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Causes Alveolar Abnormality in Transgenic Neonatal Lungs
Division of Pulmonary Biology (L.Y., A.N., C.Y.) and The Graduate Program for Molecular and Developmental Biology (C.Y.), Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45229-3039
Address all correspondence and requests for reprints to: Cong Yan, Ph.D., Children’s Hospital Medical Center, Division of Pulmonary Biology, The Children’s Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, Ohio 45229-3039. E-mail: Cong.Yan{at}chmcc.org.
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Abstract |
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was overexpressed under the control of the human surfactant protein-C 3.7-kb promoter or the rat Clara cell secretory protein 2.3-kb promoter. Overexpression of dominant negative RAR was induced by Dox in neonatal lungs from d 1–21 after birth, a critical period for alveolar maturation. This led to substantial alveolar abnormality with increased air space, larger but fewer alveoli, and the diminished alveolar surface area. In these animals, numbers of alveolar epithelial cells were significantly reduced upon Dox treatment. Expression of an RAR downstream target surfactant protein B gene, which is critical for maintaining the surfactant structure, was inhibited upon Dox treatment in alveolar type II epithelial cells. This finding supports a concept that endocrine molecule retinoic acid, and its receptor RARs play a critical role in alveolarization during the neonatal period of the lung. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RA regulates cell differentiation/proliferation and gene transcription via binding to the RA receptor (RAR)/retinoid X receptor (RXR) heterodimer. Both RARs and RXRs belong to the steroid hormone receptor superfamily and function as ligand-dependent DNA-binding transcription factors. RARs include three isotypes designated , ß, and 
. RARs and RXRs have been previously detected in respiratory epithelial cells by immunohistochemical staining (7, 8, 12). RAR is the predominant isotype identified in alveolar type II epithelial cells (8). RAR and RARß double null mutant mice died in utero and had severely hypoplastic lungs, suggesting that they are required for lung development (13). RARs activate downstream genes through recruiting nuclear receptor cofactors upon binding to RA. These coactivators possess intrinsic histone acetyltransferase activities that remodel the chromosome structure in gene activation. Although it still possesses DNA binding/dimerization activity, a dominant negative RAR (dnRAR) lacks the AF-2 domain that is the binding site for interaction with coactivators and suppresses downstream target genes. Previously, the SP-B gene transcription has been shown to be inhibited by dnRAR in the H441 epithelial cell line (14).
Because the RAR/ß double-null mutant mice died in utero, neonatal developing of the lung cannot be assessed in this model system. Therefore, to elucidate the functional role of the RA/RAR signaling pathway in alveolarization during the neonatal period, conditional double-transgenic mouse systems were established. In this system, the normal RA/RAR signaling was blocked during postnatal d 1–21 in mice by overexpressing dnRAR in distal respiratory epithelial cells under the control of the human surfactant protein-C (hSP-C) promoter or the rat Clara cell secretory protein (CCSP) promoter by doxycycline (Dox) treatment. Both promoters are distal respiratory epithelial cell specific in the lung. The morphological changes of the alveolar region in double-transgenic lungs were systematically characterized. The study supports that the RA/RAR signaling is required for normal alveolar formation in the neonatal lungs.
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Materials and Methods |
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Generation of Dox-regulatable dnRAR transgenic mice
To generate the Teto-CMV-dnRAR transgenic mouse line, the dnRAR cDNA (14) was amplified by PCR using a downstream primer (5'-CTCGCTCTAGATTATCACTTGTCATCGTCGTCCTTGTAGTC-3'), an upstream prime (5'-GCGGAATTCGCCACCATGGCCAGCAACAGCAGCTCC-3'), and a wild-type human RAR cDNA as a template. The PCR dnRAR cDNA was digested with EcoRI/XbaI and subcloned downstream of the cytomegalovirus (CMV) minimal promoter linked to seven Tet-responsive elements at the EcoRI and XbaI sites in the pUHK10-3 vector. The expression cassette containing the CMV promoter, the dnRAR cDNA, and the simian virus 40 polyadenylation signaling sequence was dissected out and purified for microinjection into FVB/N mice by the Transgenic Core Facility at University of Cincinnati, College of Medicine. Founder lines were identified by the PCR strategy using an upstream primer in the RAR cDNA coding region (5'-GAA GCG GAG GCC CAG CCG CCC-3') and a downstream primer in the pUHD 10-3 plasmid, (5'-CAT TCC CGA TGA AGA GGC CG-3'). The CCSP-reverse tetracycline responsive transactivator (rtTA) and SP-C rtTA transgenic lines were kindly provided by Dr. J. A. Whitsett and J. Tichelaar, which were genotyped with an upstream primer corresponding to the SP-C promoter (5'-GAC ACA TAT AAG ACC CTG GTC A-3') or to the CCSP promoter (5'-ACT GCC CAT TGC CCA AAC AC-3') and a downstream primer corresponding to the rtTA cDNA coding region (5'-AAA ATC TTG CCA GCT TTC CCC-3'). To detect Dox-induced expression of dnRAR mRNA in double-transgenic mice, 21-d-old young adult double-transgenic mice were treated with or without 0.5 mg/ml of Dox in drinking water for 7 d. Total RNAs were isolated from lungs using the RNA purification kit (QIAGEN, Valencia, CA). RT-PCR was used to detect the dnRAR mRNAs with the SuperScript One-Step RT-PCR kit, using the same pair of DNA oligo-primers that was used for genotyping Teto-CMV-dnRAR transgenic mice.
Analysis of lung histology and immunohistochemistry
After cross-breeding, pups were treated with Dox right after the birth by administrating mothers with drinking water containing Dox at a final concentration of 0.5 mg/ml. The Dox water was replaced three times per week. After 21 d, young animals were genotyped by tail DNA extraction. Nontransgenic, single-transgenic, and double-transgenic young animals were anesthetized, and the lungs were inflation-fixed with 4% paraformaldehyde in PBS overnight at 4 C. Lungs were washed with PBS and dehydrated through a series of ethanol followed by paraffin embedding. Five-micrometer sections were loaded onto slides for staining with hematoxylin and eosin. For morphometrical measurements, the overall proportion (percent fractional area) of the respiratory parenchyma and the airspace was determined by using a point counting method. Measurements were performed on sections taken throughout various lobes. Images were transferred by video camera to a computer screen using the METAMORPH imaging software (Universal Imaging Co., West Chester, PA). A computer-generated, 121-point lattice grid was superimposed on each field and numbers of intersections (points) falling over respiratory parenchyma (alveoli and alveolar ducts) or airspace were counted. Points falling over bronchioles, large vessels, and smaller arterioles and venules were excluded from the study. The airspace area frequency distribution of alveoli was estimated by the point-sampled intercept method using the METAMORPH imaging software. For immunohistochemical staining of SP-B, thyroid transcription factor (TTF)-1 (markers for alveolar type II epithelial cells) and cAMP response element binding protein-binding protein (CBP)/p300 (markers for both alveolar type I and type II epithelial cells) antibodies, a previously described method was followed (12). Briefly, slides were baked at 60 C for a minimum of 2 h and washed in a series of xylene and ethanol to remove paraffin from the tissues. Antigen retrieval was preformed on the tissues as described above. Endogenous peroxidase activity was removed from the tissues by incubating the tissues in methanol and hydrogen peroxide for 15 min. After washing in 0.1 M PBS with Triton X-100, nonspecific binding was blocked by incubating the slides in 0.1 M PBS with Triton X-100 with either goat or rabbit serum for 2 h depending on the antibody used. The slides were then incubated overnight at 4 C in primary antibody. The dilutions of primary antibodies followed the previous publication (12). Numbers of alveolar type I and type II epithelial cells were counted and analyzed by the METAMORPH imaging software as outlined above. Statistically significant differences were determined by using ANOVA.
Alveolar type II epithelial cell culturing and SP-B mRNA RT-PCR
As previously described (15), 2-month-old double-transgenic mice were anesthetized by ip injection. The abdominal cavity was opened and mice exsanguinated by severing the inferior vena cava and the left renal artery. The trachea was isolated and cannulated with a 20-gauge luer stub adapter. The diaphragm was cut, and the chest plate and thymus removed. Using a 21-gauge needle fitted on a 10-cc syringe, lungs were perfused with 10–20 ml 0.9% saline via the pulmonary artery. Three milliliters of dispase were rapidly instilled through the cannula in the trachea followed by 0.5 ml 1% agarose (45 C). Lungs were immediately covered with ice for 2 min to gel the agarose. Following this incubation, lungs were removed from the animals and incubated in 2 ml dispase for 45 min (25 C). Lungs were subsequently transferred to a 60-mm culture dish containing 7 ml of HEPES-buffered DMEM and 100 U/ml deoxyribonuclease I and lung tissue gently teased from the bronchi. The cell suspension was filtered through progressively smaller cell strainers (100 µm, 40 µm) and nylon gauze (20 µm). Cells were collected by centrifugation at 130 x g for 8 min (4 C) and placed on prewashed 100-mm tissue culture plates that had been coated for 24–48 h at 4 C with 42 µg CD 45 and 16 µg CD 32 in 1x PBS. Following incubation for 1–2 h at 37 C, type II cells were gently panned from the plate and collected by centrifugation. Monolayers of alveolar type II cells were cultured on Matrigel (BD Biosciences, Bedford, MA):rat tail collagen (70:30, vol:vol) in bronchial epithelial cell basal medium (minus hydrocortisone) plus 5% charcoal-stripped fetal bovine serum and 10 ng/ml keratinocyte growth factor. Cells were treated with or without 0.01 mg/ml of Dox for 2 d. For cell harvest, matrices were solubilized by incubating cultures with dispase containing 1 mg/ml of collagenase at 37 C for 60 min. Total RNAs were isolated from cells using the RNA purification kit (QIAGEN). RT-PCR was used to detect the expression level of SP-B mRNAs with the SuperScript One-Step RT-PCR kit (Invitrogen, Carlsbad, CA). To detect SP-B mRNA expression, an upstream primer (5'-TGC TGT GGA GCC TCT GAT AGA AG-3') and a downstream primer (5'-CAT AGC CTG TTC ACT GGT GTT CC-3') were used.
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Results |
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double-transgenic mice in lung epithelial cells, a double-transgenic mouse system was generated. CCSP-rtTA and SP-C-rtTA transgenic mouse lines were produced bearing the rtTA fusion protein under the control of either the 3.7-kb human SP-C gene promoter or the 2.3-kb rat CCSP gene promoter as previously described (16, 17). The CCSP promoter is nonciliated bronchiolar epithelial cell (Clara cell) specific, whereas the SP-C promoter is alveolar type II epithelial cell specific. Therefore, the rtTA fusion protein was expressed in two different populations of epithelial cells in the distal region of the lung. The Teto-CMV-dnRAR transgene was generated as described in Materials and Methods (Fig. 1A
). After cross-breeding CCSP-rtTA or SP-C rtTA transgenic mice with Teto-CMV-dnRAR transgenic mice, double-transgenic mice were selected by PCR genotyping using sequence specific oligo primers (Fig. 1B). When a pair of primers specific for the CCSP-rtTA transgene construct was used, tail DNAs from both CCSP-rtTA transgenic mice and CCSP-rtTA/Teto-CMV-dnRAR double-transgenic mice showed a specific PCR band, indicating CCSP-rtTA transgene insertion in these mice. When a pair of primers specific for Teto-CMV-dnRAR transgene construct was used, tail DNAs from both CMV-dnRAR transgenic mice and CCSP-rtTA/Teto-CMV-dnRAR double-transgenic mice showed a different specific PCR band, indicating Teto-CMV-dnRAR transgene insertion in these mice. As a control, nontransgenic mice showed no band using both pairs of primers. Genotyping of SP-C-rtTA/(Teto)-CMV-dnRAR transgenic mice was performed in the same way (data not shown). To assess if dnRAR mRNA is inducible, 21-d-old young double-transgenic mice were treated with Dox for 7 d, lungs were isolated, and total mRNAs were purified. Double-transgenic mice without Dox treatment were used as a control. As demonstrated by the RT-PCR assay, Dox treatment rapidly and significantly induced dnRAR mRNA expression in the lungs of the double-transgenic mice (Fig. 1C). In comparison, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA remained unchanged upon Dox treatment. Therefore, genomic integrated dnRAR cDNA expression is capable of responding to Dox induction in double-transgenic mice. Nontransgenic or single-transgenic mice were also used for the Dox treatment. No dnRAR mRNA was observed before and after the treatment in the lung (data not shown). Expression of dnRAR mRNA was also examined in other tissues, including the heart (Fig. 1C), the liver, and the spleen. No dnRAR mRNA was detectable after Dox induction. A similar study was performed in the SP-C-rtTA/(Teto)-CMV-dnRAR double-transgenic mice and showed the same result (data not shown).
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causes alveolar abnormality in neonatal lungs in the neonatal lungs, double-transgenic mice were bred to produce pups. Subsequently, the pups were treated with Dox from postnatal d 1–21 to induce dnRAR overexpression. After 21 d, lungs from various genotyped animals (nontransgenic, single-transgenic and double-transgenic mice) were inflated for histopathological assessment. Dox treatment of the nontransgenic lungs (Fig. 2) and the single-transgenic lungs (data not shown) showed normal alveolar maturation. In contrast, the lungs from both CCSP-rtTA/Teto-CMV-dnRAR and SP-C rtTA/Teto-CMV-dnRAR double-transgenic mice revealed marked malformation of alveoli (Fig. 2) after overexpression of dnRAR by Dox treatment. The phenotype showed characteristic focal pulmonary emphysema. To assess the survival rate of double-transgenic mice, 24 mice (including both CCSP-rtTA/Teto-CMV-dnRAR and SP-C rtTA/Teto-CMV-dnRAR transgenic mice) were left for prolonged Dox treatment. Within 1–3 months, seven of these Dox-treated mice experienced breathing difficulty and died. In comparison, no casualty was observed in nontransgenic mice group.
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or the SP-C rtTA/Teto-CMV-dnRAR double-transgenic lungs after Dox treatment (Fig. 3). The double-transgenic lungs had the increased air space and the diminished alveolar surface area. Analysis of two-dimensional frequency distribution of the alveolar region in both CCSP-rtTA/Teto-CMV-dnRAR and SP-C rtTA/Teto-CMV-dnRAR double-transgenic mice showed larger but fewer alveoli (Fig. 4). This process was irreversible because removal of Dox after treatment did not reverse the phenotype (data not shown).
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reduces alveolar epithelial cells or SP-C rtTA/Teto-CMV-dnRAR double-transgenic lungs by immunohistochemical staining. Numbers of alveolar type II epithelial cells were determined by SP-B and TTF-1 antibodies as markers. Morphometrical measurements were performed to count for positively stained cells. Compared with Dox-treated nontransgenic animals, Dox-treated CCSP-rtTA/Teto-CMV-dnRAR or SP-C rtTA/Teto-CMV-dnRAR double-transgenic animals showed a significant decrease in alveolar type II epithelial cells (Fig. 5), suggesting that overexpression of dnRAR during the neonatal period attenuated alveolar type II epithelial cell differentiation and proliferation. It is generally regarded that alveolar type II epithelial cells serve as progenitor cells for alveolar type I epithelial cells. Therefore, blocking alveolar type II epithelial cell differentiation and proliferation should also attenuate alveolar type I epithelial cell differentiation and proliferation. Using CBP antibody as markers (staining both type I and type II cells, see Ref.12), the numbers of total epithelial cells (including alveolar type I and type II epithelial cells) were also decreased in double-transgenic mice (Fig. 5).
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double-transgenic mice and cultured in vitro. After addition of Dox into the tissue culture medium for 48 h, alveolar type II epithelial cells were harvested and total RNAs were purified. The SP-B mRNA expression level was semiquantitatively analyzed by RT-PCR. The study showed that SP-B mRNA expression in double-transgenic alveolar type II epithelial cells was significantly reduced after overexpressing dnRAR by Dox treatment (Fig. 6). As a control, GAPDH mRNA expression was not changed. In addition, cells from the nontransgenic and single-transgenic mice showed no decrease in SP-B mRNA expression after Dox treatment (data not shown).
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Discussion |
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Using tissue specific CCSP or SP-C promoter controlled double-transgenic mouse systems, overexpression of dnRAR in neonatal respiratory epithelial cells resulted in abnormal alveolar formation after Dox treatment in the first 21 d postnatal period, which is characterized by the increased alveolar surface area, impaired gas exchange, inadequate oxygenation and frequent death. Interestingly, both CCSP and SP-C-rtTA double-transgenic mice caused similar destruction in the alveolar structure in neonatal lungs, indicating that in the early postnatal period, epithelial cells are not fully differentiated in which both CCSP and SP-C promoters are activated. In addition, dnRAR overexpression in one cell type may block expression of intercellular signaling molecules that are important for alveolar maturation. The pathogenic consequences were apparently a result from the decreased alveolar epithelial cell differentiation and proliferation. Both alveolar type I and type II epithelial cells were significantly reduced in Dox treated CCSP-rtTA/Teto-CMV-dnRAR or SP-C rtTA/Teto-CMV-dnRAR double-transgenic animals. Alveolar type II epithelial cells are the major sites for surfactant membrane synthesis. Decrease in alveolar type II epithelial cell numbers causes insufficient production of phospholipid and surfactant proteins that are essential components for the surfactant membranes. Alveolar walls are primarily comprised of alveolar type I epithelial cells. Reduced numbers of alveolar type I epithelial cells decrease the surface area for oxygen exchange during respiratory cycles in the lung. After Dox treatment from P1 d, pups at P4 and P10 d were also examined. No obvious lung abnormality was observed. It is possible that Dox treatment was not long enough to show the dnRAR dominant negative effect.
In addition, the RA/RAR axis controls homeostasis of molecules that are important for maintaining the surfactant structure and lung function. In SP-C rtTA/Teto-CMV-dnRAR double-transgenic mice, overexpression of dnRAR by Dox treatment reduced ability of alveolar type II epithelial cells to synthesize SP-B mRNAs. SP-B is critical for alveolar maturation and postnatal adaptation. The finding is in agreement with previous observation that the RA/RAR signaling pathway is required for transcriptional stimulation of the SP-B gene (6, 7, 20). Previously, an enhancer region that mediates RA stimulation has been identified in the 5'-flanking regulatory region of the hSP-B gene in vitro (7, 8, 18, 21). RAR and TTF-1 DNA binding sites in the enhancer region mediate RA stimulation of SP-B transcription. Deletion of this enhancer region abolishes or significantly reduces SP-B gene temporal/spatial expression in bronchiolar epithelial cells and alveolar type II epithelial cells in vivo (22). Therefore, destruction of the alveolar structure by overexpressing dnRAR is caused, at least partially, by suppressing expression of endogenous genes (e.g. SP-B gene) that are critical for maintaining the surfactant structure.
In summary, the RA/RAR signaling pathway is not only required for lung development but is also required for postnatal alveolar maturation. This is accomplished by activation of downstream target structural genes that are important for lung formation. Current studies along with previous findings will facilitate designing strategies to combat congenital and acquired respiratory diseases such as pulmonary emphysema, respiratory distress syndrome, and bronchiopulmonary displasia, the leading causes of mortality and morbidity in preterm infants.
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Acknowledgments |
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expression vector and Dr. R. DeLauro for providing the TTF-1 antibody. |
Footnotes |
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Abbreviations: CBP, cAMP response element binding protein-binding protein; CCSP, Clara cell secretory protein; CMV, cytomegalovirus; dnRAR, dominant negative RAR; Dox, doxycycline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RA, retinoic acid; RAR, RA receptor; rtTa, reverse tetracycline responsive transactivator; RXR, retinoid X receptor; SP-B, surfactant protein B; SP-C, surfactant protein C; TTF-1, thyroid transcription factor 1.
Received December 9, 2002.
Accepted for publication March 7, 2003.
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References |
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and thyroid transcription factor-1 in respiratory epithelial cells. J Biol Chem 276:21686–21691This article has been cited by other articles:
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A. C. Ross On the sources of retinoic acid in the lung: understanding the local conversion of retinol to retinoic acid Am J Physiol Lung Cell Mol Physiol, February 1, 2004; 286(2): L247 - L248. [Full Text] [PDF]
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