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Molecular Endocrinology, doi:10.1210/me.2003-0458
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Molecular Endocrinology 18 (6): 1520-1532
Copyright © 2004 by The Endocrine Society

Synergy between Signal Transducer and Activator of Transcription 3 and Retinoic Acid Receptor-{alpha} in Regulation of the Surfactant Protein B Gene in the Lung

Li Yang, Xuemei Lian, Angelynn Cowen, Huan Xu, Hong Du and Cong Yan

Division of Pulmonary Biology (L.Y., X.L., A.C., C.Y.), Division of Human Genetics (H.D.), and The Graduate Program for Molecular and Developmental Biology (H.X., C.Y.), Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45229-3039; and The Graduate Program for Pediatrics (X.L.), Chongqing University of Medical Sciences, Chongqing 400016, China

Address all correspondence and requests for reprints to: Cong Yan, Ph.D., Children’s Hospital Medical Center, Division of Pulmonary Biology, 3333 Burnet Avenue, Cincinnati, Ohio 45229-3039. E-mail: Cong.Yan{at}cchmc.org.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
During respiratory cycles, airborne particles and pathogens are inhaled into the lung, which can cause cytokine production by respiratory macrophages and inflammatory responses. Secreted cytokines affect surfactant protein expression and homeostasis in the lung. In coculturing experiments in vitro, bronchoalveolar macrophages stimulated human surfactant protein B (hSP-B) gene transcription in primary alveolar type II epithelial cells in lipopolysaccharide-independent and -dependent ways. Neutralization by IL-6 antibody abolished lipopolysaccharide-dependent macrophage stimulation of hSP-B gene transcription. IL-6 treatment enhanced signal transducer and activator of transcription (Stat)3 phosphorylation at Y705 in alveolar type II epithelial cells and Clara cells in vivo. Biochemical analysis of functional domain swapping between Stat1 and Stat3 identified that the SH2 domain and the DNA binding domain are critical for Stat3 stimulation of hSP-B gene transcription. Glutathione-S-transferase pull-down study determined functional domains required for protein-protein interaction between Stat3 and retinoic acid receptor-{alpha}. Cotransfection of Stat3 and retinoic acid receptor-{alpha} into respiratory epithelial cells resulted in synergistic DNA binding and transcriptional activation on the hSP-B gene. To assess Stat3 physiological function, overexpression of a dominant negative Stat3 in respiratory epithelial cells in a doxycycline-controlled double transgenic mouse line caused pulmonary emphysema and increase of animal death during hyperoxia. Therefore, the IL-6/Stat3 signaling axis plays an important role in surfactant protein homeostasis and respiratory inflammation in the lung.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE RESPIRATORY SYSTEM has two main functions, gas exchange and pathogen clearance. During respiratory cycles, pulmonary surfactant that is comprised of phospholipids and surfactant proteins prevents the alveolar structure from collapse at the end of expiration. Although surfactant protein A, B, C, and D (SP-A, SP-B, SP-C, and SP-D) constitute only 5% of pulmonary surfactant, they are indispensable for maintaining normal surfactant structure and function. For example, SP-B is a 79-amino acid peptide that forms oligomers in pulmonary surfactant to facilitate phospholipid spreading at the air-liquid interface. SP-B deficiency causes pulmonary surfactant malfunction and alveolar collapse during respiration. It has been shown that genetic ablation of the SP-B gene in the mouse and sporadic mutations in the human result in respiratory failure in newborns (1, 2).

SP-B gene expression is highly tissue and cell type specific. This specificity is primarily controlled at the promoter transcriptional level. A transgenic mouse model line carrying the human surfactant protein B (hSP-B) 1.5-kb 5'-flanking regulatory sequence and the lacZ reporter gene shows that transcriptional activation of the hSP-B gene starts at the onset of lung formation at embryonic d 9 and is restricted to epithelial cells throughout prenatal and postnatal lung development. In the adult lung, expression of the hSP-B 1.5-kb lacZ gene is restricted to bronchiolar epithelial cells and alveolar type II (AT II) epithelial cells (3). These studies indicate that the expression pattern of the hSP-B 1.5-kb lacZ reporter gene is similar to that of the endogenous mouse SP-B gene. Therefore, the transgenic mouse models are suitable for elucidation of molecular mechanisms that control human SP-B gene regulation. An enhancer sequence (–500 to –375 bp) has been identified in the 5'-flanking regulatory region of the hSP-B gene by deletion mutagenesis study in vitro (4). Deletion of this enhancer completely abolishes hSP-B gene expression in bronchiolar epithelial cells and dramatically reduces its expression in AT II epithelial cells in the lacZ reporter gene transgenic mice in vivo (3). This enhancer sequence is highly conserved in both human and mouse species (5).

Interactions between cis-acting genetic elements and trans-acting transcription factors are an important mechanism in controlling tissue- and cell type-specific expression of the hSP-B gene. Multiple transcription factors have been identified to bind to the hSP-B 5'-flanking regulatory region and regulate hSP-B gene transcription (4, 6, 7, 8, 9, 10, 11). Among them, thyroid transcription factor-1 (TTF-1) is an important protein factor that regulates the hSP-B gene expression (4, 6). TTF-1 is present at the onset of differentiation of respiratory epithelium and later confined to respiratory epithelial cells in the lung (12, 13, 14). Interestingly, the expression pattern of TTF-1 overlaps with that of the hSP-B 1.5-kb lacZ reporter gene in lung development (3, 14). TTF-1 contains an N-terminal transactivation domain, a DNA binding homeodomain, and a C-terminal transactivation domain (15, 16). Genetic ablation of the TTF-1 gene severely disrupts lung branching morphogenesis (17).

Retinoic acid receptor (RAR) binds to the enhancer region (–500 to –331 bp) through clustered retinoic acid (RA) response elements and stimulates SP-B gene expression upon binding to RA (8, 9, 18, 19). RAR contains multiple functional domains, including a ligand-independent transactivation AF-1 (activation function 1) domain, a DNA binding domain, a ligand-binding/dimerization domain (LBD) and a ligand-dependent transactivation AF-2 (activation function 2) domain. Double genetic ablations of RAR{alpha} and RARß genes block lung organogenesis (20). RARs transactivate target gene expression through recruitment of nuclear receptor coactivators [cAMP response element binding protein (CREB)-binding protein/p300, steroid receptor coactivator 1, activator of thyroid and retinoic acid receptor, transcriptional intermediary factor 2] that possess intrinsic histone acetyltransferase activity (21, 22, 23, 24, 25, 26, 27). RA and RAR stimulation of SP-B transcription is TTF-1 dependent. Mutations at either clustered RA response elements or TTF-1 DNA binding sites in the hSP-B enhancer region (–500 to –331 bp) abolish RA stimulation of SP-B transcription in respiratory epithelial cells (19). RAR-TTF-1 interaction has been identified by GST pull-down and mammalian two-hybrid studies (9). A protein domain mapping study demonstrates that the interaction between RAR and TTF-1 is mediated by their DNA binding domains in a synergistic fashion (9).

Transcriptional stimulation of the hSP-B gene is also mediated by members of IL-6 family cytokines and signal transducers and activators of transcription 3 (Stat3) in respiratory epithelial cells (10). Among Stat family members, only Stat3 stimulates hSP-B 500 transcription (10). This specificity and selectivity probably are determined, at least partially, by interactions with surrounding transcription factors on the hSP-B 5'-flanking regulatory region. Stat3 is originally identified as the acute phase response factor (28, 29). Subsequent studies show that Stat3 is mainly activated by IL-6 family cytokines that share the common gp130 receptor subunit (30, 31). RA and IL-6 show synergistic stimulatory effect via the hSP-B enhancer region (–500 to –331 bp) in respiratory epithelial cells, suggesting cross-talking between two pathways (10). Stat3 contains several functional domains, including an N-terminal domain, a coiled-coil domain, a DNA binding domain, a linker domain, an SH2 domain, and a transcriptional activation domain (32, 33). Upon cytokine or growth factor stimulation, Stat3 is phosphorylated on tyrosine 705 (Y705) and forms homo- or heterodimers that translocate to the nucleus to activate target gene transcription. Mutation at Y705 acts as a dominant negative mutation of Stat3 (34).

During respiratory cycles, airborne particles and pathogens, which can induce inflammatory responses and cytokine production by respiratory macrophages and epithelial cells, are inhaled into the lung. Secreted cytokines affect surfactant protein homeostasis during inflammatory responses and perturb host defenses. To elucidate how SP-B gene expression in AT II epithelial cells is affected by bronchoalveolar macrophages, an in vitro cell coculturing experiment was performed in the presence or absence of lipopolysaccharide (LPS). The functional role of IL-6 in mediating macrophage stimulation of hSP-B gene expression in AT II cells was also examined. Furthermore, functional domains critical for Stat3 stimulation of hSP-B gene transcription were identified by the domain swapping strategy between Stat3 and Stat1. The protein-protein interaction between Stat3 and RAR was identified by GST pull-down assay, chromatin immunoprecipitation (ChIP) assay, and mammalian transient-transfection assay. Peptide domains of both proteins required for the interaction were identified. To further elucidate physiological function of Stat3 in alveolar epithelial cells, a doxycycline-controlled transgenic mouse model line has been generated, in which a dominant negative Stat3 (dnStat3 Y705) was overexpressed under the control of the human surfactant protein C (hSP-C) 3.7-kb promoter in alveolar epithelial cells to suppress the endogenous Stat3 activity. Our studies support the concept that the IL-6/Stat3 signaling axis is important for SP-B homeostasis during inflammation in the lung.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Bronchoalveolar Macrophages Stimulate hSP-B Gene Transcription in AT II Epithelial Cells
The interaction between bronchoalveolar macrophages and alveolar epithelial cells is an important aspect of host defenses against airborne microorganism infection. Because bronchoalveolar macrophages and AT II cells coexist in the alveolar region, cell-cell interactions between these two cell types may play important roles in maintaining SP-B homeostasis. To assess how SP-B gene transcription in AT II cells is regulated by bronchoalveolar macrophages, in vitro macrophage-AT II epithelial cell coculturing studies were performed. Previously, we generated an FVB/N transgenic mouse line carrying the hSP-B 1.5-kb 5'-flanking regulatory sequence and the lac Z reporter gene (3). AT II epithelial cells were purified from this transgenic mouse line following a previously published procedure and cultured in dishes for 24 h (3). Bronchoalveolar macrophages were purified from wild-type FVB/N mouse bronchoalveolar lavage fluid (BALF) the next day and added to the hSP-B 1.5-kb lacZ AT II epithelial cell culture dishes in the presence or absence of LPS (10 ng/ml). AT II epithelial cells cocultured with bronchoalveolar macrophages resulted in induction of hSP-B 1.5-kb lacZ gene expression (Fig. 1Go). Addition of LPS further augmented hSP-B 1.5-kb lacZ gene expression in the coculturing system. LPS alone had a modest stimulatory effect on hSP-B 1.5-kb lacZ gene expression. This study demonstrates that there are two sets of signaling molecules made by lung macrophages that can stimulate hSP-B 1.5-kb lacZ gene expression. The first set of molecules is LPS independent and the second set of molecules is LPS dependent.



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  		Fig. 1. Bronchoalveolar Macrophages Stimulate hSP-B 1.5-kb lacZ Reporter Gene Expression in AT II Epithelial Cells

AT II epithelial cells were isolated from hSP-B 1.5-kb lacZ reporter gene FVB/N transgenic mice and cocultured with bronchoalveolar macrophages (MC) isolated from BALF. Cocultured cells were treated with or without LPS (10 ng/ml). Control represents AT II epithelial cells without macrophage coculturing. Cells were harvested for measurement of ß-galactosidase activities. Values are means ± SD; n = 3 (mice).

 
Bronchoalveolar Macrophage Stimulation of hSP-B 1.5-kb lacZ Gene Expression in AT II Epithelial Cells Is Heat Sensitive
To identify whether the signaling molecules are proteins or peptides, bronchoalveolar macrophages were purified from the wild type FVB/N mouse lungs and cultured in vitro, followed by LPS treatment. The macrophage media were collected after 48 h incubation and heated at 100 C for 5 min. The media without heat treatment were used as comparison. The heat-treated or untreated bronchoalveolar macrophage mediums (with or without LPS treatment) were added to primary monolayers of AT II epithelial cells that were isolated from hSP-B 1.5-kb lacZ transgenic mice. After 24 h incubation, cells were harvested for ß-galactosidase activity measurement. Both LPS-independent and -dependent stimulatory effects of bronchoalveolar macrophages on hSP-B 1.5-kb lacZ gene expression in AT II epithelial cells were abolished by heat treatment (Fig. 2Go). This is a clear indication that secretory paracrine peptide molecules were responsible for both LPS-independent and -dependent stimulation of bronchoalveolar macrophages in this system.



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  		Fig. 2. Heat Treatment Abolishes Stimulation of the hSP-B 1.5-kb lacZ Reporter Gene by Bronchoalveolar Macrophage Medium

AT II epithelial cells were isolated from hSP-B 1.5-kb lacZ reporter gene FVB/N transgenic mice and incubated with heat-treated or untreated bronchoalveolar macrophage (MC) mediums (LPS treated or untreated). Control represents AT II epithelial cells without addition of macrophage medium. Cells were harvested for measurement of ß-galactosidase activities. Values are means ± SD; n = 3 (mice).

 
IL-6 Is Responsible for LPS-Dependent Bronchoalveolar Macrophage Stimulation of hSP-B 1.5-kb lacZ Gene Expression in AT II Epithelial Cells
IL-6 is a major LPS-inducible cytokine in macrophages. Previously, in vitro experiments indicated that IL-6 stimulates hSP-B gene transcription in respiratory epithelial cells (10). To assess the role of IL-6 in LPS-dependent bronchoalveolar macrophage stimulation of the hSP-B 1.5-kb lacZ gene in AT II epithelial cells, IL-6 antibody neutralization experiment was performed. After coculturing AT II epithelial cells isolated from hSP-B 1.5-kb lacZ mice with wild-type mouse bronchoalveolar macrophages, IL-6 antibody was added into culture medium along with LPS (10 ng/ml). After 3 d incubation, ß-galactosidase activity was measured. IL-6 antibody neutralization abolished LPS-dependent bronchoalveolar macrophage stimulation of hSP-B 1.5-kb lacZ gene expression in AT II epithelial cells (Fig. 3Go). This study showed that IL-6 mediates, at least partially, LPS-dependent bronchoalveolar macrophage stimulation of hSP-B 1.5-kb lacZ gene expression in AT II epithelial cells.



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  		Fig. 3. IL-6 Ab Inhibits Bronchoalveolar Macrophage Stimulation of hSP-B 1.5-kb lacZ Reporter Gene Expression in AT II Epithelial Cells

AT II epithelial cells were isolated from hSP-B 1.5-kb lacZ reporter gene FVB/N transgenic mice and cocultured with bronchoalveolar macrophages (MC) with or without LPS treatment (10 ng/ml). IL-6 Ab (5 µl) was added to cocultured cells. Cocultured cells without IL-6 Ab addition served as comparison. Control represents AT II epithelial cells without macrophage coculturing. Cells were harvested for measurement of ß-galactosidase activities. Values are means ± SD; n = 3.

 
Expression of IL-6 Receptors and Phosphorylation of Stat3 in AT II Epithelial Cells
Next, expression of intracellular signaling molecules mediating the IL-6 action was investigated in mouse AT II epithelial cells. The IL-6 cell surface receptor is comprised of two distinct subunits, IL-6 R subunit and gp130 subunit (31). Colocalization of IL-6 signaling molecules with SP-B in AT II cells is important to elucidate SP-B gene regulation. To confirm expression of IL-6 receptor subunits in AT II epithelial cells, Western blot was performed. Protein extracts were prepared from AT II epithelial cells that were isolated from the wild-type FVB/N mouse lungs. Using antibodies specifically against IL-6R and gp130 in Western blot analysis, both receptor subunits were detected in AT II epithelial cells (Fig. 4AGo). It is known that Stat3 serves as a downstream transcription factor for IL-6. Treatment of IL-6 causes Stat3 phosphorylation at amino acid residue Y705. The phosphorylated Stat3 binds to downstream target genes and activates gene transcription. We have previously demonstrated expression of Stat3 in AT II epithelial cells (10). To determine whether IL-6 treatment causes Stat3 Y705 phosphorylation in AT II epithelial cells, an immunohistochemical staining study was performed using an antibody specifically against phosphorylated Stat3 at Y705. After intratracheal injection of IL-6 to the FVB/N mouse lungs, Stat3 showed increased phosphorylation at Y705 in bronchiolar and AT II epithelial cells compared with the PBS-treated mouse lungs (Fig. 4BGo). Therefore, all intracellular signaling molecules that mediate the IL-6 action are present in AT II epithelial cells, in which the SP-B gene is expressed.



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  		Fig. 4. Expression of IL-6 Receptor Subunits and Phosphorylated Stat3 Y705 in Respiratory Epithelial Cells

A, Western blot analysis of IL-6 receptor subunits in AT II epithelial cells. AT II epithelial cells were isolated from wild-type FVB/N mouse lungs. Protein extracts were prepared for SDS denaturing gel electrophoresis. Western blot analyses were performed using antibodies against IL-6R or gp130 subunits. B, Immunohistochemical staining of phosphorylated Stat3 Y705 in the lung. Lung sections from IL-6 treated (IL-6) or untreated (PBS) adult FVB/N mice were stained with an antibody specifically against phosphorylated Stat3 at Y705. More positively stained Clara cells or alveolar epithelial cells (some stained cells are represented by arrows) were detected in the IL-6-treated lungs.

 
Stat3 Domains Required for hSP-B Gene Transactivation
Previous studies showed that Stat3 stimulates hSP-B gene transcription (10). To identify which domains on Stat3 are responsible for specific activation of the hSP-B 500 reporter gene, a functional domain swapping study was performed between Stat3 and Stat1. This is because, among Stat family members, Stat1 shares the closest domain similarity with Stat3 (35). Various Stat3 peptide domains were replaced with the corresponding Stat1 domains using the two-step PCR strategy and subcloned into the mammalian expression vector PCR3.0 (Fig. 5AGo). These hybrid Stat3/Stat1 expression constructs were cotransfected into H441 cells with the hSP-B 500 luciferase reporter construct. The wild-type Stat1 and Stat3 constructs were used as negative and positive controls in the transfection assay. As shown in Fig. 5BGo, Stat3 significantly stimulated the hSP-B 500 luciferase reporter gene activity in H441 cells, whereas Stat1 did not. Replacement of the Stat3 DNA binding domain with the Stat1 DNA binding domain significantly reduced the Stat3 stimulatory effect on the hSP-B 500 luciferase reporter gene. This is in agreement with a previous finding that Stat3 and Stat1 DNA binding domains have differential DNA binding affinity (36). In addition, replacement of the Stat3 SH2 domain with the Stat1 SH2 domain abolished the Stat3-stimulatory effect on the hSP-B 500 luciferase reporter gene. Replacement of a short peptide domain around the Y705 position on Stat3 (equivalent toY701 on Stat1) with the same region on Stat1 also reduced the Stat3-stimulatory effect on the hSP-B 500 luciferase reporter gene. Replacement of the N-terminal domain (ND) and the coil-coil domain (CC) modestly reduced stimulatory effects of Stat3 on the hSP-B 500 luciferase reporter gene. To assure that decreased stimulation of the hSP-B 500 luciferase reporter gene is not a result of protein instability, Flag-tagged Stat3/Stat1 hybrid protein expression was monitored by Western blot analysis using Flag antibody after transfection into H441 cells. No significant decrease in protein expression levels of various Stat3/Stat1 hybrid proteins was observed compared with that of the wild-type Stat3 and Stat1 proteins (data not shown).



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  		Fig. 5. Functional Domain Swapping between Stat3 and Stat1 in Stimulation of hSP-B 500 Luciferase Reporter Gene in H441 Cells

A, Illustration of functional domain swapping between Stat3 and Stat1. B, Stimulation of the hSP-B 500 luciferase reporter gene by Stat3, Stat1, and various Stat3/Stat1 hybrids in H441 cells. Stimulation of the hSP-B 500 luciferase reporter gene without cotransfection was used as control. Values are means ± SD; n = 3. S1, Stat1; S3, Stat3; ND, N-terminal domain; CC, coiled coil domain; DB, DNA-binding domain; SH2, SH2 domain; Y701, Y701 domain; TAD, transcription activation domain

 
Protein-Protein Interaction between Stat3 and RAR{alpha}
Previously, a synergistic stimulation was observed between RA and IL-6 on the hSP-B (–500 to –331 bp)/SV40 luciferase reporter construct, implicating a possible interaction between Stat3 and RAR{alpha} on the hSP-B enhancer (–500 to –331 bp) (10). As determined by the Stat3/Stat1 swapping study, the SH2/Y705 region seems essential for Stat3 transactivation on the hSP-B 500 luciferase reporter gene. This domain is a strong candidate for Stat3-RAR{alpha} interaction. To test this possibility, both Stat1 SH2 and Stat3 SH2 domains were linked with GST. The GST fusion peptides were incubated with [35S]methionine-labeled RAR{alpha} for GST pull-down study. A physical protein-protein interaction between the Stat3 SH2 domain and RAR{alpha} was observed (Fig. 6AGo). The Stat1 SH2 domain showed a weak interaction compared with GST control. On the other hand, no specific interaction was observed between the Stat3 SH2 domain and TTF-1 (Fig. 6AGo). Next, to map which RAR{alpha} domains interact with the Stat3 SH2 domain, various [35S]methionine-labeled RAR{alpha} domains were incubated with the GST-Stat3 SH2 fusion peptide. Only the RAR{alpha} LBD domain was pulled down by GST-Stat3 SH2 fusion peptide (Fig. 6BGo), indicating that the Stat3 SH2 domain and the RAR{alpha} LBD domain interact with each other.



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  		Fig. 6. Interaction between the Stat3 SH2 Domain and RAR{alpha} in GST Pull-Down Study

A, Pull down of full-length RAR{alpha} by Stat3 SH2 GST fusion protein. No enhanced pull-down signal of full-length TTF-1 was observed by Stat3 SH2 GST fusion protein. B, Pull down of the RAR{alpha} LBD domain by Stat3 SH2 GST fusion protein. AF-1, RAR{alpha} ligand-independent activation domain; DBD, RAR{alpha} DNA-binding domain; LBD, RAR{alpha} ligand-binding domain; AF-2, RAR{alpha} ligand-dependent activation domain.

 
Synergistic Effect between Stat3 and RAR{alpha} in ChIP Assay and H441 Cell Transient Transfection Assay
To confirm that interaction between Stat3 and RAR{alpha} leads to synergism in stimulation of the hSP-B gene, cotransfection of Stat3 and RAR{alpha}/retinoid X receptor-{gamma} (RXR{gamma}) with the hSP-B 500 luciferase report gene into H441 cells was performed. The cotransfection showed a stimulation greater than the additive effect (Fig. 7AGo). In a ChIP assay, H441 cells were transfected with Stat3, RAR{alpha}/RXR{gamma}, or a combination of both. Chromatin partition was immunoprecipitated with the antibody specifically against Stat3. The hSP-B 5'-flanking regulatory sequence associated with Stat3 was amplified by PCR using a pair of specific primers corresponding to the hSP-B enhancer region (–500 to –331 bp) as previously described (9). As shown in Fig. 7BGo, only cotransfection of Stat3 and RAR{alpha}/RXR{gamma} showed detectable ChIP signal, indicating a synergistic DNA binding activity of Stat3 and RAR on the hSP-B 5'-flanking regulatory sequence in H441 cells. In a negative control, Flag antibody did not precipitate the hSP-B 5'-flanking regulatory sequence (data not shown). Therefore, the functional synergism between IL-6 and RA is mediated through downstream Stat3 and RAR interaction in H441 cells.



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  		Fig. 7. Synergy of DNA Binding and Transactivation between Stat3 and RAR{alpha}/RXR{gamma}

A, Stimulation of the hSP-B 500 luciferase reporter gene by Stat3, RAR{alpha}/RXR{gamma}, or combination in H441 cells. Stimulation of the hSP-B 500 luciferase reporter gene without cotransfection was used as control. Values are means ± SD; n = 3. B, H441 cells were transfected with expression vectors of Stat3, RAR{alpha}/RXR{gamma}, or combination. Soluble chromatin was immunoprecipitated with Stat3 antibody. Coprecipitated DNA was analyzed by PCR using a pair of primers corresponding to the enhancer region (–500 to –331 bp) of the hSP-B 500 5'-flanking regulatory sequence. The hSP-B 500 luciferase reporter gene construct was used as positive control. No DNA was added in negative control.

 
Generation of Lung-Specific Doxycycline-Controlled dnStat3 Mouse Model
Because Stat3 is expressed in AT II cells and controls SP-B gene expression, we reason that Stat3 plays a broad role in the lung to maintain surfactant homeostasis and influence pulmonary inflammation. To assess the functional role of Stat3 in maintaining alveolar formation, a doxycycline-controlled double transgenic mouse system was generated, in which dnStat3 was overexpressed in respiratory epithelial cells. In this system, the SP-C-reverse tetracycline responsive transactivator (rtTA) transgenic mouse line was produced bearing rtTA fusion protein under the control of the 3.7-kb human SP-C gene promoter as previously described (37, 38). The Teto-CMV-dnStat3 transgenic mouse line was generated consisting of the tet operator DNA-binding sequence linked to a minimal cytomegalovirus (CMV) promoter, dnStat3 cDNA, and simian virus 40 (SV40) polyadenylation signals (Fig. 8AGo). After cross-breeding SP-C-rtTA transgenic mice with Teto-CMV-dnStat3 transgenic mice, double-transgenic mice were identified by PCR genotyping. To assess whether dnStat3 mRNA expression is induced in double transgenic mice, double transgenic mice were treated with doxycycline for 2 months. Wild-type mice and single transgenic mice were also treated as controls. Lungs from these mice were isolated and total mRNAs were purified. As demonstrated by the RT-PCR assay, doxycycline treatment significantly induced dnStat3 mRNA expression in the lungs of the double transgenic mice (Fig. 8BGo). On the other hand, the SP-B mRNA level was reduced (Fig. 8BGo). In comparison, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA remained unchanged upon treatment (Fig. 8BGo). Control mice including wild-type and single-transgenic mice showed no changes in dnStat3 and SP-B mRNAs by doxycycline treatment (data not shown).



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  		Fig. 8. Generation of Doxycycline-Controlled dnStat3 Transgenic Mice

A, Illustration of constructs for generating SP-C-rtTA/(Teto)7-CMV-dnStat3 double-transgenic mice. B, Expression of dnStat3 mRNA, SP-B mRNA, and GAPDH mRNA in SP-C-rtTA/(Teto)7-CMV-dnStat3 double-transgenic mice. Total RNAs isolated from triplicate doxycycline-treated or untreated double-transgenic mice were used for RT-PCR analysis using a pair of specific primers for the (Teto)7-CMV-dnStat3 DNA sequence, the SP-B cDNA sequence, or the GAPDH cDNA sequence. Dox, Doxycycline.

 
Morphometric Analyses of Double-Transgenic Lungs after Doxycycline Treatment
Lungs from various groups of genotyped animals (wild-type, single-transgenic, and double-transgenic mice) were inflated for histopathological assessment. Doxycycline treatment of wild-type lungs (Fig. 9AGo) and the single-transgenic lungs (data not shown) showed the normal alveolar structure. In contrast, lungs from double-transgenic mice revealed marked malformation of alveoli (Fig. 9AGo), consistent with focal pulmonary emphysema. Morphometric measurement of the ratio between the alveolar parenchyma and the air space revealed a significant difference between wild-type and SP-C-rtTA/Teto-CMV-dnStat3 double-transgenic lungs after doxycycline treatment (Fig. 9BGo). The double-transgenic lungs had increased air space and diminished alveolar surface area. Analysis of two-dimensional frequency distribution of the alveolar region in double-transgenic mice showed larger but fewer alveoli (Fig. 9CGo). This pathophysiological phenotype is partially due to the decrease of the SP-B expression level.



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  		Fig. 9. Morphometrical Analysis of SP-C-rtTA/(Teto)7-CMV-dnStat3 Double-Transgenic Mouse

A, Disruption of the alveolar structure in the adult lung of the SP-C-rtTA/(Teto)-CMV-dnStat3 FVB/N double-transgenic mice after doxycycline treatment. The lung sections were stained with hematoxylin and eosin. The doxycycline-treated wild-type mice were used as control. WT, Wild-type mice; dnStat3, double transgenic mice. B, Ratio increase of the pulmonary airspace vs. parenchyma in double-transgenic mice after dnStat3 overexpression. Values are means ± SD; n = 3–5. ANOVA showed significant differences between wild-type and double-transgenic mice, P < 0.05. Wt, Wild-type mice; Tg, double-transgenic mice. C, Frequency distribution of the two-dimensional alveolar area in wild-type and double-transgenic mice after dnStat3 overexpression. wt, Wild-type mice; Tg, double transgenic mice

 
Overexpression of dnStat3 Reduces Animal Survival in Hyperoxic Condition
Acute lung injury is a common side effect for patients who receive high concentrations of inspired oxygen. The oxygen injury is often initiated by acute pulmonary inflammation (39). To assess the physiological and clinical relevance of Stat3 in lung oxygen injury, doxycycline-treated CCSP-rtTA/(Teto)7-CMV-dnStat3 double-transgenic and wild-type mice were exposed to 95% oxygen. Under this condition, overexpression of dnStat3 significantly accelerated animal death (Fig. 10Go). The onset of death for double-transgenic mice began at d 3.5. At d 6, more than 50% of wild-type animals still survived, compared with 0% survival of double-transgenic mice. Therefore, the Stat3 signaling in respiratory epithelial cells plays an important role in acute lung inflammation and injury during hyperoxia.



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  		Fig. 10. Survival of Doxycycline-Treated Mice under Hyperoxic Condition

Doxycycline-treated adult wild-type and SP-C-rtTA/(teto)7-CMV-dnStat3 double-transgenic mice were exposed to 95% O2. Animal mortality was recorded every 12 h. x-axis, Days exposed to 95% O2; y-axis, survival percentage of animals. The solid line represents the survival rate of wild-type animals. The dashed line represents double-transgenic mice. In each group, n = 20. Logrank test showed significant difference between survival rates of two groups. WT, Wild-type mice; dnStat3, SP-C-rtTA/(teto)7-CMV-dnStat3 double transgenic mice.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
SP-B is a critical peptide component in pulmonary surfactant for maintaining alveolar homeostasis. In pulmonary inflammation and host defenses, inhaled microorganisms (bacteria, viruses, etc.) cause respiratory inflammation and trigger host defenses in the lung. During these processes, various cytokines are secreted by resident bronchoalveolar macrophages to influence SP-B gene expression in respiratory epithelial cells. To elucidate the relationship between bronchoalveolar macrophages and epithelial cells in regulation of SP-B gene expression, a coculturing system was set up in vitro. In this system, SP-B gene stimulation by bronchoalveolar macrophages can be divided into two phases, LPS-independent and -dependent stimulations (Fig. 1Go). In both stimulations, peptide molecules play major roles in mediating hSP-B 1.5-kb lacZ reporter gene expression. Heat inactivation of media from bronchoalveolar macrophages reduced both LPS-independent and -dependent stimulations of hSP-B 1.5-kb lacZ reporter gene expression (Fig. 2Go). Therefore, bronchoalveolar macrophages secrete at least two sets of molecules to influence SP-B gene expression in the alveolar region. LPS is known to stimulate proinflammatory cytokine production, including IL-6. Therefore, it is very possible that IL-6 mediates, at least partially, LPS-dependent hSP-B 1.5-kb lacZ reporter gene expression by bronchoalveolar macrophages. Indeed, IL-6 antibody neutralization experiment in the coculturing system abolished LPS-dependent hSP-B 1.5-kb lacZ reporter gene expression by bronchoalveolar macrophages (Fig. 3Go). In Western blot analysis, IL-6 receptor subunits (IL-6R and gp130) were identified in AT II epithelial cells (Fig. 4AGo). Activation of Stat3 phosphorylation at Y705 upon IL-6 treatment in AT II epithelial cells and Clara cells was also observed (Fig. 4BGo). Taken together, these experiments clearly demonstrate that the IL-6/Stat3 signaling pathway exists in AT II epithelial cells and Clara cells and is capable of mediating SP-B gene expression upon pathogen invading. It is not clear which paracrine molecules mediate LPS-independent stimulation of hSP-B gene expression by bronchoalveolar macrophages. These molecules seem important to support the basal level expression of SP-B necessary for maintaining normal lung functions. Identification of these molecules in the future will be important to understand and elucidate SP-B homeostasis in alveoli. Our studies support a concept that various cell types in the lungs interact with and support each other through multiple molecules and mechanisms.

At the gene transcriptional level, we have previously shown that Stat3 is the only member of the Stat family stimulating hSP-B 500 luciferase reporter gene expression in respiratory epithelial cells (10). This promoter selectivity must partially rely on Stat3 interaction with the surrounding transcription factors on the hSP-B 5'-flanking regulatory sequence. These factors include TTF-1, RAR, and nuclear receptor coactivators [steroid receptor coactivator 1, activator of thyroid and retinoic acid receptor, transcriptional intermediary factor 2, cAMP response element-binding protein (CREB)-binding protein/p300, etc. (4, 6, 8, 9, 14, 19)]. We postulate that Stat3 must possess some unique amino acids in certain domains, which do not exist in other Stat members, to interact with these factors. To identify which domains are essential for Stat3 stimulation of hSP-B transcription, the functional swapping study was performed between Stat3 and Stat1. Consistent with previous observations (36), replacement of the Stat3 DNA binding domain by Stat1 DNA binding domain significantly reduced Stat3 stimulation of hSP-B 500 luciferase reporter gene expression due to DNA binding sequence selectivity (Fig. 5BGo). In addition, replacement of the Stat3 SH2 domain by the Stat1 SH2 domain abolished Stat3 stimulation (Fig. 5BGo). GST pull-down studies showed Stat3/SH2 physical interaction with RAR{alpha}, but not with TTF-1 (Fig. 6AGo). Further GST pull-down studies identified that the RAR{alpha} ligand binding domain is required for the interaction with the Stat3/SH2 domain (Fig. 6BGo). In transient cotransfection study, RAR{alpha}/RXR{gamma} significantly enhanced Stat3 DNA binding on the hSP-B 5'-flanking regulatory sequence in ChIP assay, indicating a direct interaction between the two proteins to promote DNA binding affinity in cells (Fig. 7BGo). This interaction resulted in a stimulation of hSP-B transcription greater than the additive effect of two proteins (Fig. 7AGo). Previously, we have demonstrated that the RAR{alpha} DNA binding domain interacts with the TTF-1 DNA binding domain (9). The interaction between two DNA binding domains synergistically enhanced TTF-1 DNA binding affinity to the hSP-B enhancer region (–500 to –331 bp). Therefore, RAR{alpha} interacts with surrounding transcription factors through different functional domains on the hSP-B 5'-flanking regulatory region.

In addition to regulating SP-B gene expression in respiratory epithelial cells, many other downstream target genes must be influenced by Stat3, which in turn regulate surfactant homeostasis and pulmonary inflammation. To assess the overall pathophysiological effect of Stat3 in the lung, a doxycycline-controlled dnStat3 double transgenic mouse system was established to suppress the endogenous Stat3 activity in alveolar AT II epithelial cells (Fig. 8Go). Upon doxycycline induction, severe disruption of the alveolar structure was observed, resembling characteristics of pulmonary emphysema (Fig. 9AGo). Morphometric analysis showed that the double-transgenic lungs after doxycycline treatment had larger but fewer alveoli (Fig. 9CGo). The air space was increased and the alveolar surface area was diminished (Fig. 9BGo). This directly resulted in a poor protection against lung injury caused by hyperoxia (Fig. 10Go). Although the mechanism is not entirely clear, it must be partially mediated by suppression of SP-B gene expression (Fig. 8BGo). SP-B is a critical reagent for protecting the surfactant structure in alveoli. Interestingly, inhibition of endogenous RAR in a similar doxycycline-controlled transgenic mouse system also showed pulmonary emphysema (38). Therefore, coordinated and synergistic interaction between Stat3 and RAR are not only at the gene transcriptional level, but also at the pathophysiological levels in the lung.

Whereas SP-B gene expression is up-regulated by proinflammatory molecules, such as IL-6 and Stat3, it is also down-regulated by antiinflammatory molecules. Peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) is an antiinflammatory molecule in various systems. It suppresses expression of proinflammatory cytokine IL-1ß, IL-6, and TNF{alpha} genes (40, 41). Free fatty acid derivative compounds, such as hydroxyeicosatetraenoic acids, hydroxyoctadecanoic acids, and 15-deoxy-{Delta}12,14prostaglandin J2, serve as ligands for PPAR{gamma}. Recently, it has been shown that PPAR{gamma} and its ligands negatively regulates hSP-B gene transcription in respiratory epithelial cells (11). The cis-acting element mediating PPAR{gamma} inhibition is located within the hSP-B promoter region (–218 to + 41 bp). The balance between proinflammatory molecules and antiinflammatory molecules in various physiological conditions is essential for SP-B homeostasis in maintaining surfactant function and alveolar structure. Identification of these molecules and elucidation of their working mechanisms are essential for understanding the pathogenesis of pulmonary diseases. Through these studies, new approaches for therapeutic and clinical treatment of pulmonary diseases can be identified.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animal Care
All scientific protocols involving the use of animals in this study have been approved by the Cincinnati Children’s Hospital Institution Animal Care and Usage Committee and follow guidelines established by the Panel on Euthanasia of the American Veterinary Medicine Association. Protocols involving the use of recombinant DNA or biohazardous materials have been reviewed by the Cincinnati Children’s Hospital Biosafety Committee and follow guidelines established by the National Institutes of Health. Animals were housed under Institutional Animal Care and Use Committee-approved conditions in a secured animal facility at Cincinnati Children’s Hospital Research Foundation. Animals were regularly screened for common respiratory pathogens and murine viral hepatitis. Experiments involving animal death utilize CO2 narcosis to minimize animal discomfort.

AT II Epithelial Cell and Macrophage Isolation for in Vitro Coculturing Experiment
The hSP-B 1.5-kb lacZ mouse line was generated previously (3). AT II epithelial cells were isolated from 2-month-old transgenic mice following a previously described procedure (42). Monolayers of AT II epithelial cells were cultured at a density of 4 x 105 cells per well in 12-well dishes on Matrigel-rat tail collagen (70:30, vol/vol) in bronchial epithelial cell growth medium (minus hydrocortisone) plus 5% charcoal-stripped fetal bovine serum and 10 ng/ml keratinocyte growth factor. Next day, BALF was collected from lungs of nontransgenic wild-type mice in 1 ml PBS and briefly incubated for 1 h at 37 C. The supernatant was removed, and the attached cells were collected for cell counting. Approximately 2 x 105 macrophage cells were added to each well of AT II epithelial cells and incubated for 5 d. Medium was changed every 2 d. Cells were lysed by Reporter Lysis Buffer (Promega Corp., Madison, WI) and subsequently used for ß-galactosidase assay.

Western Blot and Immunohistochemistry
To assess IL-6R and gp130 protein expression, AT II epithelial cells were isolated as mentioned above. Protein extracts were prepared in RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 1x PBS) and fractionated on a 10% polyacrylamide gel. After transferring to a nitrocellulose membrane, IL-6R antibody (1:50) and gp130 antibody (1:50) (Santa Cruz Biotechnology Inc., Santa Cruz, CA) were used to detect IL-6R and gp130 expression. To measure active Stat3 form (phosphorylation at Y705), 8-wk-old FVB/N mice received 600 U of IL-6. The control animals received PBS. After 4 h, lungs from adult mice were dissected out, and lung tissue sections were prepared as previously described (14). Tissue slides were incubated overnight at 4 C with primary phosph Stat3 Y705 antibody (1:100) (Cell Signaling, Inc.). No primary antibody was added in the negative control. The tissues were washed and treated with secondary conjugated antibodies 24 h later. The interactions were amplified with Vectastain’s Elite ABC kit (Vector Laboratories, Inc., Burlingame, CA) to visualize the signals.

Construction of Plasmids and Transfection Assays
The hSP-B 500 luciferase reporter construct was made previously (4). Stat3 and Stat1 expression vectors were kind gifts from Dr. James Darnell (Rockfeller University, New York, NY). Various Stat3/Stat1 hybrid constructs were constructed by a two-step PCR strategy. For the first PCR, sense and antisense oligonucleotide primers covering both swapping domains of Stat3 and Stat1 were synthesized. The primers were paired with an upstream primer or a downstream primer of the PCR 3.0 expression vector in the presence of Stat3 or Stat1 cDNA templates to make two sets of PCR products. The PCR products with the switched domains were subsequently purified by agarose gel electrophoresis and the QIAQuick gel extraction kit (QIAGEN, Chatsworth, CA). Purified PCR products served as templates and were mixed together with the PCR 3.0 expression vector upstream and downstream primers for the second PCR. The second Stat3/Stat1 hybrid PCR cDNA products were digested with BamH1 and NotI restriction enzymes and subcloned into the PCR 3.0 vector. In each construct, a Flag sequence was inserted at the C-terminal end of molecules.

H441 cells were seeded at a density of 2 x 105 cells per well in six-well plates. The hSP-B 500 luciferase reporter construct (0.25 µg) and pCMV-ßgal plasmid (0.5 µg) were cotransfected with various concentrations of hybrid Stat3/Stat1 constructs into H441 cells. The CMV-ß-galactosidase plasmid was included for normalization. The wild type Stat3 or Stat1 cotransfection with the hSP-B 500 luciferase reporter gene were used as positive and negative controls. Because all Stat3/Stat1 derivatives are tagged with the Flag sequence, protein expression levels of Stat3/Stat1 derivatives in H441 cells were assessed by Flag antibody and Western blot assay.

In Stat3 and RAR{alpha}/RXR{gamma} cotransfection assay, cell seeding and transfection were performed as outlined above, except that 1.5 µg Stat3 and 1.5 µg RAR{alpha} mammalian cell expression vectors were used.

GST Pull-Down Assay
The GST pull-down experiment was performed following a previous procedure (9). To make GST fusion proteins, Stat3 SH2 and Stat1 SH2 domains were subcloned into the pGEX4T-1 GST vector (Amersham Pharmacia Biotech, Arlington Heights, IL) at the EcoRI and XhoI restriction enzyme sites by PCR. The plasmids were transformed into BL21 bacterial strains for protein expression. GST-Stat3 SH2 and GST-Stat1 SH2 fusion domains were expressed and purified following a procedure previously described (9). The full-length and various truncated RAR constructs were subcloned into the PCR3.0 vector (Invitrogen, San Diego, CA) at the EcoRI and NotI sites and labeled with [35S]methionine using the In Vitro Transcription/Translation kit (Promega Corp.). Approximately 4 µg purified GST, GST-Stat3 SH2, and GST-Stat1 SH2 were incubated with 20 µl 50% Glutathione Sepharose 4B beads and 25 µl of the [35S]Met-labeled various RAR{alpha} domains for pull down.

ChIP Assay of RAR{alpha}/RXR and Stat3 in H441 Cells
H441 cells were seeded in 100-mm dishes (7.5 x 105 cells per dish). Next day, expression vectors of Stat3 (6 µg), or RAR{alpha} (6 µg)/RXR{gamma} (6 µg), or a combination were transfected into cells allowing protein expression for 3 d. Untransfected cells served as a negative control. The ChIP assay followed a previously published procedure (9). For immunoprecipitation, 25 µl Stat3 antibody (Santa Cruz Biotechnology, Inc.) were added to the supernatants and incubated overnight at 4 C. Flag antibody (Sigma Chemical Co., St. Louis, MO) was used as a negative control. Fifty microliters of 50% Protein A/G Plus Agarose Beads (Santa Cruz) were added to the samples and incubated at 4 C for 2 h followed by centrifugation. The precipitated DNA pellets were used as templates for quantitative PCR analysis with primers corresponding to the SP-B enhancer region.

Generation of Doxycycline-Controlled dnStat3 Transgenic Mice
To generate the Teto-CMV-dnStat3 transgenic mouse line, the dnStat3 cDNA (34) was amplified by PCR using a downstream primer (5'-CGCGCGGCCGCTTATCACTTGTCATCGTCGTCCTTGTAGTCTGGTGTCACACAGATG-3') and an upstream primer (5'-AGCGGATCCGCCACCATGGCTCAGTGGAACCAG-3'). The PCR product was digested with BamH1 and NotI and subcloned downstream of the CMV minimal promoter linked to seven Tet-responsive elements (7 x TRE) at the BamH1 and NotI sites in the pTRE vector (CLONTECH Laboratories, Inc., Palo Alto, CA). The expression cassette containing the CMV promoter, the dnStat3 cDNA, and the hemoglobin polyadenylation signaling sequence was dissected out and purified for microinjection into FVB/N mice by the Transgenic Core Facility at University of Cincinnati, College of Medicine. Founder lines were identified by the PCR strategy using an upstream primer in the pTRE plasmid (5'-ACGCCATCCACGATGTTTTG-3') and a downstream primer in the Stat3 cDNA coding region, (5'-GGTTGTGCTGATAGAGGACATTGG-3'). The SP-C rtTA transgenic mice were genotyped with an upstream primer corresponding to the SP-C promoter (5'-ACTGCCCATTGCCCAAACAC-3') and a downstream primer corresponding to the rtTA cDNA coding region (5'-AAA ATC TTG CCA GCT TTC CCC-3').

Total RNA Isolation and RT-PCR
To detect doxycycline-induced expression of dnStat3 mRNA in double-transgenic mice, adult double-transgenic mice were treated with or without doxycycline food for 2 months. Total RNAs were isolated from lungs using the RNA purification kit (QIAGEN, Germantown, MD). RT-PCR was used to detect the dnStat3 mRNA with the SuperScript One-Step RT-PCR Kit (Invitrogen), using an upstream primer (5'-TGACACCAACGGACTACAAGGAC-3') and a downstream primer (5'-CTGAAAACTTTGCCCCCTCC-3'). Expression of SP-B and GAPDH mRNA was used as controls as previously described (8).

Analysis of Lung Histology and Morphometric Analysis
After cross-breeding, 1-month-old double transgenic mice, single-transgenic mice, and wild-type mice were treated with doxycycline food. After 2 months on this diet, animals were anesthetized and lungs were inflation fixed with 4% paraformaldehyde in PBS overnight at 4 C. Lungs were washed with PBS and dehydrated through a series of ethanol followed by paraffin embedding. Tissue sections (5 µm) were loaded onto slides for staining with hematoxylin and eosin.

For morphometrical measurements, the overall proportion (% alveolar area) of the respiratory parenchyma and the airspace was determined by using a point-counting method as previously described (38). Measurements were performed on sections taken throughout various lobes. Images were transferred by video camera to a computer screen using METAMORPH imaging software (Universal Imaging Corp., Downingtown, PA). A computer-generated, 121-point lattice grid was superimposed on each field, and the number of intersections (points) falling over respiratory parenchyma (alveoli and alveolar ducts) or airspace was counted. Points falling over bronchioles, large vessels, and smaller arterioles and venules were excluded from the study. The airspace area frequency distribution of alveoli was estimated by the point-sampled intercept method using METAMORPH imaging software. Statistically significant differences were determined by ANOVA.

Oxygen Exposure
Wild-type and SP-C-rtTA/(Teto)7-CMV-dnStat3 double-transgenic mice (1 month old) were treated with doxycycline food for 2 months (n = 20 per group). Animals were exposed to 95% oxygen in a 70 x 55 x 25 cm airtight plastic chamber (food and water ad libitum). The oxygen concentration in the chamber was monitored with an oxygen analyzer (Mine Safety Application Co., Pittsburgh, PA), and was maintained with a constant flow of gas (3 liters/min). Survival status of the mice was checked every 3–12 h. The animals were removed from boxes right before death and humanely killed by CO2 euthanasia. Differences in survival status between two groups were assessed using Kaplan-Meier Survival Analysis in StatView 4.5 software (Abacus Concepts, Inc., Berkeley, CA).


   ACKNOWLEDGMENTS
 
We thank Dr. J. Darnell (Rockefeller University, New York, NY) for providing Stat1 and Stat3 expression vectors, Dr. P. Chambon for providing the RAR{alpha} expression vector, and Dr. R. DeLauro for providing the TTF-1 expression vector. We thank Drs. Jeffrey A. Whitsett and Jay Tichelaar for providing the SP-C rtTA transgenic mouse line. We thank Dr. H. Akinbi for proofreading the manuscript.


   FOOTNOTES
 
This work was supported by March of Dimes Grant FY02–206 (to C.Y.), National Institutes of Health Grants HL-061803 and HL-067862 (to C.Y. and H.D.).

L.Y. and X.L. contributed equally to this work and should both be considered first authors.

Abbreviations: AF, Activation function; AT II, alveolar type II; BALF, bronchoalveolar lavage fluid; ChIP, chromatin immunoprecipitation; dnStat, dominant negative Stat; CMV, cytomegalovirus; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione-S-transferase; hSP, human surfactant protein; JAK, janus family tyrosine kinase; LBD, ligand-binding domain; LPS, lipopolysaccharide; PPAR, peroxisomal proliferator-activated receptor; RA, retinoic acid; RAR, retinoid acid receptor; rtTA, reverse tetracycline-responsive transactivator; SP-A, -B, -C, and -D, surfactant proteins A, B, C, and D; RXR, retinoid X receptor; Stat, signal transducers and activators of transcription; TTF-1, thyroid transcription factor 1

Received for publication November 27, 2003. Accepted for publication March 17, 2004.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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