Retinoic acid inhibits interleukin-4-induced eotaxin production in a human bronchial epithelial cell line

Kei Takamura,1 Yasuyuki Nasuhara,1 Motoko Kobayashi,1 Tomoko Betsuyaku,1 Yoko Tanino,1 Ichiro Kinoshita,1 Etsuro Yamaguchi,1 Satoshi Matsukura,2 Robert P. Schleimer,3 and Masaharu Nishimura1

1First Department of Medicine, Hokkaido University School of Medicine, Sapporo 060-8638; 2First Department of Internal Medicine, Showa University, Tokyo 142-8555, Japan; and 3Division of Allergy and Clinical Immunology, Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland 21224-6801

Submitted 26 August 2003 ; accepted in final form 3 December 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Retinoic acid (RA) is known to accelerate wound healing and induce cell differentiation. All-trans RA (ATRA) exerts its effect by binding retinoic acid receptors, which are members of the nuclear receptor family. We investigated whether RA can alter expression of eotaxin, a potent eosinophil chemoattractant that is regulated by the transcription factors signal transducer and activator of transcription 6 (STAT6) and NF-{kappa}B. We examined the effects of RA on eotaxin expression in a human bronchial epithelial cell line BEAS-2B. ATRA and its stereodimer 9-cis retinoic acid (9-cis RA) inhibited IL-4-induced release of eotaxin at 10-6 M by 78.0 and 52.0%, respectively (P < 0.05). ATRA and 9-cis RA also significantly inhibited IL-4-induced eotaxin mRNA expression at 10-6 M by 52.3 and 53.5%, respectively (P < 0.05). In contrast, neither ATRA nor 9-cis RA had any effects on TNF-{alpha}-induced eotaxin production. In transfection studies using eotaxin promoter luciferase plasmids, the inhibitory effect of ATRA on IL-4-induced eotaxin production was confirmed at the transcriptional level. Interestingly, ATRA had no effects on IL-4-induced tyrosine phosphorylation, nuclear translocation, or DNA binding activity of STAT6. Activating protein-1 was not involved in ATRA-mediated transrepression of eotaxin with IL-4 stimulation. The mechanism of the inhibitory effect of ATRA on IL-4-induced eotaxin production in human bronchial epithelial cells has not been elucidated but does not appear to be due to an effect on STAT6 activation. These findings raise the possibility that RA may reduce eosinophilic airway inflammation, one of the prominent pathological features of allergic diseases such as bronchial asthma.

all-trans retinoic acid; 9-cis retinoic acid; eotaxin; signal transducer and activator of transcription 6


INFILTRATION OF EOSINOPHILS into the sites of inflammation is a hallmark of allergic diseases, such as bronchial asthma, and is mediated by several chemotactic factors, including eotaxin. Eotaxin was initially discovered by use of a biological assay in guinea pigs designed to identify the molecules responsible for allergen-induced eosinophil accumulation in the lung (19). Because eotaxin has been reported to be highly expressed in bronchoalveolar lavage from human asthmatics (23) and to be associated with airway hyperresponsiveness (52), it is thought to play an important role in the pathogenesis of bronchial asthma. Many kinds of cells, such as fibroblasts, alveolar macrophages, and airway smooth muscle cells have been reported to be sources of eotaxin (reviewed in Ref. 45). In human airway epithelial cell lines, Lilly et al. (26) reported the induction following stimulation with tumor necrosis factor-{alpha} (TNF-{alpha}) or interleukin (IL)-1{beta}. Mochizuki et al. (34) reported that TNF-{alpha} and IL-4 synergistically stimulate eotaxin expression in human dermal fibroblasts. A similar synergistic effect of TNF-{alpha} and IL-4 in the induction of eotaxin mRNA and protein expression has been reported in airway epithelial cells (49).

The sequence of the eotaxin promoter has been reported by two independent groups (10, 13). Overlapping elements for signal transducer and activator of transcription 6 (STAT6) and nuclear factor-{kappa}B (NF-{kappa}B) within the proximal eotaxin promoter have been shown to mediate the transcriptional induction by IL-4 and TNF-{alpha} in BEAS-2B cells (31). IL-13 also upregulates eotaxin expression by a STAT6-dependent mechanism in BEAS-2B cells (30). Glucocorticoids inhibit eotaxin mRNA expression induced with IL-1{beta}, IL-4, or TNF-{alpha} in epithelial cell lines (26, 49). Other regulators of eotaxin expression include IL-5, which was reported to inhibit TNF-{alpha}-induced eotaxin expression in eosinophils (12), and {beta}2-agonists, which were reported to inhibit TNF-{alpha}-induced eotaxin release in human airway smooth muscle cells (41).

Retinoic acid (RA) is an active metabolite of vitamin A and regulates a wide range of biological processes, including cell proliferation, differentiation, and morphogenesis (8). Predominantly all-trans retinoic acid (ATRA) and its stereodimer 9-cis retinoic acid (9-cis RA) were found to be very potent metabolites exerting pleiotropic effects. ATRA is clinically used for controlling keloid formation (18) and for treating acute promyelocytic leukemia (17). In the respiratory system, ATRA has been reported to reverse elastase-induced experimental emphysema in rats by Massaro and Massaro (29). Furthermore, Nakajoh et al. (38) showed that ATRA inhibits elastase-induced apoptosis in BEAS-2B cells, A549 cells, and primary human tracheal epithelial cells. On the other hand, ATRA was reported to activate promoter activity of the IL-8 gene through a thioredoxin-dependent NF-{kappa}B activation in human airway epithelial cells (4).

The biological response to RA is mediated by two classes of nuclear receptors. One group is the RA receptors (RARs), including three subtypes (RAR{alpha}, RAR{beta}, and RAR{gamma}) that are activated by ATRA and 9-cis RA. The second group is the retinoid X receptors (RXRs), which also include three subtypes (RXR{alpha}, RXR{beta}, and RXR{gamma}) that are activated by 9-cis RA only (21). The effects of RARs on gene activity include both transactivation (4, 9, 28) and transrepression (2, 20, 24, 47). To our knowledge, there are no reports regarding the effect of RA on the expression of eotaxin.

In the present study, we demonstrate that RA inhibits the expression of eotaxin and study the mechanisms responsible for the inhibitory effect of RA using a human bronchial epithelial cell line BEAS-2B.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and reagents. BEAS-2B cells were cultured in DMEM/F-12 medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in 5% CO2 in humidified air. ATRA and 9-cis RA were dissolved in ethanol. IL-4 (a gift from Ono Pharmaceutical) and TNF-{alpha} (PeproTech) were dissolved in deionized distilled water. Genistein, a tyrosine kinase inhibitor, and 12-O-tetradecanoylphorbol 13-acetate (TPA) were dissolved in dimethyl sulfoxide. In all experiments, ATRA and 9-cis RA were added 1 h before stimulation, and genistein was added 30 min before stimulation. Ethanol and dimethyl sulfoxide were diluted to final concentrations of <0.1% (vol/vol). All reagents were from Sigma unless otherwise indicated.

Construction of eotaxin promoter-luciferase reporter plasmids and activating protein-1-luciferase reporter plasmids. Eotaxin promoter-luciferase plasmids were constructed as previously reported (31). Briefly, a 1,363-bp fragment of the promoter region of the eotaxin gene (site -1,363 to -1), which contains CCAAT/enhancer-binding protein, activating protein-1 (AP-1), STAT6, and NF-{kappa}B response elements, was inserted into pGL3-Basic vector (Promega); the construct is referred to as pEotx.1363. Putative transcription factor binding sites in pEotx.1363 were mutated using the QuickChange site-directed mutagenesis kit (Stratagene) with pEotx.1363 as a template. Constructs pEotx.M1 and M2 were synthesized by temperature cycling using site-mutated primers and PfuTurbo DNA polymerase (Stratagene) as previously reported (31). The pEotx.M1 has a mutated STAT6 response element (TTCCCTGGAA to AGCCCTGGAA). The pEotx.M2 has a mutated NF-{kappa}B response element (GGAATCTCCC to GGAATCTGGG). The pEotx.300 plasmid was constructed by 5' end digestion of the pEotx.1363. The AP-1-luciferase reporter plasmid (AP1-Luc) is a pGL3-based vector in which two repeats of the consensus AP-1 site, TGAGTCA, were inserted. The sequence of each plasmid insert DNA was determined by the dideoxy method using the fmol DNA Sequencing System (Promega).

Transient transfections and luciferase assays. BEAS-2B cells were seeded into six-well plates and allowed to grow to 50–70% confluence. Cells were transfected with 4.5 µl Fugene 6 transfection reagent (Roche Diagnostics), 1 µg of the plasmids (pEotx.1363, pEotx.M1, pEotx.M2, pEotx.300, or AP1-Luc), and 40 ng Renilla luciferase reporter vectors, and incubated in 2 ml of medium. Several concentrations of ATRA were added 24 h after transfection, and cytokines were added 1 h after ATRA treatment. Six hours after cytokine treatment, cells were washed twice with PBS and solubilized with 100 µl of reporter passive lysis buffer (Promega). Luciferase activity was measured using Dual-Luciferase Reporter Assay Systems (Promega) and a luminometer (Turner Designs). Twenty microliters of lysate were mixed with 100 µl of Luciferase Assay Reagent II. After measurement of firefly luciferase activity, 100 µl of Stop-and-Glo Reagent were added, then Renilla luciferase activity was measured.

Measurement of eotaxin in the supernatant fluids. The concentrations of eotaxin were measured in the cell supernatant from BEAS-2B cells using a commercially available enzyme-linked immunoassay (ELISA) kit (Pharmingen). The minimum concentration detected by this kit was 15.6 pg/ml.

Evaluation of eotaxin mRNA expression. Eotaxin mRNA was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). Four hours after stimulation with IL-4 or TNF-{alpha}, total RNA was extracted by a modification of the method of Chomczynski and Sacchi (7). The RNA was reverse transcribed by using a reverse transcription reagent (PE Applied Biosystems). Quantitative PCR was carried out in duplicate using an ABI Prism 7700 Sequence Detector (PE Applied Biosystems, Foster City, CA) as described before (14). The amounts of PCR product were quantified by the Sequence Detection System using ABI PRISM 7700. Oligonucleotide PCR primer pairs and fluorescent probe for the eotaxin gene were designed from the published sequence using Primer Express software (PE Applied Biosystems) as follows: eotaxin (F): 5'-ACGCCAAAGCTCACACCT-3'; eotaxin (R): 5'-TATGAGCAGCAGCCAGAGAA-3'; probe: 5'-FAM-TCCAACCATGAAGGTCTCCGCAGGA-TAMRA-3'. Primers and labeled probe for glyceraldehyde-3-phosphatase dehydrogenase (GAPDH) as an endogenous control were purchased from PE Applied Biosystems (TaqMan human GAPDH control reagents). The PCR reactions for the target gene and GAPDH were performed in separate tubes to avoid possible competition and/or interference in a single reaction tube (11). A total of 50 µl of the PCR mixture consisted of each primer pair (0.3 µM each), each TaqMan probe (0.2 µM, Applied Biosystems Japan, Tokyo, Japan), dATP, dCTP, dGTP, and dUTP (TaqMan PCR Core Reagents Kit with AmpliTaq Gold, Applied Biosystems) each at a concentration of 200 µM, 0.5 units uracil N-glycosylase (Applied Biosystems), 1.25 units Taq DNA polymerase (AmpliTaq Gold, Applied Biosystems), 5 µl 10x PCR buffer (Applied Biosystems), MgCl2 (3.5 mM), and 0.5 µl synthesized cDNA. Amplification and detection were performed with the ABI 7700 PRISM system, with the following profile: 1 cycle of 50°C for 2 min, 1 cycle of 95°C for 10 min, 40 cycles of 94°C for 10 s, and 60°C for 1 min. The threshold cycle (CT) was standardized by that of GAPDH mRNA. Thus the amounts of target mRNA copy number, normalized to endogenous reference (GAPDH mRNA) is given by: 2-[CT(eotaxin) - CT(GAPDH)]. The values are expressed in arbitrary units by multiplying them by an appropriate constant to indicate them plainly.

Preparation of cytoplasmic and nuclear extracts. BEAS-2B cells were seeded into six-well plates. Nuclear extracts were prepared by the modified method of Schreiber et al. (46). Cells were washed twice with ice-cold PBS before resuspension in 100 µl of 10 mM Tris·HCl, pH 7.8, 1% vol/vol Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 µg/ml leupeptin, 2 mM Na3VO4, 100 mM NaF, and 2 mM dithiothreitol (DTT). After a 5-min incubation on ice, 100 µl of ice-cold H2O were added and incubated on ice for a further 2 min. After 10 passages through a 26-G needle, nuclei were separated by centrifugation at 500 g for 6 min. Supernatants (cytoplasmic extracts) were retained. Nuclear pellets were rinsed twice in 10 mM Tris·HCl, pH 7.4, containing 2 mM MgCl2. For electrophoretic mobility shift assay (EMSA), nuclei were resuspended in 20 mM HEPES, pH 7.9, containing 25% vol/vol glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and 0.2 mM PMSF. For Western blot analysis, nuclei were resuspended in 10 mM Tris·HCl, pH 7.5, containing 150 mM NaCl, 1.5 mM MgCl2, 0.65% vol/vol Nonidet P-40, 0.5 mM PMSF, and 1 mM DTT, and subjected to one freeze-thaw cycle. Whole cell extracts were prepared by resuspending cells in 10 mM Tris·HCl, pH 7.5, containing 150 mM NaCl, 1.5 mM MgCl2, 0.65% vol/vol Nonidet P-40, 0.5 mM PMSF, and 1 mM DTT.

Western blot analysis. Cytoplasmic (20 µg) or nuclear extract (10 µg) was added to an equal volume of 125 mM Tris·HCl, pH 6.8, containing 1% wt/vol SDS, 10% vol/vol glycerol, 0.1% wt/vol bromphenol blue, and 2% vol/vol 2-mercaptoethanol (2x SDS loading buffer) and boiled for 5 min. Samples were separated by electrophoresis with 8% SDS-PAGE and transferred electrophoretically to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). Membranes were probed with rabbit anti-human STAT6 antibody (SC-621, Santa Cruz) diluted 1:1,000 in 50 mM Tris, 150 mM NaCl, and 0.05% Tween 20 containing 3% nonfat dried milk for 1 h. After being washed, membranes were incubated with horseradish peroxidase-linked anti-rabbit immunoglobulin (DAKO) for 1 h and detected with the enhanced chemiluminescence detection system (ECL plus, Amersham). For detecting the tyrosine 641-phosohorylated STAT6, we used rabbit anti-human p-STAT6 antibody (SC-11762, Santa Cruz) at a dilution of 1:500, and after detecting pSTAT6, we stripped and reprobed membranes with anti-human STAT6 antibody to confirm that equal amounts of total STAT6 were transblotted.

EMSA. Nuclear proteins (10 µg) were used in binding reactions as described previously (39). Double-stranded oligonucleotides that contain both a STAT6 response element and an NF-{kappa}B response element that are partially overlapping (underlined) were designed and prepared as described previously: 5'-GGCTTCCCTGGAATCTCCCACA-3' (TTCCCTGGAA for STAT6, GGAATCTCCC for NF-{kappa}B) (31). Specificity of the complex was determined by prior addition of a 100-fold excess of unlabeled oligonucleotide. For supershift analysis, nuclear extracts were incubated on ice for 60 min with antisera raised to p50 (SC114-X), p65 (SC109-X), or STAT6 (SC621-X) (Santa Cruz) at 0.4 µg before the addition of a radiolabeled oligonucleotide. Reactions were separated by electrophoresis on 6% nondenaturing acrylamide gels in 0.25x Tris-buffered EDTA. Gels were dried, and protein-DNA complexes were visualized by autoradiography.

Statistical analysis. Analyses of data were performed using StatView (Abacus Concepts, Berkeley, CA). Data are expressed as the means ± SE. Statistical differences were determined by ANOVA with Fisher's protected least significant difference test.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ATRA and 9-cis RA inhibited eotaxin release induced with IL-4 but not with TNF-{alpha} in BEAS-2B cells. To investigate the effect of RA on eotaxin release, we added various concentrations of ATRA (10-10, 10-9, 10-8, 10-7, and 10-6 M) to BEAS-2B cells 1 h before stimulation for 24 h with IL-4 (10 ng/ml) or TNF-{alpha} (10 ng/ml). The supernatants were analyzed for eotaxin protein by ELISA. ATRA inhibited the release of eotaxin stimulated with IL-4 in a concentration-dependent manner, and the inhibition was statistically significant at 10-7 and 10-6 M (Fig. 1A, P < 0.05). However, ATRA did not affect the release of eotaxin stimulated with TNF-{alpha} (Fig. 1B). The effect of 9-cis RA on eotaxin release was also investigated under the same conditions. 9-Cis RA showed a concentration-dependent inhibition on the release of eotaxin stimulated with IL-4, and the inhibition was statistically significant at 10-7 and 10-6 M (Fig. 1C, P < 0.05), but not with TNF-{alpha} (Fig. 1D). ATRA and 9-cis RA did not affect the viability and number of cells, which was assessed by trypan blue exclusion (data not shown).



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Fig. 1. Effect of all-trans retinoic acid (ATRA) and 9-cis RA on IL-4- or TNF-{alpha}-induced eotaxin release in BEAS-2B cells. ATRA was added 1 h before stimulation for 24 h with 10 ng/ml IL-4 (A, n = 4) or 10 ng/ml TNF-{alpha} (B, n = 4). The cell supernatants were analyzed for eotaxin protein by ELISA. 9-cis RA was added 1 h before stimulation for 24 h with 10 ng/ml IL-4 (C, n = 6) or 10 ng/ml TNF-{alpha} (D, n = 6). The data are presented as means ± SE. *P < 0.05 compared with IL-4 alone.

 

ATRA and 9-cis RA inhibited eotaxin mRNA expression induced with IL-4 but not with TNF-{alpha}. To investigate whether RA inhibits eotaxin release or production, we examined the effects of RA on eotaxin mRNA expression. Various concentrations of ATRA (10-10, 10-8, and 10-6 M) were added to BEAS-2B cells 1 h before stimulation with IL-4 (10 ng/ml) or TNF-{alpha} (10 ng/ml). ATRA and 9-cis RA inhibited eotaxin mRNA expression induced with IL-4 in a concentration-dependent manner. Inhibition of eotaxin mRNA expression was statistically significant at 10-8 and 10-6 M by ATRA (Fig. 2A, P < 0.05); although of the same magnitude, the inhibitory effect of 9-cis RA was not statistically significant (Fig. 2B). In contrast, neither ATRA nor 9-cis RA had any effect on TNF-{alpha}-induced eotaxin mRNA expression (Fig. 2, A and B).



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Fig. 2. Effect of ATRA and 9-cis RA on IL-4- or TNF-{alpha}-induced eotaxin mRNA expression in BEAS-2B cells. ATRA (A, n = 6) or 9-cis RA (B, n = 4) was added 1 h before stimulation for 4 h with IL-4 (10 ng/ml) or TNF-{alpha} (10 ng/ml). Eotaxin mRNA was analyzed by RT-PCR. The results are expressed as the ratio of intensity to the GAPDH and calculated as fold increase compared with control. The data are presented as mean ± SE. *P < 0.05 compared with IL-4 alone.

 

Effect of ATRA on IL-4- or TNF-{alpha}-induced activation of the eotaxin promoter. The results described above suggest that RA may attenuate the expression of eotaxin at the transcriptional level. To test this possibility, we performed transient transfection and luciferase-driven reporter plasmid assays using a 1,363-bp fragment of the eotaxin promoter, pEotx.1363. Both IL-4 (10 ng/ml) and TNF-{alpha} (10 ng/ml) increased luciferase activity in BEAS-2B cells transfected with the pEotx.1363 (Fig. 3A). ATRA dose-dependently inhibited IL-4-induced activation of the eotaxin promoter, and maximum inhibition was observed at 10-6 M (~51%, P < 0.05). On the other hand, ATRA had no effect on TNF-{alpha}-induced activation of the eotaxin promoter (Fig. 3A). Overlapping elements for STAT6 and NF-{kappa}B within the proximal eotaxin promoter have been shown to mediate the transcriptional induction by IL-4 and TNF-{alpha}, respectively (31). A reporter containing the eotaxin promoter mutated at the binding site for STAT6, pEotx.M1, responded to TNF-{alpha} but not to IL-4 (Fig. 3B). In contrast, a reporter plasmid, pEotx.M2, which was mutated at the binding site of NF-{kappa}B, responded to IL-4 but not to TNF-{alpha} (Fig. 3C). ATRA (10-6 M) significantly inhibited IL-4-induced activation of the pEotx.M2 (~39%, P < 0.05; Fig. 3C), but not TNF-{alpha}-induced activation of the pEotx.M1 (Fig. 3B).



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Fig. 3. Effect of ATRA on IL-4- or TNF-{alpha}-induced activation of eotaxin promoter. BEAS-2B cells were transiently cotransfected with either a wild-type eotaxin promoter-luciferase reporter plasmid (pEotx.1363, A), a plasmid mutated at binding site for STAT6 (pEotx.M1, B), or a plasmid mutated at binding site for NF-{kappa}B (pEotx.M2), and an internal control plasmid encoding Renilla luciferase. Several concentrations of ATRA were added 24 h after transfection, and IL-4 (10 ng/ml) or TNF-{alpha} (10 ng/ml) was added 1 h after ATRA treatment (A, n = 7–10 for both IL-4 and TNF-{alpha}; B, n = 4; C, n = 4). Six hours after IL-4 or TNF-{alpha} treatment, cells were harvested. Values were normalized by Renilla luciferase activity to standardized transfection efficacy and calculated as fold induction compared with control. The data are presented as means ± SE from a total of 3 independent experiments. *P < 0.05 compared with IL-4 alone.

 

Effect of ATRA on IL-4-induced phosphorylation and nuclear translocation of STAT6. The above results clearly show that ATRA inhibited IL-4-induced activation of eotaxin gene transcription but not that induced by TNF-{alpha}. IL-4 stimulation results in activation of Janus-activated kinase-1 (JAK1) and JAK3 tyrosine kinases and subsequently in phosphorylation of the transcription factor STAT6 on tyrosine 641. Phosphorylated STAT6 forms dimers, translocates to the nucleus, and binds to specific recognition sequences in the promoters of IL-4-responsive genes (16, 33). Previous studies indicate that IL-4-induced eotaxin gene transcription is mediated through activation of STAT6 DNA binding activity in BEAS-2B cells (31). We therefore investigated whether ATRA has an inhibitory effect on IL-4-induced STAT6 activation. The levels of total STAT6 did not change with IL-4 treatment (Fig. 4, bottom). In the steady state (i.e., with no simulation), minimal phosphorylated STAT6 was detected (Fig. 4, top, lane 1). Treatment with IL-4 (10 ng/ml) for 10 min strongly induced phosphorylation of STAT6. Pretreatment with ATRA (10-6 M) for 1 h did not change the IL-4-induced phosphorylation of STAT6 (Fig. 4, top, lanes 2 and 3). The tyrosine kinase inhibitor genistein (200 µM) was used as a positive control for inhibiting tyrosine phosphorylation and reduced IL-4-induced the phosphorylation of STAT6 by ~50% (Fig. 4, top, lanes 2 and 4). We next assessed the effect of ATRA on nuclear translocation of STAT6 using Western blot analysis of nuclear extracts and cytosolic extracts with a pan-STAT6 antibody. STAT6 was not detected in nuclear extracts from unstimulated cells (Fig. 5, lane 4). A significant level of STAT6 was detected in nuclear extracts after treatment with IL-4 (10 ng/ml) for 20 min. Pretreatment with ATRA (10-6 M) did not change the IL-4-induced increase in the level of STAT6 (Fig. 5, lanes 5 and 6), suggesting that ATRA does not influence IL-4-induced nuclear translocation of STAT6.



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Fig. 4. Effect of ATRA on IL-4-induced phosphorylation of STAT6. Whole cell protein (20 µg) extracted from BEAS-2B cells treated with IL-4 (10 ng/ml) for 10 min was analyzed by Western blot. ATRA (10-6 M) was added 1 h before stimulation with IL-4, and genistein (200 µM) was added 30 min before stimulation with IL-4. Phosphorylated STAT6 was detected using an antibody against specific Tyr-641-phosphorylated STAT6 (top). Membranes were stripped and reprobed with an anti-pan-STAT6 antibody (bottom). Results are representative of 4 independent experiments.

 


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Fig. 5. Effect of ATRA on IL-4-induced nuclear translocation of STAT6. Cytosolic (lanes 1–3) and nuclear (lanes 4–6) protein were extracted from BEAS-2B cells treated with IL-4 (10 ng/ml) for 20 min. ATRA (10-6 M) was added 1 h before stimulation with IL-4. STAT6 was detected by Western blot analysis using an anti-pan-STAT6 antibody. Results are representative of 3 independent experiments.

 

ATRA did not block IL-4-induced STAT6 DNA binding activity. To examine the effect of ATRA on STAT6 DNA binding activity induced with IL-4, we performed EMSA using a double-stranded oligonucleotide that contains both a STAT6 response element and a partially overlapping NF-{kappa}B response element as described in MATERIALS AND METHODS. Treatment with IL-4 (10 ng/ml) for 30 min resulted in the formation of a strong binding complex (complex 1) (Fig. 6, lanes 1 and 2). This binding complex was demonstrated to contain STAT6 by inhibition with an anti-STAT6 antibody but not a normal rabbit IgG (lanes 4 and 5). Appearance of the binding complex was blocked with an excess volume of an unlabeled wild-type oligonucleotide (lane 6). Pretreatment with ATRA (10-6 M) did not affect the formation of the STAT6-DNA binding complex induced with IL-4 (compare lane 3 with lane 2). The binding complex induced with IL-4 was not inhibited with anti-p50 and anti-p65 antibodies, which recognize the NF-{kappa}B Rel family members (data not shown). We also examined the effect of ATRA on TNF-{alpha}-induced NF-{kappa}B DNA binding activity. TNF-{alpha} (10 ng/ml) resulted in the formation of strong binding complexes (complexes 1 and 2) (Fig. 7, lanes 1 and 2). This binding complex was confirmed to contain NF-{kappa}B, which is a heterodimer of p65 and p50, by supershift and competition assays (lanes 4 and 5). ATRA did not affect the formation of the NF-{kappa}B DNA binding complex induced with TNF-{alpha} (compare lane 3 with lane 2). The binding complexes induced with TNF-{alpha} were not inhibited with an anti-STAT6 antibody (data not shown).



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Fig. 6. Effect of ATRA on IL-4-induced STAT6 DNA binding activity. EMSA was performed using double-stranded oligonucleotides, which contain both a STAT6 response element and a partially overlapping NF-{kappa}B response element. Nuclear protein (10 µg) was loaded. ATRA (10-6 M) was added 1 h before stimulation with IL-4 (10 ng/ml) for 30 min. For supershift analysis, nuclear extracts were preincubated for 60 min with an anti-STAT6 antibody (lane 4) or a normal rabbit IgG (lane 5). A 100-fold excess of unlabeled oligonucleotide (cold oligo) was preincubated for 60 min (lane 6). Results are representative of 5 independent experiments.

 


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Fig. 7. Effect of ATRA on TNF-{alpha}-induced NF-{kappa}B DNA binding activity. EMSA was performed using double-stranded oligonucleotides, which contain both a STAT6 response element and a partially overlapping NF-{kappa}B response element. Nuclear protein (10 µg) was loaded. ATRA (10-6 M) was added 1 h before stimulation with TNF-{alpha} (10 ng/ml) for 30 min. Supershift analysis and competition assay were performed in a separate experiment (lanes 4 and 5). Results are representative of 2 independent experiments.

 

AP-1 is not involved in ATRA-mediated transrepression. Several studies have demonstrated that ATRA inhibits AP-1 activity by RAR-dependent mechanisms (2, 20, 47). The eotaxin promoter has AP-1 response elements (13), and pEotx.1363, which we used in luciferase assay of eotaxin promoter, also has an AP-1 response element. Together with the results described above, we speculated that ATRA may inhibit IL-4-induced transactivation of the eotaxin promoter by inhibition of AP-1 activity without reducing DNA binding activity of STAT6. To investigate the role of AP-1, we performed transient transfection assays using an eotaxin promoter (site -300 to -1) luciferase plasmid (pEotx.300), which does not contain AP-1 response elements. Both 10-8 and 10-6 M of ATRA significantly inhibited IL-4-induced luciferase activity in cells transfected with pEotx.300 (~56% at 10-8 and 70% at 10-6 M, P < 0.05; Fig. 8). This result suggests that the inhibitory effect of ATRA on eotaxin promoter activity is independent of the AP-1 response element. We next sought to determine whether ATRA influences AP-1 activity in BEAS-2B cells. A transient transfection study was performed using the AP-1-dependent reporter plasmid AP1-Luc, which contains two repeats of the consensus AP-1 site. ATRA had no effect on AP-1-dependent transactivation induced with TPA (100 nM) (Fig. 9). These results further suggest that AP-1 is not involved in the ATRA-mediated transrepression of eotaxin gene expression in BEAS-2B cells. Neither IL-4 nor TNF-{alpha} was able to activate transcription of the AP1-Luc reporter in BEAS-2B cells, suggesting that these cytokines do not activate AP-1 in bronchial epithelial cells (Fig. 9).



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Fig. 8. Effect of ATRA on IL-4-induced pEotx.300 activity. BEAS-2B cells were transiently cotransfected with 300 bp of the proximal eotaxin promoter-luciferase reporter plasmids, 300 bp of the proximal promoter sequence (pEotx. 300), and an internal control plasmid encoding Renilla luciferase. Several concentrations of ATRA were added 24 h after transfection, and IL-4 (10 ng/ml) was added 1 h after ATRA treatment (n = 4). Six hours after treatment, cells were harvested. Values were normalized by Renilla luciferase activity to standardized transfection efficacy and calculated as fold induction compared with control. The data are presented as means ± SE. *P < 0.05 compared with IL-4 alone.

 


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Fig. 9. Effect of ATRA on TPA-induced activating protein (AP)-1-dependent transactivation. BEAS-2B cells were transiently cotransfected with an AP-1-dependent promoter plasmid, AP1-Luc, which contains 2 repeats of the consensus AP-1 site, and an internal control plasmid encoding Renilla luciferase. ATRA (10-6 M) was added 24 h after transfection, and TPA (100 nM) was added 1 h after ATRA treatment (n = 3). IL-4 (10 ng/ml) or TNF-{alpha} (10 ng/ml) was added 24 h after transfection. Six hours after treatment, cells were harvested. Values were normalized by Renilla luciferase activity to standardized transfection efficacy and calculated as fold induction compared with control. The data are presented as means ± SE.

 

Effect of ATRA on IL-4- or TNF-{alpha}-induced MCP-1 production. To determine whether the effect we observed was restricted to eotaxin, we examined the influence of ATRA on IL-4- or TNF-{alpha}-induced human monocyte chemotactic protein 1 (MCP-1) in BEAS-2B cells. ATRA significantly inhibited IL-4-induced MCP-1 production, which was measured in the cell supernatant by ELISA (1,146.7 ± 17.6 pg/ml with 10 ng/ml of IL-4 vs. 648.0 ± 5.8 ng/ml with IL-4 plus ATRA 10-6 M, 43.5% inhibition; mean ± SE, P < 0.05). In contrast to the case with eotaxin, ATRA also modestly inhibited TNF-{alpha}-induced MCP-1 production (1,395.3. ± 27.8 pg/ml by 10 ng/ml of TNF-{alpha} vs. 1,147.7 ± 56.3 pg/ml by TNF-{alpha} plus ATRA 10-6 M, 17.7% inhibition; P < 0.05, n = 3).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
RA and its derivatives have been demonstrated to have anti-inflammatory effects in vivo and in vitro. In the present IL-4-induced study, we demonstrate that ATRA and 9-cis RA inhibited expression of eotaxin mRNA and protein in a bronchial epithelial cell line. These RAs inhibited the function of a reporter gene containing a fragment of the eotaxin promoter, suggesting that the inhibitory effect is transcriptional. Interestingly, neither derivative of RA had an inhibitory effect on TNF-{alpha}-induced eotaxin expression. Although we did not elucidate the mechanism of the effect of RAs, we did determine that ATRA did not inhibit IL-4-induced tyrosine phosphorylation, nuclear translocation, or DNA binding activity of STAT6, suggesting that STAT6 activation is not a target of RAs in these cells. We also demonstrated that AP-1 was not involved in the mechanism of inhibition.

The dissociation of the inhibitory effect of RA on IL-4-induced eotaxin expression and TNF-{alpha}-induced eotaxin expression suggests that the inhibitory effect on eotaxin mRNA expression is not caused by decreasing mRNA stability. This is an important observation, since glucocorticoids, which bind to a member of the nuclear receptor family that includes RARs, have been shown to destabilize eotaxin mRNA (49). The results from luciferase-driven reporter plasmid assays support the hypothesis that RA inhibits IL-4-induced eotaxin expression at the transcriptional level. Although previous studies have shown that IL-4 activates eotaxin gene transcription through STAT6, as assessed by luciferase reporter plasmid assays and EMSA (31), ATRA did not have any detectable effects on the activation or function of STAT6-DNA binding. A similar phenomenon was found by Oeth et al. (40). In their report, ATRA inhibited tissue factor (TF) expression induced with LPS but not with TNF-{alpha} or IL-8 in human monocyte without altering the binding or functional activity of the transcription factors c-Fos/c-Jun and c-Rel/p65. However, LPS induction of the cloned TF promoter was not inhibited in their study. Liganded RAR{alpha} may have an inhibitory effect through an element outside the cloned promoter region examined in their study. On the other hand, liganded RAR{alpha} may exert its inhibitory effect through an element inside the promoter region examined in our study. We found that the inhibitory effect of ATRA was not restricted to eotaxin, since ATRA inhibited the induction of MCP-1 with IL-4 and, to a lesser extent, with TNF-{alpha}. This result suggests that both eotaxin and MCP-1 are mainly induced with IL-4 through an ATRA-sensitive pathway and suggests that an additional ATRA-sensitive pathway exists, by which TNF-{alpha} induces MCP-1.

Cytokine-dependent gene expression displays burst-attenuation kinetics; for example, overexpression of corepressor, silencing mediator for retinoid and thyroid hormone receptors (SMRT, homolog to N-CoR), suppresses the expression of STAT5 target genes by facilitating their downregulation after the initial induction phase (37). Therefore, to investigate whether ATRA attenuates IL-4-induced DNA binding activity of STAT6 after the initial induction, we performed kinetic analysis by EMSA. ATRA did not inhibit DNA binding activity of STAT6 for up to 8 h after IL-4 stimulation (data not shown), which minimizes the possibility that ATRA inhibits eotaxin expression by downregulation of the DNA binding activity of STAT6.

ATRA exerts its effect by binding to RARs, members of the nuclear receptor family, and 9-cis RA exerts its effect by binding RARs and RXRs (21). In the present study, both ATRA and 9-cis RA showed the same effect on IL-4-induced eotaxin production, which indicates that RARs are most likely mediating the RA transrepression of the eotaxin gene. RARs have three subtypes, designated as RAR{alpha}, RAR{beta}, and RAR{gamma}. We confirmed the existence of RAR{alpha} and RAR{beta} in the nucleus of BEAS-2B cells by Western blot analysis (data not shown). RARs form ligand-dependent heterodimers with RXR and bind to RA response elements (RARE), specific DNA sequences in promoter regions of target genes. The thioredoxin gene and the alcohol dehydrogenase/reductase gene have been reported to be upregulated by RA in airway epithelial cells (5, 48). This raises the possibility that an RA-RAR complex may regulate eotaxin gene expression by binding to RARE. However, pEotx.1363, which we used in our reporter plasmid assays, does not contain an RARE in the inserted eotaxin promoter, suggesting that RA-mediated inhibition of eotaxin gene expression is not mediated directly by an RARE.

Transrepression of AP-1 is reported to be an important mechanism in RA-mediated repression of gene expression by which transcriptional interference with membrane receptor-controlled signaling pathways occurs independently of RARE. Examples include c-jun DNA binding inhibition (47), competitive titration by RAR and AP-1 of limiting amounts of cAMP response element binding protein (CBP) (20), downregulation of c-Jun NH2-terminal kinase activity (24), or regulation of extracellular signal-regulated kinase and CBP recruitment to an AP-1-responsive promoter (2). Our observation that ATRA inhibited IL-4-induced luciferase activity in cells transfected with pEotx.300, which does not contain AP-1 response elements, suggests that the inhibitory effect of ATRA on the eotaxin promoter is independent of AP-1 response elements.

Although our results appear to eliminate a posttranscriptional mechanism or an AP-1-dependent mechanism, we have not elucidated the mechanism of RA-mediated transrepression of IL-4-induced eotaxin gene transcription. One of us (Y. Nasuhara) (39) reported that a tyrosine kinase inhibitor significantly inhibits TNF-{alpha}-induced NF-{kappa}B-dependent transcription without inhibiting NF-{kappa}B DNA binding activity in U-937 cells. In A549 cells, inhibitors of protein kinase C or p38 mitogen-activated protein kinase inhibit IL-1{beta}-induced NF-{kappa}B-dependent transcription without inhibiting NF-{kappa}B DNA binding activity (3). Although the role of phosphorylation, which does not influence DNA binding activity, of transcription factors is not fully understood, there is a report that serine phosphorylation of c-jun increases its affinity for the coactivator CBP without altering its DNA binding activity (1). Pesu et al. (44) demonstrated that a serine/threonine kinase inhibitor inhibits IL-4-induced STAT6-dependent transcription without affecting tyrosine phosphorylation of JAK1, JAK3, and STAT6, or DNA binding activity of STAT6. It is thus possible that RA inhibits STAT6 function without inhibiting the phosphorylation, nuclear translocation, or DNA binding of STAT6.

Remodeling of chromatin structure as a result of histone acetylation is one of the critical processes in transcriptional regulation. A number of transcriptional coactivators, including CBP/p300, p300/CBP-associated factor, and steroid receptor coactivator-1 (SRC-1), which possess histone acetyltransferase activity, have been shown to associate with various transcription factors and act as general integrators of the transcription machinery (22). On the other hand, transcription is negatively regulated by corepressor complexes comprising SMRT, mSin3A/B, and histone deacetylases (36). RARs can act as either ligand-independent repressors or ligand-dependent activators, based on an exchange of SMRT-containing co-repressor complexes for coactivator complexes in response to ligands (42, 50), and RA dose-dependently releases SMRT from RAR (6). In the case of some RAR target genes, RA appears to induce transactivation by releasing corepressors from RAR and recruiting coactivators to RAR and its target elements. On the other hand, the released SMRT from RAR in response to ligands may be recruited to other activated transcription factor and repress transcription of genes. Nakajima et al. (37) reported a functional interaction of STAT5 and SMRT. They concluded that SMRT binds to STAT5A and 5B and strongly represses STAT5-dependent transcription. Although overexpression of SMRT was not found to reduce STAT6-dependent transcription in a two-hybrid system of 293 cells in their report, the interaction of STAT6 and SMRT may vary among cells. We did not detect SMRT protein in resting BEAS-2B cells, and SMRT was not coimmunoprecipitated with RAR{alpha} or RAR{beta} regardless of RA treatment, however (data not shown). The general coactivators CBP/p300 and SRC-1 have been shown to cooperate with STAT6 (27, 32). RAR{alpha} was reported to repress AP-1-dependent transcription by competitive titration of limiting amounts of CBP in response to ATRA (12). We also tried to test the possibility that RAR inhibits IL-4-induced STAT6-dependent transcription by competition for a limiting amount of coactivators. Very small amounts of CBP/p300 and SRC-1 were detected in resting BEAS-2B cells, and, as with SMRT, neither CBP/p300 nor SRC-1 was coimmunoprecipitated with STAT6 regardless of treatment (data not shown). Overexpression studies will be needed to demonstrate conclusively that RA-mediated transrepression of IL-4-induced eotaxin gene activation is independent of coactivators and corepressors.

The corepressor SMRT and coactivators CBP/p300 and SRC-1 are not only involved in STAT6-dependent transcription but also in NF-{kappa}B-dependent transcription (25, 35, 43). Therefore, if the RA-RAR complex interacts with these general corepressor or coactivators, dissociation of its inhibitory effect on IL-4- and TNF-{alpha}-induced transactivation is hard to explain. Very recently, Yang et al. (51) identified a novel coactivator for STAT6 termed p100, which mediates an interaction between STAT6 and RNA polymerase II. If this newly found coactivator is STAT6 selective, it may be an attractive candidate for the target of the effects of RA.

Eotaxin is an important mediator of eosinophil trafficking into the airway and gastrointestinal tract and is also a potent activator of eosinophils (15, 45). Our results demonstrate that RA inhibits IL-4-induced eotaxin production at least in part by inhibiting transcription. To assess the potential for clinical benefit of retinoic acid in eosinophilic inflammation, further studies will be needed in primary bronchial epithelial cells and using in vivo animal asthma models. However, these findings raise the possibility that ATRA and 9-cis RA may have a therapeutic value in reducing eosinophilic inflammation, which is important in the pathogenesis of mucosal diseases such as eosinophilic gastroenteritis, rhinitis, and asthma.


    ACKNOWLEDGMENTS
 
We thank Dr. Yasushi Shimizu, Yoko Suzuki, and Bonnie Hebden for excellent support and assistance, and Ono Pharmaceutical for providing IL-4.


    FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Nasuhara, 1st Dept. of Medicine, Hokkaido Univ. School of Medicine, North 15, West 7, Kita-ku, Sapporo 060-8638, Japan (E-mail: nasuhara{at}med.hokudai.ac.jp).

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


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