Alveologenesis is an essential developmental phase occurring postnatally in mice and during late gestation in humans. This phase is characterized by elastin deposition in the alveolar sacs leading to the formation of “secondary septa” at the very same place of elastin deposition. The cells which are responsible for the depositing ring shaped elastin structures that surround the mouths of developing alveoli are called “alveolar myofibroblasts”. The “secondary septa” form the spheroidal walls of mature alveoli. As the process of alveolarization progresses, it leads to a marked increase of the alveolar surface and therefore the area available for gas exchange increases. The process of alveologenesis mainly takes part in the period of P0-P14 in mice (approximately equivalent to the first 6 months after birth in humans). Afterwards, from P14 through P28, the maturation of the alveoli occurs [1
]. Many different signaling ligands and receptors are involved in the process of alveolarization: absence of Platelet derived growth factor alpha (Pdgfα) leads to lack of Pdgf receptor α expressing cells, which potentially form a progenitor population for the α-smooth muscle actin-expressing alveolar myofibroblasts [2
]. Sonic hedgehog (Shh) signaling is also required for proper alveolar myofibroblast differentiation [1
]. In addition, in a newborn murine hypoalveologenesis model, Perl et al. showed that the re-alveolarization induced by application of retinoic acid is dependent on Fgf signaling [4
]. Interestingly, the absence of both Fgfr3
leads to impaired alveologenesis [5
] but the endogenous Fgf ligands for these receptors are still unclear. During mouse lung development, increased Fgf signaling in the mesenchyme leads to impaired alveolar myofibroblast formation, associated with decreased elastin deposition [6
Bronchopulmonary dysplasia (BPD) is the most common chronic airway disease of prematurely born infants, whereby low gestational age and weight at birth embody important factors increasing the probability of BPD occurrence (e.g., 20% of the infants born with a gestational weight of under 1500 g and a gestational age of under 30 weeks develop BPD in the US) [7
]. Between 10,000 and 15,000 preterm infants are affected by BPD each year in the US alone [11
]. The number of BPD patients increases due to improved therapy and increased survival rate at lower gestational ages [7
]. From the pathophysiological point of view, BPD prevents alveologenesis from occurring. Oxygen toxicity associated with mechanical ventilation is considered as one of the major injurious factor in the pathogenesis of BPD. BPD interferes with the process of alveolarization, leading to a phenotype of alveolar simplification, which has been quantified as a decreased number but with an increased diameter of alveoli in rats [13
]. Furthermore, thicker alveolar walls remain and the development of the pulmonary vasculature is disrupted [8
], collectively leading to restricted gas exchange due to reduced alveolar surface and increased distance within the alveolar wall for gas diffusion between the alveolar lumen and the capillary lumen.
As more patients with BPD survive due to optimized therapy [9
], but nevertheless carry symptoms and have impaired lung function, there is an urgent need for a better understanding of the pathophysiological mechanisms underlying this complex disease. In addition, the development of new and better diagnostic approaches (e.g., miRNAs
as markers in peripheral blood [8
]) that can potentially distinguish between prematurely born infants at risk of developing BPD will enable therapy at an early stage or prevent unnecessary therapy. Finally, new therapeutic tools will be instrumental to attenuate the symptoms of impaired lung function after surviving BPD to improve the condition of these patients and to lower the high costs of caring for infants with this disease.
) are small regulatory RNAs in mammals that account for approximately 1% of the genome. They are 22- to 25-nucleotide-long single-stranded RNAs processed from hairpin transcripts, that regulate gene expression post transcriptionally in eukaryotes by binding at the 3′-UTR regions of the target mRNA, thus leading to mRNA cleavage, degradation or translational repression. The maturation of hairpin transcripts give rise to two isoforms, a 3p
guide strand and 5p
sister passenger strand. In general, only one isoform remains while the complementary isoform is degraded. But in some cases both isoforms can be produced thereby allowing the silencing of specific sets of genes through base pairing to a minimal recognition sequence [15
are involved in almost every known molecular process [16
]. Yet, only little is known about their role in late lung development nor their involvement in BPD [8
]. Although a changed expression has been described for several miRs
in BPD, a causal role in BPD remains to be established [8
in general are now known to be involved in many biological and pathological processes in the lung [17
(initially called miR-154
are both part of the human “DLK1-DIO3
genomic region”, which is located on chromosome region 14q32
(murine chromosome 12F2
]. Among the paternally expressed imprinted genes in this genomic region, DLK1
, and DIO3
and the maternally expressed imprinted genes MEG3
), and an anti-sense RTL1
) are found. In addition, this region contains a miR
cluster with 54 miRs
, thus being one of the largest miR
containing clusters in humans [18
]. The miRs
from this cluster are only expressed from the maternally inherited chromosome [20
]. Furthermore none of these miRs
binds their target mRNAs
with full complementarity, suggesting that they may act on their targets by translational repression rather than by post-transcriptional decay [20
]. The expression of the genes on the maternal chromosome 12F
in mice is regulated by so-called “DMRs
” (differentially methylated regions) [22
]. Various members of this cluster play roles in human pathologies [18
are highly conserved between mice and humans, which has been demonstrated by sequence equality [24
] (hsa-miR-154-3p = mmu-miR-154-3p
= 5′-AAU CAU ACA CGG UUG ACC UAU U-3′; hsa-miR-154-5p = mmu-miR-154-5p
= 5′- UAG GUU AUC CGU GUU GCC UUC G). Within the DLK1-DIO3
genomic region, this sequence is located in the maternally expressed imprinted intergenic region Mirg
], which is regulated by an intergenic germline-derived differentially methylated region [19
Similar expression profiles of these miR
s in embryonic and adult lung tissue were found in humans and mice, indicating evolutionary conservation of these miRs
as well as their potential functions in lung development across the two species [19
In this study, we demonstrate that the expression level of miR-154, which increases during the fetal phases of fetal lung development, normally decreases postnatally. We further demonstrate that hyperoxia treatment maintains high levels of miR-154 in alveolar type 2 cells (AT2). We therefore hypothesized that the postnatal decrease in miR-154 expression in AT2 cells is required for normal alveologenesis. To test this hypothesis, we generated a novel transgenic mouse allowing doxycycline-based miR-154 overexpression and analyzed the impact of overexpressing miR-154 in the respiratory epithelium postnatally in normoxic or hyperoxic conditions. Our results indicate that down-regulation of miR-154 in postnatal lungs may function as an important physiological switch that permits the induction of the alveolar developmental program, while conversely, failure to down-regulate miR-154 suppresses alveolarization, leading to the common phenotype of alveolar simplification. Our results support the idea that down-regulation of miR-154 within AT2 cells is an important driver of alveologenesis.
2. Material and Methods
2.1. Study Approval
Animal studies: all experiments were approved and performed in accordance with the guidelines from the Federal Authorities for Animal Research of the Regierungspraesidium Giessen, Hessen, Germany (Protocol 21/2013).
CD1 mice were crossed to generate WT pups. FVB.Cg-Tg
(thereafter called Tg(Scgb1a1-rtTA)/+
) were kindly provided by Anne Karina Perl (Jacksonlab stock number 006232). These mice have been reported to target the respiratory epithelium during embryonic and postnatal stages. They were crossed with mTg(tet(o)miR-154)gc
) (thereafter called Tg(tet(o)miR-154)/+
) generated for the need of this study by pronuclear injection of the expression cassette into the blastocysts. Mice were kept on the C57BL/6J background for at least 5 generations. Both genders were used. For induction of the transgene [Tg(Scgb1a1-rtTA)
/+] mice were fed with food containing doxycycline (concentration 625 mg/kg, Altromin Spezialfutter GmbH & Co. KG, Lage, Germany). [Tg(Scgb1a1-rtTA)/+; +/+]
littermates (negative for Tg(tet(o)miR154)
) were used as control mice. Alternatively, we also used the Rosa26rtTA/rtTA
mice (for generation of these mice see [25
]) to drive ubiquitously miR-154
expression in the lung from E7.5 to E18 [Rosa26(rtTA/rtTA); Tg(tet(o)miR154)/+
2.3. Hyperoxia Injury (BPD Mouse Model)
Newborn pups were subjected to hyperoxia (HOX) (85% O2) injury from P0-P8 in a chamber (Proox Model 110, Biospherix). To minimize oxygen toxicity and bias, nursing dams were rotated every 24 h between normoxia (NOX) and HOX. Pups and dams received food and water ad libitum.
2.4. Left Lobe Perfusion, Isolation and Tissue Processing, Alveolar Morphometry (Mean Linear Intercept, Air Space, Septal-Wall Thickness)
For newborn mice at P2, P5 and P8, the left lobe was perfused through the trachea with a pressure of 20 cm H2O with 5 mL PBS followed by 5 mL 4% PFA. For pups at E18.5 the tracheal perfusion was done by using 10 cm H2O with 1 mL PBS followed by 1 mL 4% PFA. The trachea was tied off with a string, and the lung was removed and placed in 4% PFA for max. 24 h at 4 °C. Lungs were then progressively dehydrated (30%, 50%, 70%, 99.6% ethanol, each 3 h) and embedded with a Leica embedding machine (EG 1150C, Leica, Wetzlar, Germany). Paraffin blocks were kept cold and 5 μm sections were generated.
For alveolar morphometry, lungs were flushed subsequently with PBS and 4% paraformaldehyde in phosphate-buffered saline (pH 7.0) at a vascular pressure of 20 cm H2
O. Then PBS was infused via the trachea at a pressure of 20 cm H2
O and fixed with 4% paraformaldehyde in phosphate-buffered saline (pH 7.0) via the trachea at a pressure of 20 cm H2
O. Investigations were performed using 5 μm sections of the paraffin-embedded left lobe of the lungs. The mean linear intercept, mean air space, and mean septal wall thickness were measured after staining with hematoxylin and eosin (HE). Total scans from the left lobe were analyzed using a Leica DM6000B microscope with an automated stage according to the procedure previously described [26
], which was implemented into the Qwin V3 software (Leica, Wetzlar, Germany). Horizontal lines (distance 40 μm) were placed across each lung section. The number of times the lines cross alveolar walls was calculated by multiplying the length of the horizontal lines and the number of lines per section then dividing by the number of intercepts. Bronchi and vessels above 50 μm in diameter were excluded prior to the computerized measurement. The air space was determined as the non-parenchymatous non-stained area. The septal wall thickness was measured as the length of the line perpendicularly crossing a septum. From the respective measurements, mean values were calculated.
2.5. RNA Extraction and Quantitative Real-Time RT-qPCR
After lung function measurements were taken, the right bronchus was clamped and either cranial and accessory or caudal and medial lobes were removed, placed in TRIZOL, homogenized in GentleMACs and frozen in liquid nitrogen for RNA extraction. RNA was isolated using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. RNA was reverse-transcribed (QuantiTect Reverse Transcription Kit, Cat. No. 205313, Qiagen GmbH, Hilden, Germany). cDNA was diluted to a concentration of 5 ng/μ.. Primers were designed using Roche Applied Sciences online Assay Design Tool (Roche Diagnostics Deutschland GmbH, Mannheim, Germany).
All primers were designed to span introns and blasted using NCBI software for specificity. Sybr Green Master Mix (invitrogen, Cat. No.11733-038, Carlsbad, CA, USA) was used for RT-PCR with a Roche LightCycler 480 machine (Roche Diagnostics Deutschland GmbH, Mannheim, Germany). Samples were run in triplicates using Hprt
as a reference gene. Mouse primers are listed in supplementary data
2.6. Isolation of Primary Alveolar Type II Cells and Microarray Experiments
Isolation of AT2 cells was performed as previously described [28
] with few modifications. Briefly, the whole lung was perfused with 1 mL PBS through the right ventricle to remove the intrapulmonal blood cells. Lungs were perfused with 1 mL dispase through the trachea and the trachea was tied off with a string. Lungs were digested in 2 mL dispase for 30 min at 37 °C and minced. The suspension was sequentially filtered through 70, 40, and 10 μm nylon meshes and then centrifuged at 200× g
for 10 min. The pellet was resuspended in Dulbecco’s modified eagle medium (Invitrogen, Karlsruhe, Germany), and negative selection for endothelial cells and lymphocytes/macrophages was performed by incubation on CD31- and CD45-coated Petri dishes for 45 min at 37 °C. Negative selection for fibroblasts was performed by adherence for 45 min at 37 °C on uncoated cell-culture dishes. Cell purity was analyzed in freshly isolated AT2 cells directly after isolation by epithelial cell morphology and immunofluorescence analysis with Nile red. AT2 cells used throughout this study demonstrated 95 ± 3% purity.
RNA from AT2 cells was purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany) following the kit instructions. RNA quality was assessed by capillary electrophoresis using the Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA). Purified total RNA was amplified and Cy5-labeled using the LIRAK kit (Agilent) following the kit instructions. Per reaction, 200 ng of total RNA was used. The Cy-labeled RNA was hybridized overnight to 8 × 60 K 60 mer oligonucleotide spotted microarray slides (Agilent Technologies, design ID 028005). Hybridization and subsequent washing and drying of the slides were performed following the Agilent hybridization protocol. The dried slides were scanned at 2 µm/pixel resolution using the InnoScan is 900 (Innopsys, Carbonne, France). Image analysis was performed with Mapix 6.5.0 software (Innopsys, 31,390 Carbonne, France), and calculated values for all spots were saved as GenePix results files. Stored data were evaluated using the R software and the limma package from BioConductor (free accessible software, www.r-project.org
]. Log mean spot signals were taken for further analysis. Data was quantile-normalized before averaging. Genes were ranked for differential expression using a moderated t-statistic [30
]. Pathway analyses were done using gene set tests on the ranks of the t-values. We also carried out a gene microarray between experimental [Rosa26rtTA)/rtTA; Tg(tet(o)miR154)/+
] and control [Rosa26rtTA)/rtTA; +/+
] lungs (exposed to Dox food from E7.5 to E18) for the selection of potential mRNA targets after the pull-down assay. The data of the microarray experiment are deposited in GEO and are available through the accession number GSE141300 (please note that this SuperSerie is composed of three SubSeries).
2.7. Immunofluorescence Staining
Paraffin sections were deparaffinized, blocked with 3% bovine serum albumin (BSA) and 0.4% Triton X-100 [in Tris-buffered saline (TBS)] at room temperature (RT) for 1 h and then incubated with primary antibodies against Ki67 (Thermo-Scientific; 1:200), Cdh1/Ecad (BD; 1:100), pro-SFTPC (Seven Hills; 1:100) and pro+mature SFTPB (Abcam; 1:500) at RT for 1 h or at 4 °C overnight. After incubation with primary antibodies, slides were washed three times in TBST (TBS buffer + 0.1% Tween 20) for 5 min, incubated with secondary antibodies at RT for 1 h and then washed three times in TBST before being mounted with ProLong Gold Antifade Reagent with DAPI (4,6-diamidino-2-phenylindole; Invitrogen). Fluorescent images were acquired using Leica DM5500 B fluorescence microscope connected to Leica DFC360 FX camera (Leica, Wetzlar, Germany).
2.8. Fluorescence activated Cell Sorting
Whole lungs were isolated in ice-cold Hank’s balanced salt solution (HBSS). Then, lobes were chopped finely using sterile razor blades, digested in a 10 mL solution of 0.5% collagenase in HBSS on a heating plate (40 °C) with stirring at 700 rpm for 60 min. Once the homogenate was dissociated, the cell suspension was successively passed through 20G, 24G, and 26G needles, then strained on 70 μm and 40 μm filters. One volume HBSS was added to dilute collagenase and cell suspensions were centrifuged at 1500 rpm for 5 min to remove the enzyme solution. Cells were then resuspended in 500 μL 10% FCS in DMEM and stained with fluorochrome-labeled anti-mouse antibodies for 20 min at 4 °C (please see supplementary data
), followed by washing and flow cytometric analysis with LSR Fortessa equipped with FACSDiva™ software (BD Bioscience, San Jose, CA, USA). FACS for lung epithelial progenitor cells was performed as previously described [31
2.9. Fluorescence In-Situ-Hybridization (FISH)
5 µm sections of the left lobe of the lung were deparaffinized with Xylene (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and a decreasing gradient of Ethanol. After washing the slides with DEPC-PBS the section was digested with Proteinase K (peqlab, Germany). The time of incubation and the concentration of the Proteinase K added to the Proteinase K buffer were dependent on the age of the samples (P2: 1:3000 for 4 min; P5: 1:1300 for 7 min; P8: 1:1300 for 10 min). After washing with DEPC-PBS the sections were blocked with Dual endogenous enzyme block (DAKO EnvisionTM + Dual Link System-HRP (DAB+) kit, USA) and then washed again with DEPC-PBS. Then, the sections were incubated in 0.01% Glutaraldehyde solution (Sigma-Aldrich Chemie GmbH, Germany) diluted in 4% PFA (Carl Roth GmbH & Co. KG, Germany) for 10 min followed by another washing step of DEPC-PBS. The sections were pre-incubated with miRCURY LNATM microRNA Detection Hybridization Buffer for 5 h at 54 °C before incubation with the miRCURY LNATM Detection probe (hsa-miR-154-3p, probe sequence: 5′-AATAGGTCAACCGTGTATGATT-3′) diluted in Hybridization Buffer (1:625) for 37 h at 54 °C. To protect the sections from drying out during incubation, they were covered with HybriWell Incubation chambers (Bio Cat, Germany). The sections were washed with a decreasing gradient of SSC (Sodium/Sodium citrate stock solution 1054.1, Roth, Germany) at 52 °C and incubated in a blocking solution (DIG Wash and Block Buffer Set, Roche Diagnostics GmbH, Germany) containing 72% DEPC water, 18% Maleic acid Buffer 10x and 10% blocking solution 10x for 30 min at room temperature. Anti-DIG-POD (ratio 1:400; Roche, Germany) and Sheep Serum (1:250; Dianova, Germany) were added to the blocking solution from the previous step and the section incubated for 4 h at room temperature. After a last washing step of DEPC-PBS, TSATM-plus Fluorescein System (Perkin Elmer, Boston, MA, USA) was applied to the section for 15 h. Coverslips were mounted on the slides with Prolong® Gold antifade reagent with DAPI (ProLongTM Gold antifade reagent with DAPI, P36935, Waltham, MA, USA). Slides were stored at 4 °C for further analysis.
For quantification, at least 3 histological samples and 5 areas of each sample were used. The number of miR-154-3p positive cells (miR-154-3p positive and DAPI positive) was counted and compared to the total number of DAPI positive cells for both bronchiolar and alveolar epithelium. For each sample, a mean value of the number of miR-154-3p positive cells was calculated (miR-154-3p positive/DAPI positive cells in relation to DAPI positive cells). Statistical analyses were performed as previously described.
2.10. Pull Down Assay with Biotinylated miR-154-3p
MLE12 cells were cultured in six-well plates and transfected in triplicate with 3′-biotinylated miR-154 (Bio-miR-154) or 3′-biotinylated scramble (Bio-scramble; Dharmacon), at a final concentration of 30 nM using Lipofectamine RNAimax (Invitrogen) following the manufacturer’s protocol. After 48 h, the cells were pelleted at 1000 rpm for 5 min. After washing twice, cell pellets were resuspended in 0.5 mL lysis buffer [50 mM Tris-HCl, 2 mM EDTA, 0.1% NP40, 10% glycerol, 2 mM EGTA, diethylpyrocarbonate (DEPC)-treated water, 50 U RNasin (Promega) and complete mini-protease inhibitor cocktail (Roche Applied Science)], and incubated at 4 °C for 10 min. The cytoplasmic extract was isolated by centrifugation at 10,000 rpm for 10 min. Streptavidin-coated magnetic beads (Invitrogen) were blocked for 1 h at 4 °C in blocking buffer (10 mM Tris-HCl pH 6.5, 1 mM EDTA, 1 mg/mL yeast tRNA and 1 mg/mL BSA) and washed twice with 1 mL washing buffer (10 mM Tris-HCl pH 6.5, 1 mM EDTA 0.5 M NaCl). Beads were resuspended in 0.5 mL washing buffer. Cytoplasmic extract was then added to the beads and incubated for 1 h at 4 °C with slow rotation. The beads were then washed five times with 1 mL washing buffer. RNA bound to the beads (pull-down RNA) or from 10% of the extract (input RNA), was isolated using Trizol reagent LS (Invitrogen). The level of mRNA in the Bio-miR-154 or Bio-scramble control pull-down was quantified by qPCR. mRNA levels were normalized to a housekeeping gene (Gapdh, H4). The enrichment ratio of the control-normalized pull-down RNA to the control-normalized input levels was then calculated. The data for each cell line are representative of three independent experiments. The isolated RNA was processed for gene array analysis as described above. The data of the gene array experiment are deposited in GEO and are available through the accession number GSE141300.
2.11. Statistical Analyses
Significance was determined by two-tailed Student´s t-test using GraphPad PRISM statistical analysis software. All data are presented as mean ± SEM. Values of p < 0.05 were considered significant.
Our data demonstrate that endogenous miR-154
expression is normally downregulated in the distal lung epithelium after birth. However, persistent expression of miR-154
was observed in AT2 cells isolated from postnatal lungs exposed to HOX versus NOX suggesting that downregulation of miR-154
in AT2 could be important to facilitate the induction of alveologenesis. To test this hypothesis, we generated a novel miR-154
gain of function transgenic mouse to overexpress miR-154
in the airway epithelium. miR-154
overexpression in the alveolar epithelium using the Tg
) driver line under normoxic conditions is sufficient to prevent alveolarization and triggers alveolar simplification as reflected by increased MLI. Using a pull down assay with biotinylated miR-154
, we identified Cav1
as a primary functional target for miR-154
. We further demonstrated that Cav1 protein expression is decreased in miR-154
experimental versus control lungs. This is associated with increased Tgf-β1 signaling as shown by the upregulation of P-Smad3 levels. Increased Tgf-β1 signaling in the lung at birth is associated with a BPD-like phenotype of alveolar simplification. Our conclusion that miR-154
overexpression leads to enhanced Tgf-β signaling in the lung which could be causative for the BPD-like alveolar simplification phenotype observed in these lungs is supported by the literature. We were the first to report that gain of Tgf-β1 expression in the neonatal lung led to a BPD like phenotype [40
]. This was subsequently confirmed in neonatal mice [41
]. In addition, the Tgf-β signaling pathway has been shown to be upregulated in the lung of neonatal mice exposed to hyperoxia [42
]. In summary, an intricate balance of Tgf-β signaling during prenatal and early postnatal mouse lung development seems to be essential for proper lung development and alveologenesis.
In addition, we report that the transcriptomic changes occurring following hyperoxia versus normoxia exposure in the control lungs (especially for Fgf and Tgf-β signaling as well as the epithelial markers) are suppressed in the miR-154 overexpression lungs. We therefore propose that hyperoxia may be eliciting its effect on alveologenesis by suppressing the downregulation of miR-154 expression in AT2 cells. Thus, miR-154 down-regulation may be required to release a set of gene functions that are critical for the induction of alveolarization.
Interestingly, a link between Tgf-β signalling and miR-154
was already described in the context of idiopathic pulmonary fibrosis (IPF). Milosevic and colleagues examined the involvement of miR-154
in the fibrotic phenotype of IPF patients’ lungs [43
stimulation via its downstream effector SMAD3 in vitro elicited the upregulation of various members of a miRNA
cluster, which is mapped on human chromosome 14q32
and part of the imprinted DLK1-DIO3
domain. This cluster included miR-154
was shown to induce proliferation and migration in lung fibroblasts partly via the repression of p15 (CDKN2B) protein level, a cell cycle inhibitor, and induction of the WNT/β-Catenin pathway.
locus which contains miR-154
was also described to be downstream of Histone deacetylase 3 (Hdac3). Hdac3
knockout mice display a reduction of AT1 cell spreading leading to sacculation defects [44
]. Hdac3 represses both miR-17-92
as well as the miRNAs in the Dlk1-Dio3
locus, which are targeting Tgf-β signaling. It was suggested that proper levels of Tgf-β signaling are important for AT1 cell remodeling. Interestingly, if Hdac3 controls the expression of both miR-17-92
, this will lead to the combined activation and repression of Tgf-β signaling. The final outcome in terms of Tgf-β signaling will depend on the level of expression of these miRs as well as on the expression of the downstream miR targets in the cells of interest. This situation is not uncommon as we previously described that miR-142-3p
is capable of eliciting the inhibition of Wnt signaling via the targeting of the positive regulator p300
and its activation via the targeting of the negative regulator Adenomatous polyposis coli (Apc
In conclusion, when considering the findings from the literature and taking into account our new results, we hypothesize that miR-154
could also function as an important factor for embryonic development, as its expression increases during the prenatal phases towards birth and then decreases after birth (in accordance with the findings from Williams et al. [19
]). However, the role of miR-154
during fetal development is still unknown. In order to allow proper alveologenesis after birth, we also hypothesize that miR-154
has to be decreased in expression, as both hyperoxic injury (and subsequent hyperoxia-mediated miR-154
activation) and postnatal miR-154
induction led to an impairment of alveolar formation also reflected in alveolar morphometric measurements (Figure 8
A). Thus, the injurious effect of hyperoxic injury on alveolar formation appears to be at least partly mediated by induction of miR-154
In our hypothetical model of action we hypothesize that under physiological conditions (Figure 8
must be downregulated postnatally in AT2 cells, unleashing the putative target Cav1
, which in turn leads to a downregulation of Tgf-β signaling by receptor internalization of Tgf-βr1 [39
]. However, in the context of hyperoxia injury (Figure 8
C), a hyperoxia-induced up-regulation of miR-154
occurs, which inhibits Cav1-mediated Tgf-βr1 receptor internalization, thereby maintaining Tgf-β activity in AT2 cells leading to impairment of alveologenesis.
In conclusion, when miR-154 expression in AT2 is maintained postnatally, it targets Cav1 and thereby allows increased Tgf-β1 signaling. Increased Tgf-β1 signaling in AT2 cells in turn leads to their premature transdifferentiation towards an AT1 phenotype and this interrupts alveologenesis. Our work paves the way for the possible manipulation of the miR-154-Cav1-Tgf-β signaling axis to attempt circumvention of the defective alveologenesis observed in lung diseases of human prematurity such as BPD, that are characterized by alveolar simplification.