Differential expression of forkhead box transcription factors
following butylated hydroxytoluene lung injury
Vladimir V.
Kalinichenko,
Lorena
Lim,
Brian
Shin, and
Robert H.
Costa
Department of Molecular Genetics, University of Illinois at
Chicago, College of Medicine, Chicago, Illinois 60607-7170
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ABSTRACT |
The forkhead box (Fox)
proteins are a growing family of transcription factors that have
important roles in cellular proliferation and differentiation and in
organ morphogenesis. The Fox family members hepatocyte nuclear factor
(HNF)-3
(Foxa2) and HNF-3/forkhead homolog (HFH)-8 (FREAC-1, Foxf1)
are expressed in adult pulmonary epithelial and mesenchymal cells,
respectively, but these cells display only low expression levels of the
proliferation-specific HFH-11B gene (Trident, Foxm1b). The regulation
of these Fox transcription factors in response to acute lung injury,
however, has yet to be determined. We report here on the use of
butylated hydroxytoluene (BHT)-mediated lung injury to demonstrate that
HFH-11 protein and RNA levels were markedly increased throughout the
period of lung repair. The maximum levels of HFH-11 were observed by
day 2 following BHT injury when both bronchiolar and
alveolar epithelial cells were undergoing extensive proliferation.
Although BHT lung injury did not alter epithelial cell expression of
HNF-3
, a 65% reduction in HFH-8 mRNA levels was observed during the
period of mesenchymal cell proliferation. HFH-8-expressing cells were colocalized with platelet endothelial cell adhesion molecule-1-positive alveolar endothelial cells and with
-smooth muscle actin-positive peribronchiolar smooth muscle cells.
winged helix/forkhead box DNA binding domain; hepatocyte nuclear
factor 3/forkhead homolog; alveolar endothelial cell; alveolar type II
cell; bronchiolar epithelial cells.
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INTRODUCTION |
BUTYLATED
HYDROXYTOLUENE (BHT) is a phenolic antioxidant that was used as a
food preservative, and a single dose of 400 mg/kg body wt to mice
primarily causes acute lung injury (24). The cytochrome
P-4502B enzyme is responsible for converting BHT to its
toxic hydroxylated metabolite, resulting in global lung injury (1, 11, 33). BHT-damaged pulmonary cells are replaced
through extensive cellular proliferation, which is completed within 9 days post-lung injury. At 2-4 days after BHT lung injury,
extensive damage to the bronchiolar and alveolar epithelial cells is
observed, with substantial influx of inflammatory cells
(1). Concomitantly, these pulmonary epithelial cells
undergo proliferation, which is followed by differentiation of alveolar
type II cells into type I cells. Subsequently, distal pulmonary
endothelial and interstitial cells exhibit BHT-mediated damage and
repair between 4 and 7 days after BHT exposure (1).
Moreover, biological removal of the BHT metabolites is accomplished
through increased expression of phase II detoxifying genes, which are
activated by the "cap `n' collar" Nrf1 transcription factor, as
evidenced by increased susceptibility of Nrf1-deficient mice to
BHT-induced mortality (6).
The hepatocyte nuclear factor (HNF)-3
, -3
, and -3
proteins,
which share homology in the winged helix/forkhead DNA binding domain
(8, 25), were originally identified as mediating
transcription of hepatocyte-specific genes (10, 21, 22).
They are a growing family of transcription factors that play important
roles in cellular proliferation and differentiation as well as in organ
morphogenesis (17). Recently, the nomenclature of the
winged helix/forkhead family has been revised to Forkhead box
(Fox) genes (16). Subsequent expression and
transfection studies demonstrated that HNF-3
(Foxa2) also regulates
transcription of genes required for bronchiolar and type II epithelial
cell function (2-4, 14, 28, 39). Furthermore,
HNF-3
regulates promoter expression of nkx homeodomain transcription
thyroid factor-1 (15), which is critical for branching morphogenesis of the lung (18) and regulates expression of
the surfactant protein genes (4, 5, 12, 34). Transgenic mouse studies demonstrated that increased expression of HNF-3
in the
distal respiratory epithelium blocks lung morphogenesis and
vasculogenesis through inhibition of E-cadherin and vascular endothelial growth factor gene expression (38). The
regulation of HNF-3
expression by proliferative signals following
global lung injury, however, has yet to be determined.
The human winged helix family member HNF-3/ forkhead homolog
(HFH)-11B, also known as Trident and FOXM1b, is a
potent transcriptional activator that is expressed in proliferating
cells of mouse embryos (embryonic day 16), including liver,
intestine, lung, and renal pelvis (19, 36). In adult
organs, HFH-11 expression is extinguished in the postmitotic,
differentiated cells of the liver, lung, and kidney, but its expression
continues in proliferating cells of adult tissue, primarily in thymus,
testis, small intestine, and colon (19, 36). Consistent
with a role in mediating cell cycle progression,
hfh11/Trident-deficient embryos display an abnormal polyploid phenotype in embryonic hepatocytes and cardiomyocytes, suggesting that HFH-11 expression is required to link DNA replication with mitosis (20). Reactivation of hepatic HFH-11B levels
during partial hepatectomy-induced liver regeneration occurs at the
G1/S transition of the cell cycle and continues throughout
the period of proliferation, suggesting that HFH-11 expression is a
marker for cellular propagation (36). Liver regeneration
studies with transgenic mice displaying premature HFH-11B expression
revealed that the mice exhibited an 8-h acceleration in the onset of
hepatocyte DNA replication and mitosis resulting from earlier
expression of cell cycle regulatory genes (35). These
results suggest that HFH-11B expression is limiting in proliferating
cells and that changing its kinetics of expression will accelerate
hepatocyte entry into S phase. Whether HFH-11B expression is also
induced in response to lung injury remains to be determined.
Previous in situ hybridization studies have demonstrated that HFH-8
(also known as FREAC-1 and Foxf1) expression initiates during
gastrulation in a subset of mesodermal cells, arising from the
primitive streak region that contributes to the extraembryonic mesoderm
and lateral mesoderm (26). During organogenesis, HFH-8 expression is restricted to the splanchnic mesoderm contacting the
embryonic gut, suggesting that it may participate in the
mesenchymal-epithelial induction of lung and gut morphogenesis
(23, 26). Consistent with these embryonic expression
studies, adult HFH-8 expression is restricted to the mesenchymal cells
of the alveolar sac and the lamina propria and smooth muscle of the
intestine. The regulation of HFH-8 expression in response to lung
injury and repair, however, has not yet been determined.
In this study, we used BHT-mediated lung injury to induce cellular
proliferation and examined the expression pattern of three Fox
transcription factors during the lung repair process. Although BHT lung
injury did not alter epithelial expression of HNF-3
, we show that
HFH-11 expression is markedly induced within 2 days following BHT
treatment and that its protein levels were sustained throughout the
period of cellular proliferation. We also observed a transient 65%
reduction in HFH-8 mRNA levels between 4 and 6 days following BHT
injury, suggesting that HFH-8 expression decreases during the period of
mesenchymal cell proliferation. To determine the cellular expression
pattern of the HFH-8 gene, we used heterozygous Hfh-8(+/
) mouse lungs in which the
-galactosidase
gene was knocked into the coding region of the mouse HFH-8 gene locus.
HFH-8-expressing cells, as detected by nuclear
-galactosidase enzyme
staining, were colocalized with platelet endothelial cell adhesion
molecule (PECAM)-1-positive alveolar endothelial cells and with
-smooth muscle (
-SM) actin-positive peribronchiolar smooth muscle cells.
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MATERIALS AND METHODS |
BHT treatment of mice.
BHT (3,5-di-tert-butyl-4-hydroxytoluene; Sigma, St Louis,
MO) was dissolved in corn oil (Mazola) at 40 mg/ml concentration, and a
single intraperitoneal injection of BHT (400 mg/kg body wt) was given
to BALB/c males (4-6 wk of age). To determine statistical significance of any observed differences, we used three mice per time
point following BHT administration, which included 16 h and 1, 2, 4, 6, 8, and 10 days. The mice were killed by CO2
asphyxiation, and lung tissue was used to prepare total RNA or lungs
were inflated with 4% paraformaldehyde and were then paraffin
embedded as described previously (35, 39).
Antibodies and immunohistochemical and
-galactosidase enzyme
staining.
A microtome was used to prepare 5-µm sections of lung tissue, which
were deposited onto Superfrost Plus microscope slides (Fisher) and
either stained with hematoxylin and eosin or Giemsa for morphological
examination or used for immunohistochemical staining with various
antibodies. Mouse monoclonal anti-proliferation cell nuclear antigen
(PCNA) antibody (clone PC10) was obtained from Roche Molecular
Biochemicals (Indianapolis, IN) and used at a dilution of 1:1,000;
affinity-purified rabbit polyclonal anti-mouse HFH-11 antibody was
generated and used at a dilution of 1:100 as described previously
(35, 36); rat monoclonal anti-PECAM-1 antibody (clone MEC
13.3) was purchased from PharMingen (San Diego, CA) and used at a
dilution of 1:500; mouse monoclonal HNF-3
antibody (clone 4C7) was
obtained from the University of Iowa Developmental Studies Hybridoma
Bank and used at a dilution of 1:50; and mouse monoclonal
-SM actin
antibody was purchased from Sigma (Clone 1A4) and used at a dilution of
1:400. Briefly, paraffin wax was removed from lung sections with xylene
and rehydrated with decreasing graded ethanol washes. Citrate buffer
(0.02 M, pH 6.0) was used for microwave retrieval to enhance the
antigenic activity as described previously (39). Sections
were then blocked with 2.5% normal horse serum for 1 h and
incubated at 4°C overnight with primary antibody. Staining for PCNA
was performed using horse anti-mouse antibody conjugated with alkaline
phosphatase (Vector Laboratories, Burlingame, CA). Staining for HFH-11
was developed using horse anti-rabbit antibody conjugated with biotin
followed by avidin-alkaline phosphatase conjugate (all from Vector). A 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt/nitro
blue tetrazolium kit from Vector Laboratories was used as a substrate for alkaline phosphatase. Immunohistochemical staining for PECAM-1 was
performed after trypsin retrieval (37) using biotinylated goat anti-rat antibody (PharMingen, San Diego, CA), and developed with
streptavidin-horseradish peroxidase conjugate and 3,3'-diaminobenzidine substrate kit (Vector). For colocalization studies, sections were stained with rabbit anti-HFH-11 and mouse monoclonal antibodies for
either HNF-3
or PCNA proteins. Immune complexes were detected with
swine anti-rabbit secondary antibody conjugated with
tetramethylrhodamine isothiocyanate (DAKO, Carpinteria, CA) and horse
anti-mouse secondary antibody conjugated with FITC (Vector).
All immunohistochemical reactions were carried out in parallel with
reactions lacking primary antibodies to ensure the specificity of the
observed staining. Slides were counterstained with methyl green
(Vector). Student's t-test was used to determine
statistically significant differences in percentages of
PECAM-1-positive cells in the lung. Differences of P < 0.05 were considered significant. Values are given as means ± SD.
An HFH-8 gene disruption targeting vector was generated in which the
winged helix DNA binding domain was replaced by a nuclear localizing
-galactosidase gene and phosphoglycerol kinase promoter-driven neomycin gene (see Fig. 7A). The HFH-8 gene targeting vector
replaced NH2-terminal sequences between the NcoI
and NotI sites with the nuclear localizing
-galactosidase, which was cloned in frame with the mouse HFH-8
coding region (GenBank accession number L35949). The Transgenic Mouse
Facility at the University of Cincinnati used embryonic stem (ES) cell
technology to select ES cells with the HFH-8
-galactosidase
gene-targeted locus using procedures described by Clark et al.
(7). These targeted ES cells were subsequently used to
create Hfh8(+/
) mice in which expression of the nuclear
localizing
-galactosidase gene was controlled by the HFH-8 DNA
regulatory region. To determine HFH-8-expressing cells,
Hfh8(+/
) lung tissue was stained for
-galactosidase
enzyme with 1 mg/ml X-gal substrate
(5-bromo-4-chloro-3-indolyl-
-D-galactoside) and paraffin
embedded, and a microtome was used to deposit sections on a slide as
described previously (9). Paraffin wax was removed from
lung sections with xylene and rehydrated with decreasing graded ethanol
washes followed by immunohistochemical staining (brown) with either
PECAM-1 or
-SM actin antibodies as previously described.
RNA extraction and RNase ONE protection assay.
Total mouse lung RNA was prepared by an acid
guanidium-thiocyanate-phenol-chloroform extraction method using
RNA-STAT-60 (Tel-Test "B," Friendswood, TX). RNase protection assay
was performed with [32P]UTP-labeled antisense RNA
synthesized from plasmid templates with the appropriate RNA polymerase
as previously described (10). Approximately 2 × 105 cpm of each probe was hybridized at 45°C to 20 µg
of total RNA in a solution containing 20 mM PIPES (pH 6.4), 400 mM
NaCl, 1 mM EDTA and 80% formamide overnight. After hybridization,
samples were digested 1 h at 37°C by using 10 U/sample of RNase
ONE enzyme according to the manufacturer's protocol (Promega, Madison,
WI). The RNase One protected fragments were electrophoresed on an 8% polyacrylamide-8 M urea gel followed by autoradiography. Quantitation of expression levels was determined with scanned X-ray films by using
the BioMax 1D program (Kodak). The cyclophilin hybridization signal was
used for normalization control between different lung RNA samples.
Synthesis of antisense human HFH-11B, rat HNF-3
, and rat surfactant
protein (SP) C and mouse cyclophilin RNA probes was described
previously (26, 36). Antisense mouse HFH-8 RNA probe was generated from mouse HFH-8 cDNA (nucleotides 437-816), which was cloned in pBL plasmid.
 |
RESULTS AND DISCUSSION |
Morphological changes and cellular proliferation in the lung after
BHT lung injury.
To investigate whether expression of winged helix genes was influenced
by cellular proliferation during lung injury, we administered a single,
nonlethal dose of BHT to wild-type male BALB/c mice (4-6 wk of
age). At each of the various time points after BHT injury, three mice
were killed and lung tissue was isolated and used for paraffin
embedding or preparation of total RNA as described in MATERIALS
AND METHODS. Although there was no mortality from the BHT-induced
lung injury, the mice developed shallow, rapid breathing between 2 and
6 days after treatment. Bright-field microscopy examination of paraffin
sections revealed extensive lung damage by 4 days following BHT
treatment, with increased alveolar wall thickness and influx of
leukocytes into the lung parenchyma (compare Fig.
1, A and B with
C-E). The morphological evidence of lung damage
persisted until 6 days after BHT exposure (Fig. 1, F and G) and gradually improved toward the later time points (Fig.
1H). To confirm that we have reproduced global lung
proliferation as reported by previous BHT injury studies (1, 11,
33), a commercially available PCNA antibody was used for
immunohistochemical staining. Consistent with these previous BHT
studies, injured mouse lungs exhibited elevated PCNA staining in
bronchiolar and alveolar epithelial cells by 2 days after BHT injury,
demonstrating that these cells were undergoing extensive proliferation
(Fig. 2B). An increase in
PCNA-positive cells was also detected in the alveolar region of the
lung between 4 and 8 days after BHT lung injury (Fig. 2,
C-F), correlating with proliferation of the pulmonary endothelial cells and connective fibroblasts as previously reported (1).

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Fig. 1.
Lung morphology changes after butylated hydroxytoluene (BHT)
injury. Mice were injected with a single dose of BHT, and lungs were
harvested at different time points after BHT administration, paraffin
embedded, sectioned, and then histologically stained with either both
hematoxylin and eosin (H&E) or Giemsa. Single leukocytes were already
observed in alveoli of the lung on day 2 after BHT treatment
(B) compared with untreated mouse lungs (A).
C-G: between 4 and 6 days after BHT lung injury, mouse
lungs displayed thickening of the alveolar septa, perivascular edema,
and extensive leukocyte infiltration. H: alveolar region on
day 8 after BHT treatment. Magnification for
A-C and F is ×25 and for D,
E, G, and H it is ×158.
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Fig. 2.
Induction of proliferation cell nuclear antigen (PCNA) expression
following BHT mouse lung injury. Microtome sections of
paraffin-embedded lungs were prepared at various times after BHT lung
injury and used for immunohistochemistry with anti-PCNA monoclonal
antibodies. The PCNA protein-antibody complex was visualized using
alkaline phosphatase-conjugated secondary antibody and
5-bromo-4-chloro-3-indolylphosphate p-toluidine
salt/nitro blue tetrazolium as the substrate (stains blue) and nuclei
were counterstained with methyl green (stains nuclei green).
B: PCNA-positive nuclei (blue, indicated by arrows) were
visible in bronchiolar and alveolar epithelial cells on day
2 after BHT lung injury and PCNA-negative nuclei were
counterstained with methyl green (C-F). PCNA expression
was detectable throughout the lung parenchyma between days 4 and 8 following BHT lung injury. Magnification for
A-C, E, and F is ×50 and for
D, it is ×158.
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BHT lung injury induces expression of HFH-11 (FoxM1B) during
cellular proliferation.
We previously demonstrated that HFH-11 expression is induced during
liver regeneration following partial hepatectomy, following H2O2 treatment of human microvessel endothelial
cells, and in response to tracheal administration of keratinocyte
growth factor, the latter of which causes proliferation of type II
cells (36). To investigate whether proliferative signals
following lung injury also induced expression of the HFH-11, we
performed RNase protection assays with HFH-11 antisense RNA probes and
mouse lung RNA isolated at various times after BHT lung injury (Fig.
3A). HFH-11 mRNA levels from
three distinct mouse lungs were normalized to the cyclophilin levels
and used to determine the means ± SD (Fig. 3B).
Consistent with HFH-11 involvement in cellular proliferation, injured
mouse lungs displayed a pronounced increase in HFH-11 mRNA levels by 2 days following BHT treatment, and those remained elevated until the
8-day time point (Fig. 3, A and B).

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Fig. 3.
Increased expression of hepatocyte nuclear factor
(HNF)-3/forkhead homolog (HFH)-11 mRNA after BHT lung injury. Total RNA
was prepared from mouse lungs at various times following BHT lung
injury and analyzed for HFH-11 and cyclophilin expression by RNase
protection assay. A: representative photographs of the RNase
protection assay depicting increased HFH-11 expression after BHT lung
injury. B: graphic representation showing increased
expression of HFH-11 mRNA following BHT lung injury. RNA from 3 BHT-
injured mouse lungs was normalized for cyclophilin levels and the
means ± SD were calculated and graphed.
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To determine the cellular expression pattern of the HFH-11 protein
following BHT lung injury, we used an affinity-purified HFH-11 antibody
for immunohistochemical staining of paraffin-embedded lung sections.
Consistent with previous studies, HFH-11 protein was not detected in
normal adult mouse lungs (Fig.
4A), but as early as 2 days
following BHT lung injury, abundant HFH-11 protein staining was evident
in both the alveolar (Fig. 4, B and C) and bronchial epithelial cells (Fig. 4D). Localization of the
HFH-11 protein was mainly nuclear and most of the HFH-11 staining
colocalized with PCNA-positive nuclei (Fig.
5, A and B). At 2 days following BHT injury, HFH-11 protein expression colocalizes with
HNF-3
staining (Fig. 5, C-H), which is a marker for
both bronchiolar epithelial cells (including Clara cells) and type 2 alveolar epithelial cells (39). Elevated levels of the
HFH-11 protein were also sustained in the alveolar region until
day 8 following BHT lung injury (Fig. 4, E and
H) and were detected in the mesenchymal cells of the
arteriole walls (Fig. 4, F and G). Taken
together, these results suggest an active involvement of the HFH-11
transcription factor during lung injury repair in response to BHT
cellular damage. This conclusion is supported by liver regeneration
studies with HFH-11B transgenic mice, demonstrating that premature
hepatic expression of the transcription factor HFH-11B caused
accelerated hepatocyte entry into the S phase resulting from earlier
expression of cell cycle regulatory genes (35).
Furthermore, hfh11/Trident-deficient embryos display an
abnormal polyploid phenotype in embryonic hepatocytes and
cardiomyocytes, suggesting that HFH-11 expression is required for
progression of DNA replication into mitosis (20).
Likewise, increased pulmonary expression of HFH-11 protein following
lung injury may play an important role in the induction of DNA
replication and mitosis. Other models have determined the induction of
Nrf1, CCAAT enhancer binding protein (C/EBP)-
and -
,
c-jun/c-fos, and nuclear factor (NF)-
B
transcription factors following lung injury (6, 13, 14, 29, 31,
32). This study has identified the HFH-11 protein as an
additional transcription factor that is stimulated following BHT lung
injury and that participates in cellular proliferation during lung
injury repair.

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Fig. 4.
Induction of HFH-11 protein expression in BHT-injured mouse lungs.
Microtome sections of paraffin-embedded lung were prepared at various
times after BHT injury, used for immunohistochemistry with
affinity-purified anti-HFH-11 antibody, and visualized as described in
MATERIALS AND METHODS. A-D: HFH-11-positive
nuclei (stained dark purple, indicated by arrows) is found in alveolar
(B-C) and bronchiolar epithelial (D) cells 2 days after BHT lung injury, but HFH-11 protein staining was
undetectable in untreated animals (A). At 4 days following
BHT treatment, expression of HFH-11 protein continued in alveolar
region (E), in small arteriole (F), and in large
artery (G). Alveolar expression of HFH-11 continued on
day 8 (H) following BHT lung injury. Note that
A-E and H were counterstained with methyl
green, whereas F and G were not. Magnification
for A and B is ×50 and for C-H,
it is ×158.
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Fig. 5.
Colocalization of HFH-11 with PCNA and HNF-3 proteins in BHT
lung injury. Paraffin sections of the lung were prepared 2 days after
BHT injection and used for immunohistochemistry with both affinity-
purified anti-HFH-11 antibody and monoclonal antibody against either
PCNA or HNF-3 protein and visualized using differential
immunofluorescence. Positive staining nuclei for the
proliferation-specific HFH-11 transcription factor
(tetramethylrhodamine isothiocyanate, red) displayed colocalized
nuclear staining with either PCNA (FITC, green) in alveolar cells
(A and B) or HNF-3 (FITC, green) in
bronchiolar epithelial cells (C-F) and alveolar
epithelial cells (G-H). Magnification for C
and D is ×25 and for A, B, and
E-H, it is ×100.
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BHT lung injury does not alter HNF-3
levels in proliferating
bronchiolar and alveolar epithelial cells.
Previous liver regeneration studies demonstrated that expression of the
HNF-3
is sustained in proliferating hepatocytes (27). To determine whether HNF-3
expression is also maintained in
proliferating lung cells, total RNA from BHT-injured mouse lungs was
analyzed for HNF-3
mRNA expression by RNase protection assay.
Although BHT-induced extensive proliferation of bronchiolar and
alveolar type II cells (Fig. 2), we observed no changes in either
HNF-3
or SP-C mRNA levels (Fig. 6,
A and B). To confirm that HNF-3
protein levels
were maintained during lung epithelial cell proliferation, we used
immunofluorescence to demonstrate that HNF-3
staining was maintained
in both bronchiolar (Fig. 5, D and F) and
alveolar epithelial cells (Fig. 5H). Furthermore, HNF-3
protein expression colocalizes with the proliferation-specific HFH-11
protein (Fig. 5, C, E, and G),
suggesting that pulmonary expression of HNF-3
was not influenced by
proliferative signals induced following BHT lung injury. Moreover,
previous studies demonstrated that HNF-3
protein is not altered
during hepatocyte replication in regenerating liver or following
lipopolysaccharide-induced acute-phase response (27).
Taken together, these data suggest that HNF-3
maintains
transcription of differentiated epithelial cell genes without
interfering with progression of cellular replication.

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Fig. 6.
Differential expression of HFH-8 and HNF-3 follows BHT
lung injury. Total RNA was prepared from mouse lungs at various times
following BHT lung injury, and RNase protection assay was used to
analyze for HFH-8, HNF-3 , surfactant protein (SP) C and cyclophilin
expression. Photographs of the result (A) and scanning
densitometric data (B) are shown. Relative HFH-8 and
HNF-3 mRNA expression are presented as the means ± SD.
C: decrease of HFH-8 mRNA expression was not due to reduced
number of endothelial cells. Paraffin sections of lungs were prepared
at various times after BHT injection and used for immunohistochemistry
with anti-platelet endothelial cell adhesion molecule (PECAM)-1
antibody, a marker for endothelial cells. Total number of endothelial
cells in high-power microscopic fields was counted using 3 randomly
picked sections, and the means ± SD for each time point were
calculated from 3 different mice.
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Colocalization of HFH-8 expressing cells with alveolar endothelial
and peribronchiolar smooth muscle cells.
To determine the cellular expression pattern of the HFH-8 gene, we used
heterozygous Hfh8(+/
) mouse lungs in which the normal Hfh8 gene locus was replaced by a nuclear localizing
-galactosidase gene cloned in frame with the mouse HFH-8 coding
region (Fig. 7A). Expression
of the nuclear localizing
-galactosidase gene was under the control
of the HFH-8 DNA-regulatory sequences, and thus staining for
-galactosidase enzyme activity allows identification of
HFH-8-expressing cells (a more detailed characterization of the
Hfh8(+/
) mice will be described elsewhere).
Heterozygous Hfh8(+/
) lung tissue was stained for
-galactosidase enzyme activity with X-gal substrate (blue),
paraffin embedded, sectioned, and then prepared for immunohistochemical
staining (brown) with either PECAM-1 or
-SM actin
antibodies. In the absence of primary antibody, control
immunohistochemical staining of Hfh8(+/
) lung tissue displayed only blue staining for
-galactosidase enzyme activity in
the alveolar region and in peribronchiolar smooth muscle cells (Fig. 7,
B and C). By contrast, immunohistochemical
staining of Hfh8(+/
) lung tissue with the PECAM-1
antibody demonstrated that
-galactosidase enzyme activity is
colocalized with PECAM-1 staining in the alveolar region but not in
the peribronchiolar smooth muscle cells (Fig. 7, D-F).
These results demonstrate that alveolar expression of the HFH-8 gene
resides in the PECAM-1-positive endothelial cells (30).
Pulmonary blood vessels lacked detectable HFH-8 staining, suggesting
that HFH-8 expression is restricted to alveolar endothelial cells (data
not shown). Furthermore,
-SM actin immunohistochemical staining in
Hfh8(+/
) lung tissue colocalizes with
-galactosidase-staining cells surrounding the bronchiolar region,
demonstrating that HFH-8 expression also resides in the peribronchiolar
smooth muscle cells (Fig. 7G). However, our data cannot rule
out the possibility that HFH-8 is also expressed in alveolar smooth
muscle cells.

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Fig. 7.
Colocalization of HFH-8 expressing cells with alveolar endothelial
and bronchiolar smooth muscle cells. A: diagram of the mouse
Hfh8 genomic structure with first exon (Ex1, red) containing
the winged helix DNA binding domain (WH, black box), first intron (red)
and second exons (Ex2, red). Also shown is the Hfh8 gene
targeted locus in which the NcoI to NotI
sequences (including the winged helix DNA binding domain) are replaced
by a nuclear localizing -galactosidase gene (blue, cloned in frame
with the Hfh8 coding sequence) and the phosphoglycerol
kinase (PGK) promoter-driven neomycin gene (green) cloned in the
opposite transcription direction (see MATERIALS AND
METHODS). To determine the cellular expression pattern of the
HFH-8 gene, adult Hfh8(+/ ) lung tissue was stained for
-galactosidase ( Gal) enzyme with X-gal substrate (stains blue),
paraffin embedded, sectioned, and then prepared for immunohistochemical
staining (stains brown) with either PECAM-1 or -smooth muscle actin
( -SM actin) antibodies. Expression of PECAM-1 protein is specific
for endothelial cells and -SM actin is specific for smooth muscle
cells. B and C: negative control for
immunohistochemical staining. In the absence of primary antibody, only
nuclear -galactosidase staining (blue) was found in the alveolar
endothelial cells (B) and bronchiolar smooth muscle cells
(Br; C) of the Hfh8(+/ ) adult lung (black
arrows). D-F: HFH-8-expressing cells colocalize with
PECAM-1-positive endothelial cells in the alveolar region. Nuclear
-galactosidase enzyme activity (blue) was colocalized with
PECAM-1-positive (brown) endothelial cells of the Hfh8(+/ )
adult lung (brown arrows). E-F: PECAM-1 staining
did not overlap in the -galactosidase-positive smooth muscle cells
(black arrows). G: HFH-8-expressing cells colocalize with
-SM actin-positive bronchiolar smooth muscle cells. Nuclear
-galactosidase enzyme activity was colocalized with -SM actin
immunohistochemical staining (brown) in bronchiolar smooth muscle cells
of Hfh8(+/ ) adult lung (brown arrows). Magnification for
B-G is ×158.
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BHT lung injury causes diminished expression of HFH-8 (Foxf1)
during proliferation of mesenchymal cells.
In contrast to the Fox transcription factors HFH-11 and HNF-3
, HFH-8
expression is restricted to the mesenchymal tissue of the adult
alveolar lung and intestine. To examine whether HFH-8 expression
changes following BHT lung injury, RNase protection assay was used to
examine HFH-8 mRNA levels at different time points following lung
damage. This analysis revealed a transient 65% reduction in HFH-8 mRNA
between 4 and 6 days following BHT treatment (Fig. 6, A and
B), a time period when extensive proliferation of the
mesenchymal cells is observed (1). Interestingly,
reduction in cellular proliferation correlates with the restoration of
HFH-8 expression levels by 8 days following BHT injury. The decline in
HFH-8 levels during mesenchymal cell proliferation is in contrast to
sustained expression of the winged helix HNF-3
protein in proliferating epithelial cells (Figs. 5 and 6).
Because HFH-8 is expressed in alveolar endothelial cells of the lung,
we wanted to determine whether the decrease in HFH-8 levels was due to
selective death of endothelial cells following BHT lung injury. Lung
sections for each time point were stained with anti- PECAM-1
antibodies (data not shown), and the number of PECAM-1-positive
endothelial cells (30) was counted in high-power microscope fields and plotted in Fig. 6C. This analysis
revealed that BHT lung injury did not diminish the number of
PECAM-1-positive endothelial cells but rather caused a dramatic
reduction in HFH-8 mRNA levels in response to proliferative signals.
These results suggest that reduction in HFH-8 levels coincides with the
period of mesenchymal cell proliferation.
In summary, we show that BHT lung injury induces expression of the
winged helix transcription factor HFH-11 during proliferative stages of
the repair process. Although HNF-3
expression is sustained during
pulmonary epithelial cell proliferation, BHT lung injury causes
significant reduction in HFH-8 expression, coinciding with proliferation of the pulmonary mesenchymal cells. We also used heterozygous Hfh8(+/
) mouse lungs in which the
-galactosidase gene was knocked into the coding region of the mouse
HFH-8 gene locus to determine the HFH-8 cellular expression pattern.
HFH-8-expressing cells in the adult mouse lung as detected by nuclear
-galactosidase enzyme staining were colocalized with markers
specific to alveolar endothelial cells and peribronchiolar smooth
muscle cells.
 |
ACKNOWLEDGEMENTS |
We thank Pradip Raychaudhuri, Xinhe Wang, Francisco Rausa, Mike
Major, and Doug Hughes for critically reviewing the manuscript. We also
thank Eseng Lai for the nuclear localizing
-galactosidase plasmid,
Heping Zhou for generating the HFH-8 targeting vector, and Francisco
Rausa and Jean Clark for screening the ES cells containing the targeted
HFH-8 locus. The Hfh8(+/
) mice were generated by the
Transgenic Mouse Facility at the University of Cincinnati.
 |
FOOTNOTES |
This work was supported by the National Heart, Lung, and Blood
Institute Grant R01-HL-62446-02 to R. H. Costa. The HNF-3
monoclonal antibody (4C7) developed by T. M. Jessell and S. Brenner-Morton was obtained from the Developmental Studies Hybridoma
Bank developed under the auspices of the National Institute of Child
Health and Human Development and maintained by The University of Iowa,
Dept. of Biological Sciences, Iowa City, IA 52242.
Address for reprint requests and other correspondence: R. H. Costa (E-mail: robcosta{at}uic.edu) or V. V. Kalinichenko
(E-mail: vkalin{at}uic.edu), Dept. of Molecular Genetics (M/C 669),
Univ. of Illinois at Chicago College of Medicine, 900 S. Ashland Ave, Rm. 2220 MBRB, Chicago, IL 60607-7170.
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.
Received 26 September 2000; accepted in final form 14 November 2000.
 |
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