Retinoic acid-receptor activation of SP-B gene transcription in respiratory epithelial cells

Cong Yan, Manely Ghaffari, Jeffrey A. Whitsett, Xin Zeng, Zvjezdana Sever, and Sui Lin

Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Retinoids are known to play important roles in organ development of the lung. Retinoids exert their activity by modulating the expression of numerous genes, generally influencing gene transcription, in target cells. In the present work, the mechanism by which retinoic acid (RA) regulates surfactant protein (SP) B expression was assessed in vitro. RA (9-cis-RA) enhanced SP-B mRNA in pulmonary adenocarcinoma cells (H441 cells) and increased transcriptional activity of the SP-B promoter in both H441 and mouse lung epithelial cells (MLE-15). Cotransfection of H441 cells with retinoid nuclear receptor (RAR)-alpha , -beta , and -gamma and retinoid X receptor (RXR)-gamma further increased the response of the SP-B promoter to RA. Treatment of H441 cells with RA increased immunostaining for the SP-B proprotein and increased the number of cells in which the SP-B proprotein was detected. An RA responsive element mediating RA stimulation of the human SP-B promoter was identified. RAR-alpha and -gamma and RXR-alpha but not RAR-beta or RXR-beta and -gamma were detected by immunohistochemical analysis of H441 cells. RA, by activating RAR activity, stimulated the transcription and synthesis of SP-B in pulmonary adenocarcinoma cells.

surfactant protein B; glucocorticoid receptor; thyroid transcription factor-1

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

RETINOIC ACID (RA), a derivative of vitamin A, plays highly diverse roles in cell proliferation, differentiation, and organ development. RA exerts its biological activity by binding to retinoid nuclear receptors (RARs) and retinoid X receptors (RXRs) in the nucleus of target cells. Ligand-dependent RARs and RXRs form heterodimers that bind to RA response elements (RAREs) on many genes, thereby modulating transcriptional activity. Both RARs and RXRs belong to the superfamily of nuclear receptors. RARs are activated by all trans-RA and 9-cis-RA, whereas RXRs are only activated by 9-cis-RA. Three isotypes of RAR, alpha , beta  and gamma , encoded by distinct genes (for reviews, see Refs. 15, 19) have been identified. Retinoid receptors consist of a DNA-binding domain that contains Zn2+ finger motifs, a ligand-binding domain, a ligand-independent transcriptional activation domain, a ligand-dependent transcriptional activation domain, a dimerization domain, and an F region of unknown function (for a review, see Ref. 18). Through these various domains, RARs interact with other transcriptional and signaling factors, including CBP/p300 (14), activator protein-1 (32), TFIIH (31), and TAFII135 (24).

Retinoids have pleiotropic effects in many target organs including the lung. Vitamin A deficiency is associated with squamous metaplasia of the respiratory epithelium. Clinical studies in premature infants demonstrated a correlation between low serum levels of vitamin A and chronic lung disease after respiratory distress syndrome (33). A previous study (23) demonstrated that RA influences fetal lung morphogenesis and differentiation. All three isotypes of RAR mRNAs are expressed in pulmonary tissues during fetal development (22). In situ hybridization analysis showed that RAR-beta was expressed in the epithelium of proximal bronchi in day 14.5 postconception embryos, whereas RAR-alpha and -gamma mRNAs were expressed rather weakly and homogeneously in developing lung tissue (9). RA treatment of both human and rat fetal lung explants in culture altered surfactant protein (SP) and mRNA concentrations (3, 6, 11, 25). In human lung explant, RA reduced SP-A and SP-C mRNA levels. In contrast, all trans-RA increased SP-B mRNA levels in a concentration-dependent manner, with the maximum increase observed at 3 µM (25). In vitamin A-deficient animals, fetal lung weight was significantly decreased in association with decreased phosphatidylcholine content, a marker of respiratory epithelial cell differentiation (22). Mice bearing null mutations in both RAR-alpha and RAR-beta displayed some of the organ defects, including hypoplastic lungs (23), providing further support for the role of RARs in lung morphogenesis.

Pulmonary surfactant is a complex mixture of lipids and proteins that reduces surface tension at the air-liquid interface in the alveoli. Surfactant lipids are synthesized primarily by alveolar type II epithelial cells and are stored in lamellar bodies that are secreted into the air space. SP-A, SP-B, SP-C, and SP-D are also synthesized primarily by type II or bronchiolar epithelial cells and play critical roles in maintaining stability of the surfactant layer (SP-B and SP-C) and in host defense (SP-A and SP-D). The mechanisms by which RA and its RARs/RXRs influence surfactant homeostasis in type II epithelial cells in the respiratory epithelium, especially at the level of gene transcription, are largely unknown.

SP-B is a 79-amino acid amphipathic peptide produced by the proteolytic cleavage of SP-B proprotein (proSP-B) by type II epithelial cells. The SP-B peptide is stored in lamellar bodies and secreted with phospholipids into the airway lumen (for a review, see Ref. 36). SP-B is a critical component of the surfactant complex and is essential for the formation of tubular myelin and the stability and rapid spreading of surfactant phospholipids (36). Genetic defects in SP-B cause respiratory failure after birth in both humans and SP-B gene-targeted mice (7, 26, 27).

SP-B homeostasis is modulated at multiple levels. SP-B gene transcription is influenced by thyroid transcription factor-1 (TTF-1) and hepatocyte nuclear factor-3 (HNF-3) (4, 40). SP-B gene transcription is further enhanced by cAMP and protein kinase A-dependent phosphorylation of TTF-1 (42). Glucocorticoids stimulate SP-B gene expression in both cell lines and lung explants (1, 2, 10, 34). Phorbol ester strongly inhibited SP-B gene expression (30, 35). At the posttranscriptional level, SP-B mRNA stability is enhanced by glucocorticoids and decreased by tumor necrosis factor-alpha and phorbol ester (28, 29, 38).

In the present study, both all trans- and 9-cis-RA enhanced transcription of the human and mouse SP-B promoters in human pulmonary adenocarcinoma cells (H441) and mouse lung epithelial cells (MLE-15). The H441 cell line was isolated from a human lung adenocarcinoma. The MLE-15 cell line was derived from mouse lung tumor cells immortalized by the SV40 large T antigen in vivo (37). Both cell lines have been used extensively to characterize SP-A, SP-B, and SP-C gene transcription.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell culture. H441 cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, glutamine, and penicillin-streptomycin. Cells were maintained at 37°C in 5% CO2-air and passaged weekly. The murine clonal respiratory epithelial cell line MLE-15 was propagated in HITES medium (37) containing 4% fetal bovine serum and maintained as above.

Plasmid constructs. The region from +41 to -2240 of the human SP-B (hSP-B) promoter was generated by PCR with synthetic oligonucleotide primers with the pDelta 5'-2240 SP-B chloramphenicol acetyltransferase (CAT) construct as a template as described previously (40). The upstream primer with the Mlu I site was 5'-CGCACGCGTACCTGCAGGTCAACGGATCA-3'. The downstream primer with the Xho I site was 5'-GCGCTCGAGCCACTGCAGCAGGTGTGACTC. The PCR products were digested with Mlu I and Xho I restriction enzymes and ligated with Mlu I-Xho I-digested pGL2-B luciferase reporter plasmids (Promega). The correctness of the hSP-B 2240 promotor fragment (hSP-B-2240) luciferase reporter construct was confirmed by DNA sequencing. The murine (m) SP-B promoter fragment -1797/+42 (mSP-B-1797) was subcloned in the pBLCAT6 reporter vector as described previously (5). The human TTF-1 1.7-kb luciferase reporter gene was made previously (13). Human RAR and RXR expression vectors hRAR-alpha /pSG5, hRAR-beta /pSG5, hRAR-gamma 1/pSG5, and hRXR-gamma /pSG5 were kindly provided by Dr. Pierre Chambon. Human glucocorticoid-receptor (GR) expression vector pR5-hGR-alpha was kindly provided by Dr. Ronald M. Evans.

Transfection, luciferase, and CAT assays. To determine the effects of all trans-RA and 9-cis-RA on the hSP-B promoter luciferase reporter constructs, transient transfection and luciferase assays were performed as previously described (40, 41) with minor modification. Briefly, H441 cells were seeded at a density of 2 × 105 cells/well in 6-well plates. The hSP-B reporter constructs (0.5 µg) were transfected into H441 cells by lipofectin transfection (GIBCO BRL). In each transfection, 0.5 µg of pCMV-beta -gal plasmid was included for normalization of transfection efficiency. For quantification of beta -galactosidase activity, one unit of optical density of beta -galactosidase in the protein extract was defined as hydrolysis of o-nitrophenyl-beta -D-galactopyranoside that generates absorbance to 1 optical density unit at 420 nm at 37°C. After 2 days of incubation with various concentrations of all trans- and 9-cis-RA, the cells were lysed and luciferase activity assays were performed with the luciferase assay system (Promega). The light units were assayed by luminometry (monolight 2010, Analytical Luminescence Laboratory, San Diego, CA). In RAR/RXR cotransfection assays, 0.5 µg of various RAR and RXR constructs was cotransfected with 0.5 µg of the hSP-B reporter constructs and treated with 10 µM 9-cis-RA. Each experiment was repeated at least three times. Human TTF-1 promoter studies with the luciferase reporter gene with RARs/RXR-gamma were performed as outlined above. No effect of RA and RARs/RXR-gamma on expression of beta -galactosidase activity was observed in transfection assays in H441 cells.

Transient transfection study of the mSP-B-1797 was performed with the calcium precipitation method. A mixture of mSP-B-1797 (0.67 pmol/well) and pCMB-beta -gal (1.25 µg/well) was used for transfection followed by the addition of 10 µM 9-cis-RA for 2 days. Cell extracts were prepared with three freeze-thaw cycles, and the pellets were resuspended in 50-100 µl of 0.25 M Tris, pH 7.8. CAT assays were performed as previously described (5, 41). Chromatograms of [14C]chloramphenicol and its acetylated derivatives were quantitated with a Molecular Dynamics phosphorimager (Storm 680).

RT-PCR. Total RNA was purified from H441 cells treated with 10 µM 9-cis-RA for 0 and 28 h following the procedures described previously (39). The quality of RNA samples was assessed on 1% agarose gel after ethidium bromide staining. Thirty micrograms of total RNA were reverse transcribed with the oligo(dT) primer (NEN) by superscript RT enzyme (GIBCO BRL) in the presence of deoxynucleoside triphosphate and first-strand buffer (GIBCO BRL). Five micrograms of reverse transcripts were amplified in 30 cycles of PCR with a SP-B primer pair corresponding to exons 8 and 10 (upstream primer, 5'-GGTCGCCGACAGGAGAATGGCTGC-3'; downstream primer, 5'-AAGGTCGGGGCTGTGGATACACTG-3'). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control, and the same reverse-transcribed cDNAs were used for PCR with a GAPDH primer pair (upstream primer, 5'-CAGAAGACTGTGGATGGCCCC-3'; downstream primer, 5'-GTCCACCACCCTGTTGCTGTAGCC-3'). Five micrograms of reaction products were separated on 1% agarose gel before being stained. Intensities of the PCR product bands were quantitatively analyzed by an IS-1000 Digital Imaging System (Alpha Innotech, San Leandro, CA).

Immunohistochemistry. H441 or HeLa cells were seeded onto Permanox chamber slides (Fisher) at densities ranging from 104 to 105 cells/chamber (2 chambers/slide) as described previously (12). For immunostaining of SP-B, RAR, and RXR proteins, slides were pretreated with 0.1 M PBS containing Triton X-100, pH 7.4, plus 5% goat serum for 2 h at room temperature before incubation with a 1:500 dilution of RAR and RXR rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) or a 1:100 dilution of proSP-B polyclonal antibodies overnight. The next day, the slides were washed five times in 0.1 M PBS with Triton X-100 solution, and biotinylated goat anti-rabbit IgG was added to the serum blocking solution (45 µl IgG/10 ml) for 30 min, followed by five washes in 0.1 M PBS with Triton X-100 solution. The slides were then treated with avidin-biotin reagent according to the directions in the Vectastain Elite Kit (Vector Laboratories, Burlingame, CA). The slides were visualized by a Nikon Microphot-FXA video system. For transfection studies, 2 µg of the RAR-beta expression plasmid were transfected into cells with the lipofectin transfection kit from GIBCO BRL as previously described (12). Immunohistochemical staining of the transfected cells was then performed as described above.

Electrophoretic mobility shift assay. An oligonucleotide corresponding to the hSP-B promoter -415 to -440 was synthesized, annealed, and purified. The oligonucleotide was radiolabeled by [gamma -32P]ATP and kinase and incubated with 100 ng of the purified RAR-gamma -glutathione S-transferase (GST) fusion protein as suggested by the manufacturer (Santa Cruz Biotechnology). In the absence of RXRs, a relatively higher concentration of RAR is required due to its low DNA-binding affinity. Antibody-recognizing RAR-gamma (1 µg) was used for supershift assay. An electrophoretic mobility shift assay (EMSA) was performed by following the procedures previously described (40).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Dose-dependent stimulation of hSP-B promoter activity by all trans- and 9-cis-RA. To test the effects of RA on SP-B gene transcription, the hSP-B-2240 luciferase reporter construct was transfected into H441 cells. Cells were treated with all trans- or 9-cis-RA (10-5 to 10-9 M). Both all trans- and 9-cis-RA enhanced hSP-B-2240 activity (Fig. 1). Activation was observed at 10-8 M of both 9-cis- and all trans-RA (~10% increase). Significant activation was observed at 10-5 M as assessed by one-way ANOVA (P < 0.02).


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Fig. 1.   Retinoic acid (RA) stimulates human surfactant protein (hSP) B 2240 promoter (hSP-B-2240) activity. Human pulmonary adenocarcinoma (H441) cells were transfected with hSP-B-2240 luciferase reporter vector and treated with all trans- and 9-cis-RA. Luciferase activity was measured 48 h after treatment. Activity without RA treatment was defined as 1. Activities were measured in light units of optical density (OD) of beta -galactosidase. Values are means ± SD; n = 3 experiments, each performed in triplicate. ANOVA showed significant stimulatory effect of 9-cis-RA on hSP-B promoter. * Significant difference from control values, P < 0.02.

RA induced endogenous hSP-B mRNA in H441 cells. Because the transcriptional activity of the hSP-B promoter was stimulated by RA, the effects of RA on the expression of endogenous hSP-B mRNA were assessed by RT-PCR (Fig. 2). H441 cells were treated with 9-cis-RA (10-5 M) for 0 and 28 h. Total RNAs were extracted from cells, reverse transcribed, and amplified by PCR with an hSP-B-specific primer pair, with a GAPDH-specific primer pair as a control. After 28 h of exposure to 9-cis-RA, SP-B mRNA was significantly increased (P < 0.02 by paired t-test). No significant stimulation was observed in GAPDH mRNA (P > 0.15). The H441 cells were also treated with dexamethasone (50 nM). SP-B mRNA increased 30- to 50-fold after 28 h of treatment (data not shown), consistent with previous observations (28).


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Fig. 2.   RA increases endogenous hSP-B mRNA. Total RNAs from H441 cells treated with 9-cis-RA for 0 and 28 h were prepared and reverse transcribed. Reverse-transcribed cDNA products were amplified by PCR with a primer pair for hSP-B gene and a primer pair for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as described in MATERIALS AND METHODS. GAPDH was used as a control. PCR product intensity of RA treatment at 0 h was defined as 1. Values are means; n = 2 experiments; each RT-PCR determination was performed in triplicate.

Increased proSP-B staining after treatment with 9-cis-RA. H441 cells were treated for 48 h with 9-cis-RA (10-5 M) and immunohistochemically stained with anti-human proSP-B polyclonal antibody. Intracellular proSP-B staining of H441 cells was increased by 9-cis-RA (Fig. 3).


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Fig. 3.   Stimulation of SP-B proprotein (proSP-B; arrowheads) synthesis by RA. H441 cells were treated without (A) and with (B) 10-5 M 9-cis-RA for 2 days. Cells were immunostained with rabbit proSP-B polyclonal antibody as described in MATERIALS AND METHODS.

RAR/RXR activation of the hSP-B promoter. To assess the effects of RAR on hSP-B promoter activity, three forms of human RAR (a, beta , and gamma )-RXR-gamma expression plasmids were cotransfected with the hSP-B-2240 luciferase reporter construct into H441 cells. Cotransfection of RAR-alpha /RXR-gamma , RAR-beta /RXR-gamma , and RAR-gamma /RXR-gamma into H441 cells further increased hSP-B 2.24-kb promoter activity, resulting in a seven- to ninefold induction after treatment with 9-cis-RA (P < 0.05 by one-way ANOVA; Table 1).

                              
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Table 1.   Transcriptional stimulation of the hSP-B 2.24-kb promoter by RAR/RXR and inhibition by GR in H441 cells

Glucocorticoids are known to enhance SP-B mRNA in vivo and in H441 cells. To determine the potential role of the GR in SP-B gene regulation in H441 cells, a GR expression plasmid was cotransfected with the hSP-B-2240 luciferase reporter construct (Table 1). In the absence of a GR, dexamethasone (50 nM) slightly decreased the activity of the hSP-B-2240 luciferase reporter gene. Cotransfection with a GR also slightly decreased the activity of the hSP-B-2240 luciferase reporter gene in the absence of dexamethasone. Surprisingly, cotransfection with a GR in the presence of dexamethasone significantly repressed luciferase activity of the hSP-B-2240 from 100 ± 20 to 37 ± 4.5% of luciferase activity (P < 0.02 by one-way ANOVA).

Identification of RAREs on the hSP-B promoter. In the region of -500 to -218 of the hSP-B promoter, a sequence (-440 to -415) resembling a direct repetition of RARE (core motif, A/GGG/TTCA) was identified (Fig. 4A). This oligonucleotide, Ba-wt (-415 to -440), was synthesized and incubated with the purified RAR-gamma -GST fusion protein. Interestingly, three specific RARE-RAR-gamma complexes were detected by EMSA, and an antibody recognizing RAR-gamma supershifted all three complexes (Fig. 4B). In the promoter deletion studies, activity of a hSP-B promoter (hSP-B-500) containing this RARE was significantly enhanced by cotransfection with RAR-alpha /RXR-gamma and 9-cis-RA treatment as assessed by luciferase reporter assay and one-way ANOVA (P < 0.02). A promoter construct (hSP-B-375) lacking the RARE sequence was completely unresponsive to RAR-alpha /RXR-gamma and 9-cis-RA stimulation (Fig. 4C).


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Fig. 4.   Identification of RA response element (RARE) on hSP-B promoters. A: schematic illustration of hSP-B-500, hSP-B-375, and nucleotide sequence (Ba-wt) from -415 to -440. Underlined sequences resemble direct repetition of RARE. B: electrophoretic mobility shift assay of purified RA receptor (RAR)-gamma -glutathione S-transferase protein with a nucleotide oligomer corresponding to -415 to -440 of hSP-B promoter. Antibody (Ab) recognizing RAR-gamma was included to supershift DNA-protein complexes. Arrows indicate DNA-protein complexes. C: H441 cells were transfected with hSP-B-500 or hSP-B-375 luciferase reporter vector, treated with 9-cis-RA (10-5 M), and cotransfected with RAR-alpha /retinoid X receptor (RXR)-gamma . +, Presence; -, absence. Luciferase activity was measured 48 h after treatment. Activity without RA treatment was defined as 1. Activities were measured in light units of OD of beta -galactosidase. Values are means ± SD; n = 3 experiments, each performed in triplicate. ANOVA showed significant stimulatory effect of 9-cis-RA and RAR-alpha /RXR-gamma on hSP-B-500, P < 0.02.

Immunohistochemical detection of RAR and RXR proteins in H441 and HeLa cells. Immunohistochemical staining of H441 cells was performed with distinct antibodies recognizing the alpha , beta , and gamma isotypes of RAR and RXR. RAR-alpha and -gamma and RXR-alpha were detected in the nuclei of H441 cells (Fig. 5A), whereas RAR-beta and RXR-beta and -gamma were not detected. In contrast, HeLa cells were stained by antibodies recognizing RAR-alpha and -gamma and all three forms of the RXR isotype (Fig. 5B). In both HeLa and H441 cells, RAR and RXR staining was stronger in the nuclei than in the cytoplasm. Although RAR-beta was not detected in either H441 or HeLa cells, RAR-beta was readily detected after transfection with the hRAR-beta expression plasmid in both cell lines.


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Fig. 5.   Immunohistochemical staining of RARs and RXRs in H441 (A) and HeLa (B) cells. H441 and HeLa cells were immunostained with rabbit polyclonal antibodies against RAR-alpha , -beta , and -gamma and RXR-alpha , -beta , and -gamma . As a positive control for RAR-beta protein, H441 and HeLa cells were transfected with RAR-beta expression vector for 2 days and immunostained with rabbit RAR-beta polyclonal antibody.

9-cis-RA stimulates the activity of the mSP-B promoter in MLE-15 cells. The effect of RA on the reporter construct consisting of the mSP-B-1797 (5) was tested in MLE-15 cells. 9-cis-RA (10-5 M) activated the mSP-B-CAT 1797-bp reporter construct to a similar extent as the hSP-B 2240 construct (P < 0.02 by paired t-test; Fig. 6).


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Fig. 6.   RA stimulates murine (m) SP-B 1797 promoter activity. Murine MLE-15 cells were transfected with mSP-B-CAT 1797 reporter vector and followed by treatment with 10-5 M 9-cis-RA. Chloramphenicol transferase (CAT) activity was measured 48 h after treatment. Relative CAT activity is expressed compared with promotorless pBLCAT6 construct activity after normalization to beta -galactosidase activity to correct for transfection efficiency. Values are means ± SD; n = 3 experiments, each performed in triplicate. ANOVA showed a significant stimulatory effect of 9-cis-RA on mSP-B 1797 promoter, P < 0.05.

9-cis-RA does not alter hTTF-1 promoter activity in H441 cells. SP-B gene transcription is strongly influenced by TTF-1. Previously, immunohistochemical and in situ hybridization studies (13, 17) demonstrated that TTF-1 expression was colocalized with SP-B in both human and mouse lungs. TTF-1 is also expressed in H441 and MLE-15 cells where it regulates SP-B gene transcription. To test the possibility that the effects of RA on SP-B expression were mediated by changes in TTF-1 gene transcription, the effect of RA on the activity of the human TTF-1 promoter region 1-1,700 bp was assessed. Cotransfection of RAR/RXR and GR had no effect on the hTTF-1 1.7-kb construct in the presence or absence of RA and dexamethasone (P > 0.1 by paired t-test; Fig. 7).


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Fig. 7.   Effect of RA and RARs/RXR-gamma on thyroid transcription factor (TTF)-1 promoter. H441 cells were cotransfected with human TTF-1 1.7-kb luciferase reporter vector and RARs/RXR-gamma or glucocorticoid-receptor expression plasmids. Transfected cells were treated with 10 µM 9-cis-RA or 50 nM dexamethasone. Luciferase activity was measured 48 h later. Activity of hTTF-1 1.7-kb luciferase reporter vector without 9-cis-RA treatment in absence of RARs/RXR-gamma cotransfection was defined as 1. Activities were measured in light units of OD of beta -galactosidase. Values are means ± SD; n = 3 experiments; each assay was performed in triplicate. ANOVA showed no significant stimulatory effect of 9-cis-RA, RARs/RAR-gamma , and GR on human TTF-1 1.7-kb promoter, P > 0.1.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the present work, 9-cis-RA stimulated the transcriptional activity of hSP-B and mSP-B gene promoters in both human H441 and mouse MLE-15 cell lines. The observation that relatively high doses of RA were required for hSP-B promoter activation is consistent with a previous study (25) regarding the effects of RA on SP-B mRNA expression in cultured human fetal lung explants. 9-cis-RA also significantly enhanced endogenous SP-B mRNA expression and SP-B protein accumulation in H441 cells (Figs. 2 and 3). The present study also demonstrated that cotransfection of all three RAR isotypes in combination with RXR further enhanced RA treatment-dependent transactivation on the hSP-B promoter, supporting the concept that the effects of RA are mediated by RAR/RXR activation of the SP-B promoter. Detection of RAR (alpha  and gamma ) and RXR (alpha ) expression in H441 cells and the developing respiratory epithelium in vivo also supports their potential roles in the regulation of respiratory epithelial cell gene expression.

The observation that RA enhanced SP-B mRNA and protein expression in H441 cells is consistent with previous findings from a study (25) of a human fetal lung explant culture. The RA-dependent induction in SP-B mRNA and protein is likely to be mediated, at least in part, by direct interaction of RAR with the SP-B gene promoter to enhance SP-B gene transcription. An RARE mediating the effects of RA on SP-B transcription was localized in the region -415 to -440 on the hSP-B promoter by EMSA. Deletion of this RARE resulted in loss of RA stimulation in the transient transfection studies, supporting the concept that RA and RAR/RXR heterodimers exert their effects directly on the hSP-B promoter. Although both TTF-1 and HNF-3beta stimulate the hSP-B promoter, TTF-1 had a much stronger stimulatory effect (six- to sevenfold) on the hSP-B promoter than HNF-3beta (around twofold stimulation) (42). The potential RAR sites were in close proximity to the distal TTF-1 clustered sites (40). We therefore tested whether RA enhances TTF-1 transcription, which would be more likely to enhance SP-B transcription than HNF-3beta , which is much less active on the SP-B promoter. In contrast, cotransfection of RAR/RXR with the TTF-1 promoter construct did not alter its activity, although it remains possible that RA may alter TTF-1 expression or activity by other mechanisms. For example, a recent study (16) demonstrated that the nuclear localization of TTF-1 and HNF-3beta was affected by phorbol ester.

The distribution of RARs in developing fetal mouse lung was determined by in situ hybridization (9). Although RAR-beta was expressed in the epithelium of proximal bronchi in day 14.5 postconception embryos, RAR-alpha and -gamma mRNAs were expressed homogeneously, even though weakly, in lung tissue (9). The critical roles of RAR-s in lung organogenesis were revealed by RAR gene double-knockout mice (23). In these mice, both the right and left lungs were either absent or markedly hypoplastic, indicating that RARs are required for normal lung formation. H441 cells share many features with bronchiolar respiratory epithelial cells from the distal conducting airway. The presence of RAR-alpha and -gamma and RXR-alpha in H441 cells is consistant with their origin from distal respiratory epithelium and suggests their possible roles in bronchiolar cell differentiation and cell-type lineage maintenance.

Analysis of the GR-deficient mice revealed that a GR is required for lung maturation and perinatal survival (8). The GR-deficient mice die after birth of respiratory failure that is caused by a lack of inflation of the lung. However, SPs, including SP-B, are detected in the lung from the GR-deficient animals. In a previous study (28), dexamethasone stimulated SP-B mRNA and protein in H441 cells mediated by increasing SP-B mRNA stability. It has also been reported that dexamethasone stimulated SP-B transcription, although it remains unclear whether the effects of glucocorticoids are mediated directly by interaction of a GR with the SP-B promoter (1, 2, 10, 34). In the present study, dexamethasone repressed hSP-B-2240 transcription when cotransfected with a GR in H441 cells (Table 1). Thus the hSP-B-2240 region does not mediate the stimulatory effects of dexamethasone on SP-B expression in the H441 cell line.

RA plays a critical role both in lung organogenesis and in postnatal alveolarization (20, 21). All trans-RA caused a 50% increase in the number of alveoli in postnatal rats (20). Treatment of dexamethasone inhibited the formation of alveoli. Treatment with RA prevented the inhibitory effects of dexamethasone on alveolarization (20). RA also reversed elastase-induced pulmonary emphysema in adult rats, consistent with the potential roles of RA in the growth and differentiation of lung parenchyma.

In summary, the present study demonstrates a direct stimulatory effect of RA and RAR/RXR on SP-B gene transcription. RA and RAR/RXR increased SP-B gene transcription, mRNA accumulation, and SP-B synthesis in vitro.

    ACKNOWLEDGEMENTS

We thank Dr. Susan Wert for assistance with photomicroscopy and Dr. Tim Weaver for providing proSP-B antibody. We thank Dr. Pierre Chambon for providing the retinoic acid-receptor and retinoid X-receptor plasmids and Dr. Ronald M. Evan for providing the glucocorticoid-receptor plasmid.

    FOOTNOTES

This work was supported by the American Lung Association (C. Yan) and National Heart, Lung, and Blood Institute Specialized Center of Research Grant HL-56387 (to J. A. Whitsett and C. Yan).

Address for reprint requests: C. Yan, Children's Hospital Medical Center, Division of Pulmonary Biology, TCHRF, 3333 Burnet Ave., Cincinnati, OH 45229-3039.

Received 15 October 1997; accepted in final form 14 April 1998.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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