Silencing hyperoxia-induced C/EBP? in neonatal mice improves lung
architecture via enhanced proliferation of alveolar epithelial cells
Guang Yang,1Maurice D. Hinson,1Jessica E. Bordner,1Qing S. Lin,1,2Amal P. Fernando,1Ping La,1
Clyde J. Wright,1and Phyllis A. Dennery1,2
1Division of Neonatology, Children’s Hospital of Philadelphia, and2Department of Pediatrics, University of Pennsylvania,
Submitted 11 March 2011; accepted in final form 2 May 2011
Yang G, Hinson MD, Bordner JE, Lin QS, Fernando AP, La P,
mice improves lung architecture via enhanced proliferation of alveolar epi-
thelial cells. Am J Physiol Lung Cell Mol Physiol 301: L187–L196, 2011.
lung development requires proliferation and differentiation of specific
cell types at precise times to promote proper alveolar formation.
Hyperoxic exposure can disrupt alveolarization by inhibiting cell
growth; however, it is not fully understood how this is mediated. The
transcription factor CCAAT/enhancer binding protein-? (C/EBP?) is
highly expressed in the lung and plays a role in cell proliferation and
differentiation in many tissues. After 72 h of hyperoxia, C/EBP?
expression was significantly enhanced in the lungs of newborn mice.
The increased C/EBP? protein was predominantly located in alveolar
type II cells. Silencing of C/EBP? with a transpulmonary injection of
C/EBP? small interfering RNA (siRNA) prior to hyperoxic exposure
reduced expression of markers of type I cell and differentiation
typically observed after hyperoxia but did not rescue the altered lung
morphology at 72 h. Nevertheless, when C/EBP? hyperoxia-exposed
siRNA-injected mice were allowed to recover for 2 wk in room air,
lung epithelial cell proliferation was increased and lung morphology
was restored compared with hyperoxia-exposed control siRNA-
injected mice. These data suggest that C/EBP? is an important
regulator of postnatal alveolar epithelial cell proliferation and differ-
entiation during injury and repair.
CCAAT/enhancer binding protein-?; developing lung injury; small
interfering RNA; recovery
POSTNATAL LUNG DEVELOPMENT involves a series of coordinated
events, including active cell proliferation and differentiation, to
promote proper alveolar formation. Parenchymal lung cells,
fibroblasts, endothelial cells, and epithelial type II cells have
distinct growth patterns and specific proliferation rates, begin-
ning in the neonatal period (9). Imbalance of growth factors,
inflammatory insults, and oxidative stress (reviewed in Ref. 16)
could alter these processes, resulting in impaired lung devel-
opment. Hyperoxic exposure is well known for disrupting
alveolarization in the developing lung, because it inhibits the
growth of epithelial, fibroblast, and endothelial cells forming
the alveoli (5, 19). Several studies have demonstrated that
hyperoxia results in alveolar growth arrest, induction of cell
cycle inhibitory proteins (13), altered surfactant protein (SP)
production (reviewed in Ref. 4), and disrupted extracellular
matrix signaling (1). The effect of hyperoxia on the prolifera-
tion of specific lung cell types is not well defined.
The transcription factor CCAAT/enhancer binding protein
(C/EBP) family consists of six isoforms. C/EBP? is highly
expressed in the lung (6) and plays a role in cell proliferation
and differentiation in various tissues (17, 18), as do C/EBP?
and C/EBP?. Given that the newborn lung proliferates and
differentiates rapidly, we hypothesized that C/EBP? regulates
neonatal lung cell proliferation in hyperoxia and during recov-
Here, we demonstrate that C/EBP? expression was en-
hanced in lungs of newborn mice after 72 h of hyperoxia. The
increased C/EBP? protein was predominantly localized to
alveolar type II cells, where altered proliferation was observed.
Silencing of C/EBP? with a single injection of C/EBP? small
interfering RNA (siRNA) prior to exposure did not rescue the
altered lung morphology after 72 h of hyperoxia but restored
lung morphology after 2 wk of room air recovery by enhancing
lung epithelial cell proliferation.
MATERIALS AND METHODS
Animal models and hyperoxic exposures. Pregnant C57BL/6 fe-
male mice at gestational day 18 were purchased from Charles River
Laboratories. The mice were kept in a 12:12-h light-dark cycle and
allowed access to food and water ad libitum until the time of
experimentation. Litters of neonatal (?12-h-old) pups, along with
their mothers, were randomly assigned to 21% oxygen or room air or
95% oxygen. Hyperoxic exposure was conducted in an A-chamber
(BioSpherix, Redfield, NY), which allows for continuous monitoring
and regulation of oxygen and carbon dioxide. Ambient carbon dioxide
was maintained at ?1,500 ppm by adjustment of the chamber’s
ventilation. The dams were switched every 24 h between room air and
hyperoxia to avoid injury. All procedures were reviewed and ap-
proved by the Animal Fair and Care Community of the Children’s
Hospital of Philadelphia.
Construction of siRNA. A 19-nucleotide RNA fragment, CGAC-
GAGUUCCUGGCCGAC (15), targeting mouse C/EBP? gene
transcription was synthesized in a siSTABLE format to enhance
stability of the siRNA (Dharmacon, Chicago, IL). Stock concen-
tration was made at 1 ?g/?l in RNase-free water and kept in
aliquots at ?20°C until use. A control siRNA was prepared using
siGENOME Non-Targeting Pool #1 (Dharmacon) and stored as
described above. The Non-Targeting siRNA Pool consisted of
#1–4 individual RNAs, which were characterized by genome-wide
microarray analysis and found to have minimal off-target signa-
tures. For testing efficiency of the transpulmonary delivery and
stability of the delivered siRNA, a positive control siRNA, siGLO-
cyclophilin B (siGLO), conjugated with a fluorophore Cy3
(Thermo Scientific Dharmacon), was purchased and prepared as
described above for the C/EBP? siRNA.
Intrapulmonary delivery. To increase the efficiency of delivery,
aliquots of the C/EBP? siRNA, control siRNA, or siGLO were
dissolved in saline and mixed with Lipofectamine 2000 at room
temperature for 1 h. A 30-?l (3 mg/kg body wt) aliquot of the mixture
Address for reprint requests and other correspondence: P. A. Dennery,
Division of Neonatology, Children’s Hospital of Philadelphia, 34th and Civic
Center Blvd., Philadelphia, PA 19104 (e-mail: email@example.com).
Am J Physiol Lung Cell Mol Physiol 301: L187–L196, 2011.
First published May 13, 2011; doi:10.1152/ajplung.00082.2011.
1040-0605/11 Copyright © 2011 the American Physiological Society http://www.ajplung.org L187
was injected into the left axilla of the neonatal mouse at the third
intercostal space via a 1-ml insulin syringe, as described previously,
resulting in intrapulmonary delivery (20). The mice were returned to
their mothers and kept in room air for 16 h prior to hyperoxic
exposure. The mice received only a single dose of the injected siRNA
during the exposure.
Lung morphometric evaluation: radial alveolar counts. The
lungs were inflated to a constant pressure of 25 cmH2O with 4%
paraformaldehyde in PBS and immersed in the same fixative for 24
h. Respiratory bronchioles were identified by the presence of
epithelial lining in one part of the wall. A perpendicular line was
drawn from the center of the respiratory bronchiole to the distal
acinus (the pleura or the nearest connective tissue septum). A
minimum of 40 lines were drawn on a magnified image of each
lung section, and the number of septae intersected by each line was
counted (7, 8).
Immunohistochemistry. For visualization of C/EBP? protein ex-
pression in the lung, 5-?m paraffin-embedded tissue sections were
incubated with a 1:100 dilution of polyclonal anti-C/EBP? (14AA,
sc-61, Santa Cruz Biotechnology, Santa Cruz, CA) specific to the
C/EBP? isoform overnight and then with a 1:500 dilution of anti-
rabbit IgG (Alexa Fluor 488, A11008, Invitrogen, Carlsbad, CA) for
1 h. Subsequently, sections were costained with a monoclonal anti-
body for the type II cell marker ATP-binding cassette subfamily A
member 3 (ABCA3; WMAB-ABCA3-13, Seven Hills Bioreagents,
Cincinnati, OH) at a 1:100 dilution overnight and a 1:500 dilution of
anti-mouse IgG (Alexa Fluor 594, A11005, Invitrogen) for 1 h.
Additionally, sections for evaluation of proliferating cell nuclear
antigen (PCNA) by costaining of the type II cell marker pro-SP-C
were prepared similarly. Specific antibodies were a 1:100 dilution of
a monoclonal anti-PCNA (PC10, sc-56, Santa Cruz) and a 1:100
dilution of a polyclonal anti-pro-SP-C serum (AB3786, Millipore,
Temecula, CA). After tissues were immunostained, sections were
mounted with a drop of mounting medium containing 4=,6-diamidino-
2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA) and
visualized with a fluorescence microscope (model IX70, Olympus
America, Center Valley, PA). Images were captured with a digital
camera (model C4742-95, Hamamatsu).
Quantitative immunohistochemistry for type II cell proliferation.
For quantification of proliferating type II cells, images of random
fields of terminal alveolar regions were acquired with the fluorescence
microscope. Ten fields per lung were obtained from three separate
mice in each group. For counting PCNA or pro-SP-C-positive cells,
regions for DAPI fluorescence-positive cells were randomly selected
to obviate bias toward signals generated with secondary antibodies.
Images were digitally merged to identify dual-positive cells. Quanti-
fication was achieved with Metamorph software (MetaMorph Imaging
System, Universal Imaging, West Chester, PA). Briefly, the ratios of
pro-SP-C to DAPI, PCNA to DAPI, and PCNA to pro-SP-C were
obtained from each field, and the average of 10 fields per animal was
Evaluation of protein levels in lung homogenates. Lungs were
flushed with PBS to exclude red blood cells and frozen in liquid
nitrogen. To obviate the difference in protein or mRNA levels due to
injection variability, the entire injected lung was quickly pulverized in
liquid nitrogen and mixed with 50 ?l of PBS. An aliquot was taken for
Western analysis, and the remaining lung homogenates were dis-
solved in TRIzol reagent for mRNA extraction. Western analysis was
performed to evaluate protein levels for markers of cell proliferation,
type II and type I cells, and vascular endothelial cells. The procedure
is described elsewhere (22). The antibodies and dilutions were as
follows: a 1:1,000 dilution of polyclonal anti-C/EBP? (14AA, sc-61)
for C/EBP?, a 1:1,000 dilution of hamster anti-T1? (clone 8.1.1, Iowa
Hybridoma Bank, Iowa City, IA) for the marker of type I cells (T1?),
a 1:1,000 dilution of polyclonal anti-pro-SP-B or anti-pro-SP-C serum
(Ab 3780 or Ab 3786, Millipore) for pro-SP-B or pro-SP-C, a 1:1,000
dilution of monoclonal anti-PCNA (PC10, sc-56) for PCNA, a 1:800
dilution of polyclonal anti-p21 (C-19, sc-397) for p21, a 1:200
dilution of goat anti-PECAM-1 (M-20, sc-1,506) for platelet/endothe-
lial cell adhesion molecule-1 (PECAM-1), a 1:10,000 dilution of
polyclonal anti-caveolin-1 (N20, sc-894) for caveolin-1, a 1:20,000
dilution of polyclonal anti-calnexin (SPA-860, Stressgen, Victoria,
BC, Canada) for calnexin, and a 1:10,000 dilution of monoclonal
anti-GAPDH (MAb 374, Millipore) for GAPDH.
Quantitative real-time PCR. Steady-state mRNA levels were
evaluated by quantitative real-time PCR using the TaqMan gene
expression system (Applied Biosystems). Briefly, total lung RNA
was extracted with TRIzol reagent (Invitrogen). First-strand cDNA
was synthesized with SuperScript II reverse transcriptase (Invitro-
gen) and random primers. Gene-specific mRNA levels were deter-
mined using TaqMan gene expression assays (each primer at 900
nM and probe at 250 nM; Applied Biosystems) designed over
exon-to-exon boundaries. Specific primers for each gene are listed
in Table 1. All reactions were performed in 384-well plates with a
final volume of 10 ?l. Real-time PCR plates were analyzed using
the Prism 7900HT sequence detection system with Prism SDS2.1
software (Applied Biosystems). Relative quantitation was achieved
by normalization to the value of 18S mRNA using the cycle
threshold (??CT) method.
Statistical analysis. For comparison between treatment groups,
the null hypothesis that there is no difference between treatment
means was tested by a single-factor ANOVA for multiple groups or
unpaired t-test for two groups (InStat 3, GraphPad Software).
Statistical significance (P ? 0.05, P ? 0.01, or P ? 0.001) between
and within groups was determined by Tukey’s method of multiple
Hyperoxia induces C/EBP? expression in neonatal mouse
lung. Levels of C/EBP? mRNA were increased threefold in
neonatal mouse lung exposed to 72 h of hyperoxia as measured
by quantitative real-time PCR (Fig. 1A). Protein levels of
C/EBP? were increased twofold in lung homogenates as de-
termined by Western analysis (Fig. 1B). The induced C/EBP?
appeared to be selective only in the lung, as liver and thymus
homogenates did not show increased C/EBP? protein in the
hyperoxia-exposed animal (data not shown). This induction
was not observed in similarly exposed adult animals (data not
Hyperoxia-induced C/EBP? protein is preferentially local-
ized in lung alveolar type II cells. To understand which lung
cells expressed C/EBP?, immunofluorescent staining of paraf-
fin-embedded lung tissue slides was performed. Enhanced
staining of alveolar epithelial cells was observed with antibod-
ies against C/EBP? in room air and further increased in
hyperoxia (Fig. 1C). When the lung slides were costained with
Table 1. ABI primers used for quantitative real-time PCR
Gene Symbol ABI Assay IDGene Name
CCAAT/enhancer binding protein-?
NK2 homeobox 1 or thyroid
transcription factor1 (TTF1)
Forkhead box A2 or hepatocyte
nuclear factor 3? (HNF3?)
Transforming growth factor-?1
Surfactant protein B
Surfactant protein C
C/EBP? AND NEONATAL HYPEROXIC LUNG CELL PROLIFERATION
AJP-Lung Cell Mol Physiol • VOL 301 • AUGUST 2011 • www.ajplung.org
In summary, we have demonstrated that hyperoxia increases
C/EBP? mRNA and protein expression in neonatal lung type II
cells. This was associated with disrupted lung architecture and
reduction of RAC immediately after hyperoxia and following
room air recovery. Altered type II and type I cell protein
makers were evident at 72 h of hyperoxia, and reduced type II
cell numbers were observed, despite compensatory increases in
proliferation. Inhibition of C/EBP? reversed these effects and
improved lung morphology. We speculate that C/EBP? is an
important regulator of postnatal type II cell proliferation in
hyperoxia and, thus, modulates alveolar injury and repair in the
We are grateful to Peggy McDonald and Cheryn Jarvis for administrative
assistance and Li Zhi and Nantale Nsibiwa for technical assistance.
The monoclonal antibody T1?, developed by Andrew Farr, was obtained
from the Developmental Studies Hybridoma Bank developed under the aus-
pices of the National Institute of Child Health and Human Development and
maintained by The University of Iowa, Department of Biological Sciences
(Iowa City, IA).
This work was funded by National Heart, Lung, and Blood Institute Grant
HL-58752 (P. A. Dennery).
No conflicts of interest, financial or otherwise, are declared by the authors.
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