR. RARß#2 and RARß#3, MDA-MB231 cells stably transfected with RARß. The TRE-tk-CAT reporter (250 ng) was transfected together with or without c-Jun (100 ng) and c-Fos (100 ng) into the indicated cancer cells. After transfection, cells were incubated in DMEM containing charcoal-treated 0.5% FCS for 24 h and treated with either TPA (100 ng/ml) alone or with all-trans RA (10-6 M). After 12 h, the cells were harvested, and CAT activity was determined. The activities of cotransfected ß-gal were used as reference values. Bars, SD.

Inhibition of Collagenase I Expression by Stable Expression of RARß.
We next examined the effect of RARß expression in SK-MES-1 cells on the ability of TPA to induce expression of endogenous collagenase I gene expression by RT-PCR. Expression of collagenase 1 is known to be induced by TPA through its activation of AP-1 that binds to a TRE present in the promoter (14) . As shown in Fig. 8A , TPA strongly induced expression of collagenase I by 10-fold in SK-MES-1 cells, as reported previously (14) . Treatment of cells with RA slightly repressed induction of collagenase I by TPA. For comparison, we did not observe any effect of TPA or RA on expression of the GAPDH gene. Similar results were observed in SK-MES-1 cells stably transfected with the empty vector (Vector). However, the ability of TPA to induce collagenase I expression was largely reduced in the RARß stable clones (RARß#6 and RARß#7), with only 3-fold induction. Treatment of the cells with RA completely repressed the ability of TPA to induce collagenase I expression. To further confirm the RA-independent anti-AP-1 effect of RARß, we compared the inducibility of TPA on collagenase I expression in H292 lung cancer cells and H292 cells stably expressing transfected RARß. Our result (Fig. 8B ) showed that the ability of TPA to induce collagenase I expression was significantly reduced by overexpressing of RARß also in this cell line. These data further confirm that RARß exerts a potent RA-independent anti-AP-1 activity.

View larger version (40K): [in this window] [in a new window]   Fig. 8. Overexpression of RARß reduces the ability of TPA to induce collagenase I expression in a RA-independent manner. A, effect of RARß stable transfection in SK-MES-1 cells. B, effect of RARß stable transfection in H292 cells. Confluent cells were incubated in MEM containing charcoal-treated 0.5% FCS overnight, treated with TPA (100 ng/ml) or TPA plus all-trans RA (10-6 M). Multi-RT-PCR for collagenase I and GAPDH mRNA were performed for 25 cycles using 4 pairs of primer as described in "Materials and Methods." Lane M, molecular weight marker; Lane C, a control containing the same reaction as Lane 3 without reverse transcription; Lane 1, control treated with ethanol; Lane 2, treatment with TPA; Lane 3, treatment with TPA plus all-trans RA.

View larger version (67K): [in this window] [in a new window]   Fig. 9. RA-independent inhibition of anchorage-independent growth of MDA-MB231 cells by RARß expression. A, photograph of colonies formed by parental MDA-MB231, MB231/Vector, MDA-MB231 cells transfected with the empty vector; RAR (MB231/RAR#2) and RARß (MB231/RARß#3) in the absence or presence of all-trans RA (10-7 M). B, quantitation of colonies formed by MDA-MB231, vector, MB231/RAR#2, and MB231/RARß#3 in the absence or presence of RA. Colonies were scored and expressed as percentages of the number of colonies formed by cells treated with solvent control.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The anticancer activity of retinoids is thought to be in part attributable to their direct antiproliferative effects, which have been observed in several transformed cell lines, including those for mammary, melanoma, lymphoid, and fibroblastic cancers (3) . Studies in human breast cancer cells and bronchial epithelial cells also suggest that retinoids may inhibit proliferation through inhibition of transcription factor AP-1 activity (15 , 50 , 51) , which is composed of c-Jun homodimers or c-Jun/c-Fos heterodimers. Several lines of evidence provided here demonstrate a unique RA-independent anti-AP-1 activity of RARß. In transient transfection assays, cotransfection of RARß or RARß and RXR inhibited either TPA-induced AP-1 transcriptional activity or c-Jun/c-Fos transcriptional activity in a RA-independent manner, whereas inhibition of AP-1 transcriptional activity by RAR or RAR was RA dependent (Fig. 1 ). The RA-independent repression of AP-1 transcriptional activity by RARß was observed in several cell lines, including HeLa (Fig. 1 ), SK-MES-1 lung cancer (Figs. 6 and 7A ), H292 lung cancer (Fig. 8B ), and MDA-MB231 breast cancer (Fig. 7B ) cells, suggesting that it may function in various cell types. The effect of RARß could be also observed in RARß-negative cancer cells stably transfected with RARß. Stable expression of RARß in RARß-negative SK-MES-1 and MDA-MB231 cells reduced the ability of AP-1 to induce expression of AP-1-responsive genes, including the transfected AP-1-responsive reporter genes (Fig. 7 ) and endogenous AP-1-responsive gene (Fig. 8 ).

Previous studies investigating domain requirement of AP-1/RAR interaction have shown that the ligand binding domain and DNA binding domain are required for AP-1/RAR interaction (9 , 52) . Consistent with these previous results, our data (Figs. 2 and 3 ) demonstrate that both the NH₂-terminal portion (A/B and C domains) and COOH-terminal portion (E/F domain) of RARß are involved in the inhibition of AP-1 transcriptional activity. In addition, by using various RARß mutant receptors, we demonstrate that the A/B domain of the RARß is responsible for the RA-independent repression of AP-1 transcriptional activity (Fig. 2 ). Cotransfection of the NH₂-terminal half of the RARß, but not the COOH-terminal half of RARß, showed a RA-independent repression of AP-1 activity (Fig. 2 ). In addition, deletion of the A/B domain completely abolished the RA-independent inhibition of AP-1 transcriptional activity. Furthermore, hybrid receptors containing the NH₂-terminal half of RARß, but not RAR or RAR, showed the RA-independent anti-AP-1 effects (Fig. 3 ). It is likely that the A/B domain of RARß may directly interact with AP-1. Alternatively, the A/B domain of RARß may interact with certain cofactors that mediate AP-1/RARß interaction.

Recently, it was demonstrated that competition of liganded retinoid receptors and AP-1 for common transcriptional coactivator CBP may in part contribute to the mutual inhibition of their transcriptional activity (12) . However, RARß does not possess RA-independent transactivation function on several RAREs in HeLa cells (Fig. 4 ), indicating that sequestration of a coactivator is unlikely the mechanism for RA-independent inhibition of AP-1 activity. Unliganded retinoid receptor is known to interact with receptor corepressor (7) . The fact that unliganded RARß could repress AP-1 activity suggests that RARß may inhibit AP-1 transcriptional activity through the recruitment of a receptor corepressor. Receptor corepressors are known to form a complex with histone deacetylases to mediate transcriptional repression (7) . However, TSA, a specific inhibitor of histone deacetylase (46) , failed to relieve the transcriptional repressive effect of unliganded RARß (Fig. 5 ). This observation demonstrates that histone deacetylase-associated activity is unlikely the mechanism for RA-independent inhibition of AP-1 transcriptional activity by RARß.

We have demonstrated previously that expression of RARß could induce apoptosis of certain cancer cells in response to RA treatment (20) . Our present finding suggests that inhibition of AP-1 activity may also contribute to the growth-inhibitory effect of RARß. Thus, RARß may exert its tumor-suppressive effect through different mechanisms, such as the anti-AP-1 effect and apoptosis induction, which are likely operated in a cell type-dependent manner. Our observation that RARß could inhibit AP-1 activity in the absence of RA may provide an explanation for the potent tumor-suppressive effect of RARß. Thus, unlike other RAR subtypes, RARß could act as an efficient tumor suppressor even in the absence of RA. This is clearly seen in our growth inhibition assay (Fig. 9 ), showing that expression of RARß could repress the growth of MDA-MB231 cells in soft agar in a RA-independent manner whereas the growth-inhibitory effect of RAR is RA dependent. Previous studies have demonstrated that RARß exerts tumor-suppressive effects in cancer cells (20 , 22, and 49 ) and that a low expression level of RARß may be an important contributing factor in cancer development (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35) . Our present finding suggests that the unique anti-AP-1 activity of RARß may contribute to its tumor-suppressive effect. The mitogenic stimulus, often generated by the autocrine secretion of growth factors, is transmitted to cell nucleus to activate the nuclear transcriptional factors c-Jun and c-Fos, which often trigger cell proliferation. By inhibiting AP-1 transcriptional activity, RARß may inhibit cell proliferation often associated with cancer development. Loss of this negative growth control mechanism likely plays a role in cancer development. Interestingly, overexpression of AP-1 could abrogate the growth-inhibitory effect of RA, resulting in retinoid resistance (51 , 53) . Thus, the cross-talk between AP-1 and retinoid receptor is reciprocal, and a balance between RARß and AP-1 activity may contribute to the maintenance of the proper growth of cells.

	FOOTNOTES

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

¹ This work was supported in part by NIH Grants CA60988 and CA51933 (to X-k. Z.), the Grant 6RT-0168 from the Tobacco-Related Disease Research Program of California, Grant 3PB-0018 from the California Breast Cancer Research Program, and Grant DAMD17-4440 from the United States Army Medical Research Program.

² To whom requests for reprints should be addressed, at The Burnham Institute Cancer Center, 10901 North Torrey Pines Road, La Jolla, CA 92037. Phone: (858) 646-3141; Fax: (858) 646-3195; E-mail: xzhang{at}burnham-inst.org.

³ The abbreviations used are: RAR, retinoic acid receptor; RXR, retinoid X receptor; RARE, retinoic acid response element; TPA, 12-O-tetradecanoylphorbol-13-acetate; AP, activator protein; TRE, TPA response element; CAT, chloramphenicol acetyltransferase; ß-gal, ß-galactosidase; RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; tk, thymidine kinase; TSA, trichostatin A.

Received 9/10/99. Accepted 4/18/00.

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