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First published online 3 July 2008
doi: 10.1242/dev.021964
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1 Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center
and the University of Cincinnati College of Medicine, 3333 Burnet Avenue,
Cincinnati, OH 45229-3039, USA.
2 Laboratory of Respiratory Disease, West China Hospital, Sichuan University, 37
Guo Xue Xiang, Chengdu, 610041, People's Republic of China.
* Author for correspondence (e-mail: jeff.whitsett{at}cchmc.org)
Accepted 3 June 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/ mice died of respiratory
distress immediately after birth. Abnormalities in lung maturation and
morphogenesis were observed in the respiratory epithelium, the bronchiolar
smooth muscle, and the pulmonary vasculature. Respiratory epithelial cells of
both the conducting and peripheral airways were immature. Surfactant
phospholipids were decreased and lamellar bodies, the storage form of
surfactant, were rarely found. mRNA microarray analysis demonstrated that KLF5
influenced the expression of genes regulating surfactant lipid and protein
homeostasis, vasculogenesis, including Vegfa, and smooth muscle cell
differentiation. KLF5 regulates genes controlling paracrine interactions
during lung morphogenesis, as well as those regulating the maturation of the
respiratory epithelium that is required for lung function after birth.
Key words: Pulmonary, Transcription factor, Vasculogenesis, Paracrine signaling, VEGF, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Understanding the genes and processes regulating maturation
of the respiratory epithelium has provided a basis for the diagnosis and
treatment of disorders affecting perinatal lung function.
KLF5, a member of the Kruppel-like family of transcription factors, was
first identified as an intestinal-enriched member of the zinc-finger
transcription factors of the Sp1 subfamily
(Conkright et al., 1999). In
the mouse, Klf5 mRNA is expressed in the posterior endoderm
associated with the primitive streak in the embryonic day (E) 7.5 embryo
(Moore-Scott et al., 2007).
Later in embryonic development, KLF5 is detected in epithelial cells of the
gastrointestinal tract, the outer layer of the tongue, the epidermis, the
trachea, and bronchial epithelial cells
(Ohnishi et al., 2000).
Klf5 expression is influenced by important developmental pathways,
including the WNT, retinoic acid (RA), RAS and FGF signaling pathways, which,
in turn, influence proliferation and differentiation in many organ systems,
including the lung (Chanchevalap et al.,
2004; Kawai-Kowase et al.,
1999; Nandan et al.,
2004; Ziemer et al.,
2001). Although these pathways are known to be involved in lung
morphogenesis, there is increasing evidence that they are also involved in the
pathogenesis of lung disease, being induced during inflammation, repair and
tumorigenesis (Shaw et al.,
2007).
In the mouse embryo, KLF5 is required for formation of the endoderm.
Klf5-/- mice die at approximately E8.5, well before lung
formation (Shindo et al.,
2002). Although Klf5 is expressed at relatively high
levels in epithelial cells lining the fetal and postnatal lung, the role of
KLF5 in lung development and function is unknown. In the present study, we
generated mice in which the Klf5 gene was conditionally deleted from
respiratory epithelial cells in the developing lung to assess its potential
role in lung development and function.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
kindly provided by Dr Corrinne Lobe, University of Toronto. For lung-specific,
doxycycline-induced recombination, the
FVB.Cg-Tg(SFTPC-rtTA) 5Jaw/J transgenic line was used (The
Jackson Laboratory, Bar Harbor, Maine). Triple transgenic mice, termed
Klf5/ after exposure to doxycycline, were
generated by crossing
(TetO)7-Cre-/tg;Klf5loxP/loxP and
SFTPC-rtTA-/tg;Klf5loxP/loxP mice.
Klf5loxP/loxP littermates lacking rtTA and/or Cre alleles
served as controls. Gestation was determined by identification of a vaginal
plug (E0.5). Dams bearing double- and triple-transgenic pups were maintained
on doxycycline-containing food (25 mg/g; Harlan Teklad, Madison, Wisconsin)
until E14.5. The mice were killed by injection of anesthetic and then
exsanguinated for evaluation. Transgenic mice were identified by PCR using
genomic DNA isolated from the tails as previously described
(Perl et al., 2002).
Klf5loxP/loxP PCR was carried out using primers that can
distinguish between the wild-type allele and the floxed allele: primer 1, CCT
GCG TGC AAT CCA TCT TGT TCA ATG GC; primer 2, TCA CCC TCT GCA GAT CTT AGG C;
and primer 3, GCT TGG CTC AAA ATT CCG TTC C.
Histology and immunohistochemistry
Fetal lung tissue was immersion-fixed, embedded, sectioned and
immunostained as previously described
(Davé et al., 2006).
Guinea pig anti-KLF5 antibody was raised against a His-KLF5 peptide containing
amino acids 72-245 of the mouse KLF5 protein, a region lacking sequence
identity with other KLF family members. To generate the recombinant KLF5
peptide, a fragment of Klf5 cDNA was amplified and cloned into
pTrcHis-TOPO for expression in E. coli (Invitrogen, Carlsbad, CA).
His-KLF5 peptides were purified using a His-tag protein purification kit
(Novagen, Madison, WI). The antibody was tested by ELISA, western blot and
immunohistochemistry for specificity and expression in mouse tissues.
For immunohistochemistry, CCSP, FOXJ1, phosphohistone H3, CEBP
,
SMA, and PECAM staining were performed as previously described
(Bell et al., 2008;
Davé et al., 2006;
Martis et al., 2006).
Additional antibodies used were as follows: KLF5 (1:2000), VEGFR2 (1:250,
rabbit monoclonal, 55B11 Cell Signaling Technology, Danver, MA), and
pan-cytokeratin (1:500, mouse monoclonal, C1801, Sigma-Aldrich). For dual
immunolabeling, antibodies from two different species were used: guinea pig
KLF5 (1:100); rabbit anti-CCSP (1:500); rabbit anti-proSP-C (1:200); rabbit
anti-FOXJ1 (1:1000). All experiments shown are representative of findings from
at least two independent dams, generating at least four triple transgenic
offspring that were compared with littermate controls.
Ultrastructural analysis
Electron microscopy was performed on lung tissue obtained from
Klf5/ and littermate controls at
approximately E18 (n=3 for each genotype). Tissue was fixed and
embedded as previously described (Zhou et
al., 1997).
RNA isolation and analysis
RNA was isolated from whole E18.5 lung and reverse transcribed, according
to the manufacturer's protocol (VersoTM cDNA kit, Thermo Fisher
Scientific, Waltham, MA), prior to RT-PCR analysis. Densitometric quantitation
of the PCR products was carried out using Quality One software (Bio-Rad
Laboratories, Philadelphia, PA). The relative concentrations of each mRNA were
normalized to the concentration of β-actin mRNA in each sample. Primer
sequences are available on request. Sftpb, Sftpc and Scgbla1
mRNAs were quantified by S1 nuclease protection assays using ribosomal protein
L32 as an internal control (Dranoff,
1994). Differences were assessed by Student's t-test.
RNA microarray analysis
RNAs from three different control and
Klf5/ mouse lungs were isolated using TRIzol
Reagent (Invitrogen, Carlsbad, CA), and amplified using an Ovation Biotin RNA
application and labeling system (NuGen Technologies, San Carlos, CA). Lung
cRNA was hybridized to the murine genome MOE430_2 chips, consisting of
approximately 39,000 transcripts (Affymetrix, Santa Clara, CA), using the
manufacturer's protocol. The RNA quality and quantity assessment, probe
preparation, labeling, hybridization and image scan were carried out in the
CCHMC Affymetrix Core using standard procedures. Affymetrix MicroArray Suite
version 5.0 was used to scan and quantify signals using default scan settings.
Six chips from three pair-wise experiments were used. Normalization was
performed using the Robust Multichip Average Model
(Irizarry et al., 2003a;
Irizarry et al., 2003b). Data
were further analyzed using affylmGUI from the R/Bioconductor package
(Smyth, 2004). Differentially
expressed genes were selected with a threshold of Student t-test
P-value 
0.05, False Discovery Rate (FDR) 5%, fold change
1.5 and a minimum of two present calls by Affymetrix algorithm in three
samples. Gene ontology analysis was performed using the publicly available
web-based tool DAVID (database for annotation, visualization, and integrated
discovery) (Dennis et al.,
2003). Pathways that were overly represented were identified by
comparing the overlap of differentially expressed genes and all genes in the
MOE430 mouse genome. Gene sets were associated with known pathways and disease
states from KEGG
(http://www.genome.ad.jp/kegg/),
GenMAPP
(http://www.genmapp.org/)
and GEArrays
(http://www.superarray.com/).
A pathway was considered to be overly represented when it showed a probability
P-value 0.01 and >10 gene hits.
Surfactant analysis
Saturated phosphatidylcholine (SatPC) was isolated from lipid extracts of
lung homogenates from six mice of each genotype and analyzed as previously
described (Wan et al., 2004).
Mature SP-B western blot was performed with antibody AB3426 (Chemicon,
Temecula, CA) against mature SP-B peptide.
Transient transfection assays
H441 (a human pulmonary adenocarcinoma cell line) and JEG (a human
choriocarcinoma cell line) cells were grown to 70% confluence in six-well
plates and transfected with either plasmid (0.5 µg) or siRNA (100 pmole)
using Lipofectamine 2000 (catalog number 11668-027, Invitrogen, Carlsbad, CA).
Promoter activity was determined by the measurement of luciferase activity
normalized to β-galactosidase activity 48 hours after transfection. All
experiments were done in duplicate in three independent experiments. The mean
of the control was set to 1 and relative promoter activities were shown as
mean±s.e.m. and compared by the two-tailed Student t-test
(*P<0.05). pGL2-Vegfa-Luc was kindly provided
by Dr Mukhopadhyay (Mukhopadhyay et al.,
1997). pGL3-3TP-Luc, a TGFβ responsive promoter construct,
was kindly provided by Dr Molkentin (Wrana
et al., 1992). A human HIF2 expression vector in pcDNA3 was
a gift from Dr Richard K. Bruick (Tian et
al., 1997).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KLF5 is required for normal lung morphogenesis: conditional deletion of Klf5 in the lung
LoxP sites flanking exons 2 and 3 of the Klf5 gene were introduced
into the gene-targeting vector, which was designed to delete most of the
protein-coding region, including part of the KLF5 DNA-binding domain
(Fig. 2). Germline chimeras and
mice heterozygous for the Klf5loxP allele were produced
from targeted ES cells. Heterozygous offspring were mated to generate
homozygous Klf5loxP/loxPmice. Adult
Klf5loxP/loxP mice were healthy, fertile and expressed
KLF5 normally in the lung, indicating no apparent interference with endogenous
KLF5 expression. These animals were bred into the
(TetO)7-Cre-/tg and SFTPC-rtTA-/tg
transgenic lines to enable us to create triple transgenic animals in which
Klf5 could be deleted in respiratory epithelial cells upon
doxycycline treatment. Klf5/ mice were
generated by maintaining pregnant dams on doxycycline from E0 to E14.5. Lungs
were isolated from fetuses at E15.5 and E18.5 for further analysis.
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/+ mice, KLF5 was detected in the nuclei of
both alveolar and bronchiolar epithelial cells
(Fig. 3A,C,E), whereas nuclear
staining of KLF5 was absent in most respiratory epithelial cells in the
Klf5/ mice
(Fig. 3B,D,F).
Klf5 is required for perinatal lung function at birth
When mice were maintained on doxycycline from E0 to E14.5, offspring were
born in the expected Mendelian distribution for all genotypes. At E15.5,
morphological abnormalities were not observed in the lungs of
Klf5/ mice at the light microscopic level
(Fig. 3B). At E18.5, no
morphological abnormalities were observed in the lungs of
Klf5/+ fetuses
(Fig. 3E) and these fetuses
survived normally after birth. By contrast, all
Klf5/ mice died of respiratory distress
shortly after birth. At E18.5, the lung morphology of
Klf5/ mice was perturbed
(Fig. 3D,F). The alveolar
saccules appeared hypercellular, lacking the dilated peripheral saccules that
are characteristic of the normal lung at this stage of development. The
severity of the morphological abnormalities correlated with the efficiency of
Klf5 deletion. A dramatic reduction in the number of squamous
epithelial cells were found in the lungs of
Klf5/ mice, as visualized by staining with
pan-cytokeratin antibody (Fig.
3G,H), indicating a lack of alveolar type I cell differentiation
after the deletion of Klf5. Morphometric analysis demonstrated that
the luminal area of the alveolar saccules in the lungs of
Klf5/ mice was 26.5±5.3% and that of
the controls was 37.3±3.8%. Thus, luminal area was significantly
decreased in the lungs of Klf5/ mice
(P<0.05, n=3).
KLF5 did not affect proliferation of pulmonary epithelial cells
The wet lung/body weight ratio for controls was 0.029±0.005
(n=10) and for Klf5/ mice was
0.022±0.003 (n=11). The lung/body weight ratio was slightly
but significantly decreased in Klf5/ mice
(P<0.05). Because of the failure of sacculation in lungs of the
Klf5/ mice, significant differences in the
wet lung/body weight ratio could be caused by the lack of fluid in the
alveolar saccules in the Klf5/ mice.
Previous studies indicated that KLF5 influenced the RAS/MAPK signaling pathway
and promoted cell proliferation in various cell types, including fibroblasts,
smooth muscle cells, bladder cancer and intestinal epithelial cells
(Bateman et al., 2004;
Chanchevalap et al., 2004;
Chen, C. et al., 2006;
Nandan et al., 2005;
Nandan et al., 2004;
Sun et al., 2001). In order to
determine whether KLF5 affected the proliferation of epithelial or mesenchymal
cells, phosphohistone H3 (p-histone-3) immunostaining was performed and the
average number of immunolabeled cells per mm2 in the mesenchyme and
in the epithelium was determined at E15.5, a time that active proliferation is
normally present in both of these cell types. No differences in p-histone-3
staining were observed in pulmonary epithelial or mesenchymal cells after
deletion of Klf5 (data not shown). Similarly, BrdU labeling at E15.5
revealed no differences between the lungs of
Klf5/ and control mice (data not shown).
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/ mice were immature. Alveolar type II
cells contained abundant glycogen but no lamellar bodies
(Fig. 4B). In KLF5 sufficient
(non-deleted) mice, cuboidal alveolar type II cells contained numerous
lamellar bodies, rough endoplasmic reticulum, apical microvilli, and smaller
patches of glycogen (Fig. 4A).
Secreted surfactant was observed in KLF5 sufficient mice but not in
Klf5/ mice (data not shown), and squamous
type I cells were found in close apposition to adjacent capillaries
(Fig. 4C). In
Klf5/ mice, squamous type I epithelial cells
were not observed (Fig. 4D).
The interstitial tissue was hypercellular and contained numerous capillaries
that were more centrally located (data not shown). In both control and
Klf5/ mice, bronchioles were lined by
differentiating ciliated cells containing little residual glycogen and by
immature, glycogen-containing, columnar, Clara cells (data not shown). There
were no clear differences in the ultrastructure of epithelial cells lining the
bronchioles caused by deletion of Klf5.
KLF5 is required for normal surfactant protein and lipid production
Surfactant proteins and lipids are crucial for lung function at birth,
being required for the reduction of surface tension and the maintenance of
lung expansion. Lack of surfactant proteins and lipids causes infantile
respiratory distress syndrome in preterm infants at birth. Saturated
phosphatidylcholine (SatPC) is the major surfactant phospholipid that is
crucial for lung function. Lung SatPC increases dramatically in late gestation
in most mammals and is a useful indicator of lung maturation in preterm
infants. Consistent with the lack of lamellar bodies observed by electron
microscopy, the SatPC content in the lungs of E18.5
Klf5/ mice [3.302±0.12 nmol/lung
weight (mg)] was significantly decreased (n=6, P<0.05),
compared with controls [4.86±0.37 nmol/lung weight (mg)]. Western blot
analysis was performed to detect mature SP-B in lung homogenates, as
previously described (Wan et al.,
2004). The active SP-B peptide was decreased in lung from the
Klf5/ pups
(Fig. 4E).
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, an
epithelial-specific transcription factor normally expressed in type II cells
and non-ciliated bronchiolar epithelial cells
(Fig. 5A), was dramatically
decreased in conducting airway epithelial cells, but not in peripheral
pre-type II cells (Fig. 5B).
CCSP staining, a marker of differentiated non-ciliated bronchiolar epithelial
cells, was also markedly decreased in the lungs of
Klf5/ mice
(Fig. 5D). The lack of CCSP and
the association of KLF5 expression with Clara cells suggest a potential role
of KLF5 in bronchiolar cell differentiation. By contrast, FOXJ1 staining was
not changed by deletion of Klf5
(Fig. 5F). CCSP and CEBP
staining was decreased in the bronchiolar epithelium in the
Klf5/ mice. Taken together with the robust
KLF5 staining in the Clara cells of control mice, these data suggest that KLF5
regulates differentiation or gene expression in non-ciliated cells. Promoter
constructs of pGL3-CCSP-Luc (promoter construct of Scgbla1 gene) and
pGL3-Cebpa-Luc were co-transfected with a Klf5 expression
plasmid or Klf5-targeting siRNA into H441 cells, a human pulmonary
adenocarcinoma cell line that expresses high levels of Klf5. Changes
in KLF5 expression did not significantly alter the expression of the
Scgbla1 and Cebpa gene promoters in vitro (data not shown).
As CEBP is known to regulate the Scgbla1 promoter
(Martis et al., 2006), the
lack of CEBP in the bronchial/bronchiolar epithelial cells may provide
a potential mechanism by which KLF5 influences the expression of
Scgbla1 in those cells. Aquaporin 5 (Aqp5) mRNA, an alveolar
type I cell marker, was significantly decreased in the lungs of
Klf5/ mice as analyzed by RT-PCR and
densitometric quantitation of the PCR products
(Fig. 6C), consistent with the
absence of alveolar type I cells seen morphologically.
KLF5 is required for sacculation and influences epithelial-mesenchymal interactions
Deletion of Klf5 in lung epithelial cells perturbed the
organization of the adjacent mesenchyme. Expression of SMA, a marker of
bronchiolar and vascular smooth muscle cell differentiation, was markedly
increased in the bronchioles of the lungs from
Klf5/ mice
(Fig. 5H). Although the
pulmonary vasculature was present in the lungs of
Klf5/ mice, as indicated by PECAM
(Fig. 5I,J) and VEGFR2
(Fig. 5K,L) immunostaining, the
normal alignment of epithelial cells with alveolar capillaries in the lung
periphery was perturbed. These findings support the ultrastructural changes
seen in the lungs of Klf5/ mice, in which
the normal association of epithelial and endothelial cells was disrupted.
As Vegfa is expressed in respiratory epithelial cells of the
developing lung and is essential for pulmonary vascular development, the
effects of KLF5 on the expression of Vegfa were examined by RT-PCR
using primers flanking exons 6 and 7
(Healy et al., 2000). The
splicing-variant Vegfa188, Vegfa164 and Vegfa120 mRNAs were
detected as uniquely sized products. Controls included β-actin and
Klf5 itself. Densitometric quantitation of the PCR products revealed
a significant decrease in Vegfa188 and Vegfa120 mRNA in the
lungs of Klf5/ mice
(Fig. 6D,F). Although
selectively expressed in epithelial cells, KLF5 is required for normal
morphogenesis of the capillary bed, indicating a role for KLF5 in the
regulation of paracrine interactions in the developing lung.
Genomic responses to the deletion of Klf5
To identify other genes influenced by the conditional deletion of
Klf5, mRNA expression profiles were compared in lungs from E18.5
Klf5/ mice and their littermate controls
using Affymetrix murine genome MOE 430_2 GeneChips. Genes with a fold change
of at least 1.5 were selected. Gene set enrichment analysis using the
differentially expressed genes overlapping with pathways from KEGG, GenMAPP
and Superarray indicated that KLF5 influenced the expression of genes
associated with cancer, cell cycle, MAP kinase signaling, angiogenesis, and
the TGFβ and BMP signaling pathways
(Table 1). As deletion of
Klf5 affects perinatal lung maturation and function at birth, we
chose to determine the effects of KLF5 on subsets of genes and pathways
regulating perinatal lung maturation, lipid metabolism, angiogenesis and
TGFβ signaling.
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/ mice at
E18.5 demonstrated changes in the expression of genes known to regulate
surfactant and surfactant lipid homeostasis, including Sftpa, Sftpb,
Sftpd, Abca3, Aytl2 (Lpcat1 - Mouse Genome Informatics),
Srebf1 and Srebf2
(Besnard et al., 2007;
Chen, X. et al., 2006;
Shulenin et al., 2004) (see
Table 2). Decreased expression
of Sftpb was confirmed by S1 nuclease protection assay (data not
shown), and by western blot analysis (Fig.
4). To assess whether KLF5 directly regulated the transcription of
Sftpa, Sftpb, Sftpd, Aytl2 and Abca3, promoter constructs
were co-transfected with a Klf5 expression plasmid or Klf5
siRNA into H441 cells. In contrast to previous studies demonstrating that some
of these promoters were directly regulated by NKX2.1, FOXA2 and CEBP
(Bohinski et al., 1994;
Martis et al., 2006), KLF5 did
not significantly alter the activity of these gene promoter constructs in
vitro (data not shown).
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/ mice, including VEGF, PDGF, FGF and
TGFβ (Fig. 7), supporting
the morphological findings that KLF5 regulates paracrine signaling from the
epithelium, influencing mesenchymal cell differentiation.
Table 3 lists genes altered in
the E18.5 Klf5/ lung that mediate paracrine
signaling. The increase in Tgfb2 (1.9-fold) and Acvr1c
(2.87-fold), and the decrease in Smad6 (-1.25-fold) expression were
confirmed by real-time PCR (data not shown).
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)
was co-transfected with a Klf5 expression vector in vitro. KLF5
enhanced Vegfa luciferase activity in a dose-dependent manner in
JEG-3 cells, a human choriocarcinoma cell line that does not normally express
lung-specific transcription factors (Fig.
8A) (Bachurski et al.,
2003). Whereas no change in Vegfa luciferase activity was
detectable in H441 cells, a cell line that expresses Klf5 (data not
shown). Decreasing the level of Klf5 expression by using
Klf5 siRNA inhibited Vegfa-luciferase activity in H441 cells
(Fig. 8B). As HIF2 is a
strong activator of the Vegfa promoter in H441 cells
(Maeda et al., 2008), the
effect of KLF5 on HIF2-dependent Vegfa-luciferase activity was
assessed. Klf5 siRNA significantly decreased HIF2-dependent
Vegfa-luciferase activity in H441 cells
(Fig. 8B), indicating that KLF5
might influence Vegfa expression, at least in part, via a
transcriptional mechanism.
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). TGFβ1 induced pGL2-3TP luciferase activity more than
20-fold in H441 cells. Increasing the level of KLF5 expressed in H441 cells
mildly inhibited pGL2-3TP luciferase activity at baseline and markedly
inhibited the induction of pGL2-3TP luciferase activity by TGFβ1
(Fig. 8C). Inhibition of
endogenous Klf5 mRNA levels using siRNA increased pGL2-3TP luciferase
activity and markedly enhanced pGL2-3TP luciferase activity in the presence of
TGFβ1 (Fig. 8D),
indicating that KLF5 acts as a negative regulator of TGFβ signaling in
pulmonary adenocarcinoma cells in vitro. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/ mice. Taken together, perinatal
respiratory failure in the Klf5/ mice was
mediated, in part, by decreased expression of pulmonary surfactant and the
failure to form gas-exchange structures typical of normal alveolar
saccules.
mRNA microarray studies of the lungs from
Klf5/ mice demonstrated that KLF5 was
required for the normal expression of genes regulating surfactant phospholipid
homeostasis, including Abca3, Abca4, Aytl2, Liph, Srebf1 and
Srebf2. Aytl2 (acyltransferase-like 2) is abundantly expressed in
alveolar type II cells and is required for SatPC synthesis
(Chen, X. et al., 2006).
Consistent with this observation, total SatPC was significantly decreased in
the lungs of Klf5/ mice in association with
decreased expression of Aytl2 mRNA. Expression of Abca3 in
alveolar type II cells is required for the formation of lamellar bodies, and
for surfactant storage and function
(Shulenin et al., 2004), thus
decreased expression of Abca3 may contribute to the lack of lamellar
bodies and the decreased surfactant in Klf5/
mice. Although no direct regulation of Abca3 promoter activity by
KLF5 was found in vitro, Srebf1, an important transcriptional
regulator of Abca3 (Besnard et
al., 2007), was decreased significantly in
Klf5/ mice, indicating potential, indirect
regulation of Abca3 by KLF5. Taken together, these data indicate that
KLF5 regulates type I and type II epithelial cell maturation and influences
the expression of genes that are required for surfactant protein and lipid
metabolism, which are crucial for the adaptation to air breathing.
KLF5 regulates proximal airway epithelial cell maturation
In late gestation, epithelial cells lining the conducting airways
differentiate into ciliated and non-ciliated cells. In
Klf5/ mice at E18.5, staining of CCSP and
CEBP in non-ciliated bronchiolar cells, i.e. Clara cells, was
decreased; however, ultrastructural abnormalities were not observed in the
conducting airways. The association of KLF5 expression with Clara cells in the
normal fetal and adult lung, and the loss of expression of CCSP and
CEBP (also required for CCSP expression) in the airway of
Klf5/ mice, indicates that KLF5 may
influence normal differentiation or gene expression in Clara cells.
Lung maturation is regulated by transcription factors expressed in the
developing respiratory epithelial cells. Recent studies demonstrated that
conditional deletion, or inhibition, of Gata6, Nfat, Foxa2 and
Cebpa, and mutation of Titf1, interfered with pulmonary
maturation and caused respiratory distress in newborn mice
(Davé et al., 2006;
DeFelice et al., 2003;
Martis et al., 2006;
Wan et al., 2004). As
discussed above, these transcription factors directly regulate the
transcription of many genes involved in surfactant and lipid metabolism,
including Abca3, the SFTPs, Aytl2 and others. We were unable
to demonstrate direct activation of the Sftptb, Sftptc, Abca3 and
Atyl2 gene promoters by KLF5 in vitro. Thus, the mechanisms by which
KLF5 influences the expression of these maturation-dependent genes remains
unclear. The lack of a direct effect of KLF5 on these promoter constructs
suggests that KLF5 is not a direct transcriptional regulator of these genes,
although it is possible that the cis-elements mediating the effects of KLF5
were not present in the promoter constructs used in our study. Alternatively,
the cell lines tested in our study may lack crucial transcriptional co-factors
needed for KLF5 function. Although transcription of Cebpa was not
affected by KLF5 in H441 cells, Cebpa expression was dependent on
KLF5 in bronchiolar cell types in vivo. Expression of Cebpa was
markedly decreased in the bronchiolar epithelial cells, but not in the
peripheral type II cells in the lungs of
Klf5/ mice, indicating cell-specific
regulation of Cebpa expression by KLF5. As CEBP regulates CCSP
gene expression in vivo and in vitro, an inhibitory effect of KLF5 on CCSP
expression may be mediated, at least in part, by CEBP.
KLF5 influences paracrine signaling between lung epithelium and mesenchyme
Lung morphogenesis requires precise interactions among multiple cell types
in both epithelial and mesenchymal cell compartments. Deletion of
Klf5 in epithelial cells perturbed the normal patterning and
differentiation of the mesenchyme in the saccular stage of lung development,
resulting in thickening of the mesenchyme, increased SMA staining in
bronchiolar smooth muscle, and the failure of pulmonary vessels to migrate
into close proximity to alveolar epithelial cells. Whereas previous studies
demonstrated that KLF5 enhanced SMA expression in smooth muscle cells
(Liu et al., 2003), the extent
and intensity of staining for SMA was increased in
Klf5/ mice, wherein KLF5 is selectively
deleted only in the developing respiratory epithelium. These observations
indicate that KLF5 influences paracrine interactions between pulmonary
epithelial and mesenchymal cells to regulate pulmonary smooth muscle
differentiation.
KLF5 influenced a number of mRNAs regulating morphogenesis of the lung.
Microarray analysis and extensive literature mining were used to identify the
potential pathways that were influenced by KLF5, including TGFβ, PDGF,
FGF and VEGF, all of which are known to mediate paracrine signaling during
lung development. Decreased expression of Vegfa isoforms,
Vegfr1 and Vegfr3 are consistent with a role of KLF5 in the
regulation of pulmonary vasculogenesis and may have contributed to the
abnormalities found in the lungs of the
Klf5/ mice, in which the alignment of
capillaries with the peripheral epithelium was disrupted. Consistent with the
in vivo findings, KLF5 had a transcriptional effect on the activity of the
Vegfa promoter. The changes in Vegfa expression and isoforms
might reflect direct effects of KLF5 on gene expression in respiratory
epithelial cells, but might also be influenced by more generalized effects on
lung maturational programs in various cell compartments.
The expression of Pdgfb was decreased and that of Pdgfc
was increased in the lungs of Klf5/ mice.
PDGFB is known to play an indispensable role in vasculogenesis
(Lindahl et al., 1997). During
lung development, PDGFC was detected in epithelial cells of the bronchial
tubules until E15.5, after which its expression was localized to smooth muscle
cells (Ding et al., 2000).
Enhanced expression of Pdgfc in the developing respiratory epithelium
delayed maturation, causing respiratory failure consistent with our findings
in the Klf5/ mice
(Zhuo et al., 2006).
Klf5/ mice and mice expressing an
activated form of TGFβ1 in the respiratory epithelium have morphological
similarities, including disrupted alveolar sacculation and enhanced
bronchiolar smooth muscle cell differentiation
(Zeng et al., 2001;
Zhou et al., 1996). A group of
genes known to be involved in TGFβ signaling
(Ghosh-Choudhury et al., 1994;
Nakayama et al., 1998;
Takaki et al., 2006) were
significantly altered, both negatively and positively, in the lungs of
Klf5/ mice. Increased expression of
TGFβ2 was found in the lungs of Klf5/
animals and a similar increase was observed in
Cebpa/ mice, which also exhibit delayed
perinatal lung maturation (Martis et al.,
2006). Thus, increased TGFβ2 in the lungs of
Klf5/ mice may act in a paracrine manner to
affect both epithelial and mesenchymal cell differentiation and patterning.
TGFβ2 regulates the transcription of smooth muscle cell-related genes,
including SMA, and influences (myo)fibroblast differentiation
(Wicks et al., 2006).
Inhibition of a TGFβ reporter construct by KLF5 in vitro suggests that
KLF5 influences transcriptional responses to TGFβ.
Expression of a number of genes involved in FGF signaling was altered in
the lungs of Klf5/ mice, including the
expression of Frag1, Fgfbp1 and Fgf18, which were decreased.
Deletion of Fgf18 in transgenic mice altered lung morphogenesis and
was associated with abnormal patterning of the pulmonary vasculature,
resulting in perinatal death (Usui et al.,
2004). Heparin sulfate proteoglycans bind to and modulate the
activities of various signaling molecules, including FGFs, VEGF, TGFβ1
and TGFβ2 (Bernfield et al.,
1999; Forsberg and Kjellen,
2001). Interestingly, the expression of Ndst1, a key
enzyme regulating heparin sulfate synthesis was reduced 1.56-fold in
Klf5/ animals, and
Ndst1-/- animals also exhibit respiratory failure and
pulmonary immaturity at birth (Fan et al.,
2000).
Taken together, deletion of Klf5 significantly altered the
expression of genes involved in a number of paracrine signaling processes that
are crucial for normal lung morphogenesis, including Vegfa, Tgfb and
Pdgfb. Thus, KLF5 both is regulated by diverse signaling pathways and
participates in the regulation of multiple signaling processes, including
those mediated by FGF, WNT, RAS and RA in various tissues
(Chanchevalap et al., 2004;
Kawai-Kowase et al., 1999;
Nandan et al., 2004;
Ziemer et al., 2001).
As direct-targeted gene deletion of Klf5 caused early embryonic lethality, identifying its role in the morphogenesis and differentiation of various organs has not been possible. In the present study, we generated a model for conditional deletion of Klf5, demonstrating that KLF5 is required for perinatal lung maturation and the expression of many genes crucial for lung function at birth, and that it regulates important but diverse signaling networks that influence lung morphogenesis and maturation.
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ACKNOWLEDGMENTS |
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