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Originally published In Press as doi:10.1074/jbc.M404296200 on June 1, 2004

J. Biol. Chem., Vol. 279, Issue 33, 34578-34588, August 13, 2004
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Nuclear Factor of Activated T Cells Regulates Transcription of the Surfactant Protein D Gene (Sftpd) via Direct Interaction with Thyroid Transcription Factor-1 in Lung Epithelial Cells*

Vrushank Davé{ddagger}§, Tawanna Childs{ddagger}, and Jeffrey A. Whitsett{ddagger}

From the {ddagger}Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039

Received for publication, April 19, 2004 , and in revised form, May 21, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Surfactant protein D (SP-D) plays critical roles in host defense, surfactant homeostasis, and pulmonary immunomodulation. Here, we identify a role of nuclear factor of activated T cells (NFATs) in regulation of murine SP-D gene (Sftpd) transcription. An NFAT-dependent enhancer modulated by NFATs or calcineurin and sensitive to cyclosporin was identified in the Sftpd promoter. Ionomycin and phorbol 12-myristate 13-acetate further increased the activity of this enhancer, whereas VIVIT, a potent NFAT inhibitor peptide, selectively interfered with the calcineurin-NFAT interaction and abolished enhancer function. Gel supershift and DNase I protection assays identified DNA elements that bind NFAT in the Sftpd promoter. Calcineurin and NFATc3 proteins were detected in the embryonic and adult mouse lung epithelium, and the mRNA expression profiles of the NFATs were similar in immortalized mouse lung epithelial cells and alveolar epithelial type II cells. NFATc3 and TTF-1 activated the Sftpd promoter, synergized transcription, co-immunoprecipitated from mouse lung epithelial cells, and physically interacted in vitro. Components of the calcineurin/NFAT pathway were identified in respiratory epithelial cells of the lung that potentially augment rapid assembly of a multiprotein transcription complex on Sftpd promoter inducing SP-D expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Surfactant protein D (SP-D),1 is a 43-kDa member of the calcium-dependent host defense "collectin" (collagen-lectin) family of proteins that includes surfactant protein A, serum mannose-binding protein, conglutinin, and bovine serum lectins CL-34 (1). In the lung, SP-D is synthesized and secreted primarily by type II and other nonciliated bronchiolar respiratory epithelial cells (2–4). As a component of the innate immune system, SP-D inhibits the proliferation of Gram-negative bacteria, binds and aggregates a variety of microorganisms including respiratory viruses and fungi, and enhances their presentation to resident phagocytic cells for opsonization and intracellular killing (1, 2, 5–7, 9–12). In addition, SP-D influences surfactant homeostasis, reduces alveolar macrophage apoptosis, stimulates a variety of immune cells, and enhances oxygen radical production (13–16).

Recent findings, however, implicate SP-D in immunomodulation during inflammation and allergic response. SP-D binds alveolar macrophages and circulating leukocytes (17) and stimulates the migration of human neutrophils, monocytes, and macrophages (18, 19), influencing recruitment following lung injury and inflammation. SP-D stimulates immune cell activity following exposure to pathogen-associated molecular pattern recognition. Increased expression in the lungs of transgenic mice enhanced host defense function and decreased inflammatory responses following exposure to pulmonary pathogens (7, 9, 20, 21). Conversely, Sftpd–/– mice develop emphysema associated with chronic inflammation (22) and exhibit enhanced inflammatory responses to a variety of stimuli (23). Thus, SP-D plays a role as a surveillance molecule that facilitates killing and clearance of pathogens, serving to control inflammatory responses.

Despite the important protective roles of SP-D in pulmonary homeostasis, transcriptional regulation of SP-D is poorly understood. Recently, a role for SP-1-like transcription factors has been implicated in regulation of the human SP-D gene (SFTPD) promoter, but the identity of these factors is unknown (24). AP-1 family members regulate SFTPD promoter activity. JunB and JunD enhanced, whereas c-Jun and c-Fos inhibited, transcription (24). Mutation of DNA binding sites for forkhead transcription factors FoxA1 and FoxA2 decreased transcription of SFTPD. In contrast, TTF-1 did not activate its expression in H441 cells in vitro (24).

SP-D expression is developmentally regulated and increases dramatically prior to birth. In mouse and rat lung, SP-D mRNA is first detected at midgestation and increases abruptly before birth and during the immediate postnatal period (25–27). Glu-cocorticoid treatment in utero precociously increased SP-D mRNA and accelerated lung maturation. Whereas the mechanism of SP-D induction is not known, it has been suggested that dexamethasone treatment increases promoter occupancy of CCAAT enhancer-binding protein-{beta} (C/EBP-{beta}) (24, 28). Retinoblastoma protein stimulated SFTPD activation by directly forming a complex with C/EBPs bound to the NF-IL-6 (C/EBP-{beta}) consensus site in the SFTPD promoter. Cotransfection of C/EBP-{alpha}, -{beta}, and -{delta} cDNAs increased transcription of the human SFTPD gene in H441 cells (29).

Following a variety of lung infection and injury, an acute phase response similar to that of liver is observed in the lung (4). For example, haptoglobin, C-reactive protein, serum lipopolysaccharide-binding protein, and respiratory epithelial derived fibrinogen and {alpha}1-acid glycoprotein increased following lung injury (29–33). SP-D mRNA and protein in lung lavage increased within 6 h after endotracheal endotoxin treatment in rats (32), following Pseudomonas infection in mice (34), a brief O2 exposure to rats (35), and treatment of type II cells with FGF-7 (36). SP-D mRNA was markedly increased following exposure to IL-4 and IL-13 in vivo (34, 37, 38). These observations and other data support the concept that SP-D is rapidly induced following lung injury, perhaps serving a protective role by modulating inflammation and enhancing host defense. Strikingly, Sftpd proximal promoter revealed cis-elements for C/EBPs, nuclear factor of activated T cells (NFAT), AP-1, and the signal transducers and activators of transcription family of transcription factors that are known to rapidly activate transcription of a large number of genes during an effective immune response in several organs (39–41). In addition, several TTF-1 sites were identified that may play critical roles in SP-D transcription following lung injury (42).

Since, SP-D expression is induced following lung injury and exposure to IL-4 and IL-13, we speculated that the calcineurin pathway, known to regulate T cell activation (43) mediated by dephosphorylation of NFATs, may be operative in the regulation of SP-D expression. Calcineurin is a heterodimeric Ca2+/calmodulin-dependent protein phosphatase activated by calcium ionophores such as ionomycin and inhibited by cyclosporin and tacrolimus (FK-506). It consists of one catalytic (CnA) and one regulatory (CnB) subunit (44). Changes in expression of cytokines and other immunomodulatory molecules activate T-cell receptors, increasing intracellular calcium concentrations, activating calcineurin. Once activated, calcineurin dephosphorylates NFAT and translocates them into the nucleus. NFATs bind cooperatively with other nucleoproteins to promoters of target genes to induce transcription (41, 45).

In the present work, we identified an endogenous, calcineurin/NFAT pathway in respiratory epithelial cells of the lung that assembles NFATs, AP-1, and TTF-1 on a proximal enhancer located in the 5'-region of the Sftpd gene, forming a multiprotein transcription complex inducing SP-D expression.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Construction and Mutagenesis—Sftpd promoter-reporter constructs were made in pGL3-basic vector, a luciferase reporter plasmid (Promega). Using TRANSFEC (Biobase; Biologische Datenbanken GmbH, Wolfenbüttel, Germany), a promoter sequence analysis and data base program, the Sftpd promoter was identified from a 5.7-kb Sftpd genomic clone (a kind gift from Dr. T. Korfhagen, Cincinnati Children's Hospital, Cincinnati, OH) that contained a 3-kb sequence upstream of the first exon that included a consensus TATA box. A 679-bp BtgI fragment (+79 to –600 bp) was Klenow end-filled and cloned into the SmaI site of pGL3-basic, the correct orientation was determined, and it was used to generate a series of promoter deletion mutants by PCR cloning bp +79 to –557, –357, –246, –167, and –82 into NheI and XhoI sites of pGL3-basic (see Table I for primers used). Rat TTF-1 expression plasmid pRC-CMV-TTF-1 was provided by Dr. R. DiLauro (Naples, Italy). Full-length human NFATc3 (the original clone from Nakoi Aroi), NFATc4 with a 3.1-kb cDNA containing the ORF cloned in the KpnI and NotI site in expression vector pREp4, constitutively active (CA) CA-NFATc3 and CA-NFATc4 in pAC-CMV pLpA-5+ expression vector, and constitutively active calcineurin (pECE-FLAG-{Delta}CnA) were provided by Dr. J. Molkentin (Cincinnati Children's Hospital, Cincinnati, OH). Green fluorescent protein (GFP)-VIVIT expression plasmid containing an in-frame ORF for peptide sequence MAGPHPVIVITGPHEE fused to enhanced green fluorescent protein in pEGFP.N1 (Clontech), was a kind gift from Dr. A. Rao (Harvard University, Boston, MA). pcDNA3.1 with CMV promoter (Invitrogen) and pCMV {beta}-galactosidase (Clontech) vectors were used to normalize DNA and transfection efficiency, respectively. All subcloned DNAs were confirmed by sequencing.


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  		TABLE I
List of deoxyoligonucleotides used in this study

 
Cell Culture, Transfection, and Reporter Gene Assays—HeLa cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) with 10% fetal bovine serum. Mouse lung epithelial cells, MLE-15, an immortalized mouse lung epithelial cell line that maintains some morphological and functional characteristics of type II epithelial cells, were cultured in HITES medium (46) for functional characterization of Sftpd promoter. A series of Sftpd promoter-luciferase constructs were used in transient transfection assays using Fugene 6 at a DNA/Fugene ratio of 1:2 according to the manufacturer's instructions (Roche Applied Science). Briefly, 6-well plates at 30–50% confluence were transfected with a fixed amount of Sftpd promoter-luciferase plasmid and various amounts of CMV-based cDNA expressing transactivator plasmids. For co-transfection experiments, the promoter-reporter plasmid was kept at 15 ng/well unless stated otherwise. Total DNA was normalized with corresponding CMV-empty vectors, and transfection efficiency was normalized to {beta}-galactosidase activity using 100 ng/well of pCMV {beta}-galactosidase. Two days after transfection, luciferase and {beta}-galactosidase assays were performed using 20 µl of the supernatant according to a previous protocol (47). The light units were assayed by luminometry (Monolight 2010, Analytical Luminescence Laboratory, San Diego, CA). Data obtained represent the average of three transfection experiments, each carried out in duplicate (n = 6) and depicted as means ± S.D. unless stated otherwise.

Isolation of RNA and Reverse Transcription (RT)-PCR—MLE-15 cells grown in a T75 flask to 80% confluence were washed and suspended in 10 ml of phosphate-buffered saline. 2.5 ml of cells were pelleted at 3,000 rpm and lysed in 0.5 ml of 1x RNA lysis buffer (50 mM Tris·Cl, pH 7.5, 50 mM NaCl, 5 mM EDTA, 0.5% SDS) by 2 min of vigorous vortexing. Total RNA was extracted in 100 µl of 5 M ammonium acetate (Ambion), 2 µl of glycogen and 600 µl of acid phenol/chloroform (5:1) solution, pH 4.5 (Ambion), precipitated in 2 volumes of ethanol, washed in 70% ethanol, dried, and suspended in 60 µl of diethyl pyrocarbonate-treated H2O. 10 µl of RNA suspension was reverse transcribed for first strand cDNA synthesis using SuperScript II-RT, with procedures provided by the manufacturer (Invitrogen). RNA from purified mouse alveolar epithelial type II cells (AE II) was kindly provided by Dr. John Shannon (Cincinnati Children's Hospital, Cincinnati, OH). AE II cells were prepared from 6-week-old female C57B/6 mice as described previously (48). RT products were produced by PCR using primers described in Table I. Primers and reaction conditions were optimized using cDNA made from reverse transcribed total RNA of adult mouse lung. In studies of mouse alveolar type II cells (AE II cells), PCR was performed on RT product using Amplitaq Gold DNA polymerase (Roche Applied Science) under the following conditions: 95 °C for 10 min for the denaturation cycle and 35 cycles at 95 °C (30 s), 58 °C (30 s) annealing, and 72 °C for extension, with a final extension of 10 min at 72 °C. {beta}-Actin was used as a positive control. Reactions containing RNA that was not reverse transcribed were used as a control to detect genomic DNA contamination.

Immunohistochemistry of Embryonic and Adult Lungs—Embryos were obtained at different gestational stages, processed according to previous methods (49), and embedded in paraffin. For adult lung tissues, lungs were inflation fixed with 4% paraformaldehyde at 25-cm water pressure and processed for paraffin embedding. Serial 5-µm sections were prepared for adult lung. For fetal lung, whole embryos were fixed with 4% paraformaldehyde and sectioned at 5-µm intervals. To detect the regulatory subunit calcineurin B1 (CnB1), anti-mouse CnB1 rabbit polyclonal antitibody (PA3-025; Affinity Bioreagents, Goldon, CO) at a 1:500 dilution as primary antibody and anti-rabbit IgG made in goat as secondary antibody (Vector Laboratories, Inc.) were used, respectively, for immunohistochemistry as described previously (50). Biotinylated secondary antibodies and a streptavidin-biotin-peroxidase detection system (Vector Laboratories) were used to localize the antibody-antigen complexes in the tissues, as previously described (51). A mouse-on-mouse blocking kit (Vector Laboratories) was used with primary mouse monoclonal antibodies for NFATc3, (F-1, SC-8405X) at 1:100 dilution. Antigen detection was enhanced with nickel-diaminobenzidine and Tris-cobalt, followed by counterstaining with Nuclear Fast Red.

Electrophoretic Mobility Shift Assays (EMSAs)—Nuclear extract from MLE-15 cells were prepared as previously described (52) except that nuclear extracts were stored before dialyses. 32P-Labeled double-stranded DNA probes were made by a Klenow end-filling reaction using [{alpha}-32P]dCTP. EMSAs were performed by incubating 10 nM double-stranded DNA oligonucleotide probes corresponding to the –130 (NFAT-I) and –170 (NFAT-II) sites (see Table I for oligonucleotide sequences) with 3.5 µl (~15 µg) of nuclear extract for 40 min at 4 °C in 30 µl of binding buffer (15 mM HEPES, 0.1 mM EDTA, 0.5 mM dithiothreitol, 40 mM KCl, 5% glycerol, pH 7.9) with 1 µg of poly(dI)·poly(dC) (Amersham Biosciences), and reactions were electrophoresed on a 4%, 0.5x TBE native polyacrylamide gel at room temperature for 2–3 h at 150 V. Unlabeled competitor double-stranded DNA oligonucleotides, NFAT-I and NFAT-II from the Sftpd promoter, TTF-1 (Oligo-C), with a strong TTF-1 binding site from the rat thyroglobulin gene (rTg) promoter spanning –82 to –56 bp (53), and a strong NFAT site-containing oligonucleotide from the IL-2 promoter (54) were added at a 100-fold molar excess. Antibody supershift assays were carried out in the same buffer by the further addition of 2 and 4 µl of either anti-NFATc3 antibodies (SC-1152X; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or albumin as mock, and the reaction continued at 4 °C for 1 h. Gels were autoradiographed to detect specific DNA-protein complex formation.

DNase I Footprinting—DNA fragment spanning the –246 to +79 bp region of the Sftpd promoter was subjected to DNase I reaction (see Fig. 4B) using MLE-15 nuclear extracts. The (TG)n region in the –246/+79 Sftpd promoter had four nucleotides (ACAC) less than the original promoter (GenBankTM accession number AF047742 [GenBank] ) as confirmed by sequencing. The Sftpd promoter (–246/+79) cloned in pGL3 was digested with NheI and labeled at the 3'-end using [{alpha}-32P]dCTP (6,000 Ci/mmol) in a Klenow reaction, DNA-purified, and released with XhoI. The DNase I footprinting assay contained 10 fmol (~5,000 cpm) of the end-labeled fragment in 200 µlof1x binding buffer (52) containing 150- and 300-µg nuclear extracts, and a protein binding reaction was carried out for 30 min at 4 °C. DNase I reaction conditions and purification of digested DNA were as described previously (52). Samples were heat-denatured and loaded on to an 8% sequencing gel. DNA ladders for G and A + G were made by Maxam-Gilbert chemical sequencing.



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  		FIG. 4.
DNase I footprint analysis of the proximal Sftpd promoter. A, DNase I protected sites were identified on the proximal Sftpd promoter (–246/+79). Note protections around NFAT-I, NFAT-II, AP-1, and TTF-1 binding sites in the presence of MLE-15 nuclear extracts (lanes 4 and 5, solid boxes). DNase I hypersensitivity was observed adjacent to TTF-1 (lane 4, open box) and 3' to NFAT-II binding sites (lanes 4 and 5, open box). Naked DNA was treated with DNase I as a control (lanes 3 and 6). G and A + G DNA ladders were used as markers (lanes 1, 2, and 7). Solid boxes represent DNase I footprinted regions, and open boxes show changes in DNase I hypersensitivity (decrease (HS) and increase (HS') in DNase I hypersensitivity) after incubation with MLE-15 nuclear extracts. B, Sftpd promoter fragment spanning –200 to +1 bp showing DNase I footprinted regions (solid bars). C, schematic representation of the putative transcription factor binding sites and other features of the Sftpd promoter relevant to this study. Note the purine-pyrimidine (TG)n stretch adjacent to the region that binds a number of protein complexes as judged by footprinting analysis, including putative binding elements for C/EBP and signal transducers and activators of transcription proteins. D, stimulation of Sftpd promoter activity in the presence of ionomycin and PMA (lanes 2–4). The D-246-Luc (15 ng) was transfected into MLE-15 cells. 1 µM ionomycin (Calbiochem) and 10 nM PMA (Calbiochem) in Me2SO were added to HITES medium containing 1 mM CaCl2. Cells were harvested after 16 h, and luciferase activity was measured. E, inhibition of Sftpd promoter activity in the presence of cyclosporin (lanes 2–4). The D-246-Luc (15 ng) was transfected into MLE-15 cells growing in HITES medium. Cyclosporin A (Calbiochem) in Me2SO was added to the medium at a final concentration of 2.5, 3.5, and 4 µM. After 6 h, cells were harvested, and luciferase activity was measured. A dose-dependent decrease in Sftpd promoter activity was observed. Values are means ± S.D. (n = 6).

 
Glutathione S-Transferase GST Pull-down Assays—Full-length GST-TTF-1 fusion protein was made in two steps. TTF-1 cDNA was first PCR-amplified with a stop codon from pRC-CMV-TTF-1, digested, and subcloned into EcoRI and HindIII sites (see Table I for primer details) within the multiple cloning sites of pCMV-Tag 2A vector (Clontech, Palo Alto, CA). TTF-1 cDNA was then excised from pCMV-Tag 2A with EcoRI and SalI and ligated in frame between the same enzyme sites in pGEX-4T-3, a bacterial expression vector (Amersham Biosciences), and sequenced. The GST-TTF-1 deletion constructs were made from a series of pVP16-TTF-1 plasmids (47). DNA fragments containing TTF-1 deletion derivatives were digested with XbaI and Klenow end-filled before cutting with BamHI. The DNA fragments were then subcloned into the BamHI and SmaI sites in pGEX-5X-2, a bacterial expression vector (Amersham Biosciences). The plasmids were transformed into BL21-DE3 bacterial strains for protein expression. GST fusion protein expression, cell lysis, and immobilization of proteins on glutathione-Sepharose beads were performed as described previously (55). Immobilized proteins were detected on a Tris-glycine 10–20% SDS-polyacrylamide gel by Coomassie staining and total protein-normalized for protein-protein interaction using empty glutathione-Sepharose-beads. Beads (20 µl) with immobilized proteins were washed several times with 1x phosphate-buffered saline, pH 7.4, incubated for 2hat4 °C with 15 µlof in vitro transcribed and translated radiolabeled [35S]Met-CA-NFATc3 in 1 volume binding buffer (10 mM Tris, pH 7.5, 1 mM EDTA, 100 mM NaCl and 0.1% Nonidet P-40) on a rotator. Beads were washed three times with 1.5 ml of wash buffer (same as binding buffer) and boiled in 1 volume of SDS Laemmli buffer containing 10% {beta}-mercaptoethanol. Proteins released were separated by electrophoresis under reducing conditions on SDS 10–20% Tris-glycine polyacrylamide gradient gel (Novex, San Diego, CA). Gels were dried, and radiolabeled [35S]Met-CA-NFATc3 was detected on a PhosphorImager (Storm 860; Amersham Biosciences).

Co-Immunoprecipitation Assays and Western Blotting—The 3x FLAG-tagged CA-NFATc3 expression vector used in the co-immunoprecipitation experiment was constructed as follows. CA-NFATc3 in pAC-CMV pLpA-5+ expression vector was digested with HindIII and Klenow end-filled and then excised with NotI containing the SV40 3'-untranslated region and inserted into EcoRV and NotI sites of 3x FLAG-CMV expression vector. The 3x FLAG-tagged CA-NFATc3 vector was transfected (50 µg) into 40% confluent MLE-15 cells in T75 flask, and nuclear extracts were prepared after 48 h as described (56), except that nuclear proteins were extracted by suspending nuclei for 1 h in 1 volume of high salt extraction buffer (20 mM HEPES, pH 7.9, 550 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 10 mM phenylmethylsulfonyl fluoride) with one protease inhibitor mini-tablet (Roche Applied Science). Nuclear extracts from MLE-15 cells (~15 µg/µl) were diluted in 10 volumes of 1x phosphate-buffered saline and incubated for 2 h on a rotator at 4 °C with 100 µl of mouse monoclonal anti-FLAG M2-IgG-tagged Sepharose beads (Sigma). The beads were washed three times with 1.5 ml of wash buffer (10 mM Tris, pH 7.5, 1 mM EDTA, 100 mM NaCl, and 0.1% Nonidet P-40) and boiled in 1 volume of SDS buffer (Laemmli sample buffer) containing 10% {beta}-mercaptoethanol. The co-immunoprecipitated proteins released were separated by electrophoresis under reducing conditions on SDS-10–20% Tricine polyacrylamide gradient gels (Novex) and transferred to nylon membranes (Bio-Rad). Protein blots were blocked with 5% nonfat dry milk in TBST (10 mM Tris, pH 8, 150 mM NaCl, 0.1% Tween 20) and incubated with anti-TTF-1 monoclonal antibody produced in our laboratory, followed by horseradish peroxidase-conjugated rabbit anti-mouse IgG (Calbiochem). The presence of FLAG-tagged CA-NFATc3 on beads was assessed using primary anti-FLAG mouse monoclonal antibodies directly conjugated to horseradish peroxidase (Sigma). Western blots were developed with the enhanced chemiluminescence system (Amersham Biosciences) and exposed to autoradiographic film X-Omat (Eastman Kodak Co.).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
TTF-1 and NFATs Activate Transcription from the Sftpd Promoter—A promoter-luciferase reporter plasmid (Fig. 1A) comprising –600 to +79 bp (D-600-Luc) of the mouse surfactant protein D gene (Sftpd), was transiently transfected into HeLa and MLE-15 cells. The –600/+79 bp Sftpd promoter was highly active in MLE-15 but not in HeLa cells (Fig. 1B). HeLa cells co-transfected with D-600-Luc and increasing amounts of an expression plasmid encoding TTF-1 activated the Sftpd promoter by ~15-fold (Fig. 1C). The proximal region of the Sftpd promoter contains consensus elements for both TTF-1 and NFATs. We therefore hypothesized that TTF-1- and NFAT-dependent pathways may directly influence Sftpd promoter activity in respiratory epithelial cells. Full-length NFATc3 (Fu-NFATc3), full-length NFATc4 (Fu-NFATc4), constitutively active NFATc3 (CA-NFATc3), or constitutively active NFATc4 (CA-NFATc4) expression plasmids (Fig. 1D) activated Sftpd promoter activity in MLE-15 cells. CA-NFATc3 was more active than CA-NFATc4 (Fig. 1D, lanes 4 and 5). Full-length NFATs Fu-NFATc3 and Fu-NFATc4 also activated the –600/+79 Sftpd promoter, albeit to a much lesser extent (Fig. 1D, lanes 2 and 3), suggesting endogenous but weak calcineurin activity in respiratory epithelial cells. Interestingly, this NFAT activation was not observed in HeLa cells (data not shown), suggesting the requirement of cofactors present in MLE-15 cells but not in HeLa cells.



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  		FIG. 1.
TTF-1 and NFATs activate the Sftpd promoter. A, schematic representation of the –600 bp Sftpd promoter-luciferase (D-600-Luc) construct depicting positions of the TATA-box, the 5'-untranslated region to +79 bp containing the 39 bp of exon-I. B, dose response of the transfected D-600-Luc into HeLa and MLE-15, an immortalized mouse lung epithelial cell line, measured as luciferase activity. C, effect of TTF-1 on promoter activity was assessed after co-transfection of a fixed amount of D-600-Luc (1 µg), with increasing amounts of TTF-1 expression plasmid (RC-CMV-TTF-1) at 0.1, 0.25, 0.5, 1.0, and 2 µg per well into HeLa cells in 6-well plates. Values represent two independent experiments carried out in duplicate with means ± S.D. (n = 4). D, effect of full-length (Fu-NFATc3 and Fu-NFATc4) and constitutively active (CA-NFATc3 and CA-NFATc4) NFAT expression on D-600-Luc activity. Expression vectors for NFAT cDNA (2 µg) were cotransfected with D-600-Luc (0.5 µg) into MLE cells. Values were obtained from three experiments carried out in duplicate, means ± S.D. (n = 6).

 
NFATs Are Present in the Respiratory Epithelial Cells— Since NFATs activated the –600/+79 Sftpd promoter in MLE-15 cells, we tested whether NFATs were expressed in MLE-15 and alveolar epithelial type II cells (AE-II) that are known to express SP-D (27, 57). RT-PCR was carried out using total RNA from MLE-15 and purified mouse AE-II cells. Unique sets of primers designed for murine NFATc1, NFATc2, NFATc3, NFATc4, and NFATc5 cDNA (Table I) gave PCR products of expected sizes (Fig. 2A). The expression profiles of NFATs were similar in MLE-15 and AE-II cells. NFATc1 and NFATc3 were the predominant forms (Fig. 2, lanes 2, 4, 8, and 10), NFATc4 was weakly expressed in AE-II cells (Fig. 2, lanes 5 and 11), whereas NFATc2 was not expressed in either cell type (Fig. 2, lanes 3 and 9). NFATc5, a ubiquitously expressed gene not part of the classical NFAT family (58), was detected at low levels in MLE-15 and AE-II cells (Fig. 2, lanes 6 and 12).



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  		FIG. 2.
NFATs in the respiratory epithelial cells. A, mRNA expression of NFAT genes in the respiratory epithelial cells. RT-PCR was performed on total RNA isolated from purified mouse alveolar epithelial type II (AE II) and MLE-15 cells. Unique sets of primer pairs were used to identify expression of each NFAT gene, c1, c2, c3, c4, and c5 (see Table I). NFATc1 and NFATc3 were expressed at high levels (lanes 2, 4, 8, and 10) in MLE-15 and alveolar epithelial type II cells. DNA molecular weight markers from 100 to 500 bp (lane 1, M) were used to identify correct PCR product sizes for expression of each NFAT gene. {beta}-Actin mRNA was amplified as a positive control (lanes 7 and 13). B, EMSAs revealed NFATc3 binding to proximal Sftpd promoter elements. 32P-Labeled double-stranded oligonucleotides (10 nM) derived from NFAT-I (–170/–164) and NFAT-II (–130/–124 bp) sites on the Sftpd gene were used for EMSAs (see Table I). Nuclear extracts made from MLE-15 cells transfected with NFATc3 expression vector were incubated with labeled probes. Both probes formed DNA-protein complexes of similar size as shown by the solid arrow (lanes 2 and 7). These DNA-protein complexes were abolished by 100-fold excess of unlabeled self-DNA probes (lanes 3 and 8) but not by a 100-fold excess of TTF-1 (Oligo-C) consensus probe (lanes 4 and 9). In contrast, a 100-fold excess of unlabeled NFAT site probe derived from human IL-2 promoter competed with NFAT-I and NFAT-II probes leading to loss of the DNA-protein complexes (lanes 5 and 10). Thus, specific protein complexes were identified at regions overlapping NFAT sites. C, protein complexes formed on NFAT-I and NFAT-II site oligonucleotides (lanes 2 and 7) were supershifted by NFATc3 antibody (lanes 3 and 4 and lanes 7 and 8). Note the decrease in intensity of DNA-protein complex (solid arrow) and the formation of a higher molecular weight complex in the presence of NFATc3 antibody (open arrow). Bovine serum albumin did not alter the complex (lanes 5 and 10).

 
NFAT Binds Sftpd Promoter Elements—Since NFATc3 was most active on –600/+79 Sftpd promoter and was expressed at high levels in MLE-15 and AE-II cells, we tested whether NFATc3 directly bound to the –600/+79 Sftpd promoter. Two perfect NFAT consensus sites (NFAT-I and NFAT-II) (Fig. 3B) present in the Sftpd promoter region at –170 to –164 and –130 to –124 bp from the transcription start site with a core similarity of 1.0 and a matrix similarity of 0.99 and 0.97, respectively, were identified using MatInspector 7.0 (Genomatix Software GmbH, München, Germany). Two DNA probes comprising NFAT-I or NFAT-II (see Table I) were used in EMSAs with nuclear extracts made from MLE-15 cells transfected with CA-NFATc3 plasmid. NFAT-I and NFAT-II probes formed strong DNA-protein complexes of similar size from MLE-15 nuclear extracts (Fig. 2B, lanes 2 and 7) and were competed by 100-fold excess of unlabeled self-DNA (Fig. 2B, lane 3 and 8) but not by 100-fold excess of TTF-1 (Oligo-C) consensus probe (Fig. 2B, lanes 4 and 9) designed from the rat thyroglobulin promoter (53), indicating that the protein bound specifically to NFAT sites. When a strong NFAT site probe derived from the IL-2 promoter (54) was used for competition, the DNA-protein complex could not be seen for both sites (Fig. 2B, lanes 5 and 10), further suggesting that the two NFAT sites found in the –600/+79 Sftpd promoter bound NFATs. To establish that nuclear proteins from MLE-15 cells indeed bound NFATc3 to the two NFAT sites, gel supershift experiments were carried out. Antibodies to NFATc3 supershifted DNA-protein complexes formed on NFAT-I and NFAT-II sites (Fig. 2C, lanes 3, 4, 8, and 9), whereas there was no change in the mobility of the complexes in the presence of bovine serum albumin. To further demonstrate that nuclear proteins from MLE-15 cells bound NFAT sites in the context of the native promoter, DNase I protection assays were carried out on the –246/+79 Sftpd fragment. Strong DNase I protection regions spanning two NFAT sites, NFAT-I and NFAT-II (Fig. 4A), and AP-1, TTF-1, and C/EBP sites were identified as part of the protected or hypersensitive region (Fig. 4A). The region near TTF-1 binding site was hypersensitive to DNase I at low nuclear protein concentrations (Fig. 4A, lane 4), whereas the region around the AP-1 site showed increased DNase I protection in contrast to the hypersensitivity found on naked DNA. Changes in DNase I hypersensitivity were observed 3' to the NFAT-II site (Fig. 4A, open boxes). Such changes in DNase I digestion patterns are a hallmark for the formation of specific DNA-protein complexes that induce DNA distortions leading to altered DNase I sensitivity (59), suggesting a possible multiprotein complex binding at the area of DNase I protection that may comprise NFAT, TTF-1, AP-1, and other factors.



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  		FIG. 3.
Co-expression of calcineurin B1 and NFATc3 in embryonic and adult mouse lung. CnB1 protein was detected by immunostaining in the cytoplasm of the respiratory epithelial cells at embryonic days 15.5 and 17.5 (E15.5 and E17.5) (A and B). Intense nuclear staining was observed in the bronchiolar epithelial cells in the adult mice (C). NFATc3 protein was detected in the cytoplasm of the respiratory lung epithelium of the fetal mice at embryonic day 15.5 (D). At embryonic day 17.5 and in adult mice, NFATc3 stained intensely in the nucleus in the bronchiolar epithelium (E and F). Bars, 100 µm.

 
Expression of NFATc3 and Calcineurin in the Lung Epithelium—Since TTF-1 is strictly expressed in the respiratory epithelium, it was relevant to test the expression of NFATc3 to determine whether TTF-1 and NFATc3 are co-expressed. Expression of calcineurin, a phosphatase that activates NFATs, was also assessed. NFATc3 and CnB1 were detected by immunostaining (Fig. 3). Calcineurin was detected in the cytoplasm at apical and basal regions of respiratory epithelial cells in conducting airways and peripheral regions of the lung at embryonic days 15.5–17.5. The intensity of staining was highest in the peripheral lung (Fig. 3A). CnB1 was also detected in the respiratory epithelium of the adult lung, wherein intense nuclear staining was noted in the bronchiolar region. (Fig. 3, B and C). Staining was also detected in endothelial cells of the pulmonary vasculature. At embryonic day 15.5, NFATc3 was present in the apical regions of the epithelial cells (Fig. 3D). In contrast, at embryonic day 17.5 and in the adult lung epithelium, NFATc3 became localized to the nuclei of bronchiolar cells (Fig. 3, E and F). Thus, calcineurin, NFATc3, and SP-D (27) are co-expressed in respiratory epithelial cells during lung morphogenesis and in the adult.

Calcineurin-responsive Enhancer in the Sftpd Promoter— NFAT activation and its subsequent binding to promoter elements are dependent upon its dephosphorylation by calcineurin. Therefore, the role of calcineurin in modulating Sftpd promoter activity was investigated in MLE-15 cells transfected with a proximal promoter-reporter construct (D-246-Luc) containing the two NFAT sites (Fig. 4, C and D). Treatment with ionomycin, a calcineurin activator, or ionomycin and phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator that activates AP-1 family members and synergizes with NFATs (43), activated transcription from the –246/+79 Sftpd promoter (Fig. 4C, lanes 2 and 3). Cyclosporin A, an inhibitor of calcineurin, decreased the promoter activity in a dose-dependent manner (Fig. 4D, lanes 2–4), supporting the concept that calcineurin, via NFATs influenced Sftpd promoter activity. To demonstrate that the calcineurin/NFAT pathway indeed modulated Sftpd transcription by recruiting NFAT proteins to the proximal promoter, deletion constructs of the Sftpd promoter were tested in MLE-15 cells (Fig. 5A). A proximal enhancer element was identified between –167 and –82 bp that contains a purine-pyrimidine stretch (TG)n and a 50-base pair sequence with putative NFAT, AP-1, TTF-1, and C/EBP binding sites that were footprinted (Figs. 4C and 5A). Despite having an NFAT-II site, the region between –167 and –246 repressed the enhancer activity (Fig. 5A). To identify sequences that confer NFAT-dependent activity, deletion constructs were co-transfected with CA-NFATc3 into MLE-15 cells. The promoter construct –82/+79 lacking NFAT sites did not respond to NFATc3 (Fig. 5B), whereas NFATc3 increased activity up to 11-fold for –167/+79 Sftpd promoter. No further increase in promoter activity was observed in larger promoter constructs (Fig. 5B), indicating recruitment of NFATs between –167 and –82 bp. Thus, excluding the (GT)n repetitive sequence, our data identified a 50-base pair enhancer confined to region –167/–117 bp comprising NFAT-I, AP-1, TTF-1, and C/EBP sites. The activity of –167/+79 Sftpd promoter was entirely dependent upon calcineurin and inactivation of NFATs. For example, full-length NFATc3 was inactive in the D-167-Luc construct (Fig. 5C, lane 2), whereas co-expression with constitutively active calcineurin ({Delta}Cn-A) increased promoter activity up to 10-fold (Fig. 5C, lane 4). To test whether calcineurin directly increased Sftpd promoter activity utilizing endogenously present NFAT proteins in lung epithelial cells, constitutively active calcineurin ({Delta}Cn-A) was cotransfected with D-167-Luc in MLE-15 cells. Dose-dependent expression of {Delta}Cn-A increased D-167-Luc activity (Fig. 5D, lanes 2–6). When MLE-15 cells transfected with D-167-Luc were treated with cyclosporin A, a calcineurin inhibitor, a dose-dependent inhibition of promoter activity was observed, demonstrating that –167/+79 Sftpd promoter activity was directly modulated by endogenous calcineurin (Fig. 5E).



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  		FIG. 5.
Identification of a calcineurin-responsive enhancer. A, deletion analysis of the Sftpd promoter in MLE-15 identified a transcriptional enhancer region between –167 and –82 bp in the Sftpd gene. B, co-transfection of CA-NFATc3 expression plasmid (2 µg) with a series of promoter-reporter constructs (1 µg) into MLE-15 cells revealed an NFATc3-responsive region within the –167/–82 bp of the Sftpd gene. Larger constructs were not more active. The –82/+79 Sftpd promoter construct lacking the NFAT sites did not respond to NFATc3. C, co-transfection of D-167-Luc (100 ng) with Fu-NFATc3 (1 µg) or {Delta}Cn-A (1 µg) expression plasmid alone into MLE-15 cells increased transcriptional activity by about 3-fold (lanes 2 and 3), whereas Fu-NFATc3 and {Delta}Cn-A together increased transcription by ~10-fold (lane 4). D, dose response of {Delta}Cn-A on D-167-Luc in MLE-15 cells measured as luciferase activity (lanes 2–6). A fixed amount of D-167-Luc (50 ng) was co-transfected with increasing amounts of {Delta}Cn-A expression plasmid (0.125, 0.250, 0.5, 1.0, and 2 µg) per well. Values are mean ± S.D. (n = 4). E, MLE-15 cells were transfected with D-167-Luc (15 ng) and dose-dependent effect of CsA (2.5, 3.5, and 4 µM) prepared in Me2SO (DMSO) as a vehicle was assessed after 6 h by measuring luciferase activity (lanes 2–4).

 
VIVIT Abolishes the Effects of Calcineurin/NFAT Pathway on the Minimal Sftpd Promoter Activity—A potent NFAT inhibitory peptide VIVIT docks on calcineurin and selectively interferes with the calcineurin-NFAT interaction, leaving its phosphatase activity intact (60). When plasmid expressing GFP-VIVIT was co-transfected with D-167-Luc, a dose-dependent decrease in the promoter activity was observed (Fig. 6A), indicating physical interference from GFP-VIVIT peptide on the calcineurin-NFAT interaction. Expression of GFP-VIVIT renders NFATs inactive in the phosphorylated state in the cytoplasm unable to translocate into the nucleus and activate transcription. Thus, activated NFATs are directly required for Sftpd promoter activity. Even in the presence of high levels of constitutively active calcineurin, VIVIT strongly inhibited the enhancer activity (Fig. 6B), further supporting the concept that activity of the Sftpd promoter requires direct NFAT activation and is not activated by calcineurin pathways independently of NFATs. Taken together, the activity of the proximal Sftpd promoter is modulated by the calcineurin/NFAT pathway in respiratory epithelial cells.



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  		FIG. 6.
Effect of VIVIT expression on Sftpd promoter activity. A, dose-dependent effect of plasmid expressing GFP-VIVIT (0.16, 0.32, 0.64, and 1.28 µg) on D-167-Luc promoter activity in MLE-15 cells (lanes 2–5), indicating interference by VIVIT peptide on calcineurin-mediated NFAT activation. Luciferase activity from D-167-Luc in MLE-15 cells was set to unity. B, expression of high levels of {Delta}Cn-A co-transfected (1.5 µg/well) with GFP-VIVIT did not overcome VIVIT inhibitory activity on the D-167-Luc enhancer. Values are means ± S.D. (n = 6). The value of luciferase activity obtained after co-transfection with {Delta}Cn-A was set to unity (lane 3). GFP-VIVIT plasmid expresses a GFP fused to a peptide (MAGPH-PVIVITGPHEE) containing a hydrophobic motif VIVIT that binds to calcineurin (CaN) at the NFAT binding surface, selectively excluding NFAT binding without disrupting the phosphatase activity of calcineurin required for other physiological functions. Thus, calcineurin pathways independent of NFAT do not influence Sftpd promoter activity.

 
NFATc3 and TTF-1 Physically Interact and Synergistically Activate the Sftpd Promoter—Since the functional NFAT sites are adjacent to the TTF-1 site (Fig. 4B), functional cooperativity and physical interaction of NFATc3 and TTF-1 proteins on the D-167-Luc proximal enhancer were tested. Unlike D-600m-Luc, NFATc3 activated the –167/+79 Sftpd promoter in HeLa cells, thus making it amenable for functional studies (Fig. 7A). CA-NFATc3 increased the activity of the –167/+79 Sftpd promoter and synergistically enhanced transactivation by TTF-1 in HeLa cells (Fig. 7B). To demonstrate physical association between NFATc3 and TTF-1 in respiratory epithelial cells, 3x FLAG-tagged CA-NFATc3 protein expressed in MLE-15 cells was immunoprecipitated with anti-FLAG mouse monoclonal antibodies attached to Sepharose beads (Fig. 7C, i). Co-immunoprecipitation of TTF-1 with FLAG-tagged NFATc3 was confirmed by blotting with mouse monoclonal antibody to TTF-1 (Fig 7C, ii).



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  		FIG. 7.
NFATc3 and TTF-1 physically interact and synergistically activate Sftpd promoter. A, dose response of CA-NFATc3 (0.125, 0.5, 1, and 2 µg) on D-167-Luc (0.5 µg) in MLE-15 and HeLa cells show activation of the minimal Sftpd promoter. B, synergistic activation of the murine Sftpd minimal promoter in MLE-15 cells. D-167-Luc (20 ng) co-transfected with TTF-1 expression plasmid (80 ng) RC-CMV-TTF-1 (lane 2), CA-NFATc3 (2 µg) expression plasmid (lane 3), or both plasmids (lane 4) into HeLa cells synergistically activated the minimal Sftpd promoter. C, co-immunoprecipitation (Co-IP) of 3x FLAG-tagged CA-NFATc3 and TTF-1. i, detection of immunoprecipitated 3x FLAG-tagged CA-NFATc3 from MLE-15 nuclear extracts with mouse monoclonal anti-FLAG M2 antibodies immobilized on Sepharose beads by immunoblotting (one-twentieth input). WB, Western blot; IP, immunoprecipitation. ii, co-immunoprecipitation was performed by incubating immunoprecipitated 3x FLAG-tagged CA-NFATc3 on Sepharose beads with MLE-15 nuclear extracts, and the presence of TTF-1 in the co-immunoprecipitate was detected by immunoblotting with mouse monoclonal anti-TTF-1 antibody followed by horseradish peroxidase-conjugated rabbit anti-mouse IgG (lanes 2 and 3), which also detected the mouse anti-FLAG IgG heavy and light chains immobilized on the beads (lane 1, marked with an asterisk). Recombinant TTF-1 protein expressed and purified from bacteria was run as a control (lane 4).

 
The TTF-1 Homeodomain (HD) Is Required for Direct Physical Interaction with NFATc3—To determine whether direct protein-protein interactions mediated the observed cooperation between TTF-1 and NFATc3, GST pull-down assays were performed with GST fused to full-length TTF-1 (GST-TTF-1) and in vitro transcribed and translated radiolabeled [35S]Met-CA-NFATc3. GST-TTF-1 full-length protein strongly interacted with CA-NFATc3 (Fig. 8A, lanes 2 and 3), indicating direct physical interaction between TTF-1 and NFATc3 in vitro. To identify the TTF-1 domains involved in physical interaction with NFATc3, a series of GST-TTF-1 protein deletion constructs were made (Fig. 8C) and incubated with in vitro transcribed and translated radiolabeled [35S]Met-CA-NFATc3. TTF-1 HD (residues 161–223) alone (Fig. 8B, lanes 5 and 6), TTF-1 HD with the amino-terminal (residues 1–223) region (Fig. 8B, lanes 7 and 8), and TTF-1 HD with the carboxyl-terminal (residues 161–372) region (Fig. 8B, lanes 9 and 10) strongly interacted with CA-NFATc3. In contrast, GST-TTF-1 amino-terminal (residues 1–160) region (lanes 3 and 4) and GST-TTF-1 carboxyl-terminal (residues 224–372) region (lanes 11 and 12), which do not contain the HD, did not bind [35S]Met-CA-NFATc3. TTF-1 HD was necessary and sufficient for direct physical interaction with NFATc3, whereas the flanking amino-terminal and carboxyl-terminal activation domains did not physically interact with NFATc3.



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  		FIG. 8.
TTF-1 HD is required for direct physical interaction with NFATc3. GST pull-down assays were performed with bacterially expressed GST or GST fused to full-length TTF-1 (GST-TTF-1) or various TTF-1 domains. Proteins were immobilized on glutathione-Sepharose beads and incubated with in vitro transcribed and translated radiolabeled [35S]Met-CA-NFATc3. A, beads with GST protein alone (lane 1) did not bind, whereas beads with GST-TTF-1 full-length protein (lanes 2 and 3) bound [35S]Met-CA-NFATc3. Lane 4 shows one-tenth of the total radiolabeled [35S]Met-CA-NFATc3 protein input. B, TTF-1 HD alone (lanes 5 and 6), TTF-1 HD with the amino-terminal region (lanes 7 and 8), and TTF-1 HD with the carboxyl-terminal region (lanes 9 and 10) strongly interacted with CA-NFATc3. In contrast, Sepharose beads without GST protein (lanes 1 and 2), with GST protein alone (lanes 13 and 14), with the GST-TTF-1 amino-terminal region (lanes 3 and 4), and with the GST-TTF-1 carboxyl-terminal region (lanes 11 and 12) did not bind [35S]Met-CA-NFATc3. C, schematic diagram of various GST-TTF-1 protein domains and their interaction with CA-NFATc3 protein.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Expression of SP-D is highly regulated in the respiratory epithelium, increasing prior to birth, following a variety of lung injury, exposure to pathogens, and in response to Th2-cytokines. Transcriptional pathways regulating SP-D expression are largely unknown. In this study, we identified an endogenous calcineurin/NFAT pathway in the respiratory epithelial cells that regulated Sftpd transcription. Calcineurin and NFATc3 co-expressed in the respiratory epithelial cells of the developing and postnatal lung at sites of SP-D expression (27). A calcineurin-dependent transcriptional enhancer was identified in the proximal Sftpd promoter. This region contained an NFAT·AP-1 composite element adjacent to a TTF-1 binding site. NFAT inhibitory peptide VIVIT (60) abolished the enhancer activity, demonstrating the requirement of activated NFATs to stimulate the Sftpd promoter. TTF-1 and NFATc3 physically interacted and synergistically stimulated the Sftpd promoter activity, supporting the concept that they are a part of a transcription complex regulating Sftpd transcription. A transcriptional pathway mediated by interactions between activated NFATc3 and TTF-1 regulate SP-D expression in the respiratory epithelial cells.

Whereas NFATs were first identified in T-cell development in the immune system, they are expressed in many cell types and contribute to diverse cellular functions (41). Calcineurin and NFATs have recently been implicated in development and differentiation of the nervous system, bone, cartilage, muscle, heart, skin, and fat tissues (41). Our studies demonstrate that calcineurin, NFATc3, and SP-D are coincident with TTF-1 protein expression in the respiratory epithelium during fetal lung development. Whereas neither NFATc3 nor TTF-1 expression is confined to the lung during development, these transcription factors are co-expressed in the respiratory cells of the developing lung, where they physically interacted and synergistically activated Sftpd transcription. Consistent with this finding, alveolar epithelial type II cells that co-express TTF-1 and NFATc3 mRNA secrete SP-D protein in culture (48). The role of TTF-1 and its phosphorylation mediated by several kinases play a critical role in lung development (61, 62). Thus, disparate signaling events that post-translationally influence TTF-1 and NFATc3 function may converge on target genes to initiate their spatial, temporal, and cell-specific expression. Taken together, our data support the hypothesis that the calcineurin/NFAT pathway interacts with TTF-1 to regulate SP-D expression.

Although SP-D has been regarded as constitutively expressed (1), recent studies demonstrate activation of SP-D expression following inflammation by exposure to pathogens, allergens, and pollutants (4, 32). The present study demonstrates that Sftpd promoter is regulated by the calcineurin/NFAT pathway. NFAT activates transcription of a number of genes following an effective immune response (41). NFAT binds cooperatively with transcription factors of the AP-1 family to composite NFAT·AP-1 sites found in the regulatory regions of immunomodulatory genes (43). Indeed, an NFAT·AP-1 composite site in the Sftpd proximal promoter (–139/–125) produced DNase I footprints with nuclear proteins from lung epithelial cells and was responsive to ionomycin and PMA, a hallmark of NFAT·AP-1 sites that bind activated NFAT and AP-1 family of transcription factors in target genes (41). Human SFTPD is regulated by AP-1 family members (24). Because AP-1 activity is also modulated by diverse signaling pathways (63), NFAT·AP-1-dependent transcription may integrate responses to lung injury.

TTF-1 regulates transcription of genes involved in lung morphogenesis and surfactant protein production (64). TTF-1 synergistically activates transcription of lung surfactant genes and several genes involved in lung development by direct protein-protein interaction or functional cooperation with a number of transcription factors (47, 65, 66). For example, TTF-1 interacts with GATA6 to activate SP-C and Wnt7b transcription (47, 67). Transcriptional co-activator TAZ and NFI interact with TTF-1 and synergistically activate the SP-C gene (65, 68), whereas surfactant protein A and SP-B transcription is regulated by interaction of TTF-1 with CREB-binding protein and SRC-1 (66, 69). TTF-1 also interacts with RAR/RXR and BR22 (66, 70) to activate SP-B transcription. TTF-1 is a homeodomain-containing transcription factor. In general, HD proteins have low DNA binding specificity and require partners and co-activators to bind specific regulatory regions of target genes (71). This inherent flexibility to interact with relevant cofactors allows them to execute precise spatial, temporal, and tissue-specific roles (71). Our findings demonstrate that TTF-1 acts as a cofactor for NFATc3 and synergistically activates Sftpd transcription. NFAT proteins have relatively low affinity for DNA binding sites (41). NFAT proteins interact with several families of transcription factors and cooperatively bind DNA as heterodimers on native promoters of target genes and synergize transcription (41). NFAT·AP-1 complexes have discrete and flexible polar patches providing interaction surfaces for binding of different transcription factor (72). TTF-1-NFAT interactions may be facilitated by the close apposition of the TTF-1 and NFAT binding elements in the Sftpd proximal promoter. TTF-1 HD alone interacted with NFATc3 and was required for protein-protein interaction with NFATc3. Recently, TTF-1 homeodomain interaction with the carboxyl-terminal zinc finger domain of GATA6 synergizing transcription from the Sftpc promoter was demonstrated (47). Whether GATA6 and NFATc3 interact with the same region of the TTF-1 HD is an interesting question. Nonetheless, transcriptional synergism due to physical interaction of homeodomains with other transcription factors is well documented. For example, Pitx HD interacts with basic helix-loop-helix proteins on the proopiomelanocortin promoter, whereas Pdx1 HD interacts with Pan1 on the rat insulin promoter (8, 73).

The present study identifies active components of the calcineurin/NFAT pathway in the lung epithelial cells. Calcineurin and NFATc3 are expressed coincidentally with TTF-1 and SP-D in respiratory epithelial cells of the fetal and adult lung. NFATc3 and TTF-1 physically interacted and synergistically activated Sftpd transcription.


   FOOTNOTES
 
* This work was supported by American Lung Association Grant RG-155-N (to V. D.) and NHLBI, National Institutes of Health, Grant HL 63329 (to J. A. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Division of Pulmonary Biology, 4403, Cincinnati Children's Hospital Research Foundation, 3333 Burnet Ave., Cincinnati, OH 45229-3039. Tel.: 513-636-8410 (office) and 513-636-3323 (laboratory); Fax: 513-636-7868; E-mail: davev0{at}cchmc.org.

1 The abbreviations used are: SP-D, surfactant protein D; C/EBP-{beta}, CCAAT enhancer-binding protein-{beta}; IL, interleukin; ORF, open reading frame; CA, constitutively active; CMV, cytomegalovirus; RT, reverse transcription; CnB1, calcineurin B1; EMSA, electrophoretic mobility shift assay; NFAT, nuclear factor of activated T cells; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PMA, phorbol 12-myristate 13-acetate; HD, homeodomain; GST, glutathione S-transferase; CREB, cAMP-response element-binding protein. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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