Sox17 Promotes Cell Cycle Progression and Inhibits
TGF-b/Smad3 Signaling to Initiate Progenitor Cell
Behavior in the Respiratory Epithelium
Alexander W. Lange1, Angela R. Keiser1, James M. Wells2, Aaron M. Zorn2, Jeffrey A. Whitsett1*
1Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center and the University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of
America, 2Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center and the University of Cincinnati College of Medicine, Cincinnati, Ohio, United
States of America
The Sry-related high mobility group box transcription factor Sox17 is required for diverse developmental processes including
endoderm formation, vascular development, and fetal hematopoietic stem cell maintenance. Expression of Sox17 in mature
respiratory epithelial cells causes proliferation and lineage respecification, suggesting that Sox17 can alter adult lung
progenitor cell fate. In this paper, we identify mechanisms by which Sox17 influences lung epithelial progenitor cell behavior
and reprograms cell fate in the mature respiratory epithelium. Conditional expression of Sox17 in epithelial cells of the adult
mouse lung demonstrated that cell cluster formation and respecification of alveolar progenitor cells toward proximal airway
lineages were rapidly reversible processes. Prolonged expression of Sox17 caused the ectopic formation of bronchiolar-like
structures with diverse respiratory epithelial cell characteristics in alveolar regions of lung. During initiation of progenitor cell
behavior, Sox17 induced proliferation and increased the expression of the progenitor cell marker Sca-1 and genes involved in
cell cycle progression. Notably, Sox17 enhanced cyclin D1 expression in vivo and activated cyclin D1 promoter activity in vitro.
Sox17 decreased the expression of transforming growth factor-beta (TGF-b)-responsive cell cycle inhibitors in the adult mouse
lung, including p15, p21, and p57, and inhibited TGF-b1-mediated transcriptional responses in vitro. Further, Sox17 interacted
with Smad3 and blocked Smad3 DNA binding and transcriptional activity. Together, these data show that a subset of mature
respiratory epithelial cells retains remarkable phenotypic plasticity and that Sox17, a gene required for early endoderm
formation, activates the cell cycle and reinitiates multipotent progenitor cell behavior in mature lung cells.
Citation: Lange AW, Keiser AR, Wells JM, Zorn AM, Whitsett JA (2009) Sox17 Promotes Cell Cycle Progression and Inhibits TGF-b/Smad3 Signaling to Initiate
Progenitor Cell Behavior in the Respiratory Epithelium. PLoS ONE 4(5): e5711. doi:10.1371/journal.pone.0005711
Editor: Oliver Eickelberg, Helmholtz Zentrum Mu ¨nchen/Ludwig-Maximilians-University Munich, Germany
Received January 23, 2009; Accepted May 4, 2009; Published May 27, 2009
Copyright: ? 2009 Lange et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: NIH grants HL61646 and HL090156 (J.A.W.); training grant HL007752 (A.W.L.). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Jeffrey.Whitsett@chmcc.org
Proteins in the Sry-related high mobility group box (Sox) family of
transcription factors regulate developmental processes and cell type
specification in various organ systems. Sox proteins share homology
in the HMG domain, which binds the consensus DNA sequence (A/
T)(A/T)CAA(A/T)GG to regulate gene expression and mediates
protein interactions with transcriptional cofactors . During
vertebrate embryogenesis, Sox17 is required for formation of the
endoderm, which gives rise to the liver, pancreas, and epithelium of
the gastrointestinal and respiratory tracts [2–5]. In addition,
constitutive expression of Sox17 in human embryonic stem cells is
sufficient to promote differentiation of definitive endoderm progen-
itors . Studies on mice with targeted deletion of Sox17 have
identified additional roles for Sox17 in the maintenance of fetal
hematopoietic stem cells, cardiovascular development, and angio-
genesis [7–9]. Sox17 expression is dynamically regulated during
endoderm formation and the programming of embryonic stem cells
toward respiratory epithelial cell lineages in vitro [10–12]. Although
Sox17 is highly expressed in the endoderm and is required for its
formation prior to the emergence of the lung primordium, it is not
readily detected in the respiratory epithelium thereafter. While
conditional misexpression of Sox17 in respiratory epithelial cells of
the embryonic mouse lung disrupted branching and differentiation of
proximal/distal epithelial cell types, expression of Sox17 in the adult
lung epithelium induced the formation of hyperplastic cell clusters in
the alveolar region that contained cells expressing markers
characteristic of diverse proximal airway epithelial lineages .
Together, these findings are consistent with a role for Sox17 in
regulating progenitor cell behavior and lineage specification in
The ability to reprogram mature, differentiated cell types into
alternative lineages has been the subject of significant recent
interest. Although the mechanisms underlying cell lineage
reprogramming remain poorly understood, this phenomenon is
generally associated with ectopic expression or reactivation of
genes important for embryonic development and organogenesis
. While epithelial cells in the mature lung are normally
quiescent, subpopulations of endogenous cells within the respira-
tory epithelium possess the capacity to reenter the cell cycle,
proliferate, and redifferentiate into multiple epithelial cell types
with appropriate function and location along the proximal/distal
lung axis following injury. This is consistent with the notion that
subsets of mature respiratory epithelial cells maintain remarkable
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phenotypic plasticity despite their differentiation status. In support
of this concept, several lung stem/progenitor cell candidates have
been identified in distinct niches along the airway epithelium,
including basal cells, toxicant-resistant ‘‘variant’’ Clara cells, and
ciliated cells in the conducting airway, bronchoalveolar stem cells
(BASCs) located at the bronchoalveolar duct junction (BADJ), and
type II cells in the peripheral lung [15–17]. During embryogenesis,
respiratory epithelial cells are derived from progenitors in the
foregut endoderm and are specified prior to the morphological
appearance of the lung buds . Several families of transcription
factors influence lung morphogenesis and differentiation of the
diverse respiratory epithelial cell types, including TTF-1, GATA6,
b-catenin/TCF/LEF, Forkhead (Fox), and Sox family members
. However, whether transcription factors important for lung
development can also influence progenitor cell behavior in mature
respiratory epithelial cells is not known.
The TGF-b pathway regulates diverse biological processes in
multiple cell types, including cell proliferation, differentiation,
apoptosis, and migration. Following activation of heteromeric type
II and type I kinase receptor complexes by TGF-b ligands,
intracellular signaling is initiated by phosphorylation of receptor-
activated Smad proteins. Phosphorylated Smad2 and Smad3, the
effectors of the TGF-b pathway, interact with Smad4 and translocate
to the nucleus to regulate transcription of downstream target genes
by the interaction of Smad proteins with cofactors to modulate
transcriptional activity. TGF-b signaling inhibits proliferation in
multipleepithelial celltypes by influencing the expression of cell cycle
regulatory proteins to induce arrest prior to the restriction point in
G1. For example, Smad2, 3, and 4 directly activate the promoters of
the cyclin-dependent kinase inhibitors p15 and p21, which in turn
inhibit cdk2/4/6-cyclin complex activity [21–23]. In addition to
negative regulation of branching morphogenesis in the embryonic
lung, TGF-b/Smad signaling and expression of cyclin-dependent
kinase inhibitors blocksproliferationofmature alveolartype II cellsin
culture [24–28]. Further, alveolar epithelial cells from p212/2mice
have an increased proliferation rate and lung tumorigenesis is
enhanced in adult mice heterozygous for a null mutation in TGF-b1,
supporting a role for this pathway in maintaining quiescence in the
normal respiratory epithelium in vivo [29–31]. Whether TGF-b/
Smad signaling and cyclin-dependent kinase inhibitors contribute to
negative regulation of progenitor cell activation and cell cycle reentry
in the mature respiratory epithelium has not been determined.
The present study was undertaken to determine the mechanisms
by which expression of Sox17 in mature respiratory epithelial cells
influences progenitor cell behavior. Conditional expression of
Sox17 in the adult mouse lung induced proliferation and reversibly
reprogrammed alveolar cells to form structures with phenotypic
and morphological characteristics of the proximal airway. During
the induction of progenitor cell behavior, Sox17 increased
expression of Sca-1, a progenitor cell marker, and stimulated cell
proliferation in association with altered expression of cell cycle
regulatory genes. In addition, Sox17 physically interacted with
Smad3 and negatively regulated Smad3 DNA binding and TGF-
b/Smad3 transcriptional responses. These findings provide insight
into the ability to reactivate multipotent progenitor cell behavior
and reprogram epithelial cell lineages in the lung.
Sox17 reversibly induces progenitor cell behavior in the
alveolar epithelium in vivo
Expression of Sox17 in peripheral respiratory epithelial cells
of the adult mouse lung reprogrammed mature alveolar type II
cells toward multiple proximal airway epithelial cell lineages,
supportinga rolein regulating
behavior . To determine if alveolar cells become perma-
nently respecified following ectopic Sox17 expression, Sox17
was conditionally expressed in respiratory epithelial cells of
adult mice. Lungs from CCSPrtTA/tetO-Sox17 and CCSPrtTA
single transgenic control mice were harvested after 4 weeks
exposure to doxycycline (Dox) or following removal of Dox for
1 week, and were examined for expression of Sox17 and the
proximal airway epithelial cell markers CCSP (Clara cells) and
Foxj1 (ciliated cells). Expression of Sox17 in peripheral
respiratory epithelial cells induced the formation of atypical
alveolar cell clusters which contained cells that expressed
conducting airway epithelial cell markers CCSP and Foxj1
(Fig. 1B,E,H). In lungs from CCSPrtTA/tetO-Sox17 mice in which
Dox treatment was discontinued for 1 week prior to harvest,
immunostaining for Sox17 was only detected in endothelial cells
in the peripheral lung and was indistinguishable from controls
(Fig. 1A,C). More notable, neither the alveolar cell clusters nor
peripheral expression of CCSP or Foxj1 were observed in lungs
from these mice (Fig. 1C,F,I). Together, these data demonstrate
that the Sox17-induced alveolar cell cluster formation and
lineage respecificationare reversible
progenitor cell behavior in adult CCSPrtTA/tetO-Sox17 mice,
revealing remarkable plasticity within a subset of mature
respiratory epithelial cells.
Bronchiolar-like lesions are induced in the alveoli
following prolonged Sox17 expression
To determine the effects of prolonged expression of Sox17 in
respiratory epithelial cells, adult CCSPrtTA/tetO-Sox17 mice were
maintained on Dox for 12 months. Long-term expression of
Sox17 caused the formation of organized sheets of epithelial cells
in the peripheral lung with morphological similarities to the
bronchiolar epithelium (Fig. 2). The bronchiolar-like structures
expressed Sox17 (Fig. 2B) and contained subsets of cells that
expressed proximal airway epithelial markers CCSP and Foxj1
(Fig. 2C–D), consistent with bronchiolar cell differentiation. While
CCSP+cells were detected in most of the bronchiolar-like
structures, Foxj1+cells were less frequently observed. Since the
ability of Sox17 to reprogram mature alveolar type II cells
suggests the induction of progenitor cell behavior, we examined
the bronchiolar-like structures for coexpression of CCSP, proSP-
C, and Sca-1, a property attributed to bronchoalveolar stem cells
(BASCs), a potential lung stem/progenitor population .
Expression of Sca-1, a progenitor cell marker in several tissues,
was detected in cells within the bronchiolar-like structures and
colocalized with CCSP-expressing cells (Fig. 2E–H). While a rare
subset of bronchiolar-like lesions contained cells that co-expressed
CCSP and proSP-C (data not shown), CCSP+/proSP-C+/Sca-1+
cells were never observed. Thus, the Sox17-induced bronchiolar-
like structures contained a mixed population of cells that
expressed CCSP, Foxj1, Sca-1, CCSP+/Sca-1+, and CCSP+/
proSP-C+, consistent with reprogramming of progenitor cells
along several differentiated pathways. Such bronchiolar-like
epithelial sheets were never detected in lungs from CCSPrtTA
control mice maintained on Dox for 12 months (data not shown).
Together these data show that prolonged expression of Sox17 in
the adult mouse lung dramatically influences respiratory epithelial
cell differentiation, generating ectopic structures in the peripheral
lung with characteristics of the more proximal bronchiolar
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Sox17 induces proliferation in adult respiratory epithelial
To assess the molecular mechanisms by which Sox17 initiates
cell cluster formation in respiratory epithelial progenitor cells,
Sox17 was conditionally expressed in adult CCSPrtTA/tetO-Sox17
mice for 1, 3, or 5 days. Endogenous expression of Sox17 was
detected in endothelial cells in the peripheral lung but not in
airway epithelial cells. In contrast, Sox17 staining was readily
detected in bronchioles and alveolar type II cells of CCSPrtTA/
tetO-Sox17 mice, consistent with sites of rtTA-directed gene
expression in this mouse line (Fig. 3A–F) . The formation of
cell clusters in the alveolar regions was evident 5 days after the
induction of Sox17 expression (Fig. 3F). Immunostaining for
phospho-histone H3 revealed the presence of mitotic cells in the
both the bronchioles and alveoli as early as 3 days following Sox17
expression (Fig. 3G–L), with a 4.7-fold increase in the number of
proliferative cells compared to control lungs (Fig. S1). Phospho-
histone H3 staining was also observed in a subset cells at the
bronchoalveolar duct junctions in the lungs of CCSPrtTA/tetO-
Sox17 mice (Fig. 3L; arrowhead). Although Sox17 expression and
cell proliferation were detected in the bronchioles and broncho-
alveolar duct junctions of CCSPrtTA/tetO-Sox17 mice, hyperplastic
foci within these regions were not evident. Dual immunofluores-
cence revealed colocalized expression of phospho-histone H3 with
Sox17, demonstrating that proliferation occurs within the Sox17-
expressing population of cells in CCSPrtTA/tetO-Sox17 mice
(Fig. 3M–O). Of the respiratory epithelial cells expressing Sox17,
28% coexpressed phospho-histone H3 (Fig. S1) and only a subset
of alveolar type II cells expressing Sox17 formed cell clusters.
Thus, Sox17 expression induces a subset of mature respiratory
epithelial cells in the adult mouse lung to reenter the cell cycle,
resulting in the formation of atypical cell clusters in the alveolar
region within 5 days.
Sox17 increases a Sca-1-positive cell population.
Since Sox17 induced proliferation of alveolar type II cells and
bronchiolar cells near the bronchoalveolar duct junctions, both
considered lung stem/progenitor cell niches, expression of proposed
CCSPrtTA/tetO-Sox17 mice. Increased expression of Sca-1 was
detected in the alveolar cells as well as in cells near the
bronchoalveolar duct junction after 5 days of conditional Sox17
expression in CCSPrtTA/tetO-Sox17 mice (Fig. 4A–B). Further,
immunofluorescent double labeling demonstrated that the Sca-1
positive cells colocalized with Sox17-expressing cells in lungs of
CCSPrtTA/tetO-Sox17 mice (Fig. 4C–E). While the majority of the
Sca-1-expressing cells did not show evidence of active proliferation, a
rare subset of the Sca-1 positive cells coexpressed phospho-histone
Figure 1. Sox17-induced cell clusters are reversible. Adult CCSPrtTA (A,D,G) and CCSPrtTA/tetO-Sox17 (B,C,E,F,H,I) mice were maintained on
Dox, and lungs were harvested after 4 weeks (wks) (A,B,D,E,G,H) or 1 week after discontinuing Dox (C,F,I). Immunostaining for Sox17 (A–C), CCSP (D–
F), and Foxj1 (G–I) was performed on lung sections. (A) Sox17 staining was not observed in the airway epithelium in the absence of Dox. (B,E,H)
Hyperplastic clusters of cells were observed in the alveolar region following Sox17 expression (arrows and insets). CCSP and Foxj1 staining was
detected in a subset of the Sox17-induced alveolar cell clusters (arrows and insets; E,H). (C,F,I) Neither Sox17 transgene expression nor hyperplastic
cell clusters were detected 1 week after removal from Dox. Scale bar, 50 mm.
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Figure 2. Prolonged expression of Sox17 results in formation of bronchiolar-like structures in the alveoli. Adult CCSPrtTA/tetO-Sox17
transgenic mice were maintained on Dox for 12 months. (A) H&E staining shows the presence of bronchiolar-like sheets of cells in the peripheral lung.
Arrowhead indicates the pleural surface. (B–D) Immunostaining for Sox17 (B), Foxj1 (C), and CCSP (D) was performed on lung sections. The Sox17-
induced bronchiolar-like structures contained cells expressing proximal airway markers CCSP and Foxj1. (E–H) Immunofluorescent staining for CCSP
(E), proSP-C (F), and Sca-1 (G). The bronchiolar-like structures (dotted outline) contained cells that express Sca-1 and subsets of cells expressing CCSP
or proSP-C. Arrowheads demark CCSP-expressing cells in the bronchiolar epithelium and the arrow indicates normal proSP-C expression in a type II
cell. Nuclei are stained with DAPI (H; blue) Scale bars, 50 mm.
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Figure 3. Sox17 induces proliferation of respiratory epithelial cells in the adult mouse lung. Immunohistochemistry was performed on
lung sections from adult CCSPrtTA control (A–C; G–I) and CCSPrtTA/tetO-Sox17 (D–F; J–L) transgenic mice after treatment with Dox for 1, 3, and 5 days
(d). (A–F) Immunostaining shows endogenous expression of Sox17 in endothelial cells and Sox17 transgene expression in bronchioles and alveolar
type II cells (D–F). Hyperplastic cell clusters are evident by 5 d (arrow and inset, F). (G–L) Phospho-histone H3 immunostaining shows respiratory
epithelial cell proliferation after 3 d of Dox treatment in Sox17 transgenic mice. Proliferative cells were detected in the peripheral lung (K–L; arrow
and inset) and bronchioles (K–L; arrowhead). (M–O) Colocalization of Sox17 (M) and phospho-histone H3 (pHH3; N) is shown by dual-label
immunofluorescence after 5 d Dox exposure. Nuclei are stained with DAPI (O; blue). Scale bars, 50 mm.
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Figure 4. Sox17 increases Sca-1 expression adult respiratory epithelial cells. Adult CCSPrtTA control (A) and CCSPrtTA/tetO-Sox17 (B–N)
transgenic mice were exposed to Dox for 5 d. (A–B) Immunostaining for Sca-1 was performed on lung sections. Increased Sca-1 staining was detected
in the peripheral lung and near the bronchoalveolar duct junctions after expression of Sox17 (arrowheads and inset). (C–E) Dual-label
immunofluorescence for Sox17 (C) and Sca-1 (D) demonstrated that the Sca-1 positive cells coexpressed Sox17 (arrows). (F–N) Triple-label
immunofluorescent staining was performed for CCSP, proSP-C, and Sca-1. Sca-1 colocalized with CCSP-expressing cells near the bronchoalveolar duct
junctions (open arrowhead; magnified in G–J). Sca-1 positive cells located in the peripheral lung (arrowheads; magnified in K–N) did not coexpress
proSP-C (arrow; F,L, and N). Scale bars, 50 mm (A–B; F), 20 mm (C–E).
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H3 (Fig. S2). Since Sox17 induced expression of Sca-1 in a subset of
epithelial cells located in putative lung stem/progenitor cell niches in
the alveolus and peripheral bronchioles, immunofluorescence was
performed to determine if the population of Sca-1 positive cells
coexpressed CCSP and/or proSP-C characteristic of BASCs. In
lungs from CCSPrtTA/tetO-Sox17 mice maintained on Dox for 5 days,
colocalization of Sca-1 and CCSP was observed in cells along the
bronchoalveolar duct junctions (Fig. 4F–N). In contrast, CCSP was
not detected in the Sca-1-expressing cells detected in the alveolar
region. In accordance with this observation, immunohistochemical
analysis did not reveal any CCSP-expressing cells in the peripheral
lung of CCSPrtTA/tetO-Sox17 mice exposed to Dox for 5 days (data
not shown). The Sox17-induced Sca-1 positive cells located in the
alveolar region did not coexpress proSP-C (Fig. 4F–N). Further,
CCSP+/proSP-C+/Sca-1+cells were never detected at the broncho-
3 sections from each lobe). Taken together, these studies show that
the Sox17 induced Sca-1 expression in cells near the BADJ that
coexpressed CCSP but not proSP-C, while the Sca-1 expressing cells
induced in the alveolar region expressed neither CCSP nor proSP-C.
activation, the molecular profile of the Sca-1-expressing population is
distinct from that previously describe for BASCs , emphasizing
the need for a better understanding of the phenotypic and functional
characteristics of potential stem/progenitor cells in the lung.
Sox17 influences expression of cell cycle regulatory
Since expression of Sox17 in respiratory epithelial cells in the
adult mouse lung induced cell proliferation, the effects on cell
cycle-associated gene expression was examined to identify
potential downstream targets of Sox17 that regulate this process.
Total RNA isolated from whole left lobes of adult CCSPrtTA
control and CCSPrtTA/tetO-Sox17 mice maintained on Dox for 2
and 3 days was used to analyze changes in cell cycle-related gene
expression with a commercially available oligo SuperArray (data
not shown). Subsequently, RT-PCR was used to confirm the
changes in expression of a subset of the genes identified in the
array. In addition to increased expression of Sox17 (data not
shown), mRNAs for the cyclin-dependent kinase inhibitors p15,
p21, and p57 were significantly decreased in lungs of CCSPrtTA/
tetO-Sox17 mice relative to CCSPrtTA controls following 1 day of
exposure to Dox (Fig. 5). Whereas expression of p57 was also
decreased on lungs from CCSPrtTA/tetO-Sox17 mice maintained on
Dox for 3 days, expression of p15 and p21 was similar to controls.
Further, while expression of p19 was variably decreased in lungs
from CCSPrtTA/tetO-Sox17 mice maintained on Dox for 2 days, no
differences in the expression of p16 or p27 were observed after
expression of Sox17 for 1–3 days (data not shown). Consistent with
the induction of proliferation observed by immunohistochemistry
(Fig. 3), transcripts for genes that promote cell cycle progression,
including Foxm1, cyclin A2, cyclin B1, cyclin D1, and cyclin E1, were
increased in lungs of CCSPrtTA/tetO-Sox17 mice after 3 days of
Dox treatment (Fig. 5). Together, these results demonstrate that
Sox17 expression in adult mouse lung results in decreased levels of
cyclin-dependent kinase inhibitors associated with G1 arrest and
increased expression of cell cycle-promoting genes, providing
insight into the molecular mechanisms that regulate Sox17-
induced proliferation associated with the initiation of progenitor
cell behavior in respiratory epithelial cells.
Sox17 induces cyclin D1 expression
Since cyclin D1 is a key regulator of progression through the G1
phase of the cell cycle and was increased in lungs from CCSPrtTA/
tetO-Sox17 mice, we sought to determine if it was a direct
downstream target of Sox17 during induction of respiratory
epithelial cell proliferation. Quantitative real time RT-PCR
performed using total RNA isolated from whole left lobes
demonstrated that cyclin D1 mRNA was increased 1.3-fold in
lungs from adult CCSPrtTA/tetO-Sox17 mice exposed to Dox for 1
day (data not shown). Staining for cyclin D1 was markedly
increased in bronchioles and alveolar type II cells of CCSPrtTA/
tetO-Sox17 mice following 2 days exposure to Dox (Fig. 6A–B).
Thus, cyclin D1 mRNA and protein levels were significantly
increased in lungs from CCSPrtTA/tetO-Sox17 mice just prior to the
onset of proliferation, consistent with the concept that cyclin D1
contributes to the cell cycle reentry of mature respiratory epithelial
cells following expression of Sox17.
To determine if cyclin D1 is a direct transcriptional target Sox17,
the regulatory region of cyclin D1 was examined for putative Sox
binding sites. A well-conserved consensus Sox bindingsequence was
identified in cyclin D1 proximal promoter, located at approximately
274 bp relative to exon 1 in the human and mouse genomic
sequences (Fig. 6C). The functional significance of this potential Sox
site wasassessed by reporter assay usinga human cyclin D1 promoter
deletion series. While the 247/+187 cyclin D1-luciferase reporter was
moderately responsive to Sox17, the cyclin D1-luciferase constructs
that contained the consensus Sox binding sequence were markedly
activated by Sox17 (Fig. 6C). A truncated Sox17 isoform (t-Sox17),
which lacks most of the HMG box and cannot bind DNA , did
not affect cyclin D1 promoter activity (Fig. 6C), indicating that
activation of cyclin D1-luciferase reporters by Sox17 requires DNA
binding. Together with the in vivo data, these results support the
concept that cyclin D1 is a direct downstream target of Sox17 in
respiratory epithelial cells of CCSPrtTA/tetO-Sox17 mice.
Sox17 inhibits TGF-b1-induced transcriptional activity
TGF-b is a potent inhibitor of proliferation in multiple epithelial
cell types, including alveolar type II cells . The mechanism
Figure 5. Sox17 regulates genes that control the cell cycle. RT-
PCR was used to assess expression of cell cycle-related genes in lung
tissue from adult CCSPrtTA and CCSPrtTA/tetO-Sox17 mice treated with
Dox for 1 or 3 days. Transcripts for the cyclin-dependent kinase
inhibitors p15, p21, and p57 were decreased in Sox17 transgenic lungs
after 1 day of Dox and mRNAs for genes associated with cell cycle
progression were increased by Sox17 after 3 days Dox exposure. L7 was
used as a loading control.
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underlying the anti-proliferative effects of TGF-b has been
attributed, at least in part, to Smad-dependent transcriptional
induction of cyclin-dependent kinase inhibitors including p15 and
p21, leading to G1 arrest [21–23]. Since expression of p15 and p21
was decreased in lungs of adult mice following Sox17 expression in
respiratory epithelial cells, we sought to determine if Sox17
influences TGF-b-mediated transcriptional activity in vitro using
the TGF-b/Smad responsive reporter 3TP-luciferase. In MLE-15
cells, Sox17 markedly inhibited TGF-b1-induced 3TP-luciferase
reporter activity in a dose-dependent manner relative to vector
control or t-Sox17 (Fig. 7A), demonstrating that Sox17 has an
inhibitory effect on the TGF-b pathway.
Sox17 interacts with Smad3 and inhibits Smad3-
dependent transcriptional activity
Since Smad2/3 are the transcriptional effectors of TGF-b
signaling, we examined if the inhibition of TGF-b1-induced
transcriptional activity by Sox17 was mediated by an interaction
with these Smads. GST-pulldown assays revealed a physical
interaction between Sox17 and Smad3 (Fig. 7B–C). To identify
which domains of Smad3 are required for the interaction, GST-
pulldown assays were performed using deletion constructs for
Smad3. While both the MAD homology domains (MH1 and
MH2) of Smad3 interacted with Sox17 at low stringency binding
conditions (data not shown), Smad3 1–145 did not bind to Sox17
under higher stringency conditions (Fig. 7B). Likewise, GST-
pulldowns were performed using a series of Sox17 deletions and a
point mutant, including a N-terminal deletion that removes 80%
of the HMG domain (Sox17 129–419, which is equivalent to the t-
Sox17 isoform), a C-terminal truncation that deletes the
transactivation domain (Sox17 1–359), and a point mutation in
the HMG domain (M76A), which is predicted to disrupt DNA
binding but not protein structure . Both of the Sox17
truncations and the point mutant maintained the ability to
interact with Smad3 across multiple binding stringencies (Fig. 7C).
Together, these data indicate that while multiple domains of
Smad3 are capable of interacting with Sox17, the strongest
binding is localized to the linker and MH2 regions from amino
acids 146–425. Further, the data suggest that amino acids 129–359
of Sox17 mediate the interaction with Smad3. While the
interaction observed between Sox17 and Smad3 was corroborated
using co-immunoprecipitation assays, an interaction between
Sox17 and Smad2 was not observed (data not shown).
To examine the functional significance of the interaction
between Sox17 and Smad3 on Smad3 transcriptional activity,
the 3TP-luciferase reporter was co-transfected with Smad3 in the
presence or absence of Sox17 in MLE-15 cells. Similar to its effect
on TGF-b1 activity, Sox17 significantly inhibited Smad3-induced
reporter activity (Fig. 7D). To identify the domains of Sox17 that
mediate the repression of Smad3 transcriptional activity, reporter
assays were performed using the Sox17 deletions and point
mutant. While Sox17 1–359 maintained the ability to inhibit
Smad3 activity, neither Sox17 129–419 nor Sox17 M76A
inhibited Smad3 function (Fig. 7D). Immunocytochemistry
demonstrated that full length Sox17 as well as the Sox17 deletions
and point mutant all localized to the nucleus (Fig. S3). However,
Figure 6. Cyclin D1 expression is induced by Sox17. (A–B) Immunohistochemistry for cyclin D1 was performed on lung sections from adult
CCSPrtTA (A) and CCSPrtTA/tetO-Sox17 (B) transgenic mice treated with Dox for 2 d. Cyclin D1 staining was increased in bronchioles and type II cells
after Sox17 expression. Scale bars, 50 mm. (C) The regulatory region of cyclin D1 contains a conserved Sox binding site (boxed). MLE-15 cells were
transiently transfected with human cyclin D1-luciferase reporter constructs and empty vector, t-Sox17, or full length Sox17. While the 247/+187 cyclin
D1-luciferase reporter was moderately responsive to Sox17 (2.6-fold), Sox17 strongly activated the cyclin D1 promoter constructs containing the
conserved Sox binding site (arrowheads; 296/+187; 6.4-fold and 2944/+187; 5.9-fold). Experiments were performed three times in triplicate and
representative results are shown6the standard deviation of the mean. Asterisks indicate statistical significance determined by Student’s t-test
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Figure 7. Sox17 interacts with Smad3 and inhibits TGF-b1/Smad3 transcriptional activity and Smad3 DNA binding. (A) MLE-15 cells
were transfected with the TGF-b/Smad-responsive reporter 3TP-luciferase (3TP-Luc) and increasing amounts of vector, t-Sox17, or full length Sox17 in
the presence or absence 2 ng/ml TGF-b1. Sox17 inhibited TGF-b1-mediated activation of 3TP-Luc. The graph represents average fold
activity6standard deviation. Asterisks indicate statistical significance determined by Student’s t-test (p,0.05). (B–C) Lysates from MLE-15 cells
expressing full length or mutant FLAG-Smad3 or Sox17-V5 (schematic representations) were incubated with GST only and GST-Sox17 (B) or GST-
Smad3 (C), respectively. GST-pulldowns revealed an interaction between amino acids 129–359 of Sox17 and the linker region and MH2 domain of
Smad3. MH1 and MH2, MAD homology domains; HMG, High mobility group. (D) MLE-15 cells were transfected with 3TP-Luc in the presence or
absence Smad3 and wild type or mutant Sox17. Sox17 full length and C-terminal deletion inhibited Smad3-dependent transcriptional activity.
Representative results are shown6standard deviation of the mean. Asterisks indicate statistical significance determined by Student’s t-test (p,0.05).
(E) Sox17 antagonizes TGF-b1/Smad3-mediated repression of cyclin D1 promoter activity. MLE-15 cells were co-transfected with 2944/+187 cyclin D1-
luciferase and Smad3 or Sox17 in the presence or absence of 5 ng/ml TGF-b1. The graph represents average fold activity6standard deviation. Pound
signs and asterisk indicate statistical significance determined by Student’s t-test (p,0.05). (F) Sox17 blocks Smad3 DNA binding. MLE-15 cell were
transfected with FLAG-Smad3 in the presence or absence of Sox17. After 24 h, cells were incubated with 5 ng/ml TGF-b1 for 8 h before harvesting.
Sox17 and Lung Progenitor Cell
PLoS ONE | www.plosone.org9 May 2009 | Volume 4 | Issue 5 | e5711
none of the Sox17 proteins influenced nuclear translocation of
Smad3 in the presence or absence of TGF-b1 (Fig. S3), indicating
that Sox17 does not antagonize Smad3 activity by regulating
subcellular localization. Together, these data suggest that,
although it is not required for binding to Smad3, the N-terminus
of Sox17 is important for repression of Smad3 transcriptional
To determine if the interaction between Sox17 and TGF-b/
Smad3 signaling influences regulation of cell cycle-related genes,
their effects on cell cycle gene promoters was examined. In MLE-
15 cells, Sox17 alleviated repression of cyclin D1 promoter activity
by TGF-b1 and Smad3, consistent with an antagonistic effect of
Sox17 on the TGF-b/Smad3 pathway (Fig. 7E). Since Sox17
negatively regulated Smad3 transcriptional activity in reporter
assays but did not influence Smad3 nuclear import, we sought to
determine if the repression was mediated by influencing Smad3
DNA binding. Smad3 regulates p15 gene expression by directly
binding to sites located within the first 113 bp of its promoter .
Using MLE-15 cells, Smad3 DNA binding to the p15 promoter
between 2157 and +103 in the presence or absence of Sox17 was
examined by chromatin immunoprecipitation and quantified by
real time PCR. Sox17 significantly decreased Smad3 binding to
the p15 promoter (Fig. 7F). Together, these experiments show that
Sox17 antagonizes Smad3 transcriptional activity by preventing its
ability to bind DNA, providing a potential mechanism by which
Sox17 decreased the expression of cell cycle inhibitors in the adult
mouse lung. In addition, antagonizing TGF-b1/Smad3 repression
of the cyclin D1 promoter may also contribute to the Sox17-
mediated induction of cyclin D1 expression observed in vivo.
Conditional expression of Sox17 in respiratory epithelial cells of
the adult mouse lung induced proliferation and reversibly
respecified alveolar type II epithelial cells to express markers
characteristic of the differentiated bronchiolar epithelium, sup-
porting the concept that a subset of mature respiratory epithelial
cells possesses remarkable phenotypic plasticity and progenitor cell
capabilities. Activation of this progenitor-like behavior in respira-
tory epithelial cells by Sox17 was associated with increased
expression of Sca-1 and multiple genes that promote cell cycle
reentry/progression. Sox17 decreased expression of cell cycle
inhibitors in vivo and interacted with Smad3 to inhibit TGF-b1/
Smad3-mediated transcriptional responses in vitro. Together, these
data provide insight into the mechanisms by which Sox17
stimulates respiratory epithelial progenitor cell behavior and
lineage respecification in the mature lung.
Expression of Sox17 reprogrammed a subset of mature alveolar
type II cells to ectopically express markers characteristic of diverse
conducting airway cell lineages, including ciliated, non-ciliated
secretory cells, and goblet cells that are not normally detected in
the alveolar regions of the lung , and led to the formation of
highly organized bronchiolar-like structures in the peripheral lung.
These results indicate that epithelial cells in the adult lung can
serve as multipotent progenitors capable of lineage respecification.
In support of this concept, conditional expression of SPDEF, an
ETS family transcription factor, in respiratory epithelial cells of
adult mice converts Clara cells into goblet cells . The notion
that Sox proteins have important functions in lineage reprogram-
ming is further supported by the contribution of Sox2 toward
induced pluripotency in somatic cells [37–40]. While the
mechanisms that govern progenitor cell activation and cell fate
respecification remain poorly understood, further analysis of
Sox17-induced respiratory epithelial progenitors may provide
insight into such processes in the mature lung.
Lineage reprogramming of adult cell types involves the expression
of key developmental regulatory genes . The finding from
previous studies that expression of several transcription factors
involved in regulating proximal/distal epithelial cell differentiation in
the lung, including TTF-1, Foxa1, Foxa2, and b-catenin, is also
increased in the Sox17-induced cell clusters suggests that Sox17 can
reinitiate a program typical of the developing lung . During
endoderm formation in Xenopus, Sox17 regulates expression of
Foxa1 and Foxa2, and acts upstream of GATA6 . Foxa1, Foxa2,
and GATA6 influence the expression of differentiated respiratory
epithelial cell markers, including CCSP and Foxj1 [42–44]. Taken
together, these data support the concept that Sox17, which is
necessary for endoderm formation, functions upstream of a hierarchy
of transcription factors that cooperate in specification of endodermal
cells from which respiratory epithelial lineages are derived.
Constitutive expression of Sox17 in human embryonic stem cells is
sufficient to commit cells to the definitive endoderm lineage, and
increased expression of Sox17 is associated with the differentiation of
embryonic stem cells toward cells with characteristics of the
respiratory epithelium [5,6,10–12]. Together, these findings further
support an early role for Sox17 in the establishment of endodermal
cells that later serve as precursors of lung epithelial lineages.
The induction of proliferation in a subset of bronchiolar cells
and alveolar type II cells by Sox17 was associated with increased
expression of several cyclin genes known to stimulate cell cycle
progression. Among the various cyclin genes induced by Sox17,
only cyclin D1 functions in G1 prior to the restriction point of the
cell cycle [45,46]. Notably, forced expression of cyclin D1 along
with cdk4 is sufficient to reinitiate cell cycle progression in multiple
post-mitotic cell types [47,48]. Therefore, the induction of cyclin
D1 by Sox17 is likely to play an important role in stimulating
mature respiratory epithelial cells to reenter the cell cycle. Our in
vitro studies demonstrated that Sox17 directly activated the human
cyclin D1 promoter through a conserved site located at 274 bp
relative to the first exon. Given the homology between their HMG
domains, Sox and TCF/LEF proteins have similar preferences for
consensus binding sequences, and previous studies have demon-
strated that cyclin D1 is directly regulated by b-catenin/TCF/LEF
through this same site [49,50]. In breast cancer cells, Sox2
interacts with b-catenin to cooperatively regulate the cyclin D1
promoter through the 274 bp binding site as well . In
addition, Sox17 and Sox4 respectively inhibit and enhance
proliferation of colon carcinoma cells through physical interactions
with b-catenin and TCF/LEF to modulate protein stability and
transcriptional activity, and Sox6 interacts with b-catenin and
HDAC1 to repress cyclin D1 promoter activity and proliferation in
insulinoma cells [52,53]. Taken together, these findings suggest
that Sox proteins and b-catenin/TCF/LEF complexes may
compete for common DNA binding sites and that their effects
on transcription and cell behavior may depend on relative
expression levels, protein interactions, and/or cell type. Although
it is unclear whether Sox17 is a transcriptional activator of cyclin
D1 in vivo, our data support a potential mechanism by which Sox17
Binding of Smad3 to the p15 promoter was assessed by chromatin immunoprecipitation and quantified by real time PCR. Graph represents average
fold enrichment of FLAG immunoprecipitated samples relative to IgG negative control samples. Asterisk indicates statistical significance determined
by Student’s t-test (p,0.05). Expression of Smad3 and Sox17 inputs were assessed by immunoblot.
Sox17 and Lung Progenitor Cell
PLoS ONE | www.plosone.org10 May 2009 | Volume 4 | Issue 5 | e5711
directly induces cyclin D1 to promote proliferation of a subset of
mature respiratory epithelial cells.
Recent studies have shown that post-mitotic cells can reenter
the cell cycle by blocking the expression of cyclin-dependent kinase
inhibitors, supporting a role for this protein family in maintaining
the balance between quiescence and proliferation . In the
present study, Sox17 decreased the expression of p15, p21, and p57
in adult mouse lungs, and inhibited TGF-b1/Smad3 transcrip-
tional activity in vitro. In addition, Sox17 physically interacted with
Smad3 and abrogated Smad3 binding to the p15 promoter. These
findings support a model in which Sox17 antagonizes TGF-b/
Smad-dependent expression of cell cycle inhibitors in mature
respiratory epithelial cells, facilitating G1 progression and cell
reentry. In addition, Sox17 counteracted repression of the cyclin D1
promoter by TGF-b1 and Smad3. While we favor the notion that
Sox17 directly activates the cyclin D1 promoter, it is possible that
inhibiting TGF-b/Smad3-mediated repression of cyclin D1 also
contributes to its increased expression in CCSPrtTA/tetO-Sox17
mouse lungs. By decreasing expression of inhibitors and increasing
expression of positive regulators of the G1 phase of the cell cycle,
Sox17 establishes conditions that favor reactivation of proliferation
in mature, normally quiescent respiratory epithelial cells.
In the present study, Sox17 interacted with Smad3 and repressed
TGF-b1/Smad3 transcriptional activity. Although both the MH1
and MH2 domains of Smad3 bound Sox17, the strongest
interaction was localized to the linker region and MH2 domains.
The linker region of Smad3 contains a transactivation domain and
the MH2 domain modulates transcription, whereas the MH1
domain of Smad3 regulates DNA binding and transcription. Thus,
Sox17 interaction with these domains is consistent with the
antagonistic effects of Sox17 on Smad3 DNA binding and
transcriptional activity. While that amino acids 129–359 of Sox17
appear to mediate binding to Smad3, the N-terminus of Sox17 is
necessary to antagonize Smad3 activity. Inhibition of b-catenin/
TCF transcriptional activity is also dependent on the N-terminus of
Sox17, wherein the HMG domain is required for interaction with
TCF factors to influence their protein stability [52,55]. Together,
these findings indicate that the N-terminus of Sox17 containing the
HMG domain is important for influencing the transcriptional
responses of signaling pathways and that there may be multiple
regions of Sox17 that contribute to complex protein interactions.
Sox17 initiated progenitor cell behavior in respiratory epithelial
cells when ectopically expressed at high levels in the adult lung.
While these studies provide insight into the effects of Sox17 on
reprogramming mature respiratory epithelial cells, it is unclear
whether they reflect a physiological role during development or
repair. Since Sox17 is highly expressed in the endoderm, which
gives rise to the respiratory epithelium, Sox17 likely plays an early
role in specification of endodermal precursor cells prior to
emergence of the lung primordium. In the lung, Sox17 is
expressed in mesenchymal cells during branching morphogenesis
and in pulmonary endothelial cells later in development and in the
adult . However, Sox17 mRNA was not detected in
respiratory epithelial cells and immunohistochemical staining for
endogenous Sox17 was not observed in the lung epithelium in the
present study . A number of Sox family members are
expressed at high levels in various cell types in the developing
and mature lung, including Sox2, 4, 7, 9, 11, 17, and 18 [56–60].
However, whether Sox proteins have distinct or redundant
functions during lung formation and repair remains to be
elucidated. Our data shows that the ability of Sox17 to induce
progenitor cell behavior is mediated, at least in part, by activating
cyclin D1 and decreasing TGF-b-responsive cell cycle inhibitor
expression to promote proliferation. As the transcriptional
pathways that regulate cell proliferation and differentiation during
lung formation are reactivated during regeneration of pulmonary
cell lineages following injury, determining how Sox proteins and
other transcription factors integrate signals from multiple path-
ways is important toward understanding the regulatory mecha-
nisms that control lung development and homeostasis.
Materials and Methods
rCCSPrtTA and (tetO)7CMV-Sox17-IRES-NucGFP transgenic mice
used for conditional expression of Sox17 in respiratory epithelial cells
have been previously described [13,33,61]. Adult rCCSPrtTA/
(tetO)7CMV-Sox17-IRES-NucGFP double transgenic and rCCSPrtTA
single transgenic control mice, referred to as CCSPrtTA/tetO-Sox17
and CCSPrtTA, respectively, were maintained on doxycycline-
containing food (625 mg/kg; Harlan Teklad, Madison, WI) as
free conditions according to protocols approved by the Institutional
Animal Care and Use Committee at Cincinnati Children’s Hospital
Research Foundation. Mice were sacrificed by anesthesia using a
by severing the inferior vena cava and descending aorta. All
experiments were performed using at least 3 animals per group.
Immunohistochemistry and Immunofluorescence
Lungs of adult mice were inflated with 4% paraformaldehyde/
phosphate-buffered saline (PBS), fixed by immersion overnight at
4uC, and processed according to standard protocols for paraffin
embedding. Immunohistochemistry was performed using primary
antibodies for guinea pig-anti Sox17 (1:10,000), rabbit anti-
phospho-histone H3 (1:1000; Santa Cruz), rabbit anti-CCSP
(1:5000), rabbit anti-Foxj1 (1:14,000), rabbit anti-cyclin D1 (1:400;
Abcam), and rat anti-Sca-1 (1:250; BD Pharmingen). Briefly,
sections (5 mm) were deparaffinized, rehydrated through a graded
ethanol series, and endogenous peroxidase activity was inactivated
in 1.5% H2O2 in methanol. Microwave antigen retrieval was
performed (except for Sca-1) using 10 mM citrate buffer, pH 6.0
and sections were blocked for 1–2 h in 4% normal goat or donkey
serum in PBS-0.1% Triton X-100 (PBST) followed by primary
antibody incubation overnight at 4uC. Sections were then washed
and incubated with biotinylated secondary antibodies (1:200;
Vector Labs) followed by incubation in ABC reagent (Vectastain
Elite ABC kit; Vector Labs). Antigen localization was detected
with nickel-diaminobenzidine and enhanced with Tris-Cobalt.
Sections were counterstained with 0.1% of Nuclear Fast Red and
coverslipped using Permount (Fisher Scientific).
Immunofluorescence was performed as described above with
the omission of peroxidase treatment. For immunofluorescent
double and triple labeling, primary antibodies for guinea pig anti-
Sox17 (1:1000), rabbit anti-phospho-histone H3 (1:500), rat anti-
Sca-1 (1:50), rabbit anti-proSP-C (1:500), and guinea pig anti-
CCSP (1:10,000) were used with fluorophore-conjugated second-
ary antibodies (Alexa Fluor-488, Alexa Fluor-568, and Alexa
Fluor-688; Molecular Probes). Sections were mounted with
Vectashield anti-fade reagent containing DAPI (Vector Labs).
Brightfield and fluorescent images were obtained using a Zeiss
Axioplan2 microscope equipped with AxioVision Software.
Total RNA was extracted from whole left lobes of adult mice
using TRIzol reagent (Invitrogen) and RNeasy Mini Kit (Qiagen)
according to the manufacturer’s recommendations. Mouse Oligo
GEArrays (SuperArray Bioscience Corporation) were used per the
Sox17 and Lung Progenitor Cell
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manufacturer’s protocol to examine expression of cell cycle-related
genes in lungs from adult CCSPrtTA/tetO-Sox17 (n=3) and
CCSPrtTA (n=3) mice maintained on Dox for 2 and 3 days.
Reverse transcription reactions were performed with 2 mg of RNA
and oligo(dT) primers using the SuperScript First-Strand Synthesis
kit (Invitrogen). RT-PCR was performed using the following
primers: p15 59-AGG CTT CCT GGA CAC GCT TG-39 and 59-
AGA TGG GGC TGG GGA GAA AG-39; p21 59-CGA AAA
CGG AGG CAG ACC AG-39 and 59-TCC TGA CCC ACA
GCA GAA GAG G-39; p57 59-AAG CGA ACA GGC AGG CAA
G-39 and 59-TAG AAG GCG GGC ACA GAC TC-39; Foxm1 59-
CAC TTG GAT TGA GGA CCA CTT-39 and 59-GTC GTT
TCT GCT GTG ATT CC-39; cyclin A2 59-ACC AAG AGA ATG
TCA ACC CCG-39 and 59-GGT GAA GGC AGG CTG TTT
ACT G-39; cyclin B1 59-AGG GTC GTG AAG TGA CTG GAA
AC-39 and 59-TTG GGC ACA CAA CTG TTC TGC-39; cyclin
D1 59-CAC AAC GCA CTT TCT TTC CA-39 and 59-GAC
CAG CCT CTT CCT CCA C-39; cyclin E1 59-GCA GGC GAG
GAT GAG AGC AG-39 and 59-ATA ACC ATG GCG AAC
GGA ACC-39; L7 59-GAA GCT CAT CTA TGA GAA GGC-39
and 59-AAG ACG AAG GAG CTG CAG AAC-39.
The following expression constructs have been previously
described: pCIG-Sox17 and pCIG-t-Sox17 (truncated Sox17) ;
Sox17-V5 epitope-tagged constructs encoding for wild type Sox17
(1–419), a N-terminal deletion equivalent to the t-Sox17 isoform
(129–419), C-terminal deletion prior to the transactivation domain
(1–359), and a DNA binding domain point mutant (M76A); pGEX-
mouse Sox17 encoding for glutathione S-transferase (GST)-Sox17
fusion protein . The 3TP-luciferase TGF-b/Smad-responsive
reporter plasmid  was obtained from Dr. Jeff Molkentin
(Cincinnati Children’s Hospital Research Foundation), and human
cyclin D1-luciferase promoter constructs, based on GenBank Accession
number Z29078, have been previously described and were acquired
from Dr. Karen Knudsen . The pGEX-human Smad3 construct
(Addgene plasmid 12630) encoding for GST-Smad3 was generated
in the lab of Dr. Rik Derynck (UCSF) , and the FLAG-tagged
Smad3 expression vectors were generated by Dr. Joan Massague
(Memorial Sloan-Kettering Cancer Center) (FLAG-Smad3 (1–425),
Addgene plasmid 14052; FLAG-Smad3 (1–145), Addgene plasmid
14965; FLAG-Smad3 (146–425), Addgene plasmid 14966; FLAG-
Smad3 (220–425), Addgene plasmid 14967) [65,66].
Cell culture and Reporter Assays
MLE-15 cells, an SV40 immortalized mouse lung epithelial cell
line, were maintained in HITES medium . Cells were seeded
in 6-well culture plates at 16105cells per well and transfected
using FuGENE6 (Roche). Transfections included a pCMV-b-
galactosidase expression vector as an internal control for
transfection efficiency. Recombinant human TGF-b1 (R&D
Systems) was added directly to the culture medium where
indicated. Cells were harvested 24 h post-transfection and
luciferase activity was measured using a Luciferase Assay System
kit (Promega) and normalized to b-galactosidase activity. Exper-
iments were performed three times in triplicate and statistical
significance was determined by paired Student’s t-test.
The GST-Sox17 and GST-Smad3 fusion proteins (and GST
only) were expressed in BL21 cells induced with 1 mM isopropyl
b-D-1-thiogalactopyranoside (IPTG). Cell pellets were resus-
pended in lysis buffer (25 mM Tris, pH 8.0; 0.5 mM EDTA,
0.2 M NaCl; 1 mM DTT; 15 ml/ml protease inhibitor cocktail
(Sigma); 0.1 mM PMSF), sonicated, and GST and GST fusion
proteins were purified using glutathione sepharose beads (Amer-
sham). Sox17-V5 and FLAG-Smad3 constructs were expressed in
MLE-15 cells. Whole cell protein lysates were harvested after
48 hours in cell lysis buffer (20 mM Tris, pH 8.0; 1 mM EDTA;
100 mM NaCl; 0.5% NP-40; 5 ml/ml protease inhibitor cocktail
(Sigma); 0.1 mM PMSF), and precleared using glutathione
sepharose beads. A 10% volume of the precleared lysates was
retained for experimental inputs, and the remaining lysate was
incubated with 4–8 mg of GST, GST-Sox17, or GST-Smad3
beads. Non-interacting proteins were removed by several washes
(20 mM Tris, pH 8.0; 1 mM EDTA; 300–500 mM NaCl; 0.5%
NP-40; 5 ml/ml protease inhibitor cocktail (Sigma); 0.1 mM
PMSF) and samples were eluted by boiling in Laemmli sample
buffer containing b-ME. Bound proteins were analyzed by SDS-
PAGE and immunoblot using rabbit anti-FLAG (1:6000; Sigma)
and mouse anti-V5-HRP (1:5000; Invitrogen) antibodies.
MLE-15 cells were seeded at 16106in 10 cm plates and co-
transfected with FLAG-Smad3 and pCIG or pCIG-Sox17 using
FuGENE6 (Roche). After 24 h, cells were switched to serum-free
media and treated with 5 ng/ml rhTGF-b1 (R&D Systems) for 8 h.
Crosslinking was performed by treating cells with 1% formaldehyde
for 10 min at room temperature and was terminated by addition of
0.125 M glycine. After rinsing with cold 16PBS, cells from 3 plates
per condition were pooled and incubated in hypotonic buffer
(10 mM HEPES, pH 7.8; 10 mM KCl; 1.5 mM MgCl2; 0.5% NP-
40; 5 ml/ml protease inhibitor cocktail) for 30 min at 4uC. Pellets
were collected by centrifugation and incubated in lysis buffer
(50 mM Tris, pH8.0; 10 mM EDTA; 1% SDS; 5 ml/ml protease
inhibitor cocktail) for 30 min at 4uC. Lysates were sonicated
(Diagenode bioruptor)toshearDNA andcelldebriswas removed by
centrifugation. Lysates werepreclearedwith ProteinA/G Plusbeads
(Santa Cruz), sonicated salmon sperm DNA, and BSA (1 mg/ml) for
2 h at 4uC. A 1% aliquot of precleared chromatin was removed for
experimental inputs. Equal volumes of the remaining lysate were
incubated with EZview Red anti-FLAG M2 beads (Sigma) or
Protein A/G Plus beads and normal mouse IgG (1.5 mg) overnight
at 4uC. Beads were washed once in dialysis buffer (50 mM Tris,
pH8.0; 2 mM EDTA) and 4 times with wash buffer (100 mM Tris,
pH8.0; 500 mM LiCl; 1% NP-40; 1% deoxycholic acid) followed by
elution in 200 ml (50 mM NaHCO3; 1% SDS). DNA-protein
complexes were reverse crosslinked by incubation in 0.3 M NaCl
and RNase A at 65uC overnight. DNA was isolated by phenol/
chloroform extraction and ethanol precipitation and resuspended in
50 ml H20. Genomic DNA obtained from chromatin immunopre-
cipitationswasanalyzed byrealtime PCR using SybrGreen (Roche)
and an Opticon Monitor II system (MJ Research). Primers for the
mouse p15 gene promoter were as follows: 59-CCA CCC CGC
CTA TTT GTC-39 and 59-CCG TGA GAT TGC TAC AGC C-
was calculated based on threshold cycle [C(t)] using the DC(t)
method and normalized to input samples. Results are expressed as
fold enrichment of FLAG immunoprecipitated samples relative to
IgG controls. Statistical significance was determined by paired
Student’s t-test. Expression of Smad3 and Sox17 inputs was assessed
by immunoblot using cell lysates from transfections done in parallel
to chromatin immunoprecipitations.
cells in the adult mouse lung. (A) Immunostaining for phospho-
Sox17 increases proliferation of respiratory epithelial
Sox17 and Lung Progenitor Cell
PLoS ONE | www.plosone.org 12May 2009 | Volume 4 | Issue 5 | e5711
histone H3 (pHH3) was performed on lung sections from adult
CCSPrtTA control (n=3) and CCSPrtTA/tetO-Sox17 (n=3) mice
maintained on Dox for 3 days. Total positive cells were quantified
from 21 random fields for morphometric analysis. The average
number of pHH3-positive cells per field was increased 4.74-fold in
lungs from CCSPrtTA/tetO-Sox17 mice relative to controls. Asterisk
indicates statistical significance determined by Student’s t-test
(p,0.05). (B) Dual immunofluorescence for Sox17 and pHH3 was
performed on lung sections from adult CCSPrtTA/tetO-Sox17 mice
maintained on Dox for 3 and 5 days and positive stained cells were
quantified from 20 random fields. Phospho-histone H3 was
coexpressed in 28% of the Sox17-expressing respiratory epithelial
Found at: doi:10.1371/journal.pone.0005711.s001 (0.18 MB TIF)
coexpress phospho-histone H3. Dual-label immunofluorescence
for phospho-histone H3 (pHH3; A) and Sca-1 (B) was performed
on lung sections from adult CCSPrtTA/tetO-Sox17 mice maintained
on Dox for 5 days. A rare subset of pHH3-positive cells (arrow)
colocalized with the Sca-1-expressing cells induced by Sox17
(arrow and arrowhead; B). Nuclei are stained with DAPI. Scale
bar, 20 mm.
A rare subset of Sox17-induced Sca-1 positive cells
Found at: doi:10.1371/journal.pone.0005711.s002 (0.36 MB TIF)
cells were transfected with FLAG-Smad3 and V5-tagged Sox17
full length or mutant contructs. After 24 h, cells were maintained
in the absence (A) or presence (B) of TGF-b1 (2 ng/ml) for 2 h and
dual-label immunofluorescence was performed for the V5 (green)
and FLAG (red) epitopes. Expression of all of the Sox17 constructs
colocalized with Smad3 in the nucleus. Nuclei are stained with
DAPI (blue). Scale bar, 20 mm.
Found at: doi:10.1371/journal.pone.0005711.s003 (2.27 MB TIF)
Sox17 colocalizes with Smad3 in the nucleus. MLE15
The authors would like to thank Susan Wert, Dave Loudy, Valerie
Besnard, Sheila Bell, and Debora Sinner for their technical support.
Conceived and designed the experiments: AWL. Performed the experi-
ments: AWL. Analyzed the data: AWL ARK JAW. Contributed reagents/
materials/analysis tools: JW AMZ. Wrote the paper: AWL JAW.
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