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Endocrinology Vol. 138, No. 9 3695-3703
Copyright © 1997 by The Endocrine Society

ARTICLES

Peroxisome Proliferator-Activated Receptor {gamma}1 Expression Is Induced during Cyclic Adenosine Monophosphate-Stimulated Differentiation of Alveolar Type II Pneumonocytes1

Laura F. Michael2, Mitchell A. Lazar and Carole R. Mendelson

Departments of Biochemistry and Obstetrics-Gynecology (L.F.M., C.R.M.), The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9038; and Division of Endocrinology, Diabetes and Metabolism, Departments of Medicine and Genetics (M.A.L.), University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6149

Address all correspondence and requests for reprints to: Carole R. Mendelson, Ph.D., Department of Biochemistry, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9038. E-mail: cmende{at}biochem.swmed.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The primary function of lung alveolar type II cells is to synthesize pulmonary surfactant, a lipoprotein enriched in dipalmitoylphosphatidylcholine. Because type II pneumonocytes are highly lipogenic, we considered the possible role of the adipogenic nuclear hormone receptor, peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), in their differentiation from epithelial cell precursors. A degenerate PCR-screening strategy revealed that multiple PPARs, including PPAR{gamma}, are present in differentiated type II cells. A PCR-amplified PPAR{gamma} DNA-binding domain was used to isolate a full-length PPAR{gamma}1 complementary DNA clone from a rabbit type II cell complementary DNA library. Although another PPAR{gamma} isoform, PPAR{gamma}2, is known to be highly expressed in adipocytes, only PPAR{gamma}1 was detected in rabbit type II cells by use of RT-PCR and by library screening. Rabbit PPAR{gamma}1 has 90% nucleotide sequence identity and 95% amino acid identity to mouse PPAR{gamma}1. PPAR{gamma}1 messenger RNA was readily detected in total RNA isolated from rabbit type II pneumonocytes cultured in the presence of cAMP, which causes enlargement of the prealveolar ducts, accelerates the rate of type II cell differentiation, and induces transcription of the major surfactant associated protein, surfactant protein-A. PPAR{gamma}1 messenger RNA also was detected in total RNA isolated from rabbit adipose tissue but not from whole adult or fetal lung, heart, or liver. By Western blot analysis, PPAR{gamma} protein expression was found to occur coincidentally with surfactant protein-A expression during lung type II cell differentiation. In view of the role of PPAR{gamma} in adipocyte differentiation and lipid homeostasis, we postulate that PPAR{gamma}1 induction by cAMP plays a role in the differentiation and expression of lipogenic enzymes in lung type II cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ALVEOLAR type II pneumonocyte is unique in its ability to synthesize lung surfactant, a developmentally regulated surface-active lipoprotein that reduces surface tension and prevents alveolar collapse upon exhalation of air. Surfactant, which is comprised of 80% phospholipid, 10% cholesterol, and 10% protein, is stored in type II pneumonocytes within unique lamellated organelles, termed lamellar bodies, before secretion into the alveolar lumen (1). Type II pneumonocytes constitute ~10% of the total cell population of adult lung and are first detected in human fetal lung after 20–24 weeks of gestation; however, augmented surfactant synthesis is initiated in fetal lung only after 75–85% of gestation is complete. To define the mechanisms for the regulation of type II cell differentiation and surfactant synthesis in fetal lung, we have focused on the gene encoding surfactant protein-A (SP-A), a major surfactant protein that is developmentally regulated in concert with appearance of differentiated type II cells and augmented surfactant phospholipid synthesis (2). We have observed that treatment of midgestation fetal lung in organ culture with cAMP analogs and agents that increase intracellular cAMP, such as PGE2, increases expression of the gene encoding SP-A, causes enlargement of the prealveolar ducts, and accelerates appearance of differentiated type II pneumonocytes (3, 4).

The high rate of synthesis of surfactant phospholipids by type II pneumonocytes and their storage in the cytoplasm as lamellar bodies is analogous to the synthesis of triacylglycerol and accumulation of lipid droplets by adipocytes. Although relatively little is known regarding the mechanisms involved in type II pneumonocyte differentiation, considerable progress has been made recently toward understanding the cellular and molecular mechanisms involved in differentiation of preadipocytes to adipocytes. Adipocyte differentiation has been found to be regulated by the concerted actions of insulin/insulin-like growth factor-I, glucocorticoids, cAMP, fatty acids, and peroxisome proliferators (5).

At the molecular level, families of transcription factors, including the peroxisome proliferator-activated receptors (PPAR), the CCAAT/enhancer binding proteins (C/EBP), and basic helix-loop-helix-leucine zipper proteins, respond to regulatory hormones and factors to mediate transcriptional activation of adipocyte-specific genes that create and maintain the adipocyte phenotype (6, 7, 8). PPAR{gamma}, an adipose-restricted nuclear receptor, is induced early in adipose differentiation (9, 10) and responds to lipid activators and thiazolidinediones to stimulate adipocyte differentiation and modulate the expression of target genes, including adipocyte P2 (aP2), PEPCK, and leptin (6, 9, 10, 11). Direct evidence for the critical role of PPAR{gamma} in adipose development was provided by Tontonoz et al. (6), who demonstrated that ectopic expression of PPAR{gamma} in fibroblasts treated with external inducers is sufficient to induce differentiation of fibroblasts to adipocytes.

To date, the genetic mechanisms that control differentiation of type II pneumonocytes from their epithelial cell progenitors and that regulate surfactant protein and lipid synthesis remain unknown. In previous studies to define the cis-acting elements involved in type II cell-specific and cAMP regulation of SP-A gene transcription, we transfected fetal type II cells with fusion genes containing 5'-flanking sequences from the rabbit and human SP-A genes linked to the human GH gene, as reporter (12). We observed that cAMP regulation of SP-A promoter activity in type II cells was dependent upon the concerted interactions of at least three types of regulatory elements: a cAMP-responsive element (CREsp-a)-like sequence at -260 bp (12, 13, 14); E-boxes at -80 and -1000 bp (15), which bind USF-1 and -2 (Gao, Wang, Alcorn, and Mendelson, unpublished observations); and a GT box at -65 bp, which binds Sp1 and related factors (16). We observed that CREsp-a did not bind CREB or related factors, including CREM and ATF-1. Rather, our findings suggested that this sequence serves as a binding site for a member of the nuclear receptor superfamily (13). In the present study, a degenerate PCR screen was designed to identify nuclear receptors expressed in type II pneumonocytes. Several clones were isolated that displayed homology to PPAR isoforms; a significant proportion of these were the PPAR{gamma} gene product. In consideration of the role that PPAR{gamma} plays in adipocyte differentiation and lipid synthesis, our objective was to characterize PPAR{gamma} expression patterns during type II pneumonocyte differentiation. Although PPAR{gamma} in type II cell nuclear extracts does not bind to CREsp-a in vitro, our findings indicate that PPAR{gamma}1 messenger RNA (mRNA) expression and protein synthesis are increased by cAMP analogs in alveolar type II pneumonocytes and that PPAR{gamma}1 expression is induced in association with the initiation of SP-A gene expression that occurs during type II pneumonocyte differentiation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Organ culture and type II pneumonocyte, epithelial cell, and fibroblast isolation.
Lung tissues, from 16–20-week gestation human abortuses, were obtained in accordance with the Donors Anatomical Gift Act of the State of Texas. Consent forms and protocols were approved by the Human Research Review Committee of the University of Texas Southwestern Medical Center at Dallas. Lung tissues also were obtained from 23-day gestation fetal rabbits (New Zealand white) that were treated in accordance with the NIH Guide for the Use and Care of Laboratory Animals. The human and rabbit fetal lung tissues were minced and cultured on lens paper supported by stainless steel grids in serum-free Waymouth’s MB752/1 medium (Gibco BRL Life Technologies, Grand Island, NY) in the presence of 1 mM Bt2cAMP (Boehringer Mannheim Corp., Indianapolis, IN), as described previously (17). After 4 days of culture, lung explants were digested with collagenase type I (0.5 mg/ml; Sigma Chemical Co) and collagenase type IA (0.5 mg/ml; Sigma Chemical Co.) for 15 min at 37 C with vigorous shaking. Fibroblasts were isolated from the cell suspension by plating the cells onto 100-mm tissue culture dishes in the presence of 10% FBS for 45 min at 37 C. When the supernatant was removed, the adhered fibroblasts were washed twice with serum-free medium and subsequently incubated in serum-free Waymouth’s MB752/1 medium in the absence or presence of 1 mM Bt2cAMP. To isolate type II pneumonocytes after collagenase treatment, the cell suspension was treated with DEAE-dextran (250 µg/ml, Sigma Chemical Co.) and incubated for 45 min with shaking at 37 C. Cells were pelleted at 400 x g and plated onto 60-mm tissue culture dishes that were coated with extracellular matrix prepared from MDCK cells (ATCC CRL 6253) at a density of 2–5 x 106 cells per dish (18). The extracellular matrix dishes were prepared from confluent monolayers of MDCK cells that were treated with 1% deoxycholate for 5 min. The dishes were washed three times with HBSS and stored at 37 C until needed (18). Type II pneumonocytes were incubated overnight in Waymouth’s medium with 10% FBS. Dishes were washed twice with serum-free medium and subsequently incubated in serum-free Waymouth’s MB752/1 medium in the absence or presence of 1 mM Bt2cAMP. Undifferentiated epithelial cells were isolated by the method described for the isolation of type II pneumonocytes; however, the fetal lung tissue was not cultured before epithelial cell isolation.

Degenerate PCR.
A degenerate PCR strategy was adapted from that described by Hirose et al. (19). Poly A(+) RNA was isolated from fetal rabbit lung type II cells that were treated in culture with 1 mM Bt2cAMP for 24 h. First- and second-strand complementary DNA (cDNA) was prepared from 1 µg poly A(+) RNA using Superscript Choice System for cDNA Synthesis (Gibco BRL Life Technologies). This pool of cDNA was used as template in the PCR reaction with degenerate primers that were designed according to two conserved segments of the DNA-binding domain shared by GR, RXR, RAR, NGFI-B, and Ad4BP/SF-1. PCR was performed in a 100-µl reaction containing 40 µM deoxynucleotide triphosphates (dNTPs), 180 ng of each degenerate primer [5' primer: 5'-ACGTCTGCAGTG(T/C)GA(A/G)(A/G)G(A/C/G/T)TG(T/C)AA-(A/G)(G/T)(A/C/G/T)TT(C/T)T-3'], [3' primer: 5'-ACGTGAATTC(A/C/G/T)A(A/G)(A/G)CA(C/T)TT(A/C/G/T)(C/T)(G/T)(A/C/G/T)(A/T)(A/G)(A/C/G/T)C(G/T)(A/G)CA-3'], 100 ng of cDNA template, 1x reaction buffer, and AmpliTaq polymerase (Perkin Elmer Cetus, Norwalk, CT). PCR conditions included a single 3-min 93-C melting cycle, three cycles of 93 C for 1 min, 37 C annealing for 1 min with a ramp to 72 C polymerase reaction for 2.5 min, thirty cycles of 93 C for 1 min, 53 C annealing for 1 min, and 72 C polymerase reaction for 2.5 min. The PCR products were subjected to electrophoresis through polyacrylamide, the expected band size of 154 bp was extracted, and the fragments were subcloned into the pCRII vector (Invitrogen, San Diego, CA). Subcloned PCR products were sequenced (Sequenase, USB, Cleveland, OH), and the GenBank database was searched for sequence similarity using the FASTA algorithm in the GCG software package.

Southern screen of a rabbit type II cell cDNA library.
A cDNA expression library was constructed from mRNA isolated from fetal rabbit lung type II cells that were maintained in culture in the presence of 1 mM Bt2cAMP for 3 days. The cDNAs were ligated into the Lambda ZAP II vector (Stratagene, La Jolla, CA). A PCR product, encoding the DNA-binding domain of rabbit PPAR{gamma}1, was used as probe to screen 4 x 105 plaques. Prehybridizations and hybridizations were performed at 42 C in 6x SSC (1x SSC = 0.15 M NaCl; 0.015 M Na citrate, pH 7.0), 5x Denhardt’s, 0.5% SDS, 50% formamide, and 100 µg/ml denatured salmon sperm DNA. High stringency washes were performed at 50 C using 0.1x SSC, 0.1% SDS, and 1 mM EDTA. Plaques that hybridized to the PPAR{gamma} probe were selected and purified by two rounds of screening. cDNA sequences of plaque-purified clones were subcloned into pBluescript SK (Stratagene) by in vivo excision from the {lambda}ZAPII phage, as described by the manufacturer, and sequenced using a series of internal primers.

RT-PCR.
Total RNA was isolated (20) from fetal rabbit and human fetal lung type II cells that were cultured in serum-free Waymouth’s MB752/1 medium in the absence or presence of 1 mM Bt2cAMP for 3 days and from uncultured adult rabbit and human adipose tissues. RNA (2.5 µg) was used as template for the synthesis of single-strand DNA using the First-Strand cDNA Synthesis Kit (Pharmacia Biotech Inc., Piscataway, NJ). The resulting DNA samples were templates for PCR amplification in a 50-µl reaction containing 40 µM dNTPs, 300 ng of either 5' primer with the common 3' primer [5' PPAR{gamma}2-specific primer: 5'-GAATTCATATGGGTGAAACTCTGGGA-3'] [5' PPAR{gamma}1-specific primer: 5'-GATTCATATGGTTGACACAGAGATG-3'] [3' common PPAR{gamma}1 and -{gamma}2 primer: 5'-CTAGGAATTCTATCATAAATAAGCTTCAAT-3'], 100 ng of cDNA template, 1x reaction buffer and AmpliTaq polymerase (Perkin Elmer Cetus). PCR conditions included a single 3-min 93-C melting cycle, thirty cycles of 93 C for 30 sec, 53 C annealing for 1 min, and 72 C polymerase reaction for 2 min. PCR products were separated by electrophoresis through a 1.5% agarose gel, visualized by ethidium bromide stain, and photographed. Subsequently, the PCR products were transferred to Zeta probe membrane by capillary transfer in 0.4 M NaOH and hybridized with a 32P-labeled oligonucleotide [5'-AAGACTCAGCTCTACAAT-3'] that is complementary to both PPAR{gamma} isoforms and is contained within the expected PPAR{gamma}1 and -{gamma}2 amplified PCR products. The resulting Southern blot was visualized by autoradiography. The expected PCR and Southern blot product sizes for PPAR{gamma}1 and PPAR{gamma}2 were 433 bp and 523 bp, respectively.

Northern analysis.
Thirty micrograms of total RNA, isolated using the guanidinium isothiocyanate method (20) from adult rabbit adipose, liver, heart, lung, and from fetal rabbit lung type II cells or fibroblasts cultured in the absence or presence of 1 mM Bt2cAMP, were subjected to Northern analysis (21) using radiolabeled, full-length PPAR{gamma}1 cDNA as probe. The blots were stripped and reprobed with a 32P-labeled oligonucleotide, specific for rabbit GAPDH (5'-TCCAGGCGGCAGCTCAGGTCCACG-3'), to control for loading variances.

Electrophoretic mobility shift assay.
Electrophoretic mobility shift assay was performed using a PPAR response element (PPRE) oligonucleotide that corresponds to the ARE6 sequence of the adipocyte P2 gene (5'-CTAGGATCTGTGACCTTTGTCCTAGTAAG-3') and nuclear extracts that were prepared from type II pneumonocytes, as described by Dignam et al. (22). The PPRE was radiolabeled by Klenow fill-in reaction and was incubated in a binding reaction containing 20 mM HEPES, pH 7.6, 75 mM KCl, 0.2 mM EDTA, 20% glycerol, 1 µg poly (dI-dC)-poly (dI-dC) (Pharmacia) with 2 µg of type II pneumonocyte nuclear extract. Antibodies directed against either PPAR{gamma} (23) or C/EBPß (Santa Cruz Biotechnologies, Santa Cruz, CA) were included in the binding reaction, as indicated. The binding complexes were separated by nondenaturing 5% PAGE and were visualized by autoradiography.

Western blot analysis.
Total protein extract (50 µg) from primary cultures of human fetal lung type II cells and human fetal lung epithelial cells, incubated with 1 mM Bt2cAMP for various periods of time, were subjected to Western blot analysis (ECL, Amersham, Arlington Heights, IL). Total proteins also were isolated from human adipose tissue obtained from a reduction mammoplasty as positive control.

Immunoprecipitation.
Human fetal lung type II pneumonocytes were cultured in the absence or presence of 1 mM Bt2cAMP for 5 days. The cells were washed with TBS and maintained for 1 h in DMEM minus L-methionine and L-cysteine (Gibco BRL Life Technologies). Cellular proteins were metabolically labeled for 2 h by the addition of 125 µCi/ml L[35S]methionine (DuPont-New England Nuclear, Boston, MA). After washing the cells, 1 ml RIPA (50 mM Tris, pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% Triton-X100, 1% sodium deoxycholate, 0.1% SDS), containing standard protease inhibitors, was added to each dish, and cells were disrupted completely by scraping and shearing through a 27-gauge needle. After pelleting the cellular debris, 1 x 107 cpm of lysate was precleared for 1 h at 4 C with 40 µl Protein A/G PLUS agarose (Santa Cruz Biotechnology). Precleared lysates were exposed either to 5 µl nonimmune rabbit serum or 5 µl PPAR{gamma}-specific antiserum (23) for 1 h at 4 C. Immune complexes were collected with 30 µl Protein A/G PLUS agarose for 1 h at 4 C. Pellets were washed twice with RIPA, twice with high-salt RIPA (RIPA containing 1 M NaCl), and twice more with RIPA. Pellets were resuspended in 2x SDS loading buffer and separated by electrophoresis through an 11% SDS polyacrylamide gel. The gels were treated with En3Hance (DuPont NEN), dried, and autoradiographed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Alveolar type II pneumonocytes express the nuclear receptor PPAR{gamma}
To evaluate the expression of nuclear receptor superfamily members in type II pneumonocytes, degenerate primers were designed corresponding to two conserved segments of the DNA-binding domain shared by GR, RXR, RAR, NGFI-B, and Ad4BP/SF-1. The DNA-binding domains of these receptors were chosen because they represent the three classes of receptors (homodimeric, heterodimeric, and monomeric DNA-binding proteins). The primers were used in PCR with cDNA synthesized from RNA isolated from rabbit type II pneumonocytes that were cultured in the presence of dibutyryl cAMP (Bt2cAMP), which promotes the differentiated phenotype. PCR products that were of the predicted size of 154 bp were subcloned and sequenced. Of the putative DNA-binding domains that were sequenced, approximately 56% were homologous to the DNA-binding domain of various retinoic acid receptor isoforms (24), 8% encoded ARP1/COUP-TFII (25), and 4% encoded each of NURR1 (26) and Rev-ErbA-{alpha} (27). Interestingly, 26% of the PCR products encoded the DNA-binding domain of members of the peroxisome proliferator-activated receptor subfamily (10, 28, 29, 30); 38% of these were homologous to the PPAR{gamma} isoform (10, 30). Because type II pneumonocytes actively synthesize lipids, we were intrigued to find evidence for the adipose-specific, lipid-activated PPAR{gamma} isoform in these cells.

To obtain a full-length clone of rabbit PPAR{gamma} from alveolar type II pneumonocytes, a rabbit type II pneumonocyte cDNA library was screened using the PCR-amplified DNA-binding domain of PPAR{gamma} as probe. Of the 4 x 105 independent plaques screened, three positive clones were obtained. Two of these encoded portions of the PPAR{gamma} mRNA, and one clone of 1759 bp, contained the entire open reading frame of PPAR{gamma}1, 130 bp of 5'-untranslated region, and 203 bp of 3'-untranslated region (Fig. 1Go). The mouse PPAR{gamma} (mPPAR{gamma}) gene uses alternative promoters to give rise to two different PPAR{gamma} isoforms, mPPAR{gamma}1 and mPPAR{gamma}2. The mPPAR{gamma}2 isoform differs from mPPAR{gamma}1 in that the former contains an additional 30 amino acids N-terminal to the initiating codon of mPPAR{gamma}1 and has a different 5'-untranslated region (31). The coding region of rabbit PPAR{gamma}1 (rPPAR{gamma}1) shares 90% nucleotide identity and 95% amino acid identity with mPPAR{gamma}1.



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  		Figure 1. Nucleotide and deduced amino acid sequence of the rPPAR{gamma}1 cDNA. Nucleotides are numbered to the right of the sequence. The initiation codon is located at nucleotide 131, and the termination codon is indicated as an asterisk at nucleotide 1555. Both the 5'- and 3'-untranslated regions are depicted in italics. This sequence has been assigned GenBank accession number U84893.

 
PPAR{gamma}1 mRNA is present in alveolar type II pneumonocytes
Because the mPPAR{gamma} gene can potentially yield two distinct cDNA products, PPAR{gamma}1 and PPAR{gamma}2, we sought to determine whether one or both isoforms were present in type II pneumonocytes. Although both isoforms are highly expressed in mouse and human adipose tissues, PPAR{gamma}2 expression is more restricted to adipose tissue, as compared with PPAR{gamma}1 (10, 30). To assess the expression of both isoforms in type II pneumonocytes, an RT-PCR strategy was used. Upstream oligonucleotide primers, complementary to exons I of either PPAR{gamma}1 or PPAR{gamma}2, were designed. Both upstream primers contained the nucleotide sequence encoding the respective initiating codons and the five amino acid codons that followed. Sequence comparison of the first six codons of each PPAR{gamma} isoform between mouse and human revealed 100% nucleotide identity. This suggests that these regions are strictly conserved among species. The downstream primer was complementary to internal cDNA sequences that are common to both PPAR{gamma} isoforms. The expected sizes of the PPAR{gamma}1- and PPAR{gamma}2-specific PCR products are 433 bp and 523 bp, respectively.

PPAR{gamma}2 cDNA was previously isolated from human and mouse adipocyte cDNA libraries, whereas rabbit PPAR{gamma}2 has not been described. RT-PCR analysis was, therefore, performed using RNA isolated from adipose and type II pneumonocytes from both human and rabbit. RT-PCR analysis of PPAR{gamma}1 and PPAR{gamma}2 expression in human adipose tissue revealed expression of both isoforms and thereby served as a positive control (Fig. 2Go). Surprisingly, only the PPAR{gamma}1 isoform was detected in rabbit adipose tissue. This observation may be interpreted to suggest either that the nucleotide sequence encoding the amino terminal regions of rabbit PPAR{gamma}2 is not conserved with mouse and human, or that rabbit does not express, and therefore does not require, PPAR{gamma}2 for adipocyte differentiation. Consistent with the cDNA library screen results, RT-PCR of RNA isolated from differentiated type II pneumonocytes of both human and rabbit fetal lung amplified only the PPAR{gamma}1-specific sequences (Fig. 2Go). Longer exposure of the Southern blot failed to reveal PPAR{gamma}2-specific transcripts either in human or rabbit type II pneumonocytes (data not shown).



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  		Figure 2. RT-PCR analysis of PPAR{gamma}1 and PPAR{gamma}2 expression in human and rabbit adipose and type II pneumonocytes. Total RNA was isolated from adult human (h) and rabbit (r) adipose (Ad) tissues and from fetal human and rabbit type II pneumonocytes (tII) that were incubated in the absence (C) or presence of 1 mM Bt2cAMP (Bt2) for 3 days. First-strand cDNA was synthesized from 2 µg of total RNA using random primers. PCR was targeted to amplify both PPAR{gamma} isoforms independently by use of specific primers complementary to the unique exons I of either PPAR{gamma}1 or PPAR{gamma}2 and a common internal 3' PPAR{gamma} primer. Attempted amplification of PPAR{gamma}1 is denoted (as 1) above each respective lane, and PPAR{gamma}2 is denoted (as 2). The expected sizes of the PPAR{gamma}1- and PPAR{gamma}2-specific PCR products are 433 bp and 523 bp, respectively. PCR products were separated by 1.5% agarose electrophoresis, visualized by ethidium bromide staining (upper panel) and subjected to southern blotting using a common internal radiolabeled probe (bottom panel). M, 1-kb molecular mass marker.

 
Cyclic AMP treatment of type II pneumonocytes increases PPAR{gamma}1 mRNA and protein synthesis
The RT-PCR analysis revealed that only the PPAR{gamma}1 isoform is expressed in type II pneumonocytes. To assess the relative levels of PPAR{gamma}1 mRNA expression in different rabbit tissues, aliquots of total RNA (30 µg) from adult rabbit adipose, liver and heart, from 25-day gestational age fetal rabbit lung, and from fetal rabbit type II pneumonocytes, cultured in the presence of 1 mM Bt2cAMP for 3 days, were analyzed by Northern blotting using a full-length rabbit PPAR{gamma}1 cDNA as probe (Fig. 3AGo). PPAR{gamma}1 expression was detectable in the cultured type II pneumonocytes; however, its expression level was considerably less than that of adipose tissue. By contrast, PPAR{gamma} transcripts were undetectable in whole lung and in all other tissues examined. This observed enrichment of PPAR{gamma}1 transcripts in fetal rabbit type II pneumonocytes, as compared with whole lung tissue, led us to further investigate its lung cell-specificity and cAMP-regulated expression. Fibroblasts and type II pneumonocytes were isolated from 23-day gestational age fetal rabbit lung explants that had been maintained in culture for 4 days. The type II cells and fibroblasts were cultured in the absence or presence of Bt2cAMP for 3 days. By Northern blot analysis, PPAR{gamma}1 expression was essentially undetectable in fibroblasts maintained in the absence or presence of Bt2cAMP (Fig. 3BGo). By contrast, PPAR{gamma}1 mRNA was detectable in type II cells cultured in control medium and was increased 8-fold when the type II pneumonocytes were cultured in medium containing Bt2cAMP. The level of PPAR{gamma}1 mRNA was 2.5-fold higher in adipose, as compared with the level of PPAR{gamma}1 mRNA in type II pneumonocytes maintained in medium containing Bt2cAMP. These findings indicate that PPAR{gamma}1 expression in lung is cell type-restricted and that the steady-state level of PPAR{gamma}1 mRNA in type II pneumonocytes is increased by cAMP treatment.



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  		Figure 3. Northern blot analysis of PPAR{gamma} expression in various tissues and in pulmonary fibroblast and type II pneumonocytes cultured in the absence or presence of Bt2cAMP. A. Northern blot analysis of 30 µg of total RNA isolated from adult rabbit adipose (A), liver (Li), and heart (H), from 25-day gestational age fetal rabbit lung (Lu), and from fetal rabbit type II pneumonocytes (tII) cultured in the presence of 1 mM Bt2cAMP for 3 days. B, Northern blot analysis of rabbit pulmonary fibroblasts (Fib) and type II pneumonocytes (tII) cultured either in the absence (-) or presence (+) of 1 mM Bt2cAMP for 3 days. Full-length rabbit PPAR{gamma}1 was used as probe (upper panels), as indicated, and loading variances were detected by using GAPDH as probe (lower panels).

 
To analyze the effects of cAMP on the rate of synthesis of PPAR{gamma}1 protein, type II pneumonocytes, isolated from midgestation human fetal lung explants, were cultured for 5 days in the absence or presence of Bt2cAMP. On the 5th day of culture, the cells were incubated with [35S]-methionine for 2 h, and PPAR{gamma} was immunoprecipitated. The incorporation of [35S]-methionine into PPAR{gamma}1 protein was ~3-fold higher in Bt2cAMP-treated type II pneumonocytes (Fig. 4Go, tII-B), as compared with control (Fig. 4Go, tII-C). These findings indicate that synthesis of both PPAR{gamma}1 mRNA and protein is induced by Bt2cAMP in type II pneumonocytes.



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  		Figure 4. Immunoprecipitation of PPAR{gamma}1 from metabolically labeled type II pneumonocytes cultured in the absence or presence of Bt2cAMP. Human fetal lung type II cells were cultured in the absence or presence of 1 mM Bt2cAMP for 5 days. The cells were then incubated for 1 h in medium lacking L-methionine and L-cysteine and then for 2 h with 125 µCi/ml L-[35S]methionine. Lysates precleared with Protein A/G PLUS agarose were incubated either with 5 µl nonimmune rabbit serum or 5 µl PPAR{gamma}-specific antiserum. Immune complexes were collected, washed, separated by electrophoresis through an 11% SDS polyacrylamide gel, and exposed to x-ray film.

 
The DNA-binding characteristics of type II pneumonocyte-derived PPAR{gamma}1 were evaluated by electrophoretic mobility shift assay using a PPRE from the aP2 gene as probe (10). Nuclear extracts from type II pneumonocytes bound specifically to the radiolabeled PPRE as two major complexes (Fig. 5Go). This likely comprises the binding of all PPAR isoforms in the type II pneumonocytes, as well as other proteins that have the capacity to bind to the DR1 probe, including COUP-TF. Nuclear protein binding to the PPRE was effectively competed by the presence of a 100-fold molar excess of nonradiolabeled probe. Binding to the PPRE was unaffected by a 100-fold molar excess of a nonradiolabeled consensus CRE (32) (Fig. 5Go). When added to the binding reaction, antibodies directed against PPAR{gamma} produced a distinctive supershifted complex and reduced the formation of the faster-migrating complex. Neither addition of nonimmune serum nor antibodies directed against C/EBPß disrupted or supershifted the binding complexes. These findings indicate that PPAR{gamma}1 of type II pneumonocytes binds to a PPRE, and they further substantiate that PPAR{gamma} protein is present in type II pneumonocytes. In parallel studies, we observed that the PPAR{gamma}-specific antibody failed to supershift a complex of type II cell nuclear proteins bound to CREsp-a, suggesting that PPAR{gamma} does not bind to this site (data not shown).



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  		Figure 5. Competition and supershift electrophoretic mobility shift assay (EMSA) using type II pneumonocyte nuclear extracts and a radiolabeled PPRE as probe. Nuclear proteins from type II pneu-monocytes (+tII NE) were incubated with a 32P-labeled PPRE. Nonradiolabeled competitors (PPRE and a consensus CRE from the rat GH gene) were added at 100-fold molar excess of radiolabeled probe. Rabbit antisera (2 µl) directed against PPAR{gamma} and C/EBPß (PPAR{gamma} Ab, C/EBPß Ab) and nonimmune rabbit serum were added independently to the binding reactions. To demonstrate that the PPAR{gamma} antiserum did not interact nonspecifically with the probe, radiolabeled PPRE was incubated with PPAR{gamma} Ab in the absence of type II cell nuclear proteins (far right lane). DNA-protein complexes were separated from free probe by nondenaturing PAGE and visualized by autoradiography.

 
PPAR{gamma}1 protein levels are induced during type II pneumonocyte differentiation
Because PPAR{gamma}1 is present in differentiated type II pneumonocytes, it was of interest to analyze changes in PPAR{gamma}1 expression during differentiation of alveolar epithelial cells to type II pneumonocytes. We have developed a method for isolation of undifferentiated epithelial cells from midgestation human fetal lung. Upon culture of the epithelial cells in medium containing Bt2cAMP, an induction of mRNA encoding the type II-cell specific surfactant protein, SP-A, occurs in a time-dependent manner (J. L. Alcorn, M. E. Smith, and C. R. Mendelson, unpublished observations). We used this culture system and Western blot analysis to examine the developmental expression patterns of PPAR{gamma}1 and SP-A proteins during the differentiation of lung epithelial cells to type II pneumonocytes. Before culture, the undifferentiated lung epithelial cells did not contain either immunoreactive PPAR{gamma}1 or SP-A (data not shown). Likewise, in lysates of primary alveolar epithelial cells that were cultured for 12 h in the absence or presence of Bt2cAMP, neither PPAR{gamma}1 nor SP-A proteins were detectable (Fig. 6Go, epi 0.5d lanes). By contrast, PPAR{gamma}1 expression was induced in epithelial cells maintained in control and Bt2cAMP-containing medium for 48 h (Fig. 6Go, epi 2d). SP-A protein also was detected at this time point in the epithelial cells maintained in the presence of Bt2cAMP (Fig. 6Go, epi 2d). We also analyzed immunoreactive SP-A and PPAR{gamma}1 proteins in midgestation human fetal lung explants cultured for 3 days in the absence or presence of Bt2cAMP (exp 3d) and in differentiated type II pneumonocytes that were isolated from the explants and incubated for 3 additional days in the absence or presence of Bt2cAMP (tII). Whereas, immunoreactive SP-A was detected in the untreated human fetal lung explants and was induced markedly by Bt2cAMP treatment, immunoreactive PPAR{gamma} was undetectable in control and cAMP-treated tissues. The inability to detect immunoreactive PPAR{gamma} in the cultured fetal lung explants likely is caused by its low level of expression and the fact that type II cells only comprise a fraction of the total cell population. By contrast, in the control and Bt2cAMP-treated type II cells, the levels of immunoreactive PPAR{gamma} were markedly increased. Immunoreactive SP-A levels were elevated in the cultured type II pneumonocytes and induced by Bt2cAMP (Fig. 6Go, tII). We were surprised to find that the steady-state levels of immunoreactive PPAR{gamma}1 protein in control and Bt2cAMP-treated primary alveolar epithelial cells and type II pneumonocytes were not induced by cAMP treatment. The apparent lack of stimulatory effect of cAMP on the steady-state levels of PPAR{gamma}1 protein, despite its inductive effect on PPAR{gamma}1 mRNA and de novo protein synthesis, could possibly be caused by an increased rate of turnover of PPAR{gamma}1 protein in the cAMP-treated cells. Taken together, these findings indicate that during type II pneumonocyte differentiation, the induction of PPAR{gamma}1 expression is coincident with the induction of SP-A expression, a marker of type II cell differentiation. In contrast to the stimulatory effect of cAMP on PPAR{gamma} mRNA levels and on rate of PPAR{gamma} protein synthesis, there is no apparent stimulatory effect of cAMP on the levels of immunoreactive PPAR{gamma}, either in cultured epithelial cells or in differentiated type II cells.



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  		Figure 6. Western blot analyses of PPAR{gamma}1 and of SP-A in cultured human fetal lung explants, in pulmonary epithelial cells during differentiation in culture, and in cultured type II pneumonocytes. Total protein extracts (50 µg) from midgestation human fetal lung explants incubated for 3 days in the absence or presence of Bt2cAMP (1 mM), and from primary fetal lung epithelial cells (epi), and alveolar type II cells (tII) incubated for various periods of time in the absence or presence of Bt2cAMP, were subjected to Western blot analysis using antisera directed against PPAR{gamma} (top panel) and SP-A (bottom panel). Total proteins isolated from human adipose tissue (A) also were analyzed as positive control for immunoreactive PPAR{gamma}.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In previous studies, we have found that cAMP has profound stimulatory effects on the rate of type II cell differentiation in fetal lung in culture and on expression of the gene encoding SP-A (4). In studies to define the regulatory elements of the SP-A gene that mediate cAMP-responsive transcription in type II cells, we identified a CRE-like element (CREsp-a, TGACCT) that binds type II cell nuclear proteins and is required for cAMP induction of SP-A promoter activity (13, 14). Characterization of the proteins that bind to this element revealed that they are likely members of the nuclear receptor superfamily (13). In the present study, we used degenerate PCR with cDNA, prepared from mRNA isolated from cAMP-treated rabbit type II pneumonocytes, to amplify members of the nuclear receptor superfamily that are expressed in this cell type. A large proportion of the amplified cDNAs encoded PPAR{gamma}, an adipocyte-enriched member of the PPAR family. Although we found that PPAR{gamma} does not seem to bind to CREsp-a, the finding of PPAR{gamma} expression in type II pneumonocytes was intriguing, because type II pneumonocytes, like adipocytes, synthesize and store large amounts of lipid. In consideration of the well-characterized role of PPAR{gamma} in adipocyte differentiation (10), we hypothesized that it also may play a role in the differentiation of type II pneumonocytes from their epithelial cell precursors.

The PPAR{gamma} gene in mouse and human yields two mRNA species, PPAR{gamma}1 and PPAR{gamma}2, each possessing distinct 5' exons that are spliced onto common downstream sequences. The variant 5' regions arise from alternative promoter usage (31). Although PPAR{gamma}1 is encoded by eight exons and PPAR{gamma}2 is encoded by seven exons, the last six exons are identical for both PPAR{gamma}1 and PPAR{gamma}2 (31). Both PPAR{gamma} isoforms are highly expressed in adipose tissue; however, the PPAR{gamma}2 isoform displays strict tissue-specific expression in adipose, as compared with the PPAR{gamma}1 isoform, which was originally isolated from liver (30). We observed that type II pneumonocytes from human and rabbit only express mRNA of the PPAR{gamma}1 isoform, as demonstrated both by a cDNA library screen and by RT-PCR (Figs. 1Go and 2Go). Likewise, immunoprecipitation and immunoblotting demonstrated that a protein consistent in size only with the PPAR{gamma}1 protein isoform is present in type II pneumonocytes. In studies by Tontonoz et al. (6), ectopic expression of the PPAR{gamma}2 isoform was found to be sufficient to stimulate adipose differentiation of fibroblast cell lines in a lipid-dependent manner, suggesting that this adipocyte-specific PPAR{gamma} isoform is adipogenic. The importance of the additional 30 amino acids within the amino-terminal domain of PPAR{gamma}2 was analyzed for its adipogenic potential by the creation of a PPAR{gamma}2 protein that lacks the amino-terminal 127 amino acids (6). Interestingly, overexpression of the truncated PPAR{gamma}2 caused fibroblasts to differentiate into adipocytes in the presence of activators at an even higher rate than fibroblasts expressing wild-type PPAR{gamma}2 (6). These findings indicate that the amino-terminal domain of PPAR{gamma} is not required for promoting adipogenesis and that PPAR{gamma}1 has adipogenic potential equal to or greater than PPAR{gamma}2.

By contrast, a functional DNA-binding domain is absolutely required for the adipogenic activities of PPAR{gamma}2. Mutation of the PPAR{gamma}2 DNA-binding domain was found to result in the inability of PPAR{gamma}2 to convert fibroblasts into adipocytes (6). In the present study, we found that type II pneumonocyte nuclear proteins bind to a PPRE, a direct repeat of the sequence AGGTCA with a one-nucleotide spacer (DR1), from the aP2 gene (Fig. 5Go). Supershift analysis, using a PPAR{gamma}-specific antibody, revealed the presence of PPAR{gamma} in the binding complex. On the other hand, the PPAR{gamma} antibody failed to supershift a binding complex of type II cell nuclear proteins with radiolabeled CREsp-a, suggesting that PPAR{gamma} does not bind to this nuclear receptor half-site of the SP-A gene (data not shown).

Treatment of fetal lung in organ culture with cAMP analogs or with agents that increase intracellular cAMP, such as PGE2, causes accelerated appearance of differentiated type II pneumonocytes and enlargement of the prealveolar ducts (3). Transcription of the type II pneumonocyte-specific gene, SP-A, is markedly increased in fetal lung cultured in the presence of Bt2cAMP (2). In the present study, we found that the steady-state levels of PPAR{gamma}1 mRNA were increased ~8-fold (Fig. 3Go), and de novo synthesis of PPAR{gamma}1 protein was induced ~3-fold (Fig. 4Go) in type II cells maintained in medium containing Bt2cAMP, as compared with cells maintained in control medium. These findings are the first to describe an effect of cAMP to stimulate accumulation of PPAR{gamma} mRNA and induction of PPAR{gamma} protein synthesis in any tissue. Furthermore, an increase in the levels of immunoreactive PPAR{gamma}1 was found to occur in association with differentiation of lung epithelial cells to type II pneumonocytes in culture (Fig. 6Go).

PPAR{gamma} is induced early in the time course of preadipocyte differentiation, suggesting that it may serve a role in committing the mesenchymal cell to an adipose lineage and in activation of early gene expression (10). We have observed that epithelial cells, isolated from midgestation fetal lung and maintained in primary culture in serum-free medium containing Bt2cAMP, differentiate into type II pneumonocytes. Using this system, we have found that neither PPAR{gamma}1 nor SP-A, a marker of type II cell differentiation, were detectable in freshly isolated epithelial cells or in cells that were cultured for 12 h in the absence or presence of Bt2cAMP (Fig. 6Go). However, both PPAR{gamma}1 and SP-A proteins were detected in epithelial cells after 48 h of culture in control or Bt2cAMP-containing medium. Furthermore, PPAR{gamma}1 and SP-A proteins were induced to relatively high levels in differentiated type II pneumonocytes isolated from cultured human fetal lung explants and maintained for several days in monolayer culture. These findings indicate that PPAR{gamma}1 expression is induced early in the differentiation of epithelial cells to type II pneumonocytes and is sustained in cells displaying the fully differentiated phenotype. In contrast to the stimulatory effects of Bt2cAMP on PPAR{gamma} mRNA levels and on de novo synthesis of PPAR{gamma} protein in cultured type II cells, it paradoxically was found both in lung epithelial cells and in differentiated type II cells that the levels of immunoreactive PPAR{gamma} protein were not increased by cAMP treatment. This was correlated with the finding that Bt2cAMP treatment of type II cells in culture had no effect to increase the binding of nuclear proteins to a PPRE (data not shown). This discrepancy could possibly be caused by an increased rate of turnover of PPAR{gamma} protein in cAMP-treated tissues.

In consideration of the inductive effect of cAMP on PPAR{gamma} mRNA expression and on type II cell differentiation, and the increased levels of PPAR{gamma}1 in differentiated type II cells, it is possible that PPAR{gamma}1 may mediate the regulation of type II pneumonocyte differentiation and surfactant lipoprotein synthesis. In preliminary experiments, we analyzed the effects of the PPAR{gamma} activator BRL49653 on SP-A expression in human and rabbit fetal lung in organ culture. No clear inductive effect of this agent on this marker of type II cell differentiation was observed (L. F. Michael and C. R. Mendelson, unpublished observations). These findings suggest that although PPAR{gamma} is induced in association with type II cell differentiation, it may not mediate establishment of the differentiated phenotype and the induction of SP-A gene expression in lung type II cells. The lack of effect of the PPAR{gamma} activator on SP-A gene expression was not surprising, because we have not identified a PPRE within the 5'-flanking regions of the rabbit or human SP-A genes. Therefore, it is possible that PPAR{gamma} is induced in association with type II cell differentiation and does not serve as a mediator of this process.

Alternatively, PPAR{gamma} may regulate expression of genes encoding the hydrophobic surfactant proteins (SP-B and SP-C), enzymes involved in surfactant glycerophospholipid synthesis, and/or other transcription factors in type II cells; however, these have not, as yet, been defined. In studies by Feinstein and colleagues (33), the lipogenic transcription factor C/EBP{alpha} was found to be induced in fetal rat lung toward the end of gestation and was expressed specifically in differentiated type II pneumonocytes. On the other hand, we have observed that C/EBP{delta} expression is induced in cultured human fetal lung explants before the appearance of differentiated type II cells and is stimulated by cAMP and glucocorticoids (D. R. Breed, L. R. Margraf, and C. R. Mendelson, unpublished observations). In NIH-3T3 cells, ectopic expression of C/EBPß and C/EBP{delta}, in the presence of glucocorticoids, stimulates adipogenesis and PPAR{gamma} expression (34). It has been suggested that C/EBP{alpha}, which is induced by C/EBPß and C/EBP{delta} in 3T3-L1 cells, acts synergistically with PPAR{gamma} to stimulate adipogenesis (6). We believe that identification of the factors that regulate PPAR{gamma} expression in lung type II cells, as well as the PPAR{gamma} target genes, may provide insight into the regulatory mechanisms that control type II pneumonocyte differentiation and the synthesis of surfactant lipoprotein. Elucidation of such mechanisms ultimately may lead to the development of new therapies to increase pulmonary surfactant synthesis in prematurely born infants.


   Acknowledgments
 
The authors are grateful to Margaret Smith and Jo Smith for their expert help with cell and tissue culture. We thank Dr. Serdar Bulun (University of Texas Southwestern) for the generous gifts of human adipose tissue and adipose RNA.


   Footnotes
 
1 This research was supported by NIH Grants HL-50022 (to C.R.M.) and DK-49780 (to M.A.L.). Back

2 Supported by a Predoctoral Fellowship from The Chilton Foundation, Dallas, Texas. Current address: Joslin Diabetes Center, One Joslin Place, Boston, Massachusetts 02215. Back

Received February 12, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Mendelson CR, Boggaram V 1991 Hormonal control of the surfactant system in fetal lung. Annu Rev Physiol 53:415–440[CrossRef][Medline]
  2. Boggaram V, Mendelson CR 1988 Transcriptional regulation of the gene encoding the major surfactant protein (SP-A) in rabbit fetal lung. J Biol Chem 263:19060–19065[Abstract/Free Full Text]
  3. Acarregui MJ, Snyder JM, Mitchell MD, Mendelson CR 1990 Prostaglandins regulate surfactant protein A (SP-A) gene expression in human fetal lung in vitro. Endocrinology 127:1105–1113[Abstract/Free Full Text]
  4. Odom MJ, Snyder JM, Mendelson CR 1987 Adenosine 3',5'-monophosphate analogs and ß-adrenergic agonists induce the synthesis of the major surfactant apoprotein in human fetal lung in vitro. Endocrinology 121:1155–1163[Abstract/Free Full Text]
  5. MacDougald OA, Lane MD 1995 Transcriptional regulation of gene expression during adipocyte differentiation. Annu Rev Biochem 64:345–373[CrossRef][Medline]
  6. Tontonoz P, Hu E, Spiegelman BM 1994 Stimulation of adipogenesis in fibroblasts by PPAR{gamma}2, a lipid-activated transcription factor. Cell 79:1147–1156[CrossRef][Medline]
  7. Umek RM, Friedman AD, McKnight SL 1991 CCAAT-enhancer binding protein: a component of a differentiation switch. Science 251:288–292[Abstract/Free Full Text]
  8. Kim JB, Spiegelman BM 1996 ADD1/SREBP1 promotes adipocyte differentiation and gene expression linked to fatty acid metabolism. Genes Dev 10:1096–1107[Abstract/Free Full Text]
  9. Chawla A, Schwarz EJ, Dimaculangan DD, Lazar MA 1994 Peroxisome proliferator-activated receptor (PPAR){gamma}: adipose-predominant expression and induction early in adipocyte differentiation. Endocrinology 135:798–800[Abstract]
  10. Tontonoz P, Hu E, Graves RA, Budavari AI, Spiegelman BM 1994 mPPAR{gamma}2:tissue-specific regulator of an adipocyte enhancer. Genes Dev 8:1224–1234[Abstract/Free Full Text]
  11. Kallen C, Lazar MA 1996 Antidiabetic thiazolidinediones inhibit leptin (ob) gene expression in 3T3–L1 adipocytes. Proc Natl Acad Sci USA 93:5793–5796[Abstract/Free Full Text]
  12. Alcorn JL, Gao E, Chen Q, Smith ME, Gerard RD, Mendelson CR 1993 Genomic elements involved in transcriptional regulation of the rabbit surfactant protein-A gene. Mol Endocrinol 7:1072–1085[Abstract/Free Full Text]
  13. Michael LF, Alcorn JL, Gao E, Mendelson CR 1996 Characterization of the cyclic adenosine 3',5'-monophosphate response element of the rabbit surfactant protein-A gene: evidence for transactivators distinct from CREB/ATF family members. Mol Endocrinol 10:159–170[Abstract/Free Full Text]
  14. Young PP, Mendelson CR 1996 A CRE-like element plays an essential role in cAMP regulation of human SP-A2 gene in alveolar type II cells. Am J Physiol 271:L287–L299
  15. Gao E, Alcorn JL, Mendelson CR 1993 Identification of enhancers in the 5'-flanking region of the rabbit surfactant protein A (SP-A) gene and characterization of their binding proteins. J Biol Chem 268:19697–19709[Abstract/Free Full Text]
  16. Young PP, Mendelson CR A GT box element is essential for basal and cyclic AMP regulation of the human SP-A2 gene in alveolar type II cells; evidence for the binding of lung nuclear factors distinct from Sp1. Mol Endocrinol, in press
  17. Snyder JM, Johnston JM, Mendelson CR 1981 Differentiation of type II cells of human fetal lung in vitro. Cell Tissue Res 220:17–25[CrossRef][Medline]
  18. Alcorn JL, Smith ME, Smith JF, Margraf LR, Mendelson CR Primary cell culture of human type II pneumonocytes: maintenance of a differentiated phenotype and transfection with recombinant adenoviruses. Am J Respir Cell Mol Biol, in press
  19. Hirose T, Fujimoto W, Yamaai T, Kim KH, Matsuura H, Jetten AM 1994 TAK1: molecular cloning and characterization of a new member of the nuclear receptor superfamily. Mol Endocrinol 8:1667–1680[Abstract/Free Full Text]
  20. Chirgwin JM, Przbyla AE, MacDonald RJ, Rutter WJ 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 118:5294–5299
  21. Sambrook J, Fritsch EF, Maniatis T 1989 Molecular cloning: a laboratory manual. Cold Spring Harbor Press, ed. 2, Cold Spring Harbor, NY
  22. Dignam JD, Lebovitz RM, Roeder R 1983 Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11:1475–1489[Abstract/Free Full Text]
  23. Xue J-C, Schwarz EJ, Chawla A, Lazar MA 1996 Distinct stages in adipogenesis revealed by retinoid inhibition of differentiation after induction of PPAR{gamma}. Mol Cell Biol 16:1567–1575[Abstract]
  24. Zelent A, Krust A, Petkovich M, Kastner P, Chambon P 1989 Cloning of murine alpha and beta retinoic acid receptors and a novel receptor gamma predominantly expressed in skin. Nature 339:714–717[CrossRef][Medline]
  25. Ladias JAA, Karathanasis SK 1991 Regulation of the apolipoprotein AI gene by ARP-1, a novel member of the steroid receptor superfamily. Science 251:561–565[Abstract/Free Full Text]
  26. Law SW, Conneely OM, DeMayo FJ, O’Malley BW 1992 Identification of a new brain-specific transcription factor, NURR1. Mol Endocrinol 6:2129–2135[Abstract/Free Full Text]
  27. Lazar MA, Hodin RA, Darling DS, Chin WW 1989 A novel member of the thyroid/steroid hormone receptor family is encoded by the opposite strand of the rat c-erbA alpha transcriptional unit. Mol Cell Biol 9:1128–1136[Abstract/Free Full Text]
  28. Isseman I, Green S 1990 Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature 347:645–649[CrossRef][Medline]
  29. Schmidt A, Endo N, Rutledge SJ, Vogel R, Shinar D, Rodan GA 1992 Identification of a new member of the steroid hormone receptor superfamily that is activated by a peroxisome proliferator and fatty acid. Mol Endocrinol 6:1634–1641[Abstract/Free Full Text]
  30. Zhu Y, Alvares K, Huang Q, Rao MS, Reddy JK 1993 Cloning of a new member of the peroxisome proliferator activated receptor gene family from mouse liver. J Biol Chem 268:26817–26820[Abstract/Free Full Text]
  31. Zhu Y, Qi C, Korenberg JR, Chen X, Noya D, Rao S, Reddy JK 1995 Structural organization of mouse peroxisome proliferator-activated receptor {gamma} (mPPAR{gamma}) gene: alternative promoter use and different splicing yield two mPPAR{gamma} isoforms. Proc Natl Acad Sci USA 92:7921–7925[Abstract/Free Full Text]
  32. Montminy MR, Sevarino KA, Wagner JA, Mandel G, Goodman RH 1986 Identification of a cyclic-AMP-responsive element within the rat somatostatin gene. Proc Natl Acad Sci USA 83:6682–6686[Abstract/Free Full Text]
  33. Li F, Rosenberg E, Smith CI, Notarfrancesco K, Reisher SR, Shuman H, Feinstein S 1995 Correlation of expression of transcription factor C/EBP{alpha} and surfactant protein genes in lung cells. Am J Physiol 269:L241–L247
  34. Wu Z, Bucher NLR, Farmer SR 1996 Induction of peroxisome proliferator receptor {gamma} during conversion of 3T3 fibroblasts into adipocytes is mediated by C/EBPß, C/EBP{delta}, and glucocorticoids. Mol Cell Biol 16:4128–4136[Abstract]



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