PULMONARY FGF-18 GENE EXPRESSION IS DOWNREGULATED DURING THE CANALICULAR-SACCULAR STAGES IN NITROFEN-INDUCED HYPOPLASTIC LUNGS

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1 PULMONARY FGF-18 GENE EXPRESSION IS DOWNREGULATED DURING THE CANALICULAR-SACCULAR STAGES IN NITROFEN-INDUCED HYPOPLASTIC LUNGS Hiromizu Takahashi 1,2, Florian Friedmacher 1, Naho Fujiwara 1, Alejandro Hofmann 1, Balazs Kutasy 1, Jan-Hendrik Gosemann 1, Prem Puri 1,3 1 National Children s Research Centre, Our Lady s Children s Hospital, Crumlin, Dublin, Ireland 2 Department of General Medicine, Juntendo University School of Medicine, Bunkyo-ku, Hongo, Tokyo, , Japan 3 Conway Institute of Biomolecular and Biomedical Research, School of Medicine & Medical Science, University College Dublin, Dublin, Ireland Corresponding author: Prem Puri National Children s Research Centre, Our Lady s Children s Hospital, Crumlin, Dublin 12, Ireland Tel.: Fax: prem.puri@ucd.ie 1

2 Abstract Purpose: Pulmonary hypoplasia (PH) associated with congenital diaphragmatic hernia (CDH) represents one of the major challenges in neonatal intensive care. However, the molecular pathogenesis of PH is still poorly understood. In developing fetal lungs, fibroblast growth factor 18 (FGF-18) plays a crucial role in distal airway maturation. FGF-18 knockouts show smaller lung sizes with reduced alveolar spaces and thicker interstitial mesenchymal compartments, highlighting its important function for fetal lung growth and differentiation. We hypothesized that pulmonary FGF-18 gene expression is downregulated during late gestation in nitrofen-induced hypoplastic lungs. Methods: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetuses were harvested on D18 and D21, and lungs were divided into 3 groups: controls, hypoplastic lungs without CDH [CDH(-)] and hypoplastic lungs with CDH [CDH(+)] (n=24 at each time-point). Pulmonary FGF-18 gene expression levels were analyzed by qrt-pcr. Immunohistochemistry was performed to investigate FGF-18 protein expression/distribution. Results: Relative mrna levels of pulmonary FGF-18 gene expression were significantly decreased in CDH(-) and CDH(+) on D18 and D21 compared to controls (p<0.05 and p<0.01, respectively). Immunoreactivity of FGF-18 was markedly diminished in mesenchymal cells surrounding the airway epithelium on D18 and D21 compared to controls. Conclusion: Downregulation of FGF-18 gene expression in nitrofen-induced hypoplastic lungs suggests that decreased FGF-18 expression during the canalicular-saccular stages may interfere with saccular-alveolar differentiation and distal airway maturation resulting in PH. Keywords: Fibroblast growth factor 18, Congenital diaphragmatic hernia, Pulmonary hypoplasia, Nitrofen. 2

3 Introduction Congenital diaphragmatic hernia (CDH) is a relatively common congenital malformation with an incidence of approximately 1 in 2,500 births [1,2]. Despite improvements in prenatal diagnosis and progress in postnatal management, the morbidity and mortality rates in CDH remain high due to severe pulmonary hypoplasia and persistent pulmonary hypertension [2]. The pathogenesis of pulmonary hypoplasia associated with CDH is not fully understood. Lung development begins in rats from 11 days (D11) with outpocketing of two epithelial buds from the ventral forgut into the surrounding mesoderm [3,4]. Later, lung development is divided into four stages: pseudoglandular stage (D11-18), canalicular stage (D18-19), saccular stage (D wk), and alveolar stage (week1-5) [5]. In the pseudoglandular stage, the bronchial tree develops and undifferentiated primordial system forms. In the canalicular stage, terminal sacs develop and vascularization occurs. In the saccular stage, the number of terminal sacs increases, vascularization proceeds and differentiation of type I and type II epithelial cells occur. In the alveolar stage, there is multiplication of alveoli resulting in extensive surface area [5]. Hypoplastic lungs in CDH have fewer alveoli, thickened alveolar walls, increased interstitial tissue, and markedly diminished alveolar airspace [6]. Fibroblast growth factor (FGF) signaling pathway is an essential element of the regulatory network operating between epithelium and mesenchyme at several developmental stages in the fetal lungs [7,8]. FGF-18 is a member of the FGF signaling family, playing an important role in the development of several organs, including fetal lungs [9-14]. FGF-18 genes play an essential role in distal airway 3

4 maturation during canalicular-saccular stages [15,16]. In FGF-18 knockouts, lungs were smaller in size with reduced alveolar spaces and thicker interstitial mesenchymal compartments, but distal airway branching was preserved [16]. Furthermore, the expression of marker genes for branching morphogenesis such as BMP4, Shh and FGF-10 were not impaired in FGF-18 knockout lungs [16]. These results indicate that FGF-18 genes play a key role in fetal lung development during the canalicular-saccular stages and plays no significant role in lung branching morphogenesis. The nitrofen model of CDH is an established animal model of CDH and has been widely used to investigate the pathogenesis of pulmonary hypoplasia. When nitrofen is administered to pregnant rat dams on D9, approximately 70% of offspring will have CDH and 100% hypoplasitc lungs [17]. We designed this study to test the hypothesis that pulmonary gene expression of FGF-18 is downregulated during the canalicular-saccular stages in nitrofen-induced hypoplastic lungs. Materials and Methods Animals and experimental design Adult Sprague-Dawley rats were mated and checked daily for plugging. Presence of spermatozoids in vaginal smear was considered as a proof of pregnancy and was determined as D0. On D9, rats received either 100 mg of nitrofen (WAKO Chemicals GmbH, Neuss, Germany), dissolved in 1 ml of olive oil, or only vehicle. Fetuses were harvested by cesarean delivery on D18 and D21. After thoracotomy, fetal lungs were divided into 3 groups: control, hypoplastic lungs without CDH [CDH(-)] and hypoplastic lungs with CDH [CDH(+)] (n = 24 at each time-point, respectively). Lungs were either 4

5 snap-frozen in liquid nitrogen (and stored at -70 ) for RNA extraction or fixed in 4% paraformaldehyde overnight for immunohistochemistry. The department of Health and Children approved the protocol of these animal experiments (ref. B100/4378) under the Cruelty to Animals Act, 1876; as amended by European Communities Regulations 2002 and Total RNA extraction and complementary DNA synthesis Total RNA was isolated from snap-frozen lungs at each time-point using TRIzol reagent (Invitrogen, Carlsbad, USA) according to the manufacturer s protocol and quantification was performed spectrophotometrically (NanoDrop ND-1000 UV-vis Spectrophotometer, Wilmington, USA). Synthesis of cdna was performed using Transcript High Fridelity cdna Synthesis Kit (Roche Diagnostics, Grenzach-Whylen, Germany) according to the manufacturer s protocol. All cdna samples were stored at 4. Quantitative reverse transcription polymerase chain reaction (RT-PCR) To quantify the FGF-18 mrna expression, qrt-pcr was performed using Light Cycler 480 SYBR Green I Master Mix (Roche Diagnostics, Mannheim, Germany) according to the Table 1 manufacturer s protocol. Gene-specific primer pairs used in this study are listed in Table 1. After initialization at 95 for 5 min, 55 cycles of amplification for each primer were carried out. Each cycle included a denaturation step for 10 s at 95, an annealing step for 15 s at 60 and an elongation step for 10 s at 72. Final elongate temperature was 65 for 1min. Relative gene expression of FGF-18 were 5

6 measured by Light Cycler 480 (Roche Diagnostics, West Sussex UK) and levels of target genes were normalized to the housekeeping gene β-actin. All qrt-pcr experiments were performed in duplicate for each sample at each time-point. Immunohistochemistry Formalin-fixed lungs at each time-point were fixed in 10% buffered formalin overnight, embedded in paraffin and sectioned at 5 μm. Tissue sections were deparaffined with xylene and rehydrated through graded alcohol. Then tissue sections were immersed in retrieval solution (DAKO, Cambridgeshire, UK) in microwave oven at 750 W for 15 min. After cooling down the slides on ice for 10 minutes, Peroxidase Block (DAKO, Cambridgeshire, UK) was treated for 5 min to block endogenous peroxidase activity. The sections were incubated overnight at 4 with rabbit primary antibody against FGF-18 (LOT ab9743; 1:1000, Abcam plc, Cambridge, UK) and treated in horseradish peroxidase (HRP)-labeled anti-rabbit secondary antibodies. Sections were then developed with a diaminobenzidine (DAB)-H 2 O 2 substrate complex, and counterstained with hematoxylin. After that sections were dehydrated and mounted with glass coverslips using DPX (Sigma Aldrich Ltd, Arklow, Ireland). Two investigators performed independently the examinations under light microscopy. Statistical analysis All numerical data are presented as means ± SEM. Difference between two groups at each gestational day were tested by using an unpaired Student s t-test when data had normal distribution or Mann-Whitney-U test when the data deviated from normal distribution. Statistical significance was 6

7 accepted at p < Results Relative mrna expression levels of FGF-18 in fetal rat lungs The relative mrna expression levels of pulmonary FGF-18 were significantly decreased in the nitrofen group on D18 (1.70 ± 0.16 vs 2.32 ± 0.16; *p = and 1.68 ± 0.16 vs 2.32 ± 0.16; *p = ) and D21 (2.13 ± 0.15 vs 3.40 ± 0.35; **p = and 1.81 ± 0.21 vs 3.40 ± 0.35; **p = ) Fig. 1 compared to the control group (Fig. 1). There were no significant differences between CDH(-) and CDH(+) in gene expression levels of pulmonary FGF-18 on D18 and D21. Protein expression of FGF-18 in fetal rat lungs In order to confirm the results of gene expression of pulmonary FGF-18 in qrt-pcr, protein expression and distribution of pulmonary FGF-18 were evaluated by immunohistochemistry on D18 and D21. In control lungs, FGF-18 protein expression was abundantly localized including distal airway epithelium, mesenchyme and mesothelium on D18 and D21. In the nitrofen-induced hypoplastic lungs, Fig. 2 mesenchymal FGF-18 protein expression was diminished compared to control lungs on D18 and D21 (Fig. 2). 7

8 Discussion FGFs are secreted proteins that play an important role in multiple biological processes, such as proliferation and differentiation, as well as in the morphogenesis of developing organs such as fetal lungs [18]. In fetal lungs, FGFs are essential components of the regulatory networks operating between epithelium and mesenchyme at several stages of development. Numerous studies have highlighted the importance of FGFs in epithelial-mesenchymal interactions that orchestrate fetal lung growth and differentiation [19]. FGFs seem to be sequestered and stored in the extracellular matrix, and may be released at times of need. Only a restricted number of FGF ligands are present in the embryonic lung [20]. Furthermore, it has been demonstrated that FGF signaling depends on the activation of specific cell surface receptors (FGFRs) [21]. Together, FGFs and FGFRs form a complex signaling network that is crucial for cell proliferation and airway formation [22]. Consequently, FGF up- or downregulation offers a mechanism by which the growth of specific cell populations may be controlled during fetal lung development [23]. Previous work from our laboratory has shown that FGF-10 and FGF-7 gene expression was significantly reduced in hypoplastic lungs compared with controls [24]. However, little is known about the involvement of FGF-18 in the development of pulmonary hypoplasia in CDH. FGF-18 is a novel growth factor that stimulates cell growth, proliferation and differentiation in a variety of tissues, including developing lungs [12,25]. Recently, it has been revealed that FGF-18 genes are involved in the control of various developmental events during lung development [26]. Previous studies had consistently indicated that FGF-18 expression was mainly located in interstitial tissue of distal airways, supporting the concept that FGF-18 influences cell proliferation and differentiation during lung morphogenesis [27]. Structural analysis has revealed that FGF-18 is highly conserved between humans and rats [15]. However, effects of FGF-18 were primarily limited to the fetal 8

9 lung, emphasizing its crucial role in the development of the distal lung. It has been demonstrated that FGF18 is highly involved in the control of airway differentiation during the canalicular stage and increasing the number of terminal sacs during the saccular stage as well [16,27]. Consistently, FGF-18 expression was found to be increased after prenatal tracheal ligation in fetal rats and thus stimulates lung growth [28]. FGF-18 knockout mice displayed on D18 reduced alveolar airspaces and thicker interstitial mesenchyme [16], both similar to the structural abnormalities in the nitrofen-induced rat model. Furthermore, a transient decrease in the proliferation activity has been shown in FGF-18 -/- lungs during the terminal saccular stage [16]. In the present study, we evaluated pulmonary FGF-18 gene expression levels and found that FGF-18 mrna transcripts were significantly decreased on D18 and D21 in nitrofen-induced hypoplastic lungs compared to controls. However, there were no statistical differences between CDH(-) and CDH(+), suggesting that the development of hypoplastic lungs occurs independently from the herniation of abdominal viscera into the thorax cavity. Furthermore, we provided evidence that the decreased pulmonary FGF-18 gene expression levels were also translated to the protein level. Our immunohistochemistry confirmed that FGF-18 protein expression was markedly decreased in distal epithelial and mesenchymal airway cells of nitrofen-induced hypoplastic lungs on D18 and D21. These findings may imply that downregulation of FGF-18 gene expression results in a disruption of the intracellular signal transduction cascade that ultimately results in modified protein expression. Thus, it is tempting to speculate that a decrease of FGF-18 gene and protein expression may disrupt the mesenchymal-epithelial interactions, resulting in aberrations in the tissue architecture and causing pulmonary hypoplasia in the nitrofen-induced rat model. In conclusion, downregulation of FGF-18 gene expression in nitrofen-induced hypoplastic lungs suggest that decreased FGF-18 expression during the canalicular-saccular stages may interfere with 9

10 saccular-alveolar differentiation and distal airway maturation, which finally results in pulmonary hypoplasia. Although we have previously shown that FGFR-mediated lung development is disrupted in nitrofen-induced hypoplastic lungs [29,30], further studies of FGF signaling pathway are required and will provide new insights into the pathogenesis of pulmonary hypoplasia associated with CDH. Conflicts of interest The authors report no conflicts of interest. References 1. Bohn D (2002) Congenital diaphragmatic hernia. Am J Respir Crit Care Med 166 (7): Gosche JR, Islam S, Boulanger SC (2005) Congenital diaphragmatic hernia: searching for answers. Am J Surg 190 (2): Mendelson CR (2000) Role of transcription factors in fetal lung development and surfactant protein gene expression. Annu Rev Physiol 62: Warburton D, Bellusci S, Del Moral PM et al. (2003) Growth factor signaling in lung morphogenetic centers: automaticity, stereotypy and symmetry. Respir Res 4:5. 5. Keijzer R, Puri P (2010) Congenital diaphragmatic hernia. Semin Pediatr Surg 19 (3): Brandsma AE, ten Have-Opbroek AA, Vulto IM et al. (1994) Alveolar epithelial composition and architecture of the late fetal pulmonary acinus: an immunocytochemical and morphometric study in a rat model of pulmonary hypoplasia and congenital diaphragmatic hernia. Exp Lung Res 20 (6):

11 7. Lebeche D, Malpel S, Cardoso WV (1999) Fibroblast growth factor interactions in the developing lung. Mech Dev 86 (1-2): Warburton D, Schwarz M, Tefft D et al. (2000) The molecular basis of lung morphogenesis. Mech Dev 92 (1): Dichmann DS, Miller CP, Jensen J et al. (2003) Expression and misexpression of members of the FGF and TGFbeta families of growth factors in the developing mouse pancreas. Dev Dyn 226 (4): Ellsworth JL, Garcia R, Yu J et al. (2004) Time window of fibroblast growth factor-18-mediated neuroprotection after occlusion of the middle cerebral artery in rats. J Cereb Blood Flow Metab 24 (1): Haque T, Nakada S, Hamdy RC (2007) A review of FGF18: Its expression, signaling pathways and possible functions during embryogenesis and post-natal development. Histol Histopathol 22 (1): Hu MC, Qiu WR, Wang YP et al. (1998) FGF-18, a novel member of the fibroblast growth factor family, stimulates hepatic and intestinal proliferation. Mol Cell Biol 18 (10): Liu Z, Lavine KJ, Hung IH et al. (2007) FGF18 is required for early chondrocyte proliferation, hypertrophy and vascular invasion of the growth plate. Dev Biol 302 (1): Ohbayashi N, Shibayama M, Kurotaki Y et al. (2002) FGF18 is required for normal cell proliferation and differentiation during osteogenesis and chondrogenesis. Genes Dev 16 (7): Ohbayashi N, Hoshikawa M, Kimura S et al. (1998) Structure and expression of the mrna encoding a novel fibroblast growth factor, FGF-18. J Biol Chem 273 (29): Usui H, Shibayama M, Ohbayashi N et al. (2004) Fgf18 is required for embryonic lung alveolar development. Biochem Biophys Res Commun 322 (3): Montedonico S, Nakazawa N, Puri P (2008) Congenital diaphragmatic hernia and retinoids: searching for an etiology. Pediatr Surg Int 24 (7):

12 18. Cardoso WV, Lu J (2006) Regulation of early lung morphogenesis: questions, facts and controversies. Development 133 (9): Shannon JM, Hyatt BA (2004) Epithelial-mesenchymal interactions in the developing lung. Annu Rev Physiol 66: Morrisey EE, Hogan BL (2010) Preparing for the first breath: genetic and cellular mechanisms in lung development. Dev Cell 18 (1): Ornitz DM, Xu J, Colvin JS et al. (1996) Receptor specificity of the fibroblast growth factor family. J Biol Chem 271 (25): Metzger RJ, Klein OD, Martin GR et al. (2008) The branching programme of mouse lung development. Nature 453 (7196): Han RN, Liu J, Tanswell AK et al. (1992) Expression of basic fibroblast growth factor and receptor: immunolocalization studies in developing rat fetal lung. Pediatr Res 31 (5): Teramoto H, Yoneda A, Puri P (2003) Gene expression of fibroblast growth factors 10 and 7 is downregulated in the lung of nitrofen-induced diaphragmatic hernia in rats. J Pediatr Surg 38 (7): Chailley-Heu B, Boucherat O, Barlier-Mur AM et al. (2005) FGF-18 is upregulated in the postnatal rat lung and enhances elastogenesis in myofibroblasts. Am J Physiol Lung Cell Mol Physiol 288 (1):L Franco-Montoya ML, Boucherat O, Thibault C et al. (2011) Profiling target genes of FGF18 in the postnatal mouse lung: possible relevance for alveolar development. Physiol Genomics 43 (21): Whitsett JA, Clark JC, Picard L et al. (2002) Fibroblast growth factor 18 influences proximal programming during lung morphogenesis. J Biol Chem 277 (25):

13 28. Mesas-Burgos C, Nord M, Didon L et al. (2009) Gene expression analysis after prenatal tracheal ligation in fetal rat as a model of stimulated lung growth. J Pediatr Surg 44 (4): Friedmacher F, Doi T, Gosemann JH et al. (2012) Upregulation of fibroblast growth factor receptor 2 and 3 in the late stages of fetal lung development in the nitrofen rat model. Pediatr Surg Int 28 (2): Friedmacher F, Gosemann JH, Takahashi H et al. (2013) Decreased pulmonary c-cbl expression and tyrosine phosphorylation in the nitrofen-induced rat model of congenital diaphragmatic hernia. Pediatr Surg Int 29 (1): Figure legends Fig. 1 Relative mrna expression levels of FGF-18 in fetal rat lungs on D18 and D21. Expression levels of FGF-18 genes were significantly decreased in CDH(-) and CDH(+) hypoplastic lungs compare to control lungs on D18 and D21. Fig. 2 FGF-18 immunohistochemical staining (brown) with hematoxylin counter staining (blue). Control lung on D18 (a). Nitrofen-exposed lung on D18 (b). Control lung on D21 (c). Nitrofen-exposed lung on D21 (d). Immunoreactivity of FGF-18 was markedly decreased in nitrofen-induced hypoplastic lungs compare to control lungs on D18 and D21. 13

14 Figure and table legends Table 1: Primer sequences for quantitative real-time PCR Fig. 1: The relative mrna expression levels of FGF-18 in fetal rat lungs on D18 and D21 [Control, CDH(-) and CDH(+)]. The expression levels of FGF-18 were significantly decreased CDH(-) and CDH(+) lungs compare to control on D18 and D21. Fig. 2: FGF-18 immunohistochemical staining (brown) with hematoxylin counter staining (blue). A, Control lung on D18. B, Nitrofen lung on D18. C, Control lung on D21. D, Nitrofen lung on D21. Immunoreactivity of FGF-18 were markedly decreased in nitrofen lung compare to control lung on D18 and D21.

15 Table 1: Gene Sequence (5-3 ) Product size (bp) β-actin Forward TTG CTG ACA GGA TGC AGA AG 108 Reverse TAG AGC CAC CAA TCC ACA CA FGF-18 Forward TGC GCT TGT ACC AGC TCT AC 158 Reverse CAC TCC TTG CTA GTA CCA TC Fig. 1:

16 Fig. 2: Control Nitrofen A B C D