Spatiotemporal alteration in phosphatidylinositide 3-kinase–serine/threonine protein kinase B signaling in the nitrofen-induced hypoplastic lung

Abstract

Purpose:

The pathogenesis of pulmonary hypoplasia in congenital diaphragmatic hernia (CDH) is not fully understood. The serine/threonine protein kinase B (AKT) plays important roles for lung morphogenesis through epithelial-mesenchymal interaction in phosphatidylinositide 3-kinase (PI3K)-dependent manner. It has been reported that the lung explant morphogenesis in mice is interfered by inhibitors of the PI3K-AKT pathway. We hypothesized that PI3K and AKT gene and protein expression/distribution are altered during epithelial morphogenesis in the nitrofen-induced hypoplastic lung.

Methods:

Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetal lungs were harvested on D15, D18, and D21 and divided into 3 groups as follows: control, nitrofen with CDH (CDH[−]), and nitrofen without CDH (CDH[+]) (n = 8 at each time-point, respectively). Reverse transcription polymerase chain reaction and immunohistochemistry were performed.

Results:

Messenger RNA expression levels of PI3K at D21 was significantly decreased in CDH(−) and CDH(+) group (5.71 ± 0.85 and 6.80 ± 0.88, respectively) compared to controls (8.95 ± 3.22; P < .05). Messenger RNA levels of AKT were also significantly decreased at D18 in CDH(−) and CDH(+) lungs (1.21 ± 0.16 and 1.20 ± 0.32, respectively) compared to controls (1.62 ± 0.14; P < .01). The PI3K immunoreactivity was diminished in the distal epithelium at D18 and decreased in the overall intensity at D21 in hypoplastic lungs compared to controls. The AKT immunoreactivity was decreased in mesenchyme at D18 and decreased overall intensity at D21 in CDH lungs compared to controls.

Conclusion:

Spatiotemporal alteration of pulmonary PI3K and AKT gene and protein expression during epithelial morphogenesis may interfere with epithelial-mesenchymal interaction, causing pulmonary hypoplasia in CDH by disrupting PI3K-AKT signaling pathway.

Congenital diaphragmatic hernia (CDH) remains the most life-threatening cause of severe respiratory failure in term infants [1]. Despite recent advances in prenatal diagnosis, resuscitation, and intensive care, the morbidity and mortality in CDH remain high [1]. The high mortality in newborns with CDH has been attributed to severe pulmonary hypoplasia [2]. Maternal exposure of nitrofen in both rat and mouse models during a specific period in gestation results in 100% lung hypoplasia and a high rate (40%–80%) of CDH in the offspring [3–6]. The diaphragmatic defect and associated pulmonary hypoplasia in this model is strikingly similar to the human condition. Although the nitrofen model of CDH has been broadly used, the exact pathogenesis of pulmonary hypoplasia remains unclear.

The serine/threonine protein kinase B (also known as AKT) is involved in multiple cellular functions, such as cell proliferation, differentiation, and survival [7,8]. The AKT activity is regulated by phosphatidylinositide 3-kinase (PI3K) [9]. The PI3K-AKT pathway mediates growth and survival signals through epithelial-mesenchymal interaction during development of fetal mouse lung [10]. It has also been reported that the lung explant morphogenesis in mice is interfered by inhibitors of the PI3K-AKT pathway [10]. Peptide growth factors such as fibroblast growth factors (FGFs) and bone morphogenetic protein-4, as well as morphogens such as sonic hedgehog and retinoids, mediate signaling in the lung [11]. Fibroblast growth factors, which activate the PI3K-AKT pathway, are critical to the development of fetal lung [12]. Previous study from our laboratory has shown that gene expression of FGF-7 and FGF-10 is downregulated in the nitrofeninduced hypoplastic lung [13]. Furthermore, retinoids, which can also activate PI3K, has been reported to be involved in pathogenesis of CDH and associated pulmonary hypoplasia, and recent studies have also suggested that the retinoid signaling pathway (RSP) is inhibited in the nitrofen-induced hypoplastic lung [14–17]. However, the relationship between PI3K-AKT signaling pathway and lung hypoplasia associated with CDH has not been elucidated. We designed this study to investigate the hypothesis that the pulmonary gene and protein expression/distribution of PI3K and AKT is altered during critical period of lung morphogenesis in the nitrofen-induced hypoplastic lung.

1. Materials and methods

1.1. Animals and drugs

Adult Sprague-Dawley rats were mated, and the females were checked daily for plugging. The presence of spermatozoids in the vaginal smear was considered as a proof of pregnancy; the day of observation determined gestational day 0 (D0). Pregnant female rats were then randomly divided into 2 groups. At 9 days of gestation (term, 22days), animals in the experimental group received intragastrically 100 mg of Nitrofen (WAKO Chemicals, Osaka, Japan) dissolved in 1 mL of olive oil under short anesthesia, whereas those in control group received only vehicle. Fetuses were harvested by cesarean delivery on D15, D18, and D21 of gestation. Fetuses exposed to nitrofen were divided into 2 groups as follows: nitrofen without CDH group (CDH[−]) and nitrofen with CDH group (CDH[+]) (n = 8 at each time-point, respectively). The control group (n = 8 at each time-point) consisted of animals that received only vehicle. The Department of Health and Children approved the protocol of these animal experiments (reference B100/4022) under the Cruelty to Animals Act, 1876, as amended by European Communities Regulations 2002 and 2005, and all animals were treated according to the current guidelines of animal care.

1.2. RNA isolation and real time reverse transcription polymerase chain reaction

The peripheral region of left lungs dissected from the thoracic cavity were immediately suspended in RNAlater solution (Ambion, United Kingdom) and stored at −20°C. The total RNA of each lung derived from fetuses was isolated using TRIzol Reagent (Invitrogen), according to recommended protocol. Total RNA quantification was performed spectrophotometrically (NanoDrop ND-1000 UV-Vis spectrophotometer). Total RNA (1 μg) was reverse-transcribed using RETROscript (Ambion) according to manufacturer’s instruction. After reverse transcription at 44°C for 60 minutes, polymerase chain reaction was performed using LightCycler 480 SYBR Green I Master (Roche Diagnostics, Germany) according to the manufacturer’s protocol. Gene-specific primers are listed in supplemental Table 1, and 40 cycles of amplification for each primer pair were carried out. Each cycle included a denaturation step of 15 seconds at 94°C and polymerization step of 30 seconds at 72°C. Annealing temperatures were 58°C for 30 seconds. Relative levels of gene expression were measured by LightCycler 480 (Roche Diagnostics) according to the manufacturer’s instruction. Experiments were carried out in triplicate for each data point. The messenger RNA (mRNA) expression levels of PI3K and AKT were normalized to β-actin mRNA expression levels in each sample.

1.3. Immunohistochemistry

The paraffin-embedded lungs were sectioned at a thickness of 5 μm, and the sections were deparaffined with xylene and then rehydrated through ethanol and distilled water. Tissue sections were immersed in target retrieval solution (Dako Ltd, Cambridgeshire, UK) heated for 10 minutes at 121°C followed by incubation in 0.3% hydrogen peroxide for 30 minutes to block endogenous peroxidase activity. Sections were incubated overnight at 4°C with each of the primary antibodies including a 1:100 dilution of rabbit polyclonal antibodies against PI3K p110α (Lot sc-7174; Santa Cruz Biotechnology) and AKT1/2/3 (Lot sc-8312; Santa Cruz Biotechnology). Sections were then treated in horseradish peroxidase-labeled rabbit secondary antibodies and then processed using a kit (Dako Ltd), developed with a diaminobenzidine-hydrogen peroxide substrate complex, and counterstained with hematoxylin.

1.4. Statistical analysis

All numerical data are presented as means ± SD. Differences between 2 groups at each gestational day were tested by using an unpaired Student’s or Welch’s t test when the data had normal distribution, or Mann-Whitney U test when the data deviated from normal distribution. Statistical significance was accepted at P < .05.

2. Results

2.1. Relative mRNA expression levels of PI3K and AKT in fetal rat lungs

The relative mRNA expression level of PI3K at D21 was significantly decreased in nitrofen-induced hypoplastic lungs of CDH(−) and CDH(+), compared to control lungs (P < .05) (Table 2). However, there were no significant differences between CDH(−), CDH(+), and controls in the pulmonary PI3K gene expression levels at D15 and D18. Pulmonary gene expression level of AKT at D18 was significantly decreased in CDH(−) and CDH(+) group compared to controls (P < .01), whereas there was no significant difference in AKT gene expression between hypoplastic lungs and control lungs at the other time-points (Table 3). Furthermore, there were no significant differences between CDH(−) and CDH(+) in the gene expression levels of PI3K and AKT at each time-point.

Table 2.

Relative mRNA expression levels of PI3K in lung (n = 8 for each group at each time-point)

		D15	D18	D21
Control		10.16 ± 3.37	9.46 ± 1.18	8.95 ± 3.22
Nitrofen	CDH(−)	12.39 ± 1.57	10.35 ± 2.77	5.71 ± 0.85 *
	CDH(+)		12.65 ± 6.94	6.80 ± 0.88 *

^*P < .05 vs control.

Table 3.

Relative mRNA expression levels of AKT in lung (n = 8 for each group at each time-point)

		D15	D18	D21
Control		1.95 ± 0.49	1.62 ± 0.14	1.22 ± 1.07
Nitrofen	CDH(−)	1.96 ± 0.26	1.21 ± 0.16 *	1.05 ± 0.20
	CDH(+)		1.20 ± 0.32 *	1.29 ± 0.29

^*P < .01 vs control.

2.2. Protein expression of PI3K and AKT in fetal rat lungs

To determine whether the decreased amounts of PI3K and AKT transcripts were reflected in the amount of the protein themselves in the nitrofen-induced hypoplastic lung, immunohistochemical study was performed (Figs. 1 and 2). In D15 control and CDH lungs, there was diffused expression of PI3K protein that appeared to be localized to both the epithelial and the mesenchymal compartments of the lung with no differences in immunoreactivity (Fig. 1A and B). In D18 CDH lungs, PI3K immunereactivity was decreased in the distal epithelium in CDH lungs (Fig. 1D) compared to those in control lungs (Fig. 1C), whereas there were no differences in intensity of PI3K protein expression in the mesenchyme. In D21 CDH lungs, PI3K immunoreactivity is diminished in the overall intensity (Fig. 1F), although strong PI3K protein expression is seen in control lungs (Fig. 1E).

Fig. 1.

PI3K immunohistochemistry. Pulmonary expression of PI3K protein at D15 control (A) and CDH (B) showing no difference in immunoreactivity and distribution. At D18, PI3K expression is diminished in the distal epithelium in CDH lungs (D) compared to control lungs (C). At D21, PI3K immunoreactivity is decreased in the overall intensity in CDH lungs (F), whereas strong PI3K protein expression is seen in control lungs (E).

Fig. 2.

AKT immunohistochemistry. Pulmonary AKT protein expression at D15 control (A) and CDH (B) showing no difference in immunoreactivity and distribution. At D18, AKT immunoreactivity is decreased in the mesenchyme in CDH lungs (D) compared to control lungs (C). At D21, the overall intensity of AKT protein expression is diminished in CDH lungs (F), whereas strong AKT expression is observed in control lungs (E).

In D15 control and CDH lungs, low-level pulmonary AKT protein expression was observed diffusely with no difference in immunoreactivity (Fig. 2A and B). In D18 CDH lungs, AKT immunoreactivity was decreased in the mesenchyme in hypoplastic lungs (Fig. 2D) compared to control lungs (Fig. 2C). In D21 CDH lungs, the overall immunoreactivity of AKT was diminished in CDH lungs (Fig. 2F), whereas strong AKT expression was seen in control lungs (Fig. 2E).

3. Discussion

In the present study, we examined the role of the PI3K and AKT signaling pathway in hypoplastic lungs in CDH. Immunohistochemical studies showed that the protein expression of PI3K and AKT was decreased on D18 and D21 in the nitrofen-induced hypoplastic lungs compared to control lungs. However, we observed significant downregulation of PI3K gene expression in the hypoplastic lungs only at D21 and downregulation of AKT gene expression only at D18. Furthermore, in D18 hypoplastic lungs, PI3K immunoreactivity was decreased mainly in the epithelium, whereas AKT protein expression was decreased mainly in the mesenchyme. These findings lead us to speculate that decreased interactive signaling between epithelial-PI3K and mesenchymal-AKT in D18 hypoplastic lungs interferes with epithelial-mesenchymal interaction during mid-to-late lung morphogenesis, resulting in lung hypoplasia in the nitrofen CDH model.

Apoptosis has been demonstrated to occur at several stages of lung development [18], but the significance and regulatory mechanisms of apoptosis in the development of lung have not been fully investigated. According to Stiles et al [19], the occurrence of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling-positive cells is rare on D15 and then increases up to D21. Serine/threonine protein kinase B has been reported to directly inhibit the caspase proteases, key executioners of apoptosis [20], but it is not clear how the PI3K-AKT pathway is involved in lung cell apoptosis during development. In the lung explant study, it has been reported that PI3K inhibitors, LY294002 and wortmannin, increase apoptosis in the mesenchyme of the explants [10]. Recently, it has been demonstrated that nitrofen exposure increased apoptosis in the mesenchyme of fetal rat lung [21]. It is tempting to speculate that downregulation of PI3K and AKT gene expression levels during mid-to-late lung morphogenesis may increase apoptosis in the mesenchyme, causing lung hypoplasia.

Recent studies suggest that RSP may be involved in pathogenesis of CDH and associated pulmonary hypoplasia [14–17]. It has been reported that retinoid all-trans retinoic acid activates retinoic acid receptor-mediated PI3K-AKT pathway [22]. It is well-known that RSP is inhibited in the nitrofen-induced CDH model. Furthermore, it has also reported that cellular retinol-binding protein-I inhibits, in a retinoic acid receptor-dependent manner, the PI3K-AKT pathway [23]. It has previously shown from our laboratory that mRNA expression levels of cellular retinol-binding protein-I are upregulated in the nitrofen-induced hypoplastic lung [24]. Therefore, our findings of downregulation of PI3K and AKT gene expression in hypoplastic lung are consistent with these previous studies.

In this study, we provide evidence, for the first time, that pulmonary gene expression of PI3K and AKT is downregulated in the nitrofen-induced hypoplastic lungs during mid-to-late stages of lung development. Furthermore, we also observed that the immunoreactivity of PI3K and AKT shows a tendency to decrease in late lung morphogenesis. These findings suggest that disruption of PI3K-AKT signaling during the critical period of lung morphogenesis may cause pulmonary hypoplasia in nitrofen-induced CDH rat model, interfering with epithelial-mesenchymal interaction.

Studies in the nitrofen model of CDH have showed that lung hypoplasia of the lung starts to develop early in gestation, even before the normal closure of the diaphragm take place, supported by the concept of a “dual-hit hypothesis” [25]. In our study, there were no significant differences between CDH(−) and CDH(+) in pulmonary gene expression levels of PI3K and AKT at D18 and D21. It is tempting to speculate that alteration of PI3K and AKT gene during the lung morphogenesis in the nitrofen-induced CDH model may affect the developing lung independently of mechanical compression of the lung by herniation of abdominal viscera into the thorax. This is further supported by our previous studies of gene alterations in the RSP [15,24] and in the alveolar epithelial cells type II markers in late gestation in the nitrofen model [26]. Further studies of PI3K-AKT signaling in the nitrofen-induced hypoplastic lung are required and should provide insights into the pathogenesis of altered lung morphogenesis in CDH.

Table 1.

Primers for quantitative real time reverse transcription polymerase chain reaction

Gene	Sequence (5’-3’)	Product size (base pair)
β-actin		108
Forward	TTG GAT GCC TGT GGT CTG TC
Reverse	TAG AGC CAC CAA TCC ACA CA
PI3K		113
Forward	ATG GGT TGG AAG ATC TGC TG
Reverse	TGG AAA CTT CAC CAC ACT GC
AKT		214
Forward	TGT GAT GGA GTA TGC CAA CG
Reverse	TTT GCA CAA GCC AAA GTC AG