Expression of Transcription Factor GATA-6 in Alveolar Epithelial Cells Is Linked to Neonatal Lung Disease

Background: Premature birth and respiratory distress syndrome (RDS) are risk factors for disturbed lung development and bronchopulmonary dysplasia (BPD). The molecular mechanisms related to prematurity and BPD remain largely unknown. Epithelial expression of the transcription factor GATA-6 has been implicated in normal and abnormal murine lung development. Objectives: The possible involvement of GATA-6 in the normal development and in RDS and BPD was investigated in the human and baboon lung. Methods: Immunohistochemistry was used to study the expression of GATA-6 and thyroid transcription factor 1 in lung specimens from different age groups of human and baboon fetuses and newborns with lung disease. Furthermore, the regulatory role of TGF1 in GATA-6 expression was investigated in human pulmonary epithelial cell lines using RT-PCR. Results: GATA-6 expression increased in the developing human airway epithelium along with advancing gestation, but diminished to negligible at birth. In RDS, GATA-6 expression was Received: February 11, 2010 Accepted after revision: June 22, 2010 Published online: November 12, 2010 Markku Heikinheimo, MD, PhD Children’s Hospital University of Helsinki PO Box 20, FI–00014 Helsinki (Finland) Tel. +358 9 4717 4770, Fax +358 9 4717 5299, E-Mail markku.heikinheimo @ helsinki.fi © 2010 S. Karger AG, Basel Accessible online at: www.karger.com/neo D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /2 3/ 20 17 2 :4 4: 30 A M Vähätalo et al. Neonatology 2011;99:231–240 232 components are believed to be preand postnatal inflammation, as well as oxygen toxicity, which can further damage the lung [2] . The development of BPD has been also been linked to alterations in critical signaling pathways, such as in the TGF superfamily [5] . Several factors are involved in the normal pulmonary development and pathophysiology of neonatal lung disease. Transcription factors are essential regulators of gene expression, and their abnormalities have been associated with disease in several organs. GATA transcription factors are zinc finger proteins that recognize a consensus DNA sequence (A/T) GATA (A/G), known as GATA-motif, which is an essential cis -acting element in the promoters and enhancers of a variety of genes [6] . GATA-4, GATA-5, and GATA-6 are expressed in the foregut or cardiac regions of the developing embryo, suggesting a potential role in organogenesis of the heart and lung [7–9] . These GATA proteins are also expressed in the developing mouse lung [8–11] . Out of the three GATA factors implicated in lung development, GATA-6 has been most extensively studied in the developing airway epithelium [9] . Conditional loss of function mouse models revealed that GATA-6 is required for lung maturation at the saccular stage [12] . A generalized delay in lung maturation and decreased surfactant protein B levels were observed in this model, and postnatal survival was decreased in comparison with wild-type littermates [12] . The expression of GATA-6 is normally downregulated at term in murine lung, and this seems to be essential for proper lung maturation, whereas overexpressing GATA-6 in the postnatal respiratory epithelial type II cells inhibits alveolarization and perturbs lung function [13] . Subsequent studies on murine lung have shown that GATA-6 enhances transcription of surfactant A and C genes [14, 15] . In addition, it interacts with thyroid transcription factor 1 (TTF-1), also involved in the regulation of surfactant gene expression [14, 16] . TTF-1 on the other hand regulates thyreoglobulin, the macromolecular precursor of thyroid hormones, which in turn may be involved in orchestrating the complex steps involved in lung morphogenesis [17] . Collectively, the published data on experimental models suggest that GATA-6, together with its cofactors and downstream target genes, has a central role in normal mammalian lung development and function. We postulated that abnormalities in the expression of GATA-6 might also be associated with abnormal lung development in primates, and assessed its expression during fetal and neonatal human lung development and disease. As the overexpression of TGF in the neonatal mouse lung results in histological changes similar to BPD [18] , and TGF is known to contribute to postnatal fibrosis after alveolar injury [5, 19] , the relationship of GATA-6 and TGF in pulmonary epithelial cells was studied in vitro. Materials and Methods Human Lung Tissue The human lung biopsies were obtained from autopsy material (Children’s Hospital, Helsinki University Central Hospital) of fetal or preterm/term infants, taken within 3 days post-mortem ( table 1 ). The permission to use the material in scientific purposes was obtained from the National Authority for Medicolegal Affairs. The samples were divided into three different groups according to the patient’s age and specific diagnosis: (1) fetus; (2) newborn at term; (3) preterm with RDS at 1–2 days after birth; (4) preterm with RDS at 3–7 days after birth; (5) preterm with RDS at 7 days or more after birth, and (6) preterm with BPD. BPD was defined using the published criteria [20] . Antenatal betamethasone treatment was not part of the clinical routine at the time of sample collection, and none of the patients received prophylactic glucocorticoid treatment during the early postnatal period. Premature Primate Model All animal studies were performed at the Southwest Foundation for Biomedical Research Primate Center (San Antonio, Tex., USA) and protocols and procedures were approved by the Institutional Animal Care and Use Committee. Fetal baboons (gestational controls, GCs) of varying gestational ages (125, n = 6; 140, n = 5; 160, n = 4; 175, n = 4, 8 2 days) were delivered by hysterotomy and euthanized at birth before onset of breathing. The lung samples from term animals (185 + 2 days, n = 6) were collected following term natural delivery. The preterm animals (125 + 2 days, n = 4; 125 + 6 days, n = 4; 125 + 14 days, n = 6; 125 + 21 days, n = 5), also delivered by hysterotomy, were resuscitated, placed on ventilatory support with pro re nata (PRN) O 2 , and cared for as previously described [21, 22] . 125 and 140 days of baboon gestation correspond to 27 and 32 human gestational weeks, respectively. The 125 + 14 or 21 days PRN O 2 animals develop lung histopathological lesions similar to human BPD [21] . All samples were collected during years 1996–2004, and at necropsy they were immediately fixed with 4% paraformaldehyde. Immunohistochemistry Immunoperoxidase staining of human and baboon samples for GATA-6 and human samples for TTF-1 followed the protocol previously described [23] . Rabbit polyclonal GATA-6 (Santa Cruz Biotechnology; dilution 1: 200) and mouse monoclonal antiTTF-1 (8G7G3/1; Abcam; dilution 1: 100), primary antibodies were used for the staining. The primary antibodies were incubated overnight at +4 ° C. In control experiments, nonimmune serum (Sigma; I8140-10 mg) replaced the primary antibody, or the sections were incubated with secondary antibody without primary antibody. For each sample, approximately 10 areas of stained sections were evaluated by 2 observers (R.V. and R.K.). For human samples, the proportion of positivity to each nuclear antigen was evaluated as follows: low intensity (+) for 0–20% positive nuclei or 1–80% positive cells with weak immunoreactivity, intermediate D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /2 3/ 20 17 2 :4 4: 30 A M GATA-6 and Neonatal Lung Disease Neonatology 2011;99:231–240 233 intensity (++) for 20–100% positive nuclei with at least moderate immunoreactivity, and high intensity (+++) for 80–100% positive nuclei. For baboon samples, quantitation of staining was carried out using a semiquantitative scoring system (0 = negative, 1 = mild, 2 = moderate, and 3 = intense staining). Preprepared tables were used listing the following structures and cell types: alveolar epithelium and bronchiolar/bronchial epithelium, arterial and capillary endothelium, and matrix cells (fibroblasts, smooth muscle cells). Cell Lines and TGFExposure in vitro Human A549 adenocarcinoma (American Type Culture Collection, Manassas, Va., USA) cells derived from alveolar type II cells and BEAS-2B cells (National Cancer Institute, Laboratory of Human Carcinogenesis, Dr. C. Harris, Bethesda, Md., USA), which are SV40-transformed human bronchial epithelial cells, were used for the in vitro experiments. The cell lines cells were grown in DMEM or bronchial epithelial cell growth medium supplemented with 10% FBS, 1% L -glutamine and 1% penicillinstreptomycin. The effect of TGF on GATA-6 and TTF-1 transcription factors was evaluated in both cell lines by exposing the cells to 2 or 10 ng/ml of recombinant human TGF1 (R&D Systems), and the experiments were repeated three times. The cells were seeded on 6-well plates and allowed to attach and expand to subconfluency for 48 h. The cells were serum starved for 24 h, whereafter TGF in DMEM supplemented with 1% FBS was added to the cells for 6 h. After the exposure, the cells were harvested in RNA lysis buffer for RNA isolation. RNA Extraction and RT-PCR Total RNA was extracted from the A549 and BEAS-2B cells, and contaminating genomic DANN eliminated using RNeasy Mini Kit (Qiagen: 250, Cat. No. 74106). All experiments were repeated at least three times. First strand cDNA synthesis was perTable 1. H uman patient characteristics and GATA-6 and TTF-1 immunohistochemistry scored from 1 to 3 Patient Gestational age, weeks Birth weight, g Age at death Sex Cause of death GATA-6 TTF-1 Fetus 1 14 30 – F meningomyelocele +++ +++ 2 18–19 162,43 – M meningomyelocele ++ +++ 3 20+2 346 – F HLHS +++ +++ Term 4 39+5 3,985 7 days F HLHS – – 5 40+6 3,220 25 days F heart defect – – 6 41 3,300 22 days M heart defect + – 7 41+1 3,150 16 days M heart defect – – Early RDS (1–2 days) 8 24+5 740 2 days F RDS, preterm + ++ 9 25+6 887 1 days M RDS, IVH + ++ 10 26 720 1.5 days F RDS – + 11 26+2 920 3 h F RDS, preterm – +++ Prolonged RDS (3–7 days) 12 23+6 700 3 days M RDS, IVH, preterm ++ +++ 13 24+6 812 6 days F RDS, IVH, preterm +/++ + 14 25+1 770 3 days M RDS, IVH, preterm + +++ 15 26+1 750 4 days M RDS, IVH, preterm ++ +++ 16 29+1 410 5 days M RDS – +++ 17 30+3 1,430 3 days F RDS, fetofetal transfusion, asphyxia + Late RDS (over 7 days) 18 25 3,170 4 days M IVH, BPD – ++ 19 30+4 1,070 9 days M RDS, asphyxia – – BPD 20 26 890 2 months M BPD, hydrocephalus + – 21 29+1 765 4 months M BPD, hemorrhage – – 22 25 1,070 5.5 months M BPD, Cor pulmonale –/+ + 23 41 800 9.5 months M BPD, pneumonia – – 24 40 750 10 months M BPD, pulmonary hypertension –/+ –/+ 25 28 805 7 months F BPD, pneumonia –/+ –/+ I VH = Intraventricular hemorrhage; HLHS = hypoplastic left heart syndrome. D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /2 3/ 20 17 2 :4 4: 30 A M Vähätalo et al. Neonatology 2011;99:231–240 234 formed from 1  g of total RNA using SYBR Green RT-PCR reagents and random hexamers (Applied Biosystems, Foster City, Calif., USA). The primers (Oligomer) were designed using the ABI Primer Express software (Applied Biosystems) applied to cDNA sequences derived from the UCSC Genome Bioinformatics Site web site (http://genome.ucsc.edu/) and were as follows: GATA-6 sense: 5 -ATG ACT CCA ACT TCC ACC TCT, GATA-6 antisense 5 -CAG CCT CCA GAG ATG TGT AC G, TTF-1 sense 5 -CCT GGC GCT TCA TTT TGT AG, TTF-1 antisense 5 -ACC AGG ACA CCA TGA GGA AC, GAPDH sense 5 -TCA TTT CCT GGT ATG ACA ACG, GAPDH antisense 5 -TTA CTC CTT GGA GGC CAT GT. Analysis of GATA-4, GATA-6, TTF-1 and GAPDH expression was performed using the PTC-200 Peltier Thermal Cycler (MJ Research). Densitometric analysis on the bands was performed using Media Cybernetics Image J software for Macintosh.