C. L. Scott, D. J. Walker, E. Cwiklinski, C. Tait, A. R. Tee and S. C.

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Control of HIF-1␣ and vascular signaling in fetal lung involves cross talk
between mTORC1 and the FGF-10/FGFR2b/Spry2 airway branching
periodicity clock
C. L. Scott,1 D. J. Walker,1 E. Cwiklinski,1 C. Tait,1 A. R. Tee,2 and S. C. Land1
1
Centre for Cardiovascular and Lung Biology, Division of Medical Sciences, Ninewells Hospital and Medical School,
University of Dundee, Dundee, Scotland; and 2Institute of Medical Genetics, Cardiff University, Heath Park, Cardiff, Wales,
United Kingdom
Submitted 5 October 2009; accepted in final form 1 July 2010
Scott CL, Walker DJ, Cwiklinski E, Tait C, Tee AR, Land SC.
Control of HIF-1␣ and vascular signaling in fetal lung involves cross talk
between mTORC1 and the FGF-10/FGFR2b/Spry2 airway branching
periodicity clock. Am J Physiol Lung Cell Mol Physiol 299: L455–L471,
2010. First published July 9, 2010; doi:10.1152/ajplung.00348.2009.—
Lung development requires coordinated signaling between airway and
vascular growth, but the link between these processes remains un-
clear. Mammalian target of rapamycin complex-1 (mTORC1) can
amplify hypoxia-inducible factor-1␣ (HIF-1␣) vasculogenic activity
through an NH2-terminal mTOR binding (TOS) motif. We hypothe-
sized that this mechanism coordinates vasculogenesis with the fibro-
blast growth factor (FGF)-10/FGF-receptor2b/Spry2 regulator of air-
way branching. First, we tested if the HIF-1␣ TOS motif participated
in epithelial-mesenchymal vascular signaling. mTORC1 activation by
insulin significantly amplified HIF-1␣ activity at fetal PO2 (23 mmHg)
in human bronchial epithelium (16HBE14o-) and induced vascular
traits (Flk1, sprouting) in cocultured human embryonic lung mesen-
chyme (HEL-12469). This enhanced activation of HIF-1␣ by
mTORC1 was abolished on expression of a HIF-1␣ (F99A) TOS-
mutant and also suppressed vascular differentiation of HEL-12469
cocultures. Next, we determined if vasculogenesis in fetal lung in-
volved regulation of mTORC1 by the FGF-10/FGFR2b/Spry2 path-
way. Fetal airway epithelium displayed distinct mTORC1 activity in
situ, and its hyperactivation by TSC1⫺/⫺ knockout induced wide-
spread VEGF expression and disaggregation of Tie2-positive vascular
bundles. FGF-10-coated beads grafted into fetal lung explants from
Tie2-LacZ transgenic mice induced localized vascular differentiation
in the peripheral mesenchyme. In rat fetal distal lung epithelial
(FDLE) cells cultured at fetal PO2, FGF-10 induced mTORC1 and
amplified HIF-1␣ activity and VEGF secretion without induction of
ERK1/2. This was accompanied by the formation of a complex
between Spry2, the cCBL ubiquitin ligase, and the mTOR repressor,
TSC2, which abolished GTPase activity directed against Rheb, the G
protein inducer of mTORC1. Thus, mTORC1 links HIF-1␣-driven
vasculogenesis with the FGF-10/FGFR2b/Spry2 airway branching
periodicity regulator.
lung development; epithelium; mesenchyme; hypoxia; rheb; tuberous
sclerosis complex
HYPOXIA IS AN IMPORTANT PHYSIOLOGICAL context for fetal devel-
opment, but its role in organ morphogenesis is not well under-
stood. In the embryonic lung, low PO2 favors airway branching
and bud formation (18, 19, 54), yet a critical morphogenic role
Address for reprint requests and other correspondence: S. C. Land, Centre
for Cardiovascular and Lung Biology, Division of Medical Sciences,
MACHS2 Level 4 Mailroom, Ninewells Hospital and Medical School, Univ.
of Dundee, Dundee, DD1 9SY Scotland, United Kingdom (e-mail: s.c.land
@dundee.ac.uk).
http://www.ajplung.org 1040-0605/10 Copyright © 2010 the American Physiological Society
Am J Physiol Lung Cell Mol Physiol 299: L455–L471, 2010.
First published July 9, 2010; doi:10.1152/ajplung.00348.2009.
for hypoxia is unlikely, as branched airway growth occurs at
higher oxygen tensions (7, 37, 46, 49, 54). Vascular branching,
by contrast, is almost completely dependent on the low PO2 of
the fetal lung and is abolished by experimental increases in
Downloaded from ajplung.physiology.org on September 30, 2010
oxygen tension (1, 22, 54). Despite these different sensitivities
to oxygen, airway and vascular systems develop in tandem to
form an interwoven tubular network with a shared semifractal
pattern of branching to the level of the respiratory acinus (57).
Thus, although hypoxia is necessary for proper lung develop-
ment, its effects are secondary to the signaling network that
links airway and vascular growth together.
Airway branching morphogenesis begins by evagination of
the laryngo-tracheal groove into the surrounding mesenchyme
tissue. After dividing into two distal buds, a series of sequential
branching events occurs to form the conductive airways of the
respiratory tree. Outgrowth of the airways is induced by
fibroblast growth factor-10 (FGF-10), which is transiently
expressed in the mesenchyme tissue surrounding each prospec-
tive bud. This is believed to activate Ras/Raf/MEK1 signaling
to the extracellular-signal regulated kinases 1 and 2 (ERK1/2)
through its receptor, FGFR2b, expressed in the airway epithe-
lium (7, 52, 53). Negative feedback of this pathway is
mediated by Sprouty2 (Spry2), whose activity is induced as
an additional, but time-delayed, response to FGFR2b/
ERK1/2 activation (37, 53). Thus, the temporal, spatial, and
concentration-dependent activation of FGFR2b by FGF-10
and inhibition by Spry2 control the length of each branch
and the location of bifurcations. This mechanism acts as an
airway branching periodicity regulator or “clock” that de-
fines the semifractal branching pattern of the conducting
airways in the lung (40, 55).
The mechanism that links vasculogenesis to this process
remains unknown. The differentiation of mesenchyme progen-
itor cells into vascular tissue is initiated by the secretion of
vascular endothelial growth factor (VEGF) from the airway
epithelium. VEGF expression is regulated by hypoxia-induc-
ible factors (HIFs), a family of transcription factors whose
hypoxia-activated ␣-subunit is required for transcriptional ac-
tivity. Three HIF-␣ isoforms are expressed in the fetal lung
(21, 47). HIF-1␣ occurs in the branching epithelium, and its
homozygous knockout in mice causes severe vascular defects
and death at embryonic day (E) 10.5 (29). HIF-2␣ occurs in the
epithelium and the interstitium. Its knockout does not affect the
growth of the conducting vasculature but produces vascular
defects during alveolar septation (10). HIF-3␣ is a truncated
isoform that lacks the COOH-terminal transactivation domain
present in HIF-1␣ and 2␣ and so may act as a repressor of these
L455
L456 mTORC1 AND VASCULAR SIGNALING IN FETAL LUNG
transcription factors (6). Although it is present in the develop-
ing lung, its function remains unknown.
Of these, HIF-1␣ plays the major role in early pulmonary
vasculogenesis and so essentially defines the growth of the
conducting vasculature (21, 29, 47). HIF-1␣ activity is regu-
lated by O2-dependent prolyl-hydroylase domain proteins
(PHD1, 2, 3) and the asparagine hydroxylase factor inhibiting
HIF (FIH-1) (reviewed in Ref. 28). In normoxia, these en-
zymes silence HIF signaling by catalyzing the hydroxylation of
proline residues 402 and 564, and so allow the proteasomal
degradation of HIF-1␣ by the von Hippel-Lindau E3 ubiquitin
ligase. In addition, FIH-1 catalyzes the hydroxylation of aspar-
agine 803 and blocks the interaction of the HIF-1␣ transacti-
vation domain with the p300 transcriptional coactivator. In
hypoxia, degradation of PHD proteins by another E3 ubiquitin
ligase, SIAH (seven in absentia homolog), allows rapid stabi-
lization, nuclear translocation, and transactivation of HIF-1␣
leading to the expression of hypoxia-regulated proteins (45).
PHD proteins critically regulate HIF-1–3␣ stability during
lung development, as their inhibition augments HIF target gene
expression and promotes blood vessel growth and formation of
the blood gas barrier (2, 3, 22). This effect is so profound that
vascular signaling and growth can be sustained in explants
cultured in extreme hyperoxia [95% O2, (20)]. These studies
fail to explain, however, the temporal and spatial regulation of
HIF activity that would be necessary to form a branched
vascular and airway tubule network. One possibility is that
oxygen gradients radiating from the fetal pulmonary vascula-
ture may result in subtle differences in PHD1–3 activity that
coordinate HIF transcription between the two systems. In the
fetal lung, however, any such gradient would be shallow [⬃15
mmHg (20 ␮M)]: difference between the PO2 of the umbilical
vein (⬃30 mmHg) and the PO2 of the amniotic fluid (⬃15
mmHg; Ref. 31) and ⬃10-fold below the Km O2 of the PHD
enzymes (230 –250 ␮M; Ref. 26). A second, more plausible,
scenario suggests that morphogens that are transiently ex-
pressed in the growing tip of the airway bud locally regulate
HIF-1␣ and vascular signaling. Thus, the low PO2 of the fetal
lung maintains a baseline level of HIF-1␣ stability and activity
that is regionally amplified by growth-regulating signals. Such
events might be considered to confer contextual regulation
over HIF proteins, directing their activity subsequent to PHD
and FIH induction.
We have discovered that the mammalian target of rapamycin
(mTOR), a nutrient-sensitive regulator of growth and differen-
tiation, amplifies PHD/FIH-induced HIF-1␣ activity by inter-
acting with a conserved mTOR signaling (TOS) motif within
the HIF-1␣ PAS-A domain (33). This interaction, which re-
quires the mTOR complex 1 (mTORC1) adaptor protein,
raptor, promotes the binding of the p300 transcription initiation
complex to the HIF-1␣ COOH-terminal transactivation domain
(CTAD) and substantially amplifies HIF-1␣-evoked expres-
sion of vascular genes such as VEGF. HIF2␣ lacks this TOS
motif, and HIF-3␣ lacks the CTAD, thus the positive effect of
this interaction on hypoxic gene expression is specific to
HIF-1␣. This complements a growing number of studies that
show HIF-1␣ transactivation to be similarly regulated by other
kinases (9, 20, 41, 48). Here, we aimed to determine if
mTORC1 could, in principle, regulate HIF-1␣ and vasculo-
genic signaling in a cell line culture model of the lung epithe-
lial-mesenchymal interface. We then investigated if this inter-
AJP-Lung Cell Mol Physiol • VOL
action influenced vasculogenesis in the fetal lung in vivo and
whether it was subject to regulation by airway branching
morphogens in primary cultures of fetal distal lung epithelium
(FDLE). Our results demonstrate that FGF-10 induces
mTORC1 activity via Spry2 in fetal airway epithelium and that
this amplifies HIF-1␣-evoked vascular signaling beyond the
level induced by exposure to fetal PO2 alone. We postulate a
model whereby mTORC1 coordinates HIF-1␣ vascular signal-
ing with the FGF-10/FGFR2b/Spry2 airway branching period-
icity clock.
MATERIALS AND METHODS
Chemicals and reagents. Recombinant human FGF-10 was from
R&D Systems (Abingdon, UK). Rapamycin, LY-294002, and U0126
were obtained from Merck Biosciences (Nottingham, UK). Antibod-
ies against Flk1 (A-3), -Flt-1, and VEGF (A-20) were from Santa
Cruz Biotechnology; Tie2 (ab24859) and Spry2 (ab50317) were from
Abcam (Cambridge, UK); and S6K1, S6K1 phospho-T389, ERK1/2,
Downloaded from ajplung.physiology.org on September 30, 2010
ERK1/2 phospho-(T202/Y204, T183/Y185), TSC1, and TSC2 were
from Cell Signaling Technology (Danvers, MA). Antibodies against
epitope tags and all other reagents (unless stated) were purchased
from Sigma-Aldrich (Dorset, UK).
Plasmids and molecular biology. NH2-terminal flag-tagged pRK7-
Rheb, pRK7-HA-HIF-1␣, and pRK7-HA-HIF-1␣(F99A) and the
HIF-1␣ luciferase reporter gene, pHRE3-TK-GL2, were generated as
described (33). pRL incorporating the minimal thymidine kinase (TK)
promoter was from Promega. Kinase dead (D2357E) mTOR was
kindly provided by Dr. Stuart Schreiber (Harvard Univ.), and pXJ40-
FLAG-human(h)Spry2, pXJ40-FLAG-Spry2 (Y55F), and pXJ40-HA-
Rheb were kindly provided by P. Yusoff and G. Guy (Proteos,
Singapore).
Culture of cell lines. The diploid human embryonic lung mesen-
chymal fibroblast (HELMF) cell line, HEL-12469, was obtained from
the European Collection of Cell Cultures. An immortalized human
bronchial airway epithelial cell line [16HBE14o-, termed HBE here
(12)] was obtained from Dr. D. Gruenert, California Pacific Medical
Center, San Francisco, CA. Both were routinely cultured in DMEM
supplemented with Ham’s F-12 nutrient mix (DMEM/F-12) and 8.5%
FCS from Invitrogen (Paisley, UK) in a humidified atmosphere
containing 21% O2 and 5% CO2. For coculture experiments, HELMF
were seeded into Costar six-well culture dishes at a density of 5 ⫻ 105
cells/ml⫺1 and allowed to establish for 4 days before each experiment.
HBE, seeded separately at a density of 2.5 ⫻ 105 cells·ml⫺1 onto
2.4-cm diameter Costar Transwell permeable supports, were other-
wise treated identically. Sixteen hours before the start of each exper-
iment, the cells were placed into serum-free MEM (SFM), and then
the HBE and HELMF cells were combined under the experimental
conditions described in each figure by placing Transwell filters into
the wells containing the HELMF cells.
Primary culture of rodent fetal lung cells and organ explants.
FDLE were isolated from gestation day 19 rat fetuses as described
previously (5, 32). All procedures conformed with the Animals
(Scientific Procedures) Act, 1986, UK. FDLE were seeded in DMEM/
F-12 containing 8.5% FCS onto Transwell permeable supports at a
density of 5 mg of packed cell wt·ml⫺1, whereas fetal mesenchymal
lung fibroblasts (FMLF) obtained as a byproduct of the FDLE isola-
tion procedure were seeded in the same media at a density of 1 ⫻ 106
cells/well in six-well plastic dishes. Twenty-four hours after isolation,
FDLE and FMLF were placed into DMEM/F-12 supplemented with
2% charcoal and ion-exchange resin-stripped serum (stripped serum)
and placed at fetal PO2 until use.
Lung explants from gestation day 13 or 14 Sprague-Dawley rat
fetuses or gestation day 12 Tie2-LacZ mouse fetuses were cultured in
DMEM/F-12 supplemented with 2% stripped serum on Whatman
polycarbonate filter supports and cultured at fetal PO2. For bead
299 • OCTOBER 2010 • www.ajplung.org
 mTORC1 AND VASCULAR SIGNALING IN FETAL LUNG
experiments, heparin-coated acrylic beads (Sigma) were prepared as
described in Supplemental Fig. S1 and grafted into an incision in the
peripheral mesenchyme (Supplemental material for this article is
available online at the Journal website). In all cases, digital images
were captured at the start and end of each experiment using a Leica
DC 300F digital camera mounted on an MZ FLIII binocular micro-
scope under identical magnification settings (Leica Camera, Milton
Keynes, UK).
All cultures were maintained at 37°C in a humidified atmosphere
containing 5% CO2. The oxygen content of the atmosphere was
maintained at the level stated in each figure using a MACS VA500
Microaerophilic Workstation (Don Whitley Scientific, Shipley, West
Yorkshire, UK).
Transfection and cell lysis. HBE, HELMF, and FDLE were trans-
fected at 60 – 80% confluency using Lipofectamine 2000 (Invitrogen,
Paisley, UK) according to the manufacturer’s instructions. To mea-
sure transfection efficiency, HBE and HELMF cells were transfected
with a CMV-driven GFP reporter gene, and the number of GFP
fluorescing cells was expressed as a proportion of DRAQ-5-stained
nuclei. Transfection efficiency was routinely ⬎95% for both HELMF
and HBE. pRL was used to assess even transfection of FDLE and was
not found to vary significantly between experiments. To create ly-
sates, cells or explants were washed twice in PBS and then harvested
in kinase lysis buffer containing Complete Protease Inhibitor Cocktail
tablets (Roche) as described previously (33).
Measurement of HIF-1␣ transcriptional activity. HIF-1␣ transcrip-
tional activity in HBE was measured by luciferase assay as described
previously (33). For HBE and HELMF cells, medium was replaced
after transfection with fresh SFM, and cells were allowed to acclima-
tize to the respective experimental PO2 for 16 h. Subsequent treat-
ments with insulin or rapamycin were then performed for 6 h before
lysis. Experiments involving FGF-10 and/or Spry2 overexpression
varied from this format as described in the legend to the relevant
figures. In all cases, luciferase activity was corrected for protein
content and expressed as change relative to the control group stated in
the figure legend. HIF-1␣ transcriptional activity in FDLE was mea-
sured using the same procedure in conjunction with pRL (transfection
ratio of 8:1, pHRE3-TK-GL2:pRL) to control for potential variation in
transfection efficiency between preparations. These results were ad-
justed for protein content, normalized against Renilla expression, and
expressed as fold change relative to the stated control group.
Quantitative PCR. Detection of pluripotency marker genes in
HELMF (Oct4, Sox2, and NANOG) and FGFR2b and Spry2 in HBE
was performed as described in Supplemental Figs. S2 and S4.
Immunoprecipitation and Western blotting. Total protein content of
cell lysates was determined using the BioRad protein assay. For
immunoprecipitations (IP), 150 ␮g of total protein was precleared for
1 h at 4°C in nonconjugated protein G-sepharose. IP was then
performed for a further 3 h using cCBL or FLAG antibodies conju-
gated to protein G-sepharose beads as appropriate. Beads were then
washed twice with buffer A [50 mM HEPES (pH 7.4), 100 mM NaCl,
10 mM MgCl2, 1 mg·ml⫺1 BSA, 1 mM dithiothreitol, and 1% Triton]
and buffer B [50 mM HEPES (pH 7.4), 100 mM NaCl, 10 mM MgCl2,
and 0.1% Triton] with Complete Protease Inhibitor Cocktail (Roche)
included in all buffers. IP complexes eluting with the beads were
dissolved in Laemmli sample buffer and processed by Western blot-
ting. Western blots were performed on either cell lysates or IP samples
by SDS-PAGE and transferred to nitrocellulose as described before
(33). Even transfer onto nitrocellulose was confirmed using Ponceau
S and by reference to actin.
VEGF ELISA. ELISA for human or rat VEGF-A was performed as
described before (29) using the DuoSet ELISA system from R&D
Systems.
Sprouting assay. HELMF were induced to form spheroids by
nonadherent culture on soft agar. Spheroids of 400- to 600-␮m
diameter were placed into growth factor-reduced Matrigel (BD Bio-
sciences), and HBE-conditioned medium was laid above the collagen
AJP-Lung Cell Mol Physiol • VOL
L457
dome. Images of spheroids were collected at 24-h intervals, and the
compound sprout length was calculated at 48 h. Spheroid images were
digitally separated into quadrants of equal size, and sprout length was
measured using Scion Image 4.0.2 software at five evenly spaced
intervals per quadrant. Measurements were pooled from four sphe-
roids for each treatment, and each series of treatments was derived
from independent experiments.
Measurement of airway surface complexity and epithelial cell
height. Airway surface complexity [ASC; perimeter (mm)/公area
(mm2)] was determined from digital images by tracing the dimensions
of the airway perimeter as described (Ref. 46 and Refs. therein).
Epithelial height was measured from images of hematoxylin and
eosin-stained sections generated from paraformaldehyde-fixed and
paraffin wax-embedded gestation day 14 explants after 48 h of culture.
Ten measurements were taken at even intervals around each airway
cross-section using an Openlab camera-calibrated micrometer. Fre-
quency distribution of epithelial cell height is represented as box plots
and are compared with equivalent measurements from gestation day
16 fetal rat lungs.
Immunohistochemistry. Paraformaldehyde-fixed fetuses of wild-
Downloaded from ajplung.physiology.org on September 30, 2010
type (WT) and homozygous TSC1⫺/⫺ mice (E12.5) were kindly
provided by Prof. Jeremy Cheadle (Univ. of Cardiff, Wales, UK). A
Histostain Plus broad spectrum DAB kit was used to visualize anti-
body-specific staining. Sections incubated in preimmune serum were
treated as controls. Antigen retrieval and quenching of endogenous
peroxide activity were performed according to the manufacturer’s
instructions. Images were obtained using a Zeiss Axiovert microscope
and Improvision 5 software.
Rheb-GAP assay. The ratio of GTP-to-GDP-bound Rheb was
determined in FDLE as described by Tee et al. (51). Briefly, FDLE
transfected with FLAG-Rheb were incubated at fetal or ambient PO2
for 6 h in medium containing FGF-10 at the indicated concentrations
and 125 kBq/cm⫺2 [32P]O4 in DMEM containing 2% charcoal-
stripped medium. After lysis, FLAG-Rheb was immunoprecipitated,
and the proportion of eluted GTP or GDP was determined by TLC on
PEI cellulose using 0.5 MKH2PO4 (pH 3.3) as the mobile phase. The
migration position of radiolabeled spots (Rf; ratio of spot migration:
mobile phase migration) was visualized using a phosphorimager and
were 0.65 (GTP) and 0.81 (GDP), matching equivalent values calcu-
lated from Tee et al. (51).
Statistics. All values are expressed as means ⫾ SE unless otherwise
stated in the figure legend. The value of n for HBE/HELMF studies
was determined as the number of experimental repeats on cells of
different passage. For FDLEs and explants, n was determined as the
number of pregnant dams used to generate independent preparations
unless otherwise stated in the figure legend. In all cases, Student’s
t-test was used to assess significance between paired single treatments,
and one-way ANOVA with post hoc significance assessed with the
Tukey or Dunn test was used for multiple comparisons. A P value ⬍
0.05 was considered to be statistically significant.
RESULTS
Part 1: Role of the HIF-1␣ TOS motif in vascular signaling.
The initial goal of our study was to establish a culture model
that could be used to explore the requirement for the HIF-1␣
TOS motif in vascular signaling. For ease of culture, transgene
expression, and relevance to the airway environment, we se-
lected HBE cells as a well-characterized model of the bronchial
epithelium (12) and HELMF cells as a fetal lung-derived
mesenchymal cell line with progenitor cell characteristics (con-
stitutive expression of the pluripotency markers, Oct4, Sox2,
and NANOG, under basal conditions. See Supplemental Fig.
S2). A bicameral chamber was used to determine if HBE
grown on a permeable support in the upper half of the chamber
displayed vasculogenic activity (HIF-1␣ activity, VEGF re-
299 • OCTOBER 2010 • www.ajplung.org
L458 mTORC1 AND VASCULAR SIGNALING IN FETAL LUNG
lease) when maintained at the PO2 of the fetal lung (23 mmHg).
HELMF cells were maintained beneath the permeable support
and probed for the appearance of vascular markers (Flk-1
expression, sprouting) (Fig. 1A). HIF-1␣ activity was deter-
mined by transfecting each culture with a HIF-driven reporter
gene, and mTOR-dependent modulation of this response was
determined by treating cells for 6 h with 20 nM insulin in
combination with the selective mTOR inhibitor, rapamycin.
LY-294002 (50 ␮M) was used to block the activity of PI3-
kinase, which lies upstream of the mTORC1 complex. Con-
centration of insulin and LY-294002 were based on the effec-
tive doses shown to respectively induce and inhibit a PI3K-
regulated Na⫹ current in H441 human bronchial epithelial cells
(27). Choice of 100 nM rapamycin is based on the recom-
mended concentration for these compounds to achieve selec-
tive and complete inhibition of mTORC1 (4). Culture of
HELMF at fetal PO2 induced HIF-1␣ activity 2.5-fold above
that at alveolar PO2. Insulin had no observable effect; however,
rapamycin and LY-294002 each produced a significant block-
ade of this endogenous activity (Fig. 1B). The scope for
HIF-1␣ activation at fetal PO2 (25-fold) was substantially
greater in HBE compared with HELMF and was inhibited by
⬃50% by either rapamycin or LY-294002. Insulin augmented
this activity by a further fourfold. Rapamycin and LY-294002
suppressed this induced activity to the level observed without
insulin treatment (Fig. 1C). VEGF-A secretion was signifi-
cantly induced by culture at fetal PO2 and was augmented
further by insulin. Rapamycin and LY-294002 lowered the
insulin-evoked VEGF-A secretion to the spontaneous level
induced by culture at fetal PO2 alone (Fig. 1D).
The VEGF-A receptor, Flk1, promotes angioblast differen-
tiation and is a critical signaling component of epithelial-
endothelial cross talk, which is necessary for proper branching
morphogenesis (14). We therefore examined whether HELMF
maintained in coculture with HBE displayed Flk-1 expression
in response to culture at fetal PO2 or insulin (Fig. 1E). Both
conditions were found to induce Flk-1 protein expression in
HELMF, and this corresponded with increased ␣S6K1 (T389)
phosphorylation, a specific substrate of mTORC1. Rapamycin
and LY-294002 abolished this activation and led to a reduction
in Flk-1 expression. HELMF cells subjected to the same
treatment but in the absence of HBE did not express Flk-1
protein.
An intact HIF-1␣ TOS motif is required for vasculogenic
trophic signaling between HBE and HELMF. mTORC1 can
amplify HIF-1␣ activity by interacting with a mTOR signaling
(TOS) motif within the HIF-1␣ PAS-A domain. This effect is
independent of the prolyl hydroxylase regulation of HIF-1␣ but
promotes the binding of the p300 translation initiation complex
to its COOH-terminal transactivation domain (33). We there-
fore determined if mutation of the first phenylalanine residue of
the TOS motif (F99A) interfered with vasculogenic signaling
from HBE to HELMF. Overexpression of the upstream acti-
vating G protein of mTOR, Rheb, together with rapamycin
treatment were used to further specify observed effects to the
mTOR pathway. Flk-1 expression and mTORC1 activity
(␣S6K1-T389 phosphorylation) was induced in HELMF ex-
posed to conditioned medium from the HBE cells coexpressing
WT HA-HIF-1␣ and Rheb (Fig. 2, A and B). Although this
Flk-1 expression was insensitive to rapamycin, HELMF treated
with conditioned medium from HBE expressing the HIF-1␣
AJP-Lung Cell Mol Physiol • VOL
TOS mutant failed to express Flk-1 despite mTORC1 activa-
tion by Rheb. Reporter gene assays conducted in parallel
showed that Rheb augmented HIF-1␣ activity in cells overex-
pressing WT but not TOS-mutant HA-HIF-1␣.
Sprouting assays were used to measure the potentiation of
vascular outgrowth from HELMF spheroids induced by con-
ditioned medium from HBE treated as in Fig. 2A. Sprout
branch length was significantly elevated from spheroids ex-
posed to medium from HBE transfected with both WT-HIF-1␣
and Rheb (Fig. 2C). Exposure to medium from HBE express-
ing the HIF-1␣ TOS mutant suppressed sprouting, and, in both
cases, was almost completely abolished by rapamycin.
Part 2: mTOR-dependent regulation of vascular signaling in
the fetal lung. Experiments in Part 1 establish that HBE/
HELMF cells can be used to explore vascular signaling in vitro
and establish the principle that mTORC1 positively modulates
HIF-1␣-mediated vasculogenesis through the NH2-terminal
TOS motif. We next explored the role of mTORC1 in fetal
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lung development and asked if it could act as a critical point of
coordination between vascular signaling with the FGF-10/
FGFR2b/Spry2 airway branching periodicity regulator.
mTORC-1 is active in airway epithelium and participates in
airway morphogenesis. The pattern of mTORC1 activity in
gestation day 19 fetal rat lungs in situ was determined from the
distribution of ␣S6K1-T389 phosphorylation. Nonimmune se-
rum was used as a control. Intense staining was apparent in the
nascent airway endodermal epithelium and in the mesothelium
lining the lung periphery (Fig. 3). ␣S6K1-T389 phosphoryla-
tion was not uniform around the endodermal tubes appearing
more intense within the airway tip region.
These results suggest that endogenous factors induce
mTORC1 in the endodermal epithelium of the developing
airway and that mTORC1 may participate in lung morphogen-
esis. This was confirmed in isolated pseudoglandular stage
(gestation day 14) rat lung explants where inhibition of endog-
enous mTORC1 activity with rapamycin (0.1 ␮M) signifi-
cantly augmented airway epithelial surface folding complexity
(ASC, a measure of branching tendency) and promoted epithe-
lial flattening (an indicator of epithelial cell maturation) at fetal
PO2 (Fig. 4, A–C). The rise in ASC appeared to be accompa-
nied by a loss of mesenchyme mass; therefore, we examined
the effect of PO2 and mTOR activity on rat fetal mesenchymal
lung fibroblast (FMLF) proliferation. Growth rate was greater
at fetal compared with alveolar PO2 and was inhibited by either
rapamycin or transfection with a kinase dead (D2357E) mutant
of mTOR (Fig. 4D) suggesting an additional role for both
hypoxia and mTOR in regulating mesenchyme cell density.
FGF-10 induces discrete expression of Tie2 in fetal
mouse lung explants. These experiments demonstrate that
mTORC1 participates in lung morphogenesis, and our ex-
periments in HBE/HELMF predict that growth factor activa-
tion of mTORC1 will induce vasculogenic signaling in the fetal
lung. We therefore explored whether FGF-10, a central inducer
of airway outgrowth, induced vascular differentiation in mu-
rine fetal lung organ explants. Beads soaked in FGF-10 (Sup-
plemental Fig. S1) were grafted into the peripheral mesen-
chyme of gestation day 12 fetal lung explants from mice
bearing a Tie2-driven LacZ reporter gene. Explants were
cultured at fetal PO2 for 48 h and then stained to reveal the
pattern of Tie2 gene activation. This expression was absent
from the peripheral mesenchyme surrounding the distal airway
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Fig. 1. mTOR amplifies hypoxia-inducible factor-1␣ (HIF-1␣) activity and vasculogenic signaling in HBE-HELMF cocultures. A: the bicameral culture method
for examining epithelial-mesenchymal trophic signaling. Human bronchial epithelial cells (HBE) were cultured on permeable supports suspended above a
monolayer of HEL-12469 human embryonic lung fibroblast cells (HELMF) in serum-free MEM (SFM). Single culture was performed by culturing HBE and
HELMF in SFM separately. B: HIF-1␣-driven luciferase reporter gene expression in HELMF at alveolar (100 mmHg) or fetal (23 mmHg) PO2. Values are
expressed as fold change over untreated cells at alveolar PO2, n ⫽ 4, *P ⬍ 0.05. C: HIF-1␣ activity in HBE; n ⫽ 4, *P ⬍ 0.05. D: VEGF secretion in HBE
is elevated at fetal PO2 and is inhibited by rapamycin and LY-294002. VEGF production was undetectable from HELMF cells (data not shown); *P ⬍ 0.05, n ⫽
4. E: Flk-1 expression at fetal PO2 in HELMF cocultured with HBE (top) or alone (HELMF single culture, bottom). Representative of 3 independent experiments.

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Fig. 2. The HIF-1␣ F99A TOS mutation suppresses trophic signaling between HBE and HELMF. A, top blots: conditional phenotype of HBE cells used to
generate conditioned medium for HELMF culture. Rapamycin (100 nM) was used to suppress mTOR signaling. Bottom blots: expression of Flt1 and Flk1
[nascent (150 kDa) and glycosylated (200 kDa) forms shown] in HELMF following 18-h culture in HBE-conditioned medium. Blots are representative of 4
experiments. B: top histogram shows the actin-corrected change in Flk1 protein abundance in HELMF cultured in HBE-conditioned SFM. *P ⬍ 0.05, n ⫽ 5.
Bottom histogram shows the activity of HIF-1␣ in the HBE used to generate the conditioned medium. Activity is expressed as fold change over empty vector
(pRK7) control. *P ⬍ 0.05 relative to control group; n ⫽ 5. C: sprouting from HELMF spheroids in Matrigel following 48-h culture in medium obtained from
the HBE in A. Scale bar: 182 ␮m. Box plots at bottom show the data distribution of sprout length. (From bottom: 5th percentile, smallest nonoutlier, lower
quartile, median, top quartile, 95th percentile. Bars indicate the data range.) *P ⬍ 0.05 relative to wild-type transfected control group; **P ⬍ 0.001 relative to
wild-type rheb-transfected group; n ⫽ 4. One-way ANOVA on ranks with Dunn’s post hoc test.

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Fig. 3. mTORC1 activity in the pseudoglandular rat lung in vivo. Gestation day 19 fetal rat lungs were fixed and stained with a phospho-antibody against the
mTORC1 substrate, p70S6K1-T389. A: low-power (⫻40) image of fetal lung in situ. p70S6K-T389 phosphorylation is apparent in the nascent airway epithelium
(brown stain). B: control section treated with nonimmune IgG at same magnification. C: mid-power (⫻200) image showing airway (a), mesenchyme (m), and
endothelial tube (et) structures. Note apparent polarization of mTORC1 activity around the tube (white arrows). D: high-power (⫻400) image showing mTOR
activity extending laterally from the epithelium indicating activity within a possible branching node. Localized activity in cells of the mesenchyme is evident
(black arrows). E: high mTOR activity in the mesothelial lung lining (mth), a site of FGF9 expression (56). White arrow indicates regional activity in an epithelial
lateral branch node. F and G: ⫻200 and ⫻400 images of sections treated with nonimmune IgG.

tips but was evident around the proximal airway and in the
nexus of airway branches. FGF-10-coated beads grafted into
the peripheral mesenchyme induced Tie2 gene activation
around the bead, which was not observed with PBS-soaked
control beads (Fig. 5). Note that the distortion of airway and
peripheral mesenchyme tissue around the bead position indi-
cates intimate contact between bead and tissue. This estab-
lishes the principle that a localized FGF-10 signal can promote
vascular differentiation of mesenchyme tissue at fetal PO2.
Homozygous knockout of TSC1⫺/⫺ causes aberrant vascular
growth in the fetal lung in situ. TSC1⫺/⫺ produces a hyperac-
tive mTOR phenotype that is fatal in mice from gestation day
13.5 (58). To determine if mTOR hyperactivity produces a
hypervascular lung, we performed immunohistochemistry on
fixed sections of gestation day 12.5 from WT and TSC1⫺/⫺
mouse fetuses. As with pseudoglandular stage rat lung,
mTORC1 phosphorylation of ␣S6K1-T389 in WT fetal lungs
was distinct in airway epithelium and was particularly pro-
nounced at the growing tips of airway buds. VEGF staining
was confined to airway epithelium, and Tie2 angiopoietin
receptor expression was confined to discrete regions of mes-
enchymal tissue corresponding to nascent vascular bundles
(Fig. 6, A–C). TSC1⫺/⫺ mouse lungs also displayed apparently
normal airway growth; however, mTORC1 phosphorylation of
␣S6K1-T389 was homogenously distributed in the airway
epithelium. Similarly, VEGF expression was widely distrib-
uted throughout the lung and was accompanied by disaggre-
gation of Tie2-positive vascular bundles (Fig. 6, D–F).
FGF-10 regulates HIF-1␣ activity via mTORC1 in FDLE.
Having established that endogenous factors sustain mTORC1
activity in the fetal lung and that localized release of FGF-10
promotes vascular differentiation in mesenchyme, we next
tested the hypothesis that FGF-10 regulates mTORC1-depen-
dent HIF-1␣ and vascular signaling using primary cultures of
FDLE at either fetal or alveolar PO2. By culturing FDLE on
permeable supports, we aimed to maintain apical/basolateral
polarization of these cells as shown before (5, 32, 42). FGF-10
produced a dose-dependent, rapamycin-sensitive activation of
HIF-1␣ and VEGF secretion at fetal PO2, which was absent at
alveolar PO2 (Fig. 7A). mTORC1 activity was dose depen-
dently induced by FGF-10 at fetal PO2 but was not sustained at
FGF-10 concentrations ⱖ0.01 ␮g·ml⫺1 at alveolar PO2 (Fig. 7,
B and C). As FGF-10 is reported to induce ERK1/2 signaling
in adult murine airway epithelia (53) and can, in principle,

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L462 mTORC1 AND VASCULAR SIGNALING IN FETAL LUNG

Fig. 4. Endogenous mTOR activity is critical

for morphogenesis of fetal rat lung explants.
A: influence of culture PO2 and rapamycin on
airway surface complexity [ASC; perimeter/
公airway area (46)] in lung explants from
gestation day (G) 14 fetal rats. Circles, Con-
trol; triangles, 0.1 ␮M rapamycin. *P ⬍ 0.05
23 mmHg control vs. 100 mmHg control,
n ⫽ 15; †P ⬍ 0.05 rapamycin vs. control at
23 mmHg, n ⫽ 15. B: box plots showing
data distribution of epithelial thickness in

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G14 explants maintained for 48 h in the
presence or absence of rapamycin (0.1 ␮M)
at the indicated PO2. Measurements from
G16 lung in situ are included for comparison
(from bottom: 5th percentile, smallest non-
outlier, lower quartile, median, top quartile,
95th percentile. Bars indicate the data
range). *P ⬍ 0.05, n ⫽ 4. One-way ANOVA
on ranks with Dunn’s post hoc test. C: en-
dogenous activity of mTOR [␣S6K-(P)-
T389] in explants cultured in SFM for 48 h
at fetal or alveolar PO2. Insulin (20 nM) was
used as a positive control, and rapamycin
(0.1 ␮M) was used to block mTOR activity.
Representative of 4 replicates. D: growth
rate of fetal rat lung fibroblasts determined
over 48 h at fetal or alveolar PO2 in the
presence of rapamycin or following overex-
pression of a kinase dead (D2357E) mutant
of mTOR. *P ⬍ 0.05 relative to 23 mmHg
treatment, n ⫽ 6.

activate mTORC1 by repressing TSC1/2, we measured
ERK1/2 phosphorylation in parallel with mTORC1. Basal
ERK1/2 phosphorylation was low in FDLE, and enhanced blot
images showed it to be further suppressed by FGF-10 (Fig. 7,
B and C). ERK1/2 activity was not required for mTORC1
activation by FGF-10 at fetal PO2 as S6K1-T389 phosphoryla-
tion was sustained in the presence of the MEK1 inhibitor,
U0126 (Fig. 7D). Notably, the low endogenous activity of
ERK1/2 in FDLE was echoed in perinatal rat lung, which
showed little measurable ERK1/2 activity in gestation days 19
and 21 homogenates but a significant activation during the
early postnatal period (Supplemental Fig. S3).
Parallel studies in HBE cells revealed a similar induction of
HIF-1␣ activity by FGF-10, which was significantly inhibited
by rapamycin but declined at the highest concentrations of
FGF-10 (Supplemental Fig. S4). In contrast to our findings in
FDLE, the induction of mTORC1 by FGF-10 was accompa-
nied by a transient activation of ERK1/2 and was significantly
repressed by U0126 (Supplemental Fig. S5).
FGF-10 destabilizes Spry2 and promotes interaction be-
tween cCBL and TSC2 in FDLE. Current models of lung
development suggest that FGF-10-FGFR2b signaling is antag-
onized by Spry2 and that this mechanism regulates airway
branching periodicity. Given the lack of apparent control of
mTORC1 by ERK1/2 in FDLE, we focused on the role of
Spry2 as a potential regulator of the mTORC1-HIF-1␣ inter-
action. Spry2 was resolved in FDLE as a double band with
masses of 38.9 (full length) and 35.6 kDa (truncated) using an
NH2-terminal epitope antibody (Abcam, ab50317). FGF-10
induced a dose-dependent degradation of full-length Spry2 at

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 mTORC1 AND VASCULAR SIGNALING IN FETAL LUNG
both alveolar and fetal PO2 that correlated with the rise in
mTORC1 activation, HIF-1␣ reporter gene expression, and
VEGF production (Figs. 7, A and B, and 8A). The cleavage of
full-length Spry2 occurred by proteolysis as blockade of de
novo synthesis using cycloheximide led to its clearance within
3 h of addition of 0.1 ␮g/ml⫺1 FGF-10. This cleavage was
proteasomal as addition of MG132 abolished the truncated
form of Spry2 and stabilized the full-length form in the
presence of FGF-10 (Fig. 8, B and C).
To explain the association between Spry2 clearance and
mTORC1 activation, we investigated whether repression of
mTORC1 by TSC1/2 was dependent on cCBL, an E3 ubiquitin
ligase that binds to the phosphorylated Y55 residue of Spry2
(17). TSC2 consistently immunoprecipitated with cCBL, and
this association increased significantly on exposure to 0.1
␮g/ml⫺1 FGF-10 and the proteasomal inhibitor, MG132. Sur-
prisingly, Spry2 interaction with cCBL also increased with
FGF-10 treatment, suggesting that TSC2 preferentially associ-
ates with the intact Spry2-cCBL complex (Fig. 9A). When in
complex with TSC1, TSC2 inhibits mTORC1 assembly by
acting as a GTPase activating protein (GAP) towards Rheb;
therefore, we determined if the interaction between TSC2 and
cCBL/Spry2 corresponded with a decrease in Rheb-GAP ac-
tivity and a subsequent rise in GTP-bound Rheb (Fig. 9B).
FGF-10 tended to increase guanine nucleotide binding to
FLAG-Rheb expressed in FDLE at fetal PO2; however, the
proportion of radioactivity eluting with GTP was significantly
greater at 0.1 and 1 ␮g·ml⫺1 FGF-10 compared with controls
and is consistent with decreased TSC1/2 GAP activity.
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L463
Fig. 5. Vascular differentiation is induced by
FGF-10 in E12 fetal murine lung explants.
Beads coated in FGF-10 or PBS (control)
were grafted onto E12 murine fetal lung
explants from Tie2-LacZ transgenic mice.
Explants were maintained at fetal PO2 for 48
h, fixed, and then stained for LacZ reporter
expression. Grayscale images on left show
bead location and morphogenesis over 48 h;
color images on right show subsequent ex-
pression of Tie2-LacZ reporter gene with
bead excised. Bead position is indicated as: F
(FGF-10), P (PBS). Combined treatment
indicates PBS and FGF-10-coated beads
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grafted onto the same explant. Note lack of
Tie2-LacZ expression in the peripheral mes-
enchyme (PM) shown in bottom image.
Findings shown in these images were repli-
cated across explants isolated from 3 inde-
pendent litters.
FGF-10 and Sprouty2 regulate HIF-1␣ activity via
mTORC1 in HBE. The preceding results are perplexing be-
cause they suggest that Spry2 cleavage is necessary for
mTORC1 activation but that FGF-10 promotes a stable inter-
action between Spry2, cCBL, and TSC2. To discriminate
between these outcomes, we reasoned that overexpression of
WT Spry2 would quench the cleavage of Spry2 by FGF-10 and
thus abolish mTORC1 and its induction of HIF-1␣ and VEGF
secretion. Conversely, overexpression of a mutant form of
Spry2, which cannot interact with cCBL [Spry2(Y55F); Ref.
17], would promote cCBL-mediated clearance of TSC2 and
thus Rheb-GTP binding, HIF-1␣ activity, and VEGF expres-
sion. FDLE transfected with empty vector (EV) alone dis-
played Spry2 clearance in response to 0.1 ␮g·ml⫺1 FGF-10,
which corresponded with increased Rheb-GTP binding,
HIF-1␣ activation, and VEGF production (Fig. 10). Overex-
pression of WT-Spry2 sustained Spry2 abundance in the pres-
ence of FGF-10, prevented Rheb-GTP binding, and did not
augment HIF-1␣ activity or VEGF secretion. Cells transfected
with the Spry2(Y55F) mutant, however, showed marked in-
duction of Rheb-GTP binding, HIF-1␣ activation, and VEGF
secretion. Densitometry revealed that although TSC2 abun-
dance changed little with FGF-10 treatment in the EV-trans-
fected cells, overexpression of WT-Spry2 significantly induced
TSC2 stability, whereas Spry2(Y55F) promoted TSC2 clear-
ance. These data provide independent confirmation that Spry2
regulates mTORC1 at the level of the TSC1/2 complex and that
this regulation is mediated through the Y55 binding site for
cCBL. Moreover, our data illustrate that this effect is sufficient
299 • OCTOBER 2010 • www.ajplung.org
L464 mTORC1 AND VASCULAR SIGNALING IN FETAL LUNG
Fig. 6. Knockout of the upstream repressor of
mTOR, TSC1, alters vasculogenic signaling in
the murine lung. Wild-type (A–C) and TSC1⫺/⫺
(D–F) embryonic day (E) 12.5 murine fetuses
were sectioned and stained for mTOR activity
[S6K1 (T389) phosphorylation], VEGF, and
Tie2 expression. Arrows in A indicate raised
mTOR activity at sites of airway epithelial out-
growth in wild-type animals. Arrows in C indi-
cate Tie2 receptor expression corresponding to
nascent vascular bundles. Images are represen-
tative of 4 fetuses from independent litters.
Bar ⫽ 200 ␮m.
to significantly alter vasculogenic signaling through HIF-1␣
and VEGF.
DISCUSSION
mTOR complex 1 is a central regulator of protein synthesis
and cell growth and so acts as a hub where nutrient, metabolic,
hormonal, and cytokine signals are integrated towards a de-
fined pattern of growth. In mice, mTOR expression arises at
E8.5 in the mid-thoracic region, coinciding with the initiation
of lung development. Its inhibition by mutation or rapamycin
treatment is lethal by E12.5 and is linked to forebrain defects,
loss of somites, and an inability to turn around the embryonic
axis (25). Ontogenic studies of mTOR function in rat fetuses
show its activity is high during the early stages of develop-
ment, declining towards term, but rebounds during the early
neonatal period and corresponds with proportionate changes in
translation efficiency during gestation and birth (42, 43).
Our study aimed to determine how the activity of HIF-1␣, a
critical regulator of vasculogenesis, is coordinated with the
signals that control airway morphogenesis. We show that in
both immortalized adult and primary fetal lung epithelia, ex-
posure to fetal PO2 induces the activity of HIF-1␣ and VEGF.
This is markedly amplified by growth-inducing factors such as
insulin and FGF-10 and is sensitive to mTORC1 inhibition by
rapamycin. In HBE cells, we demonstrate that mTORC1-
dependent regulation of HIF-1␣ via the NH2-terminal TOS
motif is sufficient to induce the appearance of vascular traits in
AJP-Lung Cell Mol Physiol • VOL
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cocultured mesenchyme cells (Figs. 1 and 2). In FDLE primary
cultures, we show that stimulating this interaction using FGF-
10, a critical initiator of airway budding, induces VEGF secre-
tion and promotes localized differentiation of mesenchyme into
vascular tissue in fetal lung explants. Although our study did
not demonstrate that endogenous FGF-10 induces mTORC1 in
fetal airway epithelium in situ, we do show mTORC1 activity
is present in developing airway and that its hyperactivation
strongly induces VEGF expression in fetal lung. Taken as a
whole, these data establish that mTORC1 is a potent positive
modulator of epithelial/mesenchymal vascular signaling that
has the potential to coordinate HIF-1␣ activity with FGF-10, a
growth factor that controls airway branch outgrowth.
These data imply that mTORC1 activity is insensitive to the
low PO2 of the fetal environment. In tumor cell lines, hypoxic
induction of HIF-1␣ regulated genes such as Redd1, Redd2 (8,
11), and Bnip3 (35) repress mTORC1 activity resulting in a
suppression of tumor growth. Of these genes, Redd1 is ex-
pressed in fetal distal lung epithelial cells; however, it does not
appear to mediate hypoxic repression of mTOR in these cells
(42). We used immunohistochemistry to determine the distri-
bution pattern of ␣S6K1-T389 phosphorylation as a readout of
mTORC1 activity in pseudoglandular stage fetal rat and mice
lungs in vivo. Our data show that mTORC1 activity was
evident in airway endodermal growth buds and in the mesothe-
lium lining the periphery of the lung, a site of FGF-9 expres-
sion (56) (Figs. 3 and 5). Experiments in isolated fetal rat lung
299 • OCTOBER 2010 • www.ajplung.org
 mTORC1 AND VASCULAR SIGNALING IN FETAL LUNG
explants, cultured at the PO2 of the fetal lung, demonstrate that
this activity was necessary for balanced airway growth because
exposure to rapamcyin produced significant morphological
changes including accelerated airway branching (raised ASC in
Fig. 4), epithelial flattening, and mesenchymal thinning. Over-
all, these data show mTOR signaling participates in tissue-
specific growth events despite the relative hypoxia of the fetal
environment.
This raises the question of whether mTOR is involved in
controlling cell pluripotency/differentiation in the developing
lung. In cancer, mTOR hyperactivation sustains cell prolifer-
ation and represses differentiation by positive induction of the
Notch signaling pathway (36). Elsewhere, the role of mTOR in
cell differentiation appears to be quite specific to cell type. For
example, rapamycin promotes terminal differentiation of vas-
cular smooth muscle cells (39) but maintains pluripotency of
bone marrow-derived mesenchymal stem cells (24). Rapamy-
AJP-Lung Cell Mol Physiol • VOL
L465
Fig. 7. FGF-10 positively modulates HIF-1␣
activity via mTORC1 in polarized, primary
FDLE cells. A: top graph shows HIF-1␣
activity measured from FDLE transfected
with pHRE3-TK-GL2 and pRL (8:1) and
cultured at fetal (23 mmHg; closed symbols)
or alveolar (100 mmHg; open symbols) PO2
for 6 h in the presence of increasing concen-
trations of FGF-10. Rapamycin (0.1 ␮M; tri-
angles) was added to a parallel set of cells to
determine mTORC1-sensitive HIF-1␣ activ-
ity. Values are normalized to 0 ␮g·ml⫺1
FGF-10 treatment at fetal PO2. Bottom graph
shows the level of VEGF secreted into the
medium beneath the permeable support from
the same cells. *P ⬍ 0.05 relative to control
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at fetal PO2; †P ⬍ 0.05 relative to paired
rapamycin treatment, n ⫽ 5. B: representa-
tive blots showing mTORC1 [␣S6K-(p)-
T389 phosphorylation] and ERK1/2 activa-
tion (T202/Y204 and T185/Y187 phosphor-
ylation) and Spry2 protein abundance
measured in samples from A. *Contrast is
enhanced separately from total ERK blot to
show decline in ERK1/2 phosphorylation
with FGF-10. C: densitometry of pooled
experimental blots represented in B. Open/
closed symbols are as described in A; trian-
gles represent ERK2 values. *P ⬍ 0.05 rel-
ative to control, n ⫽ 4. D: the MEK1 inhib-
itor U0126 (10 ␮M) does not prevent
mTORC1 activation (S6K1-T389 phosphor-
ylation) by 0.1 ␮g/ml⫺1 FGF-10 in FDLE at
fetal PO2. Rapamycin (0.1 ␮M) was added to
show inhibition of mTORC1. Histogram
(right) shows pooled densitometry data for
mTORC1 activity and ERK1/2 phosphory-
lation. *P ⬍ 0.05 relative to control; †not
significantly different from FGF-10 treat-
ment. ND, not detectable; n ⫽ 5.
cin also abolishes transforming growth factor-␤-evoked in-
creases in cell size and migration/invasion during epithelial-
to-mesenchymal transition in murine mammary epithelial cells
(30). In the developing lung, mTOR-directed translation is
active during early and mid-stages of development and de-
clines in late gestation, but peaks again on postnatal day 1 (42,
43). This late-gestation decline in translation efficiency is
accompanied by a switch to mTOR-independent transcript
expression involving genes linked to the development of im-
munity, antioxidant defense, and the acquisition of a terminally
differentiated epithelial phenotype [e.g., SCGB3A1, (44)] and
may be necessary to prepare the lung for air breathing and
exposure to airborne pathogens.
mTOR is recognized as an important inducer of vascular
growth by modulating angiogenic signaling as well as endo-
thelial cell proliferation and differentiation (reviewed in Ref.
34). To determine if mTOR signaling was necessary for vas-
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L466 mTORC1 AND VASCULAR SIGNALING IN FETAL LUNG

Fig. 7 —Conitnued

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culogenesis in the fetal lung in vivo, we examined how tar-
geted knockout of the upstream repressor of mTOR, TSC1,
altered the distribution and expression of VEGF and the an-
giopoietin receptor, Tie2. Mice lacking the TSC1 allele display
constitutively active mTOR activity, develop exencephaly and
abnormal vacuolarization of myocardial cells in utero, and fail
to develop beyond E13 (58). We found that airway growth
proceeds up to at least E12.5 in TSC1⫺/⫺ fetal lungs and that
gross airway morphology (epithelial thickness, airway patency,
mesenchyme thickness) differs little from age-matched WT
lungs (Fig. 6). However, mTOR activity appeared to be uni-
form in the airway epithelium of TSC1⫺/⫺ lungs and did not
show the distinct localization at growth tips observed in WT
lungs. TSC1⫺/⫺ lungs also showed excessive staining for

Fig. 8. FGF-10 destabilizes Spry2 in FDLE.

A: abundance of Spry2 declines with in-
creasing FGF-10 concentration at fetal
(closed circles) and alveolar (open circles)
PO2. Densitometry performed on full-length
Spry2 in ratio to actin. *P ⬍ 0.05 relative to
control, n ⫽ 4. B: Spry2 becomes unstable in
the presence of FGF-10. FDLE were treated
with 100 ␮M cycloheximide (CHX) and 0.1
␮g·ml⫺1 FGF-10 as indicated at fetal PO2.
Representative blot is accompanied by graph
showing full-length Spry2 abundance mea-
sured in ratio to actin. Circles, control; tri-
angles, 0.1 ␮g·ml⫺1 FGF-10. *P ⬍ 0.05,
n ⫽ 4 relative to time 0. Error bars may be
within symbols. C: Spry2 cleavage is protea-
somal. FDLE were treated with 10 ␮M
MG132 and 0.1 ␮g·ml⫺1 FGF-10 at fetal
PO2 for 3 h as indicated. Proteins were sep-
arated by 10% SDS-PAGE to resolve both
full-length and cleaved Spry2 product. Blot
is representative of n ⫽ 3.

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 mTORC1 AND VASCULAR SIGNALING IN FETAL LUNG L467

Fig. 9. Spry2 associates with cCBL and

TSC2. A: immunoprecipitation (IP) of cCBL
from FDLE at fetal PO2 treated with FGF-10
(0.1 ␮g·ml⫺1) or MG132 (10 ␮M) as indi-
cated. Immunoblots (IB) were performed to
detect TSC2, cCBL, and Spry2. Histograms
on right show ratio of TSC2 or Spry2 to
cCBL. *P ⬍ 0.05 relative to control, n ⫽ 5.
B: effect of FGF-10 on TSC1/2 GAP activity
towards the Rheb guanine nucleotide bind-
ing protein in FDLE at fetal or ambient PO2.
TLC phosphorimage shows resolution of
32
P-GTP and -GDP immunoprecipitating
with FLAG-Rheb in cells treated with FGF-

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10. Immunoblot beneath shows even recov-
ery of FLAG-Rheb by IP. Histogram shows
ratio of GTP:GDP as a percentage of total
radioactivity eluting with Rheb. The super-
imposed line graph shows fold change in
total [32P]-labeled guanine nucleotide phos-
phates eluting with Rheb, *P ⬍ 0.05 relative
to control at fetal PO2, n ⫽ 4.

VEGF and an absence of discretely organized Tie2-positive that mTOR activation amplifies HIF-1␣ transcriptional activity
vascular bundles around the airways. VEGF is a HIF-regulated and VEGF expression. The reason for the decline in coordi-
target gene, and its raised expression in TSC1 knockout mouse nated vasculogenesis, which follows, may arise from dysfunc-
lungs is consistent with our observations in Figs. 1, 2, and 7 tional regulation of Tie2 signaling caused by hyperexpression

Fig. 10. Overexpression of Spry2 in FDLE

abolishes FGF-10-evoked Rheb-GTP bind-
ing and is relieved by mutation of the Spry2
(Y55) cCBL docking site. A: TSC1/2 GAP
activity (ratio of GTP:GDP bound Rheb),
HIF-1␣ activity, and VEGF secretion in
FDLE transfected with empty vector, wild-
type, or Y55F Spry2. For GAP assays, the
transfection mix included FLAG-Rheb (ratio
of 1:4 Rheb to test vector), whereas for
HIF-1␣ activity assays and VEGF secretion,
the transfection mix included pHRE3-
TK-GL2 (1:4). All experiments were per-
formed at fetal PO2. *P ⬍ 0.05 relative to EV
control; n ⫽ 4 (Rheb GAP assay), n ⫽ 6
(HIF-1␣ luciferase and VEGF secretion).
B: representative blot showing TSC2, Spry2,
and actin expression levels. Note that the
Spry2 blot shows bands for the endogenous
full-length and cleaved forms of Spry2 in
EV lanes, which are superimposed by over-
expressed Spry2 in other lanes. Histogram
shows TSC2 abundance corrected for actin.
*P ⬍ 0.05 relative to respective EV and
Spry2 (Y55F) treatment, n ⫽ 6 independent
transfections.

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L468 mTORC1 AND VASCULAR SIGNALING IN FETAL LUNG

of VEGF. Tie2 and its ligands, angiopoietin 1 and 2 (Ang1 and
2), control the proliferation and maturation of nascent blood
vessels. Proteolytic cleavage of Tie2 results in the shedding of
a truncated soluble form of this receptor, sTie2, which is
capable of binding Ang1 and 2 and so competitively inhibits
the uncleaved membrane-bound Tie2 receptor. VEGF acceler-
ates the cleavage and shedding of sTie2 and so its elevated
expression decreases Tie-2-dependent blood vessel wall as-
sembly but promotes the proliferation of vascular endothelial
cells (16).
The final part of this study determined how mTORC1-
dependent regulation of HIF-1␣ and VEGF secretion is linked
to the FGF-10/FGFR2b/Spry2 pathway, a central regulator of
airway outgrowth and branching. FGF-10-coated beads pro-
duced a localized increase in Tie2-promoted reporter gene
expression in fetal murine lung explants. More detailed exam-
ination of the link between mTORC1 and FGF-10 in FDLE
(Figs. 7–10) and HBE (Supplemental Figs. S4 and S5) showed
tion would arise by ERK1/2-dependent phosphorylation and
subsequent inactivation of the TSC1/2 complex. We were
therefore surprised to discover that FGF-10 actually suppressed
ERK1/2 activity in FDLE cultured at fetal PO2 and that signal-
ing through the mTORC1 pathway was unaltered by inhibition
of the upstream kinase to ERK1/2, MEK1. Induction of
ERK1/2 by FGF-10 was originally reported in an immortalized
adult murine lung epithelial cell line (MLE15) that was cul-
tured, unpolarized, at ambient PO2 (53). We repeated these
experiments using unpolarized, immortalized adult lung-de-
rived HBE cells and observed transient induction of ERK1/2
by FGF-10, which was not sustained with time or increased
FGF-10 dosage (Supplemental Figs. S4 and S5). Although the
magnitude of ERK1/2 signaling was greater at alveolar com-
pared with fetal PO2, mTORC1 induction by FGF-10 was,
nevertheless, ERK1/2 dependent, leading us to suggest that
ERK1/2 signaling induces, but does not sustain, mTORC1
activity in HBE. These differences in FGF-10 signaling re-

Downloaded from ajplung.physiology.org on September 30, 2010

a clear induction of HIF-1␣ activity and VEGF secretion with
increasing FGF-10 dosage, which was suppressed to the basal
hypoxic level by rapamycin. Given that FGF-10 has been
reported to induce ERK1/2 activation and proliferation of
airway epithelium (53), we anticipated that mTORC1 activa-
sponses between FDLE and MLE15/HBE probably reflect
variation in tyrosine kinase receptor expression linked to de-
velopment, culture, and oxygen exposure; however, our finding
that overall ERK1/2 activity is low in FDLE as well as in the
late-term fetal lung, but is augmented by oxygen in HBE and

Fig. 11. Model of integrated airway and vasculogenesis in the developing lung. Model is represented next to a late embryonic stage (E12) rat lung showing
formation of airway branches extending into the mesenchyme tissue surrounded by blood-filled vascular structures proximal to the airway tip. Foregut (Fg) is
shown displaced to left of lung. Zone A: FGF-10 expressed in the mesenchyme ahead of nascent airway buds induces Spry2 cleavage by an unknown ubiquitin
ligase (possibly SIAH2 or cCBL) in the progenitor epithelial cells of the airway tip. This cleavage event, although necessary for mTORC1 activation, is
accompanied by the formation of a complex between Spry2, cCBL, and TSC2 (Figs. 7 and 8), which dislocates the TSC1/2 complex through the proteolytic
cleavage of TSC2 (Fig. 10). This disrupts TSC1/2 GTPase activating protein (GAP) activity allowing GTP interaction with Rheb and activation of mTORC1.
Zones B and C: trophic signaling, involving VEGF release by airway epithelium into the mesenchyme and subsequent vasculogenesis, proceeds proximal to the
airway tip by binding of mTORC1 to the HIF-1␣ TOS motif and subsequent interaction with the p300 transcriptional initiation complex. This interaction locally
amplifies the transcriptional activity of HIF-1␣ causing increased release of VEGF and the appearance of vascular structures behind the growing tip of the airway.
Our model acknowledges that there are 2 oxygen-dependent points of control that are critical for proper lung morphogenesis. These are: 1) the proliferation of
mesenchyme tissue that requires low-oxygen tension and mTOR activity for sustained growth, and 2) hypoxic priming of HIF-1␣ transcriptional activity, which
can be positively modulated by locally expressed growth factors. The ERK1/2 activation by FGF-10 reported by Tefft et al. (53) and observed by us in HBE
(Supplementary Figs. S4 and S5) is represented by a dotted line. Our study shows that the magnitude of this response is also oxygen sensitive as it is suppressed
at fetal compared with alveolar PO2, leading us to speculate a secondary role for oxygen in regulating ERK1/2 signaling in the fetal lung. This phenomenon could
be linked to the conservation of progenitor cell populations during lung morphogenesis (50).

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 mTORC1 AND VASCULAR SIGNALING IN FETAL LUNG
increases in postnatal lung with the onset of breathing (Sup-
plementary Data Figs. S3–S5), suggests that low oxygen ten-
sions could have a role to play in constraining ERK1/2 signal-
ing during fetal development. Numerous studies in embryonic
stem cells have established that low oxygen tensions conserve
pluripotency, and it is now shown that temporal activation of
ERK1/2 induces differentiation to a defined phenotype (50). It
is possible, then, that the constrained ERK1/2 activity we
observed in fetal tissue may be critical to conserve populations
of progenitor cells in the fetal lung.
Our studies thus highlight mTORC1, and not ERK1/2, as an
important downstream target of FGF-10 in FDLE. Given that
Spry2 antagonism of the FGF-10 receptor, FGFR2b, is be-
lieved to be an important regulator of airway branching peri-
odicity, it follows that mTORC1 activity and its regulation of
HIF-1␣ should be closely governed by Spry2. We found the
activation of mTORC1 by FGF-10 was accompanied by three
key events: 1) proteasomal cleavage of Spry2 to a truncated
form, 2) formation of a stable complex between Spry2, cCBL,
and TSC2, and 3) decreased TSC1/2 GAP activity towards
Rheb as indicated by increased Rheb-GTP binding. Our Spry2
overexpression experiments demonstrated that enforced main-
tenance of Spry2 levels during FGF-10 treatment sustained
TSC2 expression levels and suppressed Rheb-GTP binding,
whereas overexpression of the Spry2 Y55F mutant, which
lacks a functional cCBL binding domain (17), caused TSC2
clearance, Rheb-GTP binding, and induced HIF-1␣ activity
and VEGF secretion. Together, these results suggest that Spry2
is a potent regulator of mTORC1 activation and that this
regulation depends on an interaction between cCBL and TSC2.
Spry2 cleavage in hand with the formation of an apparently
stable Spry2-cCBL-TSC2 complex accords with several pro-
posed physiological roles for Spry2 (reviewed in Ref. 23). One
scenario suggests that Spry2 sequesters cCBL activity. Cleav-
age of Spry2 by another ubiquitin ligase (SIAH2), or cCBL
itself, targets downstream elements of RTK signaling pathways
for proteasomal clearance. Two variations on this theme sug-
gest that Spry2 performs either a targeting or adaptor-protein
function that is necessary for cCBL to interact with its substrate
proteins. Our finding that Spry2 was consistently cleaved by
FGF-10 treatment and that relief of the Spry2-cCBL interaction
by overexpression of the Spry2-Y55F mutant led to TSC2
clearance and Rheb-GTP binding suggests that a cCBL seques-
tering role for Spry2 is possible in unstimulated cells. How-
ever, immunoprecipitations performed against endogenous
cCBL showed that FGF-10 treatment does not lead to the
complete cleavage of Spry2 with some remaining associated
with cCBL when in complex with TSC2, thus a targeting or
adaptor-protein role is also possible. Spry2 binds protein phos-
phatases (SHP2; PP2A) that can mediate its own dephosphor-
ylation as well as that of other phosphoproteins. As TSC2 is a
substrate of both Shp2 and PP2A (38, 59) and Spry2 has been
shown to associate with Shp2 in fetal airway epithelium (52),
it is conceivable that the decline in Spry2 abundance with
FGF-10 disrupts phosphatase activity directed towards TSC2
thereby promoting Rheb-GTP binding and mTORC1 activa-
tion (38, 59). Finally, we acknowledge that Spry2 can regulate
HIF-1␣ independently of RTKs by targeting prolyl hydroxy-
lases 1 and 3 for degradation via the Siah2 ubiquitin ligase
(45); however, this mechanism was not investigated further
here.
AJP-Lung Cell Mol Physiol • VOL
L469
We have incorporated these results into a model that links
vascular signaling with the FGF-10/FGFR2b/Spry2 signaling
pathway in the developing fetal lung (Fig. 11). Our model
differs from the classic view of branched airway morphogen-
esis in that we exclude a significant role for ERK1/2 and
highlight, for the first time, the central importance of mTORC1
as a coordinator of airway and vascular morphogenesis. This
model also highlights the importance of the low oxygen tension
of the fetal environment for priming key developmental events,
particularly, establishing a basal activation of HIF-1␣ and the
maintenance of a mesenchymal cell population. By implica-
tion, therapies that target the HIF signaling system to treat
developmental vascular diseases such as bronchopulmonary
dysplasia need to take into account the role of the mTOR
signaling pathway as a major determinant of temporal and
spatial vascular signaling.
ACKNOWLEDGMENTS
Downloaded from ajplung.physiology.org on September 30, 2010
We thank Claire Cockburn for assistance with qPCR determinations of
pluripotency markers in HELMF, Prof. Jerry Cheadle and Dr. Cleo Bonett
(Cardiff Univ.) for provision of fixed TSC1⫺/⫺ fetuses, and Drs. Graeme Guy
and Permeen Yusoff for provision of Spry2 vectors and related advice.
GRANTS
This work was supported by grants to S. C. Land from The Wellcome Trust
(088032/z/08/z), TENOVUS (Scotland), and the Anonymous Trust. A. R. Tee
is supported by the Association of International Cancer Research through a
Career Development Fellowship (06-914/915).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
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