Mouse ATF-2 Null Mutants Display Features of a Severe Type of
Meconium Aspiration Syndrome*
Toshio
Maekawa
§,
François
Bernier
,
Motohiko
Sato¶,
Shintaro
Nomura¶§,
Mandavi
Singh
,
Yoshiro
Inoue
,
Tomoyuki
Tokunaga**,
Hiroshi
Imai**,
Minesuke
Yokoyama
,
Andreas
Reimold§§,
Laurie H.
Glimcher§§, and
Shunsuke
Ishii
§¶¶
From the
Laboratory of Molecular Genetics, Tsukuba
Life Science Center, RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki
305-0074, Japan, the ¶ Department of Pathology, Osaka
University Medical School, 2-2 Yamada-oka, Suita,
Osaka 565-0871, Japan, the
Department of Anatomy, Hokkaido
University School of Medicine, Sapporo, Hokkaido 060-0815, Japan, the
** National Institute of Animal Industry, 2 Ikenodai, Kukisaki,
Inashiki, Ibaraki 305-0901, Japan, the

Mitsubishi Kagaku Institute of Life
Sciences, 11 Minami-Ohya, Machida, Tokyo 194-0031, Japan, the
§§ Department of Cancer Biology, Harvard School
of Public Health, and Department of Medicine, Harvard Medical
School, Boston, Massachusetts 02115, and the § CREST (Core
Research for Evolutional Science and Technology), JST (Japan Science
and Technology Corporation)
 |
ABSTRACT |
Mouse null mutants of transcription factor ATF-2
were generated by the gene targeting method. They died shortly after
birth and displayed symptoms of severe respiratory distress with lungs filled with meconium. These features are similar to those of a severe
type of human meconium aspiration syndrome. The increased expression of
the hypoxia inducible genes suggests that hypoxia occurs in the mutant
embryos and that it may lead to strong gasping respiration with
consequent aspiration of the amniotic fluid containing meconium. A
reduced number of cytotrophoblast cells in the mutant placenta was
found and may be responsible for an insufficient supply of oxygen prior
to birth. Using the cDNA subtraction and microarray-based
expression monitoring method, the expression level of the
platelet-derived growth factor receptor
gene, which plays an
important role in the proliferation of trophoblasts, was found to be
low in the cytotrophoblasts of the mutant placenta. In addition, ATF-2
can trans-activate the PDGF receptor
gene promoter in
the co-transfection assay. These results indicate the important role of
ATF-2 in the formation of the placenta and the relationship between
placental anomalies and neonatal respiratory distress. The ATF-2 null
mutants should enhance our understanding of the mechanism of severe
neonatal respiratory distress.
 |
INTRODUCTION |
Meconium aspiration syndrome
(MAS)1 is a common neonatal
problem that results in acute and chronic respiratory morbidity (for review, see Refs. 1 and 2). Unfortunately, our understanding of this
entity is incomplete. Aspiration of meconium particles may occur
before, during, or after delivery and is associated with deep
inspiratory movements because of fetal respiratory depression. Aspiration of meconium may cause mechanical obstruction of the airways,
chemical pneumonitis, and surfactant inactivation. Although MAS can be
prevented in the majority of infants by appropriate suctioning at birth
or by early administration of surfactant, the severe form of MAS is
still a neonatal problem that remains to be resolved (3).
A number of transcription factors of the ATF/CREB family, all of which
contain a DNA-binding domain consisting of a cluster of basic amino
acids and a leucine zipper region (b-zip), have been identified (for
review, see Ref. 4). They bind to the cAMP response element (CRE) as
homodimers or heterodimers formed through the leucine zipper. Among the
numerous transcription factors of the ATF/CREB family, three factors,
ATF-2 (also called CRE-BP1), ATF-a, and CRE-BPa form a subgroup (5-9).
These factors are capable of forming homodimers or heterodimers with
c-Jun (10). A common characteristic of this group of factors is their
activation by the stress-activated protein kinases such as the Jun
amino-terminal kinase and p38 (11-13). The stress-activated protein
kinases phosphorylate this group of factors at sites close to the
NH2-terminal transcriptional activation domain containing
the metal finger structure and stimulate their
trans-activating capacity. Because a group of factors of the
ATF/CREB family including CREB are activated via direct phosphorylation by cAMP-dependent protein kinase (14), the CREB and ATF-2
subgroups are linked to the distinct signaling cascades involving the
cAMP-dependent protein kinase and stress-activated protein
kinase pathways. Within its subgroup ATF-2 has been most extensively
studied and shown to be ubiquitously expressed with the highest level
of expression being observed in the brain (15). ATF-2 mutant mice
generated by gene targeting exhibited decreased postnatal viability and growth with a defect in endochondral ossification and ataxia
accompanied by a decreased number of cerebellar Purkinje cells (16).
Here we report that independently generated mouse null mutants of ATF-2 show features of severe respiratory distress, which are similar to
those of a severe type of human MAS (3). This phenotype is different
from that previously reported for ATF-2-deficient mice (16), which we
show here to contain small amounts of a mutant protein and hence refer
to as ATF-2m/m.
 |
EXPERIMENTAL PROCEDURES |
Construction of the Targeting Vector--
The ATF-2 genomic
clones were isolated from a library derived from TT2 cells by the
standard plaque hybridization procedure. A 10.0-kb genomic DNA
subfragment, which contains the exon encoding amino acids 327-395, was
used to generate the targeting vector. A neomycin cassette driven by
the phosphoglycerate kinase gene promoter was inserted into the newly
generated BglII site between amino acids 378 and 379 in this
exon. To increase the frequency of gene targeting, the diphtheria
toxin-poly(A) signal cassette for negative selection was fused to the
short arm as described (17).
Generation of ATF-2-deficient Mutant Mice--
The embryonic
stem (ES) cells used were TT2 cells derived from an F1 embryo resulting
from a cross between C57BL/6 and CBA mice (18). The
NotI-linearized targeting vector (100 µg) was electroporated into 1.0 × 107 TT2 cells. Targeted
clones were selected after 7-10 days growth in the presence of G418
(150 µg/ml) and were then expanded in duplicate 24-well plates. The
homologous nature of the recombination was confirmed by Southern blot
analysis using several restriction enzymes and several probes located
either inside or outside the targeting vector. In addition, three
different primers, shown in Fig. 1A, were used to amplify a
1100-base pair fragment from the wild type allele or a 1000-base pair
fragment from the mutant allele. Chimeras were produced by injecting
about 10 ES cells into 40 ICR 8-cell embryos and transplanting the
embryos into the uterus of pseudopregnant females. Six- to
eight-week-old male progeny with a high degree of chimerism were
derived from three clones and were bred with BALB/c, C57BL/6, or ICR
females to produce heterozygous mice capable of transmitting the
targeted allele through the germ line. The mice were maintained by the
Division of Experimental Animal Research, RIKEN.
Genotyping of ES Cells, Embryos, and Animals--
Genomic DNA
was isolated from cultured cells, embryos, and tail clippings by
digestion overnight at 55 °C in lysis buffer (10 mM
Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5%
SDS, 0.5 mg/ml proteinase K) followed by RNase A treatment,
phenol-chloroform extraction, and ethanol precipitation. For Southern
blot analysis, genomic DNA (about 20 µg) was digested with
BglII and resolved on 0.8% agarose gels.
Detection of ATF-2 Proteins--
The brains of 18.5-days
postcoitum (dpc) fetuses were washed in PBS, resuspended in 300 µl of
lysis buffer consisting of 45 mM Tris-HCl (pH 7.4), 135 mM NaCl, 2.2 mM EDTA, 0.9% Triton X-100, 0.9%
sodium deoxycholate, 0.09% SDS, and 1% Trasylol, and homogenized. After centrifugation, samples of lysates were resolved on a 10% SDS-polyacrylamide gel electrophoresis. Proteins were transferred onto
a polyvinylidene difluoride membrane, and ATF-2 protein was detected
using anti-ATF-2 polyclonal antibodies, which were raised against the
full-length recombinant ATF-2 protein and ECL detection reagents
(Amersham Pharmacia Biotech).
Histological Analysis, Immunohistochemistry, and in situ
Hybridization--
Embryos and placentas were fixed in 4%
paraformaldehyde, dehydrated, and embedded in paraffin. Sections (5 µm) were stained with hematoxylin and eosin according to standard
procedures. Paraffin sections of 4 µm were cut and used for
immunohistochemistry. Rabbit polyclonal antibody raised against the
bacterially expressed full-length ATF-2 and anti-PDGF receptor
antibody C-20 (Santa Cruz) were used. ATF-2 immunoreactivity was
enhanced by treatment for 15 min in an autoclave at 120 °C, and PDGF
receptor
immunoreactivity was enhanced by microwave treatment for
25 min at 60 °C. Primary antibody-antigen complexes were detected by
peroxidase-conjugated anti-rabbit IgG (Cappel Co.), and the signal was
amplified by biotin-tyramide (NEN Life Science Products). In
situ hybridization using the digoxigenin-labeled PDGF receptor
RNA was performed essentially as described (19).
RNase Protection Assay--
RNase protection assays using the
vascular endothelial growth factor (VEGF) and tyrosine 3-hydroxylase
probes, which were prepared by the PCR-based method, were performed as
described (20).
cDNA Subtraction and Microarray-based Expression
Monitoring--
cDNA subtraction was done using the
PCR-SelectTM cDNA subtraction kit
(CLONTECH) according to the protocol supplied by
the supplier. Microarrays containing 18,378 nonredundant mouse cDNA
clones chosen from the I.M.A.G.E. collection (Genome Systems, Gene
Discovery Array mouse version 1.0) were used to identify the putative
target genes of ATF-2 according to the procedure described by the supplier.
Effect of PDGF on Proliferation of Trophoblast-derived
Cells--
Jar cells, a human choriocarcinoma cell line, were
maintained in Ham's F12 medium supplemented with 10% fetal bovine
serum. PDGF-AA or PDGF-BB (Pepro Tech Inc.) was added to the cells
directly at a final concentration of 10 ng/ml. The degree of BrdUrd
incorporation was examined using the cell proliferation enzyme-linked
immunosorbent assay system (Amersham Pharmacia Biotech) according to
the protocol from supplier.
The Co-transfection Assay--
The luciferase reporter, in which
the 2.2-kb human PDGF receptor
promoter (from
2120 to +118) was
linked to the luciferase gene, was provided by Dr. S. Mosselman (21). A
mixture containing 0.2 µg of the luciferase plasmid
pPDGFR
-2120-luc, 0.5 µg of the plasmid to express various forms of
ATF-2, and 1 µg of the internal control plasmid pact-
-gal, in
which the chicken cytoplasmic
-actin promoter is linked to the
-galactosidase gene, was transfected into Chinese hamster ovary
cells, and luciferase assays were performed. The total amount of
plasmid DNA was adjusted to 3 µg by adding the control plasmid DNA pact1.
 |
RESULTS |
Respiration Defects of the ATF-2o/o
Mutant--
ATF-2o/o mutant mice were generated by
homologous recombination in TT2 ES cells. The gene was disrupted by
inserting the neomycin resistance (neor)
gene into the exon (amino acids 327-395) that encodes the DNA-binding domain consisting of the basic amino acid region between amino acids
378 and 379. Homologous recombinants were characterized by the
appearance of a 2.9-kb BglII fragment with the 3'-probe and
a 1000-base pair PCR-amplified fragment with the two primers A and C
(Fig. 1, A and B).
Chimeras were obtained from three independent mutant ES clones and
mated with BALB/c females to generate F1 heterozygous mutant mice.
Intercrosses between heterozygotes yielded the homozygous mutants. Wild
type mice, heterozygotes, and homozygotes were born with the expected
1:2:1 ratio, but the homozygous mutant mice died immediately after
birth. After birth, of a total of 326 offspring, 243 breathed
regularly, made movements, arched their backs, and turned pink after
ventilation of their lungs. Genotype analyses of these mice by Southern
blot analysis indicated that 85 were wild type and 158 were
heterozygous for the ATF-2 mutated allele. However, none were
homozygous for the mutated allele. In contrast, 83 pups exhibited
respiratory distress, cyanosis, and subsequently died within 10 min
(Fig. 2A). The shape, size, and body weight of these pups were similar to those of the wild type.
Genotyping of the dead pups revealed that these pups were all
homozygous for the ATF-2 mutated allele. Thus, the ATF-2 mutation results in perinatal recessive lethality. The same result was obtained
with homozygous mutants generated from two other independently derived
ATF-2-defective ES cell lines, regardless of whether they had been
crossed with BALB/c, C57BL/6, or ICR mice. From these results, we
conclude that homozygous ATF-2o/o mutant mice are able to
develop to term but die shortly after birth as a result of respiratory
failure.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 1.
Generation of null ATF-2-deficient mice.
A, diagrammatic representations of the ATF-2 locus and
targeting construct. The DNA-binding domain-containing exon (amino
acids 327-395) is shown by a box. The
neor gene (neo) was inserted into the
exon encoding the DNA-binding domain and is flanked by 9.0- and 1.2-kb
homologous sequences on its 5'- and 3'-side, respectively. The location
of the probes used for Southern blot analyses is given along with the
expected sizes of the hybridizing fragments. The three primers used for
PCR are also indicated. B, BamHI; Bg,
BglII; N, NcoI. B, examples
of genomic Southern blots (left panel) and PCR (right
panel) analyses of wild type (+/+), heterozygous
(+/ ), and homozygous ( / ) mutant mice.
Genomic DNAs were isolated from the tails of mice and digested with
BglII for Southern blot analysis. bp, base pairs.
C, loss of ATF-2 protein in ATF-20/0 embryos.
Upper panel, immunodetection of the ATF-2 protein. Extracts
(100 µg of protein) from the brain of wild type, ATF-2m/m
homozygous, and ATF-2o/o homozygous mutant fetuses of 18.5 dpc were used for Western blotting with the anti-ATF-2 antibody. The
bands indicated by an asterisk are nonspecific
signals. The two ATF-2 proteins (1 and 2) were
detected in the wild type, whereas the protein indicated by the number
3 was observed only in the ATF-2m/m fetuses.
Lower panel, wild type ATF-2 protein and the mutant form
expressed in ATF-2m/m embryos are schematically indicated.
SAPK, stress-activated protein kinases.
|
|

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 2.
Respiration defects of the
ATF-2o/o homozygous mutant. A, cyanosis in
the newborn mutant pups. The newborn pups generated by mating between
ATF-2o/+ heterozygotes (+/ ) are indicated. The color of
the ATF-2o/o homozygotes ( / ) is pale, indicating
cyanosis. B, presence of meconium in the mutant lung. The
lung sections were prepared from the wild type (+/+) and the
ATF-2o/o homozygous mutant and analyzed by microscopy. The
mutant lung contains meconium. C, absence of air in the
mutant lung. The lungs were prepared from newborn pups generated by
mating between ATF-2o/+ heterozygotes and were then put
into PBS solution. The lungs from the wild type or ATF-2o/+
heterozygote floated on PBS, whereas those from the homozygous mutants
sank to the bottom of container containing PBS.
|
|
This phenotype of the ATF-2o/o homozygotes differed from
that we2 previously reported
for the ATF-2m/m mice. Approximately half of
ATF-2m/m mice survived over one month and exhibited complex
anomalies including a defect in endochondral ossification at the
epiphyseal plates, an ataxic gait, and reduced numbers of cerebellar
Purkinje cells (16). The ATF-2m/m mutant was generated by
inserting the neomycin cassette into the exon encoding amino acids
277-326. However, domain analyses of the ATF-2 protein indicated that
the region between amino acids 155 and 338 can be deleted without a
loss of trans-activating capacity (22). The difference in
the phenotypes of the two independently generated lines of ATF-2 mutant
mice suggested that a certain amount of a splice form of ATF-2 protein
that we2 had failed to detect originally may be generated
in the ATF-2m/m mice. Reverse transcriptase-PCR analysis of
ATF-2m/m RNA revealed a transcript that, upon sequencing,
contained an in-frame splicing that removed the exon disrupted by the
neomycin cassette. The mRNA species expressed in
ATF-2m/m encoded the ATF-2 protein lacking the region
between amino acids 277 and 326 (Fig. 1C, lower
panel). To examine whether a mutant protein was made from this
transcript, whole cell lysates were prepared from the brains of wild
type, ATF-2m/m, and ATF-2o/o homozygous mutant
fetuses of 18.5 dpc and used for Western blotting (Fig. 1C,
upper panel). In the wild type lysates, the two forms of
ATF-2 protein, consisting of 70- and 62-kDa polypeptides (Fig. 1C, bands 1 and 2), were detected. Although these
two forms were not detected in either the ATF-2m/m or
ATF-2o/o homozygotes, a novel 66-kDa protein was detected
in the ATF-2m/m lysate (Fig. 1C, band 3). These
results indicate that the ATF-2o/o mutant described here is
a null mutant, whereas the previously described ATF-2m/m
mutant contains a mutant protein. It is likely that the use of much
larger amounts of extract permitted the detection of this mutant
protein, which we2 had previously failed to detect. We
conclude that small amounts of a mutant ATF-2 protein are sufficient to
protect at least some ATF-2m/m mice (but not all, because
the viability of the ATF-2m/m mice is only 50%) from death
shortly after birth by respiratory failure, and the surviving
ATF-2m/m mice allowed an assessment of the role of ATF-2 in
other organ systems. Interestingly, when ATF-2m/+ and
ATF-2o/+ mice were mated, the compound ATF-2m/o
heterozygotes displayed the same phenotype (neonatal lethality from
respiratory failure) as the ATF-2o/o homozygotes,
demonstrating a gene dosage effect of the ATF-2m allele.
Thus, a 50% reduction in levels of the ATF-2 mutant protein in the
compound heterozygote results in the emergence of neonatal lethality,
demonstrating exquisite sensitivity of the phenotype to levels of
ATF-2.
To understand the mechanism of impaired respiratory function, the lungs
prepared from neonates were analyzed. Careful histological analyses of
the ATF-2o/o mutant lung sections indicated the presence of
meconium in the alveoli, showing that prenatal aspiration of the
amniotic fluid containing meconium had probably occurred (Fig.
2B). Consistent with this, the wild type lung floated on
PBS, but the mutant lung sank, indicating absence of air in the alveoli
of the mutant lung at birth (Fig. 2C). Pulmonary histology
showed normal architecture of the mutant lung including the presence of
branching, alveolization, septation, and alveolar volume. We examined
the expression level of the four surfactant proteins, which are
important for the mechanical stability of the lung (for review, see
Ref. 23). Although the level of surfactant protein B expression was
decreased to half of that of the wild type in the ATF-2o/o
mutant, the other three surfactant genes were expressed at the same
level as that of the wild type (data not shown).
Hypoxia and a Reduced Number of Cytotrophoblast in Mutant
Placentas--
It has been observed that hypoxia in embryos may lead
to strong gasping respiration with consequent aspiration of the
amniotic fluid containing meconium (1-3, 24). Therefore, we examined whether hypoxia occurs in the mutant embryos. RNA was prepared from
various tissues of the wild type and ATF-2o/o embryos at
18.5 dpc, and the expression levels of VEGF and tyrosine 3-hydroxylase
genes, which are known to be induced by hypoxia (25, 26), were examined
by RNase protection (Fig. 3A).
The levels of VEGF mRNA in the mutant brain and liver were 4- and 23-fold, respectively, higher than those of the wild type. In addition,
the tyrosine 3-hydroxylase mRNA level in the mutant brain was
26-fold higher than that of the wild type. These results suggest that
hypoxia occurs in the mutant embryos. In the case of
ATF-2m/m embryos, a similar induction of VEGF and tyrosine
hydroxylase mRNA was observed only in the limited number of embryos
(data not shown). This is consistent with the fact that the limited number of ATF-2m/m mutant mice also died immediately after
birth like ATF-20/0.

View larger version (61K):
[in this window]
[in a new window]
|
Fig. 3.
Hypoxia and a reduced number of
cytotrophoblast in mutant placentas. A, increased
levels of hypoxia inducible mRNAs in the mutant embryos. Total RNA
was prepared from various tissues of wild type and ATF-2o/o
homozygous mutants. RNase protection was performed using probes from
the VEGF and tyrosine hydroxylase (TH) genes whose
expression is induced by hypoxia. The 181- and 192-nucleotide bands
were detected with the VEGF probe, whereas the tyrosine hydroxylase
probe generated the 161-nucleotide band. The cytoplasmic -actin
probe that gave rise to the 250-nucleotide band was used as a control.
The density of the protected bands was normalized with respect to that
of the -actin gene, and the relative amounts are indicated as a bar
graph. The shaded bar indicates the data obtained with
mutant RNA. B, reduced number of cytotrophoblasts in the
18.5-dpc mutant placentas. Left panels, low power
magnification (×5.2) of comparable sections through the wild type
(+/+) and mutant ( / ) placentas at 18.5 dpc.
L, labyrinth region. Middle panels, higher
magnification (×200) views of the left panels. The number
of cytotrophoblast cells is reduced in the mutant. Right
panels, high power magnification (×200) views of sections through
the 14.5-dpc placentas. C, expression of ATF-2 in the wild
type cytotrophoblast cells. Expression of ATF-2 in cytotrophoblast
cells of 18.5-dpc wild type (+/+) or mutant
( / ) placentas was examined by immunostaining
(×50).
|
|
To investigate the mechanism of hypoxia in 18.5-dpc embryo, we
histologically analyzed the wild type and mutant embryos. No detectable
differences in the histological structures of any of the organs could
be detected, including the central nervous system, where ATF-2 is
expressed at the highest level (15), and the cardiovascular system
(data not shown). To examine whether some abnormality in the central
nervous system leads to the phenotype of the ATF-2o/o
mutants, we made transgenic mice expressing ATF-2 from the
neurofilament promoter in the central nervous system. However,
transgenic ATF-2o/o mice were not rescued from postnatal
lethality (data not shown).
A histological comparison of E18.5 placentas from wild type mice and
homozygous mutants revealed a slight but significant alteration in the
labyrinth region of the mutant placentas. The labyrinth region in the
normal placenta has numerous fine embryonic vessels surrounded by
trophoblast epithelial cells that are bathed in maternal blood. In the
labyrinth region of the mutant placenta, the number of cytotrophoblast
cells was clearly lower, although the size of the region was almost the
same as that of the wild type (Fig. 3B). The average number
of cytotrophoblast cells/mm2 obtained by examining the 18 placentas was 465 ± 52, 483 ± 40, and 216 ± 17 in the
wild type, heterozygous, and homozygous placenta, respectively.
Furthermore, the network of embryonic vessels and maternal sinuses was
poorly developed, and fibrinoid had accumulated around the fetal blood
vessels (data not shown). The number of spongiotrophoblast cells of the
junctional zone, which produces hormones and growth factors, was
approximately the same in the mutant and normal placentas. The
expression of ATF-2 in the trophoblast was confirmed by immunostaining
using the anti-ATF-2 polyclonal antibody (Fig. 3C),
supporting the idea that ATF-2 is required for the normal proliferation
of trophoblasts. In most of the ATF-2m/m placentas, a
decrease in number of cytotrophoblast cells was not evident, and the
faint but significant signal of ATF-2 immunostaining was detected (data
not shown). However, the placental defect similar to that of
ATF-20/0 was also detected in the limited number of
ATF-2m/m placentas (data not shown). This is consistent
with the fact that some ATF-2m/m pups died immediately
after birth like the ATF-20/0 pups. From approximately E11
onwards, the labyrinth region of the placenta starts to function as a
nutrient and oxygen transport unit (27). A decrease in the number of
cytotrophoblastic cells was not observed at early stages such as E14.5
(Fig. 3B, right panels). This is consistent with
the observation that the sizes of the mutant embryos were almost the
same as those of the wild type. The reduced number of cytotrophoblast
cells at the late stage may lead to an insufficient oxygen supply,
resulting in gasping respiration and aspiration of the amniotic fluid
containing meconium.
PDGF Receptor
Is a Target of ATF-2--
To identify the
gene(s) regulated by ATF-2 in cytotrophoblast cells, we employed a
cDNA subtraction method. Total RNA was prepared from the placenta
of wild type and ATF-2o/o homozygous embryos of 18.5 dpc
and used for cDNA subtraction analysis. The expression levels of
the genes identified by the cDNA subtraction method were further
examined by Northern blotting. Five genes were found to be
down-regulated in the mutant, and one of them was the PDGF receptor
gene. PDGF receptor
has been reported to be expressed in a
subpopulation of cytotrophoblasts and to be required for their
proliferation (28). On the other hand, six genes up-regulated in the
ATF-2o/o mutant were identified, and two of them, such as
the glucose transporter and cytokeratin genes, are known to be hypoxia
inducible (29, 30). Among them, however, there was no obvious candidate that might be expected to block the proliferation of trophoblasts.
In addition to cDNA subtraction analysis, we also used the
microarray-based expression monitoring. Microarrays containing 18,378 nonredundant mouse cDNA clones chosen from the I.M.A.G.E. collection, which is thought to be 20% in the mouse genome, were hybridized with the cDNA probes prepared using the wild type and ATF-2o/o placenta RNAs. Because the placental tissue
prepared from ATF-2o/o homozygous embryos should contain a
significant amount of ATF-2o/+ maternal tissue, we
speculated that the difference in the expression level of ATF-2 target
genes detected by this method is not so high. Therefore, we picked up
the genes whose expression level is different more than 2.5-fold
between the wild type and ATF-2o/o homozygous mutant. Two
hundred fifty-three genes were found to be down-regulated in the
mutant, and 78 genes among them encoded the known proteins. One of them
was the PDGF receptor
gene, and the difference in the PDGF receptor
expression level between wild type and mutant was 2.7-fold.
Although some other genes in this group, including the hepatocyte
growth factor activator-like protein and the ect2 oncogene,
also appear to be involved in the regulation of cellular proliferation,
no evidence was reported so far indicating that these genes are
involved in the proliferation control of cytotrophoblast cells. On
the other hand, 213 genes up-regulated in the ATF-2o/o
mutant were identified, and 54 genes among them encoded the known proteins. This group contains the hypoxia inducible genes encoding glucose transporter-3, epidermal growth factor-like growth factor, GADD45, and insulin-like growth factor-II. Among them, however, there
was no obvious candidate that might be expected to block the
proliferation of trophoblasts.
Because the PDGF receptor
gene was identified as a candidate of the
target gene of ATF-2 by using two different methods, we examined the
expression of the PDGF receptor
gene in wild type and mutant
placentas using immunostaining and in situ hybridization (Fig. 4A). The expression
level of PDGF receptor
was significantly decreased in the mutant,
although some PDGF receptor
expression still remained. This partial
decrease in PDGF receptor
expression may explain the mild defects
of the placenta in ATF-2o/o homozygotes.

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 4.
PDGF receptor is a
target of ATF-2. A, decreased expression of the PDGF
receptor in the mutant. Expression of the PDGF receptor in
cytotrophoblast cells of 18.5-dpc. Wild-type (+/+) or mutant
( / ) placentas were examined by immunostaining
(upper panels) or in situ hybridization
(lower panels). The antisense and sense probes used are
indicated. B, stimulation of DNA synthesis by PDGF in
trophoblast-derived cells. In the presence or absence of recombinant
PDGF-AA or PDGF-BB, Jar cells were incubated with BrdUrd
(BrdU), and the incorporation of BrdUrd was determined.
Values of a representative experiment are given as the mean ± S.E. (n = 3). BrdUrd incorporation in the absence of
PDGF is 100%. C, activation of the PDGF receptor promoter by ATF-2. A mixture containing the luciferase plasmid
pPDGFR -2120-luc, which contains the 2.2-kb PDGF receptor promoter, the plasmid to express various forms of ATF-2, and the
internal control plasmid pact- -gal were transfected into Chinese
hamster ovary cells, and luciferase assays were performed. The two
ATF-2 deletion mutants, BR and 271-338, lack the basic region of
the b-zip DNA-binding domain and the region between amino acids 271 and
338, respectively. The C32S mutant is a point mutant of the metal
finger structure in the NH2-terminal activation domain. The
average of relative levels of luciferase activities of four experiments
is indicated by a bar graph along with standard deviations. The
shaded bar indicates the significant activation.
|
|
Although PDGF receptor
has been reported to be expressed in a
subpopulation of cytotrophoblasts (28), we examined whether PDGF really
stimulates the proliferation of cytotrophoblast-derived cells using a
human choriocarcinoma cell line, Jar. PDGF-AA and PDGF-BB stimulated
BrdUrd incorporation into Jar cells by 40.5 ± 7.5% and 72.0 ± 6% (mean ± S.E.), respectively (Fig. 4B). Although this degree of stimulation is not high, it is significant compared with
the published data (31).
To further examine whether the promoter activity of the PDGF receptor
gene is directly regulated by ATF-2, we performed co-transfection experiments. A luciferase reporter plasmid, in which
the human PDGF receptor
promoter was linked to the luciferase gene,
(21) was co-transfected into Chinese hamster ovary cells along with the
ATF-2 expression plasmid (Fig. 4C). ATF-2 increased luciferase gene expression about 2.7-fold. In contrast, the ATF-2 mutant (
BR), in which the basic region of the b-zip DNA-binding domain was deleted, did not enhance the activity of the PDGF receptor
promoter. Another mutant (C32S), in which the metal finger
structure in the NH2-terminal activation domain was
disrupted by changing the cysteine residue at amino acid 32 into
serine, also did not stimulate the luciferase expression. The modest
activation of the PDGF receptor
promoter by wild type ATF-2 is
consistent with the partial decrease in PDGF receptor
expression in
the ATF-2o/o homozygotes. Consistent with the phenotype of
ATF-2m/m embryos, the ATF-2 protein lacking the region
between amino acids 271 and 338 (
271/338), which is similar to the
ATF-2 protein expressed in ATF-2m/m mutant, retained the
capacity to enhance the PDGF receptor
promoter activity. These
results indicate that ATF-2 activates the PDGF receptor
promoter.
Although the PDGF receptor
promoter region contains the sequence
suitable for binding to ATF-2, we cannot exclude the possibility that
ATF-2 indirectly activates this promoter.
 |
DISCUSSION |
All of the ATF-2 null mutants examined displayed symptoms of
severe respiratory distress with the lungs filled with meconium and had
the reduced number of cytotrophoblast cells in the placenta. Although
both PDGF receptor
and ATF-2 are expressed in various tissues other
than placenta, only the placental defect was evident during
embryogenesis. CRE-BPa and ATF-a, which have a striking homology with
ATF-2 and form a subgroup in the ATF/CREB gene family (8, 9), may
compensate for the lack of ATF-2 in other tissues. Aspiration of the
amniotic fluid containing meconium is observed in MAS, which is a
common neonatal problem that results in acute and chronic respiratory
morbidity (1, 2). Although human MAS has been speculated to be caused
by multiple mechanisms, no direct evidence exists to show that a
deficiency in any one gene leads to MAS. MAS exhibits a varying
severity, but mortality occurs rarely in the human infant and not
usually within minutes of birth. In this sense, the phenotype of ATF-2
null mutant is apparently different from a typical type of MAS.
However, it should be noted that the human infants can be treated by
appropriate suctioning at birth or by early administration of
surfactant, whereas the ATF-2 mutant mice could not be treated because
of a lack of appropriate systems. In addition, a severe form of human
MAS, which cannot be prevented by these treatments, shows features
similar to those of ATF-2 null mutants. Interestingly, the placentas of
some severe MAS infants showed significant abnormalities (3),
suggesting that at least some type of MAS may be a prenatal rather than
a postnatal disease. In addition to the presence of fluid, the level of
surfactant protein B in the ATF-2 mutant lungs was decreased to half of
the level of the wild type. The decrease in the surfactant protein
level is associated with respiration distress syndrome (23). Thus, the
ATF-2 null mutants appear to assume the aspects of multiple syndromes
of respiration failure. The ATF-2 null mutants described here will be
useful to understand the mechanism of severe neonatal respiration
distress and should lead, eventually, to the development of new
diagnostic tools and therapies for this type of disease.
 |
ACKNOWLEDGEMENTS |
We are grateful to C. Torigata for
histological analysis of meconium, S. Aizawa, M. Nagata, T. Watanabe,
and E. Momotani for an earlier contribution to this work, S. Nishikawa
for the anti-PDGF receptor
cDNA, S. Mosselman for the human
PDGF receptor
promoter construct, and Y. Ogawa, M. Nakayama, and M. Kitaoka for helpful discussions.
 |
FOOTNOTES |
*
This work was supported in part by the ACR Arthritis
Investigator Award (to A. R.) and by National Institutes of Health
Grant AI32412 (to L. H. G).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶¶
To whom correspondence should be addressed. Tel.:
81-298-36-9031; Fax: 81-298-36-9030; E-mail:
sishii{at}rtc.riken.go.jp.
2
A. Reimold and L. H. Glimcher, unpublished results.
 |
ABBREVIATIONS |
The abbreviations used are:
MAS, meconium
aspiration syndrome;
VEGF, vascular endothelial growth factor;
PBS, phosphate-buffered syndrome;
PCR, polymerase chain reaction;
ES, embryonic stem;
dpc, days postcoitum;
PDGF, platelet-derived growth
factor;
kb, kilobase pairs;
CRE, cAMP response element;
BrdUrd, bromodeoxyuridine.
 |
REFERENCES |
-
Greenough, A.
(1995)
Early Hum. Dev.
41,
183-192[CrossRef][Medline]
[Order article via Infotrieve]
-
Katz, V. L.,
and Bowes, W. A., Jr.
(1992)
Am. J. Obstet. Gynecol.
166,
171-183[Medline]
[Order article via Infotrieve]
-
Thureen, P. J.,
Hall, D. M.,
Hoffenberg, A.,
and Tyson, R. W.
(1997)
Am. J. Obstet. Gynecol.
176,
967-975[Medline]
[Order article via Infotrieve]
-
Karin, M.,
and Smeal, T.
(1992)
Trends Biochem. Sci.
17,
418-422[CrossRef][Medline]
[Order article via Infotrieve]
-
Maekawa, T.,
Sakura, H.,
Kanei-Ishii, C.,
Sudo, T.,
Yoshimura, T.,
Fujisawa, J.,
Yoshida, M.,
and Ishii, S.
(1989)
EMBO J.
8,
2023-2028[Abstract]
-
Hai, T.,
Liu, F.,
Coukos, W. J.,
and Green, M. R.
(1989)
Genes Dev.
3,
2083-2090[Abstract]
-
Kara, C. J.,
Liou, H.-C.,
Ivashikiv, L. B.,
and Glimcher, L. H.
(1990)
Mol. Cell. Biol.
10,
1347-1357[Medline]
[Order article via Infotrieve]
-
Gaire, M.,
Chatton, B.,
and Kedinger, C.
(1990)
Nucleic Acids Res.
18,
3467-3473[Abstract]
-
Nomura, N.,
Zu, Y.-L.,
Maekawa, T.,
Tabata, S.,
Akiyama, T.,
and Ishii, S.
(1993)
J. Biol. Chem.
268,
4259-4266[Abstract/Free Full Text]
-
Hai, T.,
and Curran, T.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
3720-3724[Abstract]
-
Gupta, S.,
Campbell, D.,
Dérijard, B.,
and Davis, R. J.
(1995)
Science
267,
389-393[Medline]
[Order article via Infotrieve]
-
Livingstone, C.,
Patel, G.,
and Jones, N.
(1995)
EMBO J.
14,
1785-1797[Abstract]
-
van Dam, H.,
Wilhelm, D.,
Herr, I.,
Steffen, A.,
Herrlich, P.,
and Angel, P.
(1997)
EMBO J.
14,
31798-31811
-
Gonzalez, G. A.,
and Montminy, M. R.
(1989)
Cell
59,
675-680[Medline]
[Order article via Infotrieve]
-
Takeda, J.,
Maekawa, T.,
Sudo, T.,
Seino, Y.,
Imura, H.,
Saito, N.,
Tanaka, C.,
and Ishii, S.
(1991)
Oncogene
6,
1009-1014[Medline]
[Order article via Infotrieve]
-
Reimold, A. M.,
Grusby, M. J.,
Kosaras, B.,
Fries, J. W. U.,
Mori, R.,
Maniwa, S.,
Clauss, I. M.,
Colins, T.,
Sidman, R. L.,
Glimcher, M. J.,
and Glimcher, L. H.
(1996)
Nature
379,
262-265[CrossRef][Medline]
[Order article via Infotrieve]
-
Yagi, T.,
Nada, S.,
Watanabe, N.,
Tamemoto, H.,
Kohmura, N.,
Ikawa, Y.,
and Aizawa, S.
(1993)
Anal. Biochem.
214,
77-86[CrossRef][Medline]
[Order article via Infotrieve]
-
Yagi, T.,
Tokunaga, T.,
Furuta, Y.,
Nada, S.,
Yoshida, M.,
Tsukada, T.,
Saga, Y.,
Takeda, T.,
Ikawa, Y.,
and Aizawa, S.
(1993)
Anal. Biochem.
214,
70-76[CrossRef][Medline]
[Order article via Infotrieve]
-
Holmgren, L.,
Glaser, A.,
Pfeifer-Ohlsson, S.,
and Ohlsson, R.
(1991)
Development
113,
749-754[Abstract]
-
Umenishi, F.,
Verkman, A. S.,
and Gropper, M. A.
(1996)
DNA Cell Biol.
15,
475-480[Medline]
[Order article via Infotrieve]
-
Afink, G. B.,
Nistér, M.,
Stassen, B. H. G. J.,
Joosten, P. H. L. J.,
Rademakers, P. J. H.,
Bongcam-Rudloff, E.,
Van Zoelen, E. J. J.,
and Mosselman, S.
(1995)
Oncogene
10,
1667-1672[Medline]
[Order article via Infotrieve]
-
Matsuda, S.,
Maekawa, T.,
and Ishii, S.
(1991)
J. Biol. Chem.
266,
18188-18193[Abstract/Free Full Text]
-
Floros, J.,
and Kala, P.
(1998)
Annu. Rev. Physiol.
60,
365-384[CrossRef][Medline]
[Order article via Infotrieve]
-
Block, M. F.,
Kallenberger, D. A.,
Kern, J. D.,
and Nepveux, R. D.
(1981)
Gynecol. Obstet.
57,
37-40
-
Shweiki, D.,
Itin, A.,
Soffer, D.,
and Kesher, E.
(1992)
Nature
359,
843-845[CrossRef][Medline]
[Order article via Infotrieve]
-
Czyzyk-Krzeska, M. F.,
Bayliss, D. A.,
Lawson, E. E.,
and Millhorn, D. E.
(1992)
J. Neurochem.
58,
1538-1546[Medline]
[Order article via Infotrieve]
-
Cross, J. C.,
Werb, Z.,
and Fisher, S. J.
(1994)
Science
266,
1508-1518[Medline]
[Order article via Infotrieve]
-
Holmgren, L.,
Claesson-Welsh, L.,
Heldin, C. H.,
and Ohlsson, R.
(1992)
Growth Factors
6,
219-231[Medline]
[Order article via Infotrieve]
-
Ebert, B. L.,
Firth, J. D.,
and Ratcliffe, P. J.
(1995)
J. Biol. Chem.
270,
29083-29089[Abstract/Free Full Text]
-
Genbacev, O.,
Zhou, Y.,
Ludlow, J. W.,
and Fisher, S. J.
(1997)
Science
277,
1669-1672[Abstract/Free Full Text]
-
Battegay, E. J.,
Rupp, J.,
Iruela-Arispe, L.,
Sage, E. H.,
and Pech, M.
(1994)
J. Cell Biol.
125,
917-928[Abstract]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.