Permissive Effects of Oxygen on Cyclic AMP and Interleukin-1 Stimulation of Surfactant Protein A Gene Expression Are Mediated by Epigenetic Mechanisms

Article (PDF Available)inMolecular and Cellular Biology 26(8):2901-12 · May 2006with8 Reads
DOI: 10.1128/MCB.26.8.2901-2912.2006 · Source: PubMed
Abstract
Surfactant protein A (SP-A) is important for immune defense within the alveolus. Cyclic AMP (cAMP) stimulation of SP-A expression in lung type II cells is O2 dependent and mediated by increased phosphorylation and binding of thyroid transcription factor 1 (TTF-1) to an upstream response element (TTF-1-binding element [TBE]). Interleukin-1 (IL-1) stimulation of SP-A expression is mediated by NF-κB (p65/p50) activation and increased binding to the TBE. In this study, we found that O2 also was permissive for IL-1 induction of SP-A expression and for cAMP and IL-1 stimulation of type II cell nuclear protein binding to the TBE. Using chromatin immunoprecipitation, we observed that when type II cells were cultured in 20% O2, cAMP and IL-1 stimulated the recruitment of TTF-1, p65, CBP, and steroid receptor coactivator 1 to the TBE region of the SP-A promoter and increased local acetylation of histone H3; these effects were prevented by hypoxia. Hypoxia markedly reduced global levels of CBP and acetylated histone H3 and increased the expression of histone deacetylases. Furthermore, hypoxia caused a global increase in histone H3 dimethylated on Lys9 and increased the association of dimethyl histone H3 with the SP-A promoter. These results, together with findings that the histone deacetylase inhibitor trichostatin A and the methyltransferase inhibitor 5′-deoxy(5′-methylthio)adenosine markedly enhanced SP-A expression in lung type II cells, suggest that increased O2 availability to type II cells late in gestation causes epigenetic changes that permit access of TTF-1 and NF-κB to the SP-A promoter. The binding of these transcription factors facilitates the recruitment of coactivators, resulting in the further opening of the chromatin structure and activation of SP-A transcription.
MOLECULAR AND CELLULAR BIOLOGY, Apr. 2006, p. 2901–2912 Vol. 26, No. 8
0270-7306/06/$08.000 doi:10.1128/MCB.26.8.2901–2912.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Permissive Effects of Oxygen on Cyclic AMP and Interleukin-1
Stimulation of Surfactant Protein A Gene Expression
Are Mediated by Epigenetic Mechanisms
Kazi Nazrul Islam
and Carole R. Mendelson*
Departments of Biochemistry and Obstetrics and Gynecology, University of Texas Southwestern Medical Center at Dallas,
5323 Harry Hines Boulevard, Dallas, Texas 75390-9038
Received 9 May 2005/Returned for modification 17 June 2005/Accepted 17 January 2006
Surfactant protein A (SP-A) is important for immune defense within the alveolus. Cyclic AMP (cAMP)
stimulation of SP-A expression in lung type II cells is O
2
dependent and mediated by increased phosphorylation
and binding of thyroid transcription factor 1 (TTF-1) to an upstream response element (TTF-1-binding
element [TBE]). Interleukin-1 (IL-1) stimulation of SP-A expression is mediated by NF-B (p65/p50) activa-
tion and increased binding to the TBE. In this study, we found that O
2
also was permissive for IL-1 induction
of SP-A expression and for cAMP and IL-1 stimulation of type II cell nuclear protein binding to the TBE. Using
chromatin immunoprecipitation, we observed that when type II cells were cultured in 20% O
2
, cAMP and IL-1
stimulated the recruitment of TTF-1, p65, CBP, and steroid receptor coactivator 1 to the TBE region of the
SP-A promoter and increased local acetylation of histone H3; these effects were prevented by hypoxia. Hypoxia
markedly reduced global levels of CBP and acetylated histone H3 and increased the expression of histone
deacetylases. Furthermore, hypoxia caused a global increase in histone H3 dimethylated on Lys9 and increased
the association of dimethyl histone H3 with the SP-A promoter. These results, together with findings that the
histone deacetylase inhibitor trichostatin A and the methyltransferase inhibitor 5-deoxy(5-methylthio)ad-
enosine markedly enhanced SP-A expression in lung type II cells, suggest that increased O
2
availability to type
II cells late in gestation causes epigenetic changes that permit access of TTF-1 and NF-BtotheSP-A
promoter. The binding of these transcription factors facilitates the recruitment of coactivators, resulting in the
further opening of the chromatin structure and activation of SP-A transcription.
To define mechanisms involved in lung cell-specific, devel-
opmental, and hormonal regulation of pulmonary surfactant
synthesis, we have focused on surfactant protein A (SP-A), the
major surfactant protein, which is lung specific and synthesized
in type II cells. SP-A, a member of the collectin subfamily of
C-type lectins, plays an important role in immune defense
within the lung alveolus by binding to a variety of bacterial,
viral, and fungal pathogens to facilitate their uptake by alveolar
macrophages and to enhance macrophage migration and cyto-
kine production (see reference 53 for a review). SP-A is an
excellent marker of type II cell differentiation and surfactant
synthesis in the fetal lung, since it is developmentally regulated
with surfactant phospholipid synthesis (see reference 36 for a
review). Recently, we obtained compelling evidence that SP-A
secreted by the fetal lung into amniotic fluid acts as a hormone
that stimulates the migration of amniotic fluid macrophages to
the uterus, where they initiate an inflammatory response lead-
ing to parturition (13).
In studies using midgestation human fetal lung explants
cultured in a 20% O
2
environment, we found that type II cell
differentiation and SP-A gene expression were enhanced by
hormones and factors that increase cyclic AMP (cAMP)
(43). Interestingly, cAMP stimulation of type II cell differ-
entiation and SP-A expression are dependent upon a critical
atmospheric O
2
tension (1). When fetal lung explants were
cultured ina1to2%O
2
-containing environment, type II
cells failed to differentiate, and no stimulatory effects of
cAMP on SP-A expression were apparent. These findings led
us to postulate that increased vascularization of the fetal
lung during the third trimester of gestation and enhanced
O
2
availability to type II cell precursors may facilitate cAMP
induction of type II cell differentiation and surfactant pro-
tein gene expression (1).
To define genomic regions and response elements involved
in lung cell-specific, developmental, and hormonal regulation
of SP-A expression, we have utilized transgenic mice and trans-
fected type II cells. In transgenic mice, we found that as little
as 400 bp of DNA flanking the 5 end of the rabbit SP-A gene
directed appropriate lung cell-specific and developmental reg-
ulation of expression (3). This region contains four response
elements that are individually required for basal and cAMP
stimulation of SP-A promoter activity in transfected type II
cells (2, 17, 32, 39, 56, 57). The finding that mutagenesis of any
one of these elements markedly reduced basal and cAMP
induction of SP-A promoter activity suggests a cooperative
interaction of transcription factors binding to these sites. One
of these response elements, termed thyroid transcription factor
1 (TTF-1)-binding element (TBE), at 170 bp binds the homeo-
domain factor, thyroid transcription factor 1 (TTF-1/Nkx2.1/
T/EBP) (31, 32). Cyclic AMP enhances the binding of type II
cell nuclear proteins to the TBE as well as TTF-1 phosphory-
lation, acetylation, and transcriptional activity (31, 55). TTF-1
* Corresponding author. Mailing address: Department of Biochem-
istry, The University of Texas Southwestern Medical Center at Dallas,
5323 Harry Hines Boulevard, Dallas, TX 75390-9038. Phone: (214)
648-2944. Fax: (214) 648-3214. E-mail: cmende@biochem.swmed.edu.
2901
also regulates the expression of the mouse SP-A gene (9) as
well as genes encoding SP-B (8) and SP-C (25). TTF-1 is
expressed from the earliest stages of lung development (30),
where it plays an essential role in branching morphogenesis
(26). On the other hand, SP-A and the other surfactant protein
genes are developmentally induced at later gestational time
points (36). We therefore postulate that these temporally dis-
tinct actions of TTF-1 may be mediated by selective changes in
TTF-1 posttranslational modification and interaction with
other critical transcription factors and coactivators.
In addition to the observed effects of cAMP, we found that
treatment of human fetal lung type II cells with the cytokines
interleukin-1 (IL-1) and IL-1 increased SP-A expression
and binding of type II cell nuclear proteins to the TBE; IL-1
also had an additive stimulatory effect with cAMP (20). In
characterizing the TBE, we noted that it contains an inverted
binding site for the transcription factor NF-B, which is acti-
vated by cytokines and protein kinase A (PKA) (7, 58). In its
inactive state, NF-B, a heterodimer of p50 and p65, exists in
the cytoplasm in a complex with “inhibitor of B” (IB). Ex-
posure of cells to cytokines causes phosphorylation and pro-
teolytic degradation of IB, with subsequent activation and
translocation of the p50/p65 heterodimer to the nucleus, where
it binds to response elements in target genes (7). Reactive
oxygen species (ROS) serve a permissive role in cytokine ac-
tivation of NF-B by facilitating cytokine-mediated serine
phosphorylation of IBbyIB kinase, with consequent IB
degradation via the proteasome pathway (33). We observed
that NF-B p50 and p65 interact with TTF-1 at the TBE and
act synergistically with TTF-1 to increase SP-A promoter ac-
tivity (20). Although direct binding of NF-B to the TBE has
not been established, we observed that TTF-1 interacts with
NF-B p65 in vivo (20). Furthermore, antioxidant NF-B in-
hibitors as well as dominant-positive forms of IB (which block
NF-B activation) reduced type II cell nuclear protein binding
to the TBE and blocked inductive effects of cAMP and IL-1
on SP-A expression (20). These findings suggest that NF-B
acts together with TTF-1 at the TBE to activate SP-A gene
expression.
The coactivators CREB-binding protein (CBP) and steroid
receptor coactivator 1 (SRC-1) synergistically interact with
TTF-1 at the TBE to increase SP-A promoter activity (55).
TTF-1 binds to CBP and SRC-1 in vitro (55). NF-B p65 also
interacts with CBP and its homologue, p300 (19, 59); phospho-
rylation of p65 by PKA increases transcriptional activity by
enhancing its association with CBP (59). On the other hand,
SRC-1 interacts with p50 to enhance NF-B-mediated tran-
scription (40). In light of these findings, we propose that TTF-1
and NF-B proteins may activate SP-A gene transcription by
forming a transcriptional activation complex at the TBE that is
stabilized by multivalent interactions with coactivators. In the
present study, we tested the hypothesis that increased O
2
avail
-
ability plays a permissive role in cAMP and cytokine induction
of SP-A expression by facilitating the binding of TTF-1 and
NF-B to the TBE as well as in promoting the recruitment of
essential coregulators, which in turn mediate changes in his-
tone posttranslational modifications and the opening of the
chromatin structure.
MATERIALS AND METHODS
Preparation and culture of human fetal lung explants and type II cells.
Midgestation human fetal lung tissues were obtained from Advanced Bioscience
Resources, Inc., in accordance with the Donors Anatomical Gift Act of the State
of Texas; protocols were approved by the Human Research Review Committee
of the University of Texas Southwestern Medical Center at Dallas. Type II cells
were isolated from cultured midgestation human fetal lung explants, as described
previously (4). Briefly, lung explants were maintained in organ culture in serum-
free Waymouth’s MB752/1 medium (Life Technologies, Inc.) in the presence of
dibutyryl cAMP (Bt
2
cAMP) to promote type II cell differentiation (47). After 3
days of culture, tissues were digested with collagenase; isolated cells were treated
with DEAE-dextran and plated at a density of 5 10
6
to 9 10
6
cells/60-mm
dish. The cells were maintained overnight in Waymouth’s medium containing
10% fetal bovine serum and then placed in serum-free Waymouth’s medium
(control medium) for 24 h at 37°C in a humidified atmosphere of 95% air and 5%
CO
2
(20% O
2
) or placed in a modular incubator chamber (Billups-Rothenberg,
Inc., Del Mar, CA) in an atmosphere containing 2% O
2
, 93% N
2
, and 5% CO
2
,
followed by incubation in these environments for up to 2 days in control medium
in the absence or presence of Bt
2
cAMP (1 mM) (Roche/BMB), IL-1 (10 ng/ml)
(Sigma), or Bt
2
cAMP and IL-1 in combination. In other experiments, type II
cells were cultured in a 20% O
2
environment for 24 h in the absence or presence
of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) (50 nM; Sigma)
(14) or the protein methylation inhibitor 5-deoxy-5-(methylthio)adenosine (d-
MTA) (300 M; Sigma) (48). TSA and d-MTA were dissolved in dimethyl sulfoxide
(DMSO), and an equivalent amount of DMSO vehicle was added to the untreated
dishes. Bt
2
cAMP was then added to half of the dishes, and the incubation was
continued in the absence or presence of these inhibitors for an additional 24 h.
Electrophoretic mobility shift assays (EMSAs). Nuclear extracts were pre-
pared from lung type II cells as described previously (57). Protein concentrations
were determined by a modified Bradford assay (Bio-Rad). Double-stranded
oligonucleotides corresponding to the TBE (underlined) and flanking sequences
(5-GTGCTCCCCTCAAGGGTCCT-3) (Integrated DNA Technologies, Inc.)
were end labeled using [
32
P]ATP (Perkin-Elmer, NEN) and used as probes.
Nuclear proteins (7 g) were incubated with a
32
P-labeled TBE oligonucleotide
for 30 min at room temperature in reaction buffer (20 mM HEPES, pH 7.6, 75
mM KCl, 0.2 mM EDTA, 20% glycerol) and 1 g of poly(dI-dC)-poly(dI-dC)
(Pharmacia) as a nonspecific competitor. Protein-DNA complexes were sepa-
rated from the free probe on 4% nondenaturing polyacrylamide gel and visual-
ized by autoradiography.
Immunoblot analysis. Human fetal type II cells were cultured for up to 48 h
in control medium, in medium containing Bt
2
cAMP (1 mM), or in medium
containing IL-1 (10 ng/ml) alone or in combination with Bt
2
cAMP. In some
experiments, type II cells were also incubated in the absence or presence of TSA
or d-MTA, as described above. The cells were scraped from the dishes and
homogenized in ice-cold phosphate-buffered saline (PBS) containing a protease
inhibitor cocktail (1 tablet/10 ml) (Roche Laboratories). Proteins (15 g) from
nuclear and cytoplasmic (supernatant) fractions obtained during the isolation of
nuclei for EMSA (47) were separated on 10% sodium dodecyl sulfate (SDS)-
polyacrylamide gels and transferred onto nitrocellulose membranes as described
previously (38). The membranes were then analyzed for SP-A, TTF-1, p50, p65,
and CBP by immunoblotting using specific antisera (antibodies for SP-A [43] and
TTF-1 [32] were generated in our laboratory; antibodies for p50, p65, and CBP
[Santa Cruz Biotechnology, Inc.] and histone H3, histone H3 acetylated on
lysines 9 and 14, and histone H3 dimethylated on lysine 9 [Upstate Biotechnol-
ogy, Inc.] were obtained commercially) and an enhanced chemiluminescence
system (ECL), according to the manufacturer’s recommendations (Amersham).
Real-time reverse transcriptase PCR. Total RNA was isolated from human
fetal lung type II cells cultured in the absence or presence of Bt
2
cAMP (1 mM)
or IL-1 for 24 h in an atmosphere of 2% or 20% O
2
by the one-step method
described previously Chomczynski and Sacchi (12) (TRIzol; Invitrogen). RNA
was treated with DNase to remove any contaminating DNA, and 4 g was
reversed transcribed using random primers and Superscript II RNase H reverse
transcriptase (Invitrogen). Validated primer sets directed against HDACs 1 to
11, NCoR1, and SMRT along with the constitutively expressed cyclophilin were
utilized (Table 1). All primer sets produced amplicons of the expected sizes and
sequences. For the quantitative analysis of mRNA expression, the ABI Prism
7700 detection system (Applied Biosystems) was employed with the DNA bind-
ing dye SYBR green (PE Applied Biosystems) for the detection of PCR prod-
ucts. The cycling conditions were 50°C for 2 min and 95°C for 10 min followed
by 40 cycles of 95°C for 15 s and 60°C for 1 min. The cycle threshold was set at
a level where the exponential increase in PCR amplification was approximately
parallel between all samples. To correct for differences in RNA quantity among
2902 ISLAM AND MENDELSON MOL.CELL.BIOL.
samples, data were normalized by using the ratio of the target cDNA concen-
trations to that of cyclophilin. We have compared expression of cyclophilin to
that of 18S rRNA and have found both to be unaffected by treatment or changes
in O
2
tension.
ChIP. Human fetal type II cells were cultured for up to 48 h in serum-free
medium, in medium containing Bt
2
cAMP (1 mM), or in medium containing
IL-1 (10 ng/ml) alone or in combination with Bt
2
cAMP. Chromatin immuno
-
precipitation (ChIP) was performed using a modification (11) of previously
published methods (29). Briefly, the type II cells (1 10
7
to 1.5 10
7
cells) were
washed once with PBS and then incubated with 1% formaldehyde (in control
medium) for 10 min at room temperature to cross-link proteins and DNA.
Cross-linking was terminated by the addition of glycine (0.125 M final concen-
tration). The cells were then washed twice with cold PBS and placed in 500 lof
“lysis buffer” (25 mM Tris, pH 8.1, 140 mM NaCl, 1% Triton X-100, 0.1% SDS,
protease inhibitor cocktail [Roche], and 3 mM EDTA). The lysates from several
dishes were combined and sonicated on ice to produce sheared, soluble chro-
matin. The soluble chromatin was then aliquoted into 500-l amounts and
incubated with antibodies for TTF-1 (31), p65, p50 (Santa Cruz), acetylated
histone H3 (lysines 9 and 14; Upstate Biotechnology, Inc.), dimethyl histone H3
(lysine 9), SRC-1, or CBP (Santa Cruz Biotechnology, Inc.) and incubated at 4°C
overnight. Two aliquots were reserved as controls: one was incubated without
antibody, and the other was incubated with nonimmune immunoglobulin G.
Protein A/G Plus agarose beads (60 l) were then added to each tube, the
mixtures were incubated for2hat4°C, and the immune complexes were col-
lected by centrifugation. The beads containing the immunoprecipitated com-
plexes were then washed sequentially for 5 to 10 min in wash buffer I (20 mM
Tris-HCl, pH 8.1, 2 mM EDTA, 0.1% SDS, 1% Triton X-100, 150 mM NaCl),
wash buffer II (same as wash buffer I except that it contained 500 mM NaCl),
wash buffer III (10 mM Tris-HCl, pH 8.1, 1 mM EDTA, 1% NP-40, 1% deoxy-
cholate, 0.25 M LiCl), and 2 Tris-EDTA buffer. The beads were then eluted
with 250 l of elution buffer (1% SDS, 0.1 mM NaHCO
3
plus 20 g salmon
sperm DNA [Sigma]) at room temperature. This procedure was repeated once,
and eluates were combined. Cross-linking of the immunoprecipitated chromatin
complexes and “input controls” (5% of the total soluble chromatin) was reversed
by heating the samples at 65°C for 4 h. Proteinase K (15 g; Invitrogen) was then
added to each sample in buffer (50 mM Tris-HCl, pH 8.5, 1% SDS, 10 mM
EDTA) and incubated for1hat45°C. The DNA was purified by phenol-
chloroform extraction and precipitated in ethyl alcohol for at least 30 min
at 20°C. Samples and “input” controls were diluted in 10 to 100 l Tris-EDTA
buffer just prior to PCR. Real-time PCR (14) was carried out using 5 (5-TTT
TTCTTTACCAGGTTCTGTGCTGCTC-3) and 3 (5-CACATTTCCCTGCA
GAACACTA-3) primers that amplify a 75-bp region of the hSP-A2 5-flanking
region surrounding the TBE.
RESULTS
Cyclic AMP and IL-1 enhance recruitment of TTF-1 and
NF-B p65 to the SP-A gene promoter. Previously, we observed
that Bt
2
cAMP and IL-1 increased SP-A expression in human
fetal type II cells and that these effects were mediated by
increased binding of TTF-1 and NF-B in type II cell nuclear
extracts to the TBE (20). In the present study, ChIP was uti-
lized to analyze the time-dependent effects of cAMP and IL-1
on in vivo binding of TTF-1 and NF-B p65 to this element.
Human fetal lung type II cells were cultured in the absence
or presence of Bt
2
cAMP or IL-1 for 1, 6, or 24 h. After
immunoprecipitation of chromatin complexes, quantitative
real-time PCR was used to amplify a 75-bp genomic region
surrounding the TBE within the 5-flanking region of the SP-A
gene (Fig. 1A). As can be seen in Fig. 1B and C, IL-1 stim-
ulated the recruitment of TTF-1 and p65 to the TBE, and this
effect was maximal within 6 h. The stimulatory effect of IL-1
on the recruitment of p65 to the TBE was observed as early as
1 h after the start of treatment. Cyclic AMP also increased the
recruitment of TTF-1 and p65 to the TBE; however, the
cAMP-stimulated recruitment of TTF-1 was first observed at
24 h, while cAMP stimulation of p65 recruitment was evident
by6h.
Hypoxia inhibits cAMP and IL-1 induction of SP-A expres-
sion and binding of type II cell nuclear proteins to the TBE. In
previous studies, we found that a critical O
2
tension was re
-
quired for type II cell differentiation and cAMP induction of
SP-A gene expression in human fetal lung maintained in organ
culture; these effects of hypoxia were reversible (1). To deter-
mine whether hypoxia also prevented IL-1 stimulation of SP-A
expression and to begin to define mechanisms for these effects,
human fetal type II cells were incubated for 6, 24, or 48 h in the
absence or presence of Bt
2
cAMP or IL-1 ina2%or20%
O
2
-containing environment. In this experiment, all type II cells
were incubated for 48 h in the serum-free culture medium;
Bt
2
cAMP and IL-1 were added to the medium at 6, 24, or
48 h prior to harvesting of the cells for analysis. Type II cell
nuclear proteins were analyzed for DNA-binding activity by
EMSA using the radiolabeled TBE as a probe. Cytoplasmic
fractions from the same cells were analyzed for SP-A protein
levels by immunoblotting.
As can be seen in the immunoblot in Fig. 2B, when human
fetal lung type II cells were cultured in a 20% O
2
environment,
equivalent inductive effects of Bt
2
cAMP and IL-1 on SP-A
expression were evident at 6 and 24 h. By 48 h, the stimulatory
effect of Bt
2
cAMP was greater than that of IL-1. On the other
hand, when the cells were cultured in 2% O
2
, effects of
Bt
2
cAMP and IL-1 were barely detected. As shown in the
EMSA in Fig. 2A, when type II cells were cultured in a 20% O
2
environment, cAMP and IL-1 had pronounced stimulatory
effects on the binding of type II cell nuclear extracts to the TBE
at all time points. In contrast to the SP-A immunoblot, after
48 h of incubation, IL-1 had a more pronounced stimulatory
effect on TBE-binding activity than Bt
2
cAMP. It should be
noted that the effects of cAMP on SP-A promoter activity are
due to the cooperative interaction of transcription factors
binding to a number of different response elements, including
a nuclear receptor response element (2, 39, 56), an E box that
binds USF1/2 (18), and a GT box, which binds Sp1 (57). Mu-
tagenesis of any one of these elements abrogates cAMP induc-
tion of SP-A gene expression. Thus, whereas the effect of IL-1
TABLE 1. Primers used in quantitative real-time PCR
Primer name NCBI accession no. Forward primer Reverse primer
HDAC-1 BC000301 5-GAACCTTAGAATGCTGCCGC-3 5-CAGGGTCGTCTTCGTCCTCA-3
HDAC-2 BC031055 5-GGAGCCCATGGCGTACAGT-3 5-TTCATGGGATGACCCTGTCC-3
HDAC-4 AF132607 5-TACATGTCCCTCCACCGCTAC-3 5-AGCCATGTTGACGTTGAAACC-3
HDAC-11 BC009676 5-TCATGGATGTCTACAACCGCC-3 5-CTCATCATCCTCTGTGCCCC-3
Cyclophilin NM021130 5-TTTCATCTGCACTGCCAAGA-3 5-TTGCCAAACACCACATGCT-3
NCoR1 NM006311 5-TTCGCAGTCCCTGATTATCGT-3 5-AAGGAAGGTCGCCTTCGAAG-3
SMRT NM006312 5-CATGAACGGGCTTATGGCC-3 5-TGCATGAACTTCTCCCGGA-3
V
OL. 26, 2006 MECHANISMS IN O
2
REGULATION OF SP-A EXPRESSION 2903
may be due primarily to NF-B binding to the TBE, the effect
of cAMP is exerted through a number of different transcription
factors and response elements. The stimulatory effects of cAMP
and IL-1 on TBE-binding activity were decreased when type II
cells were cultured under hypoxic conditions, compared to their
effects in cells cultured in 20% O
2
.
TTF-1 and NF-B protein levels in fetal lung type II cells
are not altered by hypoxia. To determine whether the effects of
O
2
tension on cAMP and IL-1 stimulation of TBE-binding
activity were correlated with nuclear levels of TTF-1 and NF-
B, we analyzed nuclear levels of TTF-1, p65, and p50 by
immunoblotting. Human fetal type II cells were incubated for
6 h in control medium or in medium containing Bt
2
cAMP or
IL-1 in either a 2% or 20% O
2
-containing environment. As
can be seen in Fig. 3 and as observed previously (20), treatment
of type II cells with IL-1 caused an increase in nuclear levels of
p50 and p65, whereas TTF-1 levels were unaffected; Bt
2
cAMP
had no effect to alter nuclear localization of TTF-1 or NF-B
proteins. Importantly, the actions of IL-1 to increase nuclear
levels of p50 and p65 after 6 h of incubation were unaffected by
O
2
tension (Fig. 3). Collectively, these findings suggest that the
inhibitory effects of hypoxia on the binding of type II cell
nuclear proteins to the TBE are likely mediated by actions
independent of expression or nuclear translocation (in the case
of NF-B) of these transcription factors. Such effects could
include alterations in the posttranslational modification of
these proteins with associated changes in the recruitment of
coactivators.
Increased O
2
tension is required for cAMP- and IL-1-in
-
duced recruitment of TTF-1 and NF-B to the TBE. ChIP was
used to analyze the effects of O
2
tension on cAMP- and IL-1-
FIG. 1. Cyclic AMP and IL-1 enhance recruitment of TTF-1 and NF-B p65 to the SP-A gene promoter. Type II cells were cultured for 24 h
in control medium in a 2% or 20% O
2
environment and then incubated for 1, 6, or 24 h with Bt
2
cAMP (1 mM) or IL-1 (10 ng/ml) in 2% or 20%
O
2
. The cells were then treated with 1% formaldehyde followed by lysis and sonication to shear and solubilize the cross-linked chromatin, which
was immunoprecipitated using antibodies specific for NF-B p65 and TTF-1. DNA was purified, and the relative abundance of a 75-bp region
surrounding the TBE was quantified by real-time PCR using primers indicated by the arrows in panel A. Panel B is representative of four
independent experiments with comparable results. The data are expressed as arbitrary units. The bars represent the means standard errors of
the means (SEM) of values from three sets of culture dishes.
FIG. 2. Hypoxia inhibits cAMP and IL-1 induction of SP-A expres-
sion and binding of type II cell nuclear proteins to the TBE. Human
fetal lung type II cells were cultured for 24 h in control medium and
then incubated for 6, 24, or 48 h in the absence or presence of
Bt
2
cAMP or IL-1 in either a 2% or 20% O
2
-containing environment.
Nuclear proteins (7 g) from the type II cells were analyzed for
binding activity by EMSA using the radiolabeled TBE as a probe (A).
Cytoplasmic fractions (15 g protein) from the same cells were
analyzed for SP-A protein levels by immunoblotting (B). Shown are
autoradiograms of a representative immunoblot and gel shift from
an experiment that was repeated four times with similar results.
F.P., free probe.
2904 ISLAM AND MENDELSON M
OL.CELL.BIOL.
induced recruitment of TTF-1 and NF-B p65 to the region of
the SP-A promoter surrounding the TBE in vivo. Type II cells
were incubated for 24 h in control medium in a 2% or 20% O
2
environment and then cultured for 6 or 24 h with cAMP or
IL-1, alone or in combination. As we also observed in the
experiment shown in Fig. 1, when type II cells were cultured in
a 20% O
2
environment, cAMP and IL-1 enhanced recruit
-
ment of TTF-1 and p65 to the TBE. By contrast, when cells were
cultured under hypoxic conditions, cAMP and IL-1 stimulation of
in vivo binding activity was prevented (Fig. 4A and B). A similar
effect of O
2
tension was observed for the recruitment of p50 to the
TBE (data not shown). These findings indicate that a critical O
2
tension is essential for cAMP and IL-1 stimulation of binding of
TTF-1 and NF-BtotheSP-A promoter.
Increased O
2
tension is required for cAMP- and IL-1-in
-
duced recruitment of CBP and SRC-1 to the TBE. Previously,
we observed that TTF-1 synergistically interacted with NF-B
(20) and with the coactivators CBP and SRC-1 (55) at the TBE
to increase SP-A promoter activity. TTF-1 also was found to
physically interact with NF-B in vivo (20) and with these
coactivators in vitro (55). CBP and SRC-1 have also been
reported to interact with p65 (19) and p50 (40), respectively. In
the present study, ChIP was used to define the role of O
2
tension on in vivo binding of CBP and SRC-1 to the region of
the SP-A promoter containing the TBE. Human fetal lung type
II cells were cultured in control medium or in medium con-
taining Bt
2
cAMP or IL-1 for 6 or 24 h at 2% and 20% O
2
.As
can be seen in Fig. 5A and B, when the type II cells were
cultured in 20% O
2
, cAMP and IL-1 enhanced the recruit
-
ment of CBP and SRC-1 to the TBE. A greater stimulatory
effect of cAMP plus IL-1 was also evident at both time points.
Differential effects of cAMP and IL-1 on the recruitment of
CBP and SRC-1 were observed at the 6- and 24-h time points;
this may be due, in part, to differences in the kinetics of cycling
of transcription factors and coactivators on and off the DNA.
Notably, when the cells were cultured in a 2% O
2
environment,
the stimulatory effects of cAMP on CBP and SRC-1 recruit-
ment were greatly reduced.
Increased O
2
tension facilitates cAMP induction of histone
H3 acetylation at the TBE. In light of the fact that both CBP
and SRC-1 have histone acetylase activity, the effects of O
2
tension on the acetylation of histones at the SP-A promoter in
the region of the TBE were analyzed. ChIP assays were carried
out using antibodies to histone H3 acetylated on lysines 9 and
14. Human fetal lung type II cells were cultured in the absence
or presence of Bt
2
cAMP or IL-1 for6or24hina2%or20%
O
2
environment. When type II cells were cultured in a 20%
oxygen environment, cAMP and IL-1 treatment caused a
marked increase in local acetylation of histone H3 after 6 and
24 h of incubation (Fig. 6). By contrast, when the cells were
cultured in 2% O
2
, no stimulatory effects of cAMP or IL-1
were evident (Fig. 6). On the other hand, basal levels of acety-
lated histone H3 were essentially unaffected by changes in O
2
tension. Taken together, these findings suggest that O
2
serves
a permissive role in cAMP- and IL-1-stimulated recruitment of
coactivators with histone acetylase activity to the SP-A pro-
moter. The cAMP-induced recruitment of coactivators likely
mediates increased local histone acetylation and an opening of
the chromatin structure leading to increased SP-A gene tran-
scription.
Increased O
2
tension enhances nuclear levels of CBP and of
acetylated histone H3 in human fetal lung type II cells. In
consideration of the permissive effects of O
2
on cAMP-induced
recruitment of CBP, SRC-1, and acetylated histone H3 to the
genomic region containing the TBE in cAMP-treated type II
cells, nuclear levels of immunoreactive CBP and of acetylated
FIG. 3. TTF-1, p65, and p50 levels in human fetal lung type II cells are not altered by hypoxia. Human fetal type II cells were incubated for
24 h in control medium in 2% or 20% O
2
followed by incubation for6hintheabsence (control) or presence of Bt
2
cAMP or IL-1 in either a
2% or 20% O
2
-containing environment. Nuclear proteins (15 g) were analyzed by immunoblotting using antisera to TTF-1 (A), p50 (B), and p65
(C). Shown in the upper panel are autoradiograms of immunoblots from a representative experiment. Shown in the lower panel are summarized
results of densitometric scans of the immunoblots. Scanned values for the treated samples in each set are plotted relative to those of the control
sample in that set. Data are the means SEM values from three independent scans.
V
OL. 26, 2006 MECHANISMS IN O
2
REGULATION OF SP-A EXPRESSION 2905
and total histone H3 were analyzed in type II cells cultured in
the absence or presence of Bt
2
cAMP or IL-1 in 2% or 20%
O
2
. As can be seen in Fig. 7,
nuclear levels of immunoreactive
CBP and acetyl-histone H3 were increased in cells cultured in
20% O
2
compared to levels in cells cultured in 2% O
2
. The
stimulatory effects of O
2
were independent of treatment. By
contrast, levels of total histone H3 were unaffected by O
2
tension. This suggests that permissive effects of increased O
2
tension on cAMP and cytokine stimulation of SP-A gene ex-
pression are mediated, in part, by global increases in histone
acetylase activity that are independent of hormonal treatment
as well as hormone-dependent effects on the binding of TTF-1,
NF-B, and coactivators to the SP-A promoter with local en-
hancement of histone acetylation.
Hypoxia increases corepressor mRNA levels in cultured
type II cells. Real-time reverse transcriptase PCR was used to
analyze mRNA levels for HDACs 1 to 11, NCoR1, and SMRT
in human fetal type II cells cultured for 24 h in control medium
followed by incubation for 24 h in the absence (control) or
presence of Bt
2
cAMP or IL-1 in 2% or 20% O
2
. Remarkably,
the effects of O
2
tension and hormonal treatment were essen
-
tially the same for all corepressors analyzed. Findings for
HDACs 1 and 2 (representative of class I), HDAC-4 (repre-
sentative of class II), HDAC-11 (representative of class IV),
NCoR1, and SMRT are shown in Fig. 8. mRNA levels of these
corepressors were markedly induced by hypoxia in type II cells
cultured in control medium. Interestingly, cAMP and IL-1
partially antagonized the inductive effects of hypoxia on HDAC
expression. These findings suggest that hypoxia promotes a
closed chromatin structure through global induction of histone
deacetylation.
Hypoxia increases nuclear levels of histone H3 dimethylated
on Lys9 and enhances levels of dimethyl (Lys9) histone H3
associated with the TBE region of the SP-A promoter. Human
fetal type II cells were incubated for 24 h in control medium
followed by incubation for 24 h in the absence (control) or
presence of Bt
2
cAMP or IL-1 in either a 2% or 20%
O
2
-containing environment. Nuclear and cytoplasmic frac
-
tions were analyzed for histone H3 dimethylated on Lys9
and for SP-A, respectively, by immunoblotting. As can be
seen in Fig. 9, nuclear levels of dimethyl (Lys9) histone H3
were increased in cells incubated in 2% O
2
compared to
levels in cells incubated in 20% O
2
for all treatment groups
(Fig. 9A, upper panel), while levels of total histone H3 were
unaffected (Fig. 9A, lower panel). Effects of hypoxia on
levels of dimethylated H3 were correlated with the inhibi-
tory effects of hypoxia on cAMP and IL-1 induction of
SP-A expression (Fig. 9A, middle panel). The hypoxia-in-
duced increase in total nuclear levels of dimethyl (Lys9) H3
was associated with increased levels of dimethyl (Lys9) H3
associated with the 75-bp genomic region surrounding the
hSP-A TBE, as revealed by ChIP (Fig. 9B).
The histone deacetylase inhibitor TSA and the protein
methylation inhibitor d-MTA increase SP-A expression in cul-
tured type II cells. Human fetal lung type II cells were cultured
for 24 h in 20% O
2
in the presence of DMSO vehicle or with
FIG. 4. Effects of cAMP and IL-1 to increase recruitment of TTF-1 and NF-B to the TBE are inhibited by hypoxia. Type II cells were
incubated for 24 h in control medium in a 2% or 20% O
2
environment and then treated for another 6 or 24 h with Bt
2
cAMP or IL-1, alone or
in combination. After cross-linking, lysis, and sonication, the solubilized chromatin was subjected to immunoprecipitation using specific antibodies
to NF-B and TTF-1. After immunoprecipitation, DNA was purified and analyzed for the 75-bp region containing the TBE (Fig. 1A) using real-time
PCR. The data are expressed as arbitrary units. Shown is a representative of four independent experiments. The bars represent the means SEM
of values from three sets of culture dishes.
2906 ISLAM AND MENDELSON M
OL.CELL.BIOL.
TSA (50 nM) or d-MTA (300 M) dissolved in DMSO fol-
lowed by coincubation with or without (control) Bt
2
cAMP for
an additional 24 h. Cytoplasmic fractions were analyzed for
SP-A by immunoblotting. As can be seen, both TSA and d-
MTA dramatically increased the levels of SP-A in the absence
or presence of Bt
2
cAMP compared to levels in cells incubated
with DMSO alone (Fig. 10A and B). These findings suggest
that SP-A expression is enhanced by increased acetylation and
decreased methylation of histone H3 (Lys9).
DISCUSSION
The activating signals and molecular mechanisms that me-
diate temporal and spatial regulation of SP-A gene expression
in the fetal lung remain to be elucidated. We previously ob-
served that cAMP and IL-1 stimulation of SP-A gene expres-
sion in human fetal lung is mediated via TTF-1 and NF-B
binding to a response element (TBE) 175 bp upstream of the
transcription initiation site of the hSP-A gene (20, 31). Since
TTF-1 is expressed from the very earliest stages of lung devel-
opment (30), where it plays a role in branching morphogenesis
(26), while SP-A gene expression is induced only after 80%
of gestation is complete (37), we suggest that temporally dis-
tinct, gene-specific actions of TTF-1 could be mediated by
selective changes in its posttranslational modification and as-
sociation with other transcriptional regulators. Our finding
that cAMP stimulation of type II cell differentiation and SP-A
gene expression is dependent upon an O
2
tension of 10% (1)
has led us to consider the possibility that increased O
2
avail
-
FIG. 5. Cyclic AMP- and IL-1-induced recruitment of CBP and SRC-1 to the TBE is inhibited by hypoxia. Type II cells were incubated
for 24 h in control medium in a 2% or 20% O
2
-containing environment and then incubated for another 6 or 24 h with cAMP or IL-1, alone
or in combination. The soluble cross-linked chromatin was immunoprecipitated using specific antibodies to CBP and SRC-1. After
immunoprecipitation, DNA was analyzed for the 75-bp region containing the TBE (Fig. 1A) using real-time PCR. The data are expressed
as arbitrary units. Shown is a representative of four independent experiments. The bars represent the means SEM of values from triplicate
dishes of type II cells.
FIG. 6. O
2
is required for cAMP induction of histone H3 acet
-
ylation at the TBE. Human fetal lung type II cells were cultured in
control medium for 24 h in a 2% or 20% O
2
-containing environ
-
ment followed by incubation in the absence (control) or presence of
Bt
2
cAMP or IL-1 for6or24hin2%or20%O
2
. The solubilized
chromatin was subjected to immunoprecipitation using an antibody
to acetylated histone H3. After immunoprecipitation, DNA was
purified and analyzed for the 75-bp region containing the TBE (Fig.
1A) by real-time PCR. Data are expressed as arbitrary units. Shown
is a representative of three independent experiments. The bars
represent the means SEM of data from triplicate culture dishes.
V
OL. 26, 2006 MECHANISMS IN O
2
REGULATION OF SP-A EXPRESSION 2907
ability to the alveolar epithelium as a result of vascularization
of the developing lung may play a permissive role in the acti-
vation of TTF-1 and NF-B.
Transcriptional activation of TTF-1 and NF-B proteins is
known to be regulated by phosphorylation. Cyclic AMP stim-
ulation of type II cell nuclear protein binding to the TBE was
found to be associated with increased TTF-1 phosphorylation;
moreover, phosphatase treatment of type II cell nuclear ex-
tracts abolished DNA-binding activity (31). Furthermore, the
PKA catalytic subunit increased TTF-1 transcriptional activa-
tion of the SP-A (31) and SP-B (54) promoters. Cytokines (42)
and PKA (58, 59) have also been reported to increase phos-
phorylation and DNA-binding and transcriptional activities of
NF-B p65. Phosphorylation of p65 by PKA increased its tran-
scriptional activity by enhancing p65 association with CBP (59).
CBP and SRC-1 also act synergistically with TTF-1 to increase
SP-A promoter activity, and this was enhanced by cotransfection
of the PKA catalytic subunit (55). Our finding that cAMP treat-
ment of type II cells caused a marked increase in TTF-1 acetyla-
tion (55) suggests that PKA-mediated phosphorylation of TTF-1
enhances its interaction with coactivators with HAT activity in
vivo. The resulting increase in TTF-1 acetylation may further
increase DNA-binding and transcriptional activity (21). In light of
these collective findings, we postulate that phosphorylation of
TTF-1 and NF-B proteins in response to activating hormones
and factors may facilitate their TBE binding and recruitment of
the histone acetylases CBP and SRC-1.
By use of ChIP, we observed in the present study that cAMP
and IL-1 stimulation of SP-A gene expression in human fetal
lung type II cells was associated with enhanced in vivo recruit-
ment of TTF-1 and NF-B p65 to the genomic region contain-
ing the TBE. IL-1 had a rapid (as early as 1 h) effect to
promote increased binding of p65 and TTF-1 to the TBE,
reaching maximal levels by 6 h. Cyclic AMP also stimulated in
vivo binding of p65 and TTF-1 to the TBE-containing region,
but this was evident after a longer latency period. These find-
ings agree with previously published in vitro studies using
EMSA in which IL-1 caused a marked increase in the binding
of type II cell nuclear proteins to the TBE within1hof
treatment, whereas a stimulatory effect of cAMP on TBE-
binding activity was first detectable after 6 h (20).
FIG. 7. Levels of immunoreactive CBP and acetylated histone H3 in human fetal type II cells are reduced by hypoxia. Human fetal type II cells
were incubated for 24 h in control medium in a 2% or 20% O
2
-containing environment followed by incubation for 24 h in the absence or presence
of Bt
2
cAMP or IL-1 in 2% or 20% O
2
. Nuclear proteins (15 g) were analyzed by Western blotting using antibodies to CBP (A), acetyl-histone
H3 (B), and total histone H3 (C). Shown in the upper panel are autoradiograms of immunoblots from representative experiments. Shown in the
lower panel are densitometric scans of the immunoblots. Scanned values for the treated samples in each set are plotted relative to those of the
control sample in that set. Data are the means SEM from three independent scans.
FIG. 8. Hypoxia increases corepressor mRNA levels. Human fetal
lung type II cells were cultured in control medium for 24 h in a 2% or 20%
O
2
-containing environment followed by incubation in the absence (con
-
trol) or presence of Bt
2
cAMP or IL-1 for24hin2%or20%O
2
. Total
RNA prepared from these cells was analyzed for levels of HDACs 1 to 11
and for NCoR1 and SMRT by quantitative PCR using SYBR green (PE
Applied Biosystems) for detection of PCR products. Shown are data for
HDACs 1 and 2 (representative of class I), HDAC-4 (representative of
class II), HDAC 11 (representative of class IV), NCoR1, and SMRT.
Data were normalized by analyzing the ratio of the target cDNA concen-
trations to that of cyclophilin. Shown is a representative of three indepen-
dent experiments. The bars represent the means SEM of data from
triplicate culture dishes.
2908 ISLAM AND MENDELSON M
OL.CELL.BIOL.
Cyclic AMP and IL-1 stimulation of SP-A expression and in
vitro binding of nuclear proteins to the TBE were inhibited
when the cells were cultured in a hypoxic (2% O
2
) environ
-
ment. Using ChIP, we also observed that when the type II cells
were cultured in 2% O
2
, cAMP and IL-1 stimulation of in vivo
recruitment of TTF-1 and p65 to the TBE was prevented.
These findings support the concept that ROS serve a permis-
sive role in transcriptional activation of NF-B in response to
cytokine signaling (33) and suggest that ROS are also permis-
sive for TTF-1 transcriptional activation. Our previous obser-
vation that the antioxidant pyrrolidine dithiocarbamate, a
known NF-B inhibitor, blocked IL-1 and cAMP induction of
type II cell nuclear protein binding to the TBE and reduced
SP-A expression (20) further indicates a requirement for ROS
in NF-B and/or TTF-1 activation of SP-A gene transcription.
As mentioned above, phosphorylation appears to be required
for the binding of type II cell nuclear proteins to the TBE (31).
Thus, the finding that cAMP and IL-1 stimulation of TTF-1
and NF-B binding to the TBE region was inhibited when type
II cells were cultured in 2% O
2
compared to 20% O
2
suggests
that phosphorylation of these proteins may be reduced by
hypoxia. This could be due a decreased rate of phosphorylation
and/or an increased rate of dephosphorylation of these pro-
teins. It should be noted that hypoxia has been reported to
increase activities of the protein phosphatases protein phos-
phatase 1 (28), calcineurin (34), and tyrosine phosphatase (16)
in a number of cell types.
TTF-1 (55), NF-B p50 (40), and NF-B p65 (19, 59) were
previously found to interact with the coactivators CBP and
SRC-1 in vitro. In the present study, we observed using ChIP
that cAMP and IL-1 stimulated in vivo recruitment of CBP and
SRC-1 to the SP-A genomic region containing the TBE in type
II cells cultured in 20% O
2
; this was prevented when type II
cells were cultured in a hypoxic (2% O
2
) environment. The
O
2
-dependent increase in the recruitment of CBP to the
genomic region surrounding the TBE was also associated with
global effects on overall nuclear levels of CBP protein. Immu-
noreactive levels of CBP were elevated in nuclei of type II cells
cultured in a 20% O
2
environment compared to those in cells
cultured in a 2% O
2
environment; however, in contrast to our
findings using ChIP, this inductive effect of O
2
was indepen
-
dent of hormonal treatment. On the other hand, mRNA levels
for HDACs 1 to 11, NCoR1, and SMRT were markedly in-
duced by hypoxia in type II cells incubated in control medium.
Treatment with Bt
2
cAMP and IL-1 partially antagonized the
inductive effect of hypoxia on HDAC mRNA expression,
whereas these factors had little or no effect on HDAC mRNA
levels in cells cultured in 20% O
2
. In this regard, oxidative
stress has been reported to increase acetylation of core his-
tones (44) through the inhibition of histone deacetylases (22).
Both CBP and SRC-1 coactivators catalyze the acetylation
of specific lysine residues in the N-terminal tails of the nucleo-
somal core histones (35), resulting in their decreased affinity
for DNA and local unwinding of DNA around the nucleosome.
This opening of the chromatin structure facilitates recruitment
to the promoter of transcription factors and of RNA polymer-
ase II, resulting in the stabilization of the preinitiation complex
and activation of transcription initiation (35). Corepressors, on
the other hand, inhibit transcriptional activation by mediating
deacetylation of histones to promote the formation of a closed
FIG. 9. Nuclear levels of dimethyl histone H3 (K9) and association
of dimethyl H3 (K9) with the TBE-containing region of the SP-A
promoter are increased by hypoxia. Human fetal lung type II cells were
cultured in control medium for 24 h in a 2% or 20% O
2
-containing
environment followed by incubation in the absence (control) or pres-
ence of Bt
2
cAMP or IL-1 for24hin2%or20%O
2
. (A) Nuclear and
cytoplasmic proteins were analyzed by immunoblotting using antisera
to dimethyl histone H3 (K9) (upper panel), SP-A (middle panel), and
total histone H3 (lower panel). (B) Solubilized chromatin isolated
from the cells was subjected to immunoprecipitation using an antibody
to dimethyl histone H3 (K9). After immunoprecipitation, DNA was
purified and analyzed by real-time PCR for the 75-bp region contain-
ing the TBE. The data are expressed as arbitrary units. Shown is a
representative of three independent experiments. The bars represent
the means SEM of data from triplicate culture dishes.
FIG. 10. The histone deacetylase inhibitor TSA and the protein
methylation inhibitor d-MTA increase SP-A expression in lung type II
cells. Human fetal lung type II cells were cultured in medium in the
presence of DMSO vehicle or with TSA (50 nM) or d-MTA (300 M)
dissolved in DMSO for 24 h in a 20% O
2
environment followed by
coincubation in the absence (control) or presence of Bt
2
cAMP for an
additional 24 h. Cytoplasmic fractions were analyzed for SP-A by
immunoblotting. Shown is a representative immunoblot of this exper-
iment, which was repeated three times with comparable results.
V
OL. 26, 2006 MECHANISMS IN O
2
REGULATION OF SP-A EXPRESSION 2909
chromatin structure. Histones are subject to a number of other
posttranslational modifications, including methylation, phos-
phorylation, ubiquitylation, and ADP-ribosylation (see refer-
ence 23 for a review). It has been suggested that the combina-
torial nature of these covalent modifications of the histone tails
reveals a “histone code,” which provides a unique regulatory
system that dictates the transition between transcriptionally
silent (heterochromatin) and active (euchromatin) chromatin
(23). Whereas euchromatin is generally associated with his-
tones acetylated on specific lysine residues (e.g., lysine 9 of
histone H3 [H3-K9] and H3-K14), heterochromatin contains
predominately hypoacetylated histones.
In the present study, using ChIP, we observed in cultured
type II cells that the cAMP- and IL-1-induced binding of
TTF-1 and p65 as well as of CBP and SRC-1 coactivators to the
SP-A promoter was associated with an O
2
-dependent increase
in the in vivo binding of H3-K9, a marker of active chromatin
(23). By contrast, in cells cultured under hypoxic conditions,
cAMP and IL-1 had no effect to enhance recruitment. Global
levels of acetylated H3-K9 were also markedly reduced in type
II cells cultured in 2% O
2
. This inhibitory effect of hypoxia on
H3-K9 acetylation was associated with increased expression of
HDACs 1 to 11 as well as NCoR1 and SMRT mRNAs. Im-
portantly, incubation of type II cells with the HDAC inhibitor
TSA enhanced SP-A expression in type II cells cultured in the
absence or presence of Bt
2
cAMP. Based on these findings, we
postulate that permissive effects of increased O
2
tension on
cAMP and cytokine stimulation of SP-A gene expression are
mediated, in part, by global inhibition of HDAC expression. In
this “permissive” environment, cAMP- and IL-1-induced phos-
phorylation of TTF-1 and NF-B results in their increased
binding to the TBE, facilitating the recruitment of histone
acetylases such as CBP and SRC-1. This in turn causes acety-
lation of core histones (e.g., H3-K9) in the TBE-containing
region of the SP-A promoter, resulting in a further opening of
the chromatin structure.
Methylation of specific lysine residues in histones (e.g., H3-
K9) has been found to play a role in transcriptional silencing
(23) by forming a binding site for heterochromatin protein 1;
this subsequently mediates the formation of the condensed
structure of heterochromatin (15, 27). In Schizosaccharomyces
pombe, methylation of H3-K9 occurs after K9 is deacetylated
by the action of an HDAC complex (41). On the other hand,
methylation of H3-K4 (23) and methylation of arginine resi-
dues (49) have been implicated in transcriptional activation. In
the present study, we found that hypoxia increased the recruit-
FIG. 11. Proposed mechanisms for the developmental induction of SP-A gene transcription in the fetal lung. During the first two-thirds of
gestation, the fetal lung is poorly vascularized and relatively hypoxic. Based on the findings of this study, we propose that under hypoxic conditions,
there is increased expression of HDACs and enhanced methylation of histones on residues known to mediate a repressed chromatin state (e.g.,
K9 of histone H3). The SP-A gene is therefore rendered inaccessible to activating transcription factors. During the latter third of gestation, capillary
networks form within the fetal lung, resulting in increased O
2
availability to the developing pulmonary epithelium. The elevated O
2
tension
facilitates the inhibition of HDAC expression and the increased acetylation of histones on lysine residues known to mediate an active chromatin
state (e.g., K9 of histone H3). This may be further enhanced by demethylation of core histones and DNA. Increased O
2
tension also promotes
increased phosphorylation, activation, and binding of TTF-1 and NF-B to the TBE with subsequent recruitment of CBP and SRC coactivators
containing intrinsic histone acetylase activity. This results in a local increase in histone acetylation (e.g., K9 of histone H3), resulting in a further
opening of the chromatin structure, the formation of a stable basal transcription complex at the promoter with recruitment of RNA polymerase
II, and enhanced SP-A gene transcription.
2910 ISLAM AND MENDELSON M
OL.CELL.BIOL.
ment of histone H3 dimethylated on lysine 9 to the SP-A
TBE-containing region. Hypoxia also caused a global increase
in dimethylated H3-K9. Notably, d-MTA, an S-adenosylmethi-
onine metabolite that inhibits histone H3 methylation (refer-
ence 48 and data not shown), markedly increased SP-A expres-
sion in type II cells cultured in the absence or presence of
Bt
2
cAMP. This further supports the inhibitory role of histone
H3 (Lys9) methylation in SP-A gene expression. Unlike acet-
ylation, methylation does not remove the positive charge of the
lysine but increases bulkiness by the addition of one, two, or
three methyl groups to its ε-amino group. The significance of
the number of methyl groups is not understood, but it has been
suggested to add a level of complexity to the histone code (23).
Although histone acetylation, phosphorylation, and ubiqui-
tylation are dynamic and reversible, methylation has long been
thought to be essentially irreversible and involved in long-term
maintenance of a repressed transcriptional state (23). Re-
cently, it has been found that methylation of certain histone
residues (e.g., H3-K9) in a number of inflammatory response
genes is also subject to rapid changes (45). Interestingly, a
lysine-specific histone demethylase, Epe1, that alters hetero-
chromatin integrity in yeast has been described (6). Epe1 is
structurally related to the 2-OG-Fe(II)-dependent dioxygenase
superfamily and has been proposed to catalyze hydroxylation
of the methyl groups on histone tails, resulting in oxidative
demethylation (51). These findings are of interest in light of
the proposed role of O
2
and ROS as modulators of cell differ
-
entiation (5, 46). It has been postulated that increased vascu-
larization of tissues with associated enhanced O
2
delivery plays
a role in cellular differentiation with the associated loss of
mitotic activity (1, 10, 24). Cellular differentiation in a number
of organisms has been found to be associated with an increase
in O
2
free radicals (5), an induction of the mangano isoform of
superoxide dismutase, and a reduction in cellular levels of
reduced glutathione (5). In the developing fetal rat lung, a
marked decline in levels of glutathione (52) and induction of
the mangano isoform of superoxide dismutase activity (50)
were observed, suggestive of an increase in tissue O
2
tension.
Based on our findings, we suggest that the hypoxic environ-
ment that exists within the fetal lung during the first and
second trimesters of gestation promotes increased HDAC ac-
tivity, hypoacetylation, and increased methylation of core his-
tones, resulting in a closed chromatin structure (Fig. 11). Dur-
ing that latter third of gestation, the increased vascularization
of the fetal lung and enhanced O
2
availability to the pulmonary
epithelium may promote histone demethylation and increased
histone acetylation, resulting in a “permissive” chromatin struc-
ture. The increased O
2
tension also facilitates cAMP and IL-1
induction of NF-B and TTF-1 transcriptional activation and
binding to the SP-A promoter. These activated transcription
factors in turn recruit CBP and SRC coactivators, which cata-
lyze the acetylation of core histones in the genomic region
surrounding the TBE and a further opening of the chromatin
structure. This would facilitate increased access of basal tran-
scription factors and polymerase to the SP-A promoter, leading
to the developmental activation of gene transcription (Fig. 11).
We propose that the present findings may provide a paradigm
for transcriptional regulation of other genes that are develop-
mentally regulated in association with increased tissue vascu-
larization and O
2
availability.
ACKNOWLEDGMENTS
This research was supported in part by NIH grants 2-U01-HL-
052647 and 5-R37-HL-050022. K.N.I. was supported by NIH grant
5-T32-HD-07190.
We are grateful to Meg Smith for her expert assistance in the
preparation of primary cultures of fetal lung type II cells.
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2912 ISLAM AND MENDELSON MOL.CELL.BIOL.
    • "revealed that amongst the seven, KIAA1324, NET1, NTN3, RPL10 and TFPI2 were epigenetically regulated through DNA methylation. In the remaining two, SFTPA1 was epigenetically regulated565758. However, the experimental evidence was lacking the epigenetic related data for CRISP3. "
    [Show abstract] [Hide abstract] ABSTRACT: Background The tissue-specific Unigene Sets derived from more than one million expressed sequence tags (ESTs) in the NCBI, GenBank database offers a platform for identifying significantly and differentially expressed tissue-specific genes by in-silico methods. Digital differential display (DDD) rapidly creates transcription profiles based on EST comparisons and numerically calculates, as a fraction of the pool of ESTs, the relative sequence abundance of known and novel genes. However, the process of identifying the most likely tissue for a specific disease in which to search for candidate genes from the pool of differentially expressed genes remains difficult. Therefore, we have used ‘Gene Ontology semantic similarity score’ to measure the GO similarity between gene products of lung tissue-specific candidate genes from control (normal) and disease (cancer) sets. This semantic similarity score matrix based on hierarchical clustering represents in the form of a dendrogram. The dendrogram cluster stability was assessed by multiple bootstrapping. Multiple bootstrapping also computes a p-value for each cluster and corrects the bias of the bootstrap probability. Results Subsequent hierarchical clustering by the multiple bootstrapping method (α = 0.95) identified seven clusters. The comparative, as well as subtractive, approach revealed a set of 38 biomarkers comprising four distinct lung cancer signature biomarker clusters (panel 1–4). Further gene enrichment analysis of the four panels revealed that each panel represents a set of lung cancer linked metastasis diagnostic biomarkers (panel 1), chemotherapy/drug resistance biomarkers (panel 2), hypoxia regulated biomarkers (panel 3) and lung extra cellular matrix biomarkers (panel 4). Conclusions Expression analysis reveals that hypoxia induced lung cancer related biomarkers (panel 3), HIF and its modulating proteins (TGM2, CSNK1A1, CTNNA1, NAMPT/Visfatin, TNFRSF1A, ETS1, SRC-1, FN1, APLP2, DMBT1/SAG, AIB1 and AZIN1) are significantly down regulated. All down regulated genes in this panel were highly up regulated in most other types of cancers. These panels of proteins may represent signature biomarkers for lung cancer and will aid in lung cancer diagnosis and disease monitoring as well as in the prediction of responses to therapeutics.
    Full-text · Article · Nov 2012
    • "The centralized homeodomain is known to bind to other transcription factors, including retinoic acid receptors (RARs) (Yan et al., 2001), zinc finger GATA transcription-6 (GATA-6) (Liu et al., 2002; Weidenfeld et al., 2002), and nuclear factor of activated T cells (NFAT) (Dave et al., 2004). Finally, the COOH-terminus functionally interacts with the DNA repair protein thymine DNA glycosylase (TDG) (Missero et al., 2001 ), other transcriptional coactivators such as p300 (Bachurski et al., 2003; Grasberger et al., 2005), SRC-3, SRC-2, BR22 (amino acid 6–206) (Yang et al., 2001; 2003), poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1), PARP-2 (Maeda et al., 2006), and other transcription factors such as nuclear factor κB (NF-κB) (Islam and Mendelson, 2006 ), signal transducers and activators of transcription 3 (STAT3) (Yan et al., 2002 ), Drosophila mothers against decapentaplegic 3 (SMAD3) (Li et al., 2002), nuclear factor I (NFI) (Missero et al., 2001 ), and ezrinradixin-moesin (ERM) (Lin et al., 2006). In human, calf, rat, and mouse, Nkx2-1 protein has been characterized as a single polypeptide chain of between 371 and 378 amino acids (aa) and with a molecular mass ranging from 38–42 kDa. "
    [Show abstract] [Hide abstract] ABSTRACT: Nkx2-1 (Nkx homeobox-1 gene), also known as TTF-1 (thyroid transcription factor-1), is a tissue-specific transcription factor of the thyroid, lung, and ventral forebrain. While it has been shown to play a critical role in lung development and lung cancer differentiation and morphogenesis, molecular mechanisms mediating Nkx2-1 cell- and tissue-specific expression in normal and cancerous lungs have yet to be fully elucidated. The recent identification of prognostic biomarkers in lung cancer, particularly in lung adenocarcinoma (ADC), and the different reactivity of patients to chemotherapeutic drugs have opened new avenues for evaluating patient survival and the development of novel effective therapeutic strategies. The function of Nkx2-1 as a proto-oncogene was recently characterized and the gene is implicated as a contributory factor in lung cancer development. In this review, we summarize the role of this transcription factor in the development, diagnosis, and prognosis of lung cancer in the hope of providing insights into the utility of Nkx2-1 as a novel biomarker of lung cancer.
    Article · Nov 2012