Development of Cytosolic Hypoxia and Hypoxia-inducible Factor Stabilization Are Facilitated by Aquaporin-1 Expression

Laboratorio de Investigaciones Biomédicas, Departamento de Fisiología Médica y Biofísica, Hospital Universitario Virgen del Rocío, Universidad de Sevilla, Sevilla, 41013 Spain.
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2007; 282(41):30207-15. DOI: 10.1074/jbc.M702639200
Source: PubMed
ABSTRACT
O(2) is essential for aerobic life, and the classic view is that it diffuses freely across the plasma membrane. However, measurements of O(2) permeability of lipid bilayers have indicated that it is much lower than previously thought, and therefore, the existence of membrane O(2) channels has been suggested. We hypothesized that, besides its role as a water channel, aquaporin-1 (AQP-1) could also work as an O(2) transporter, because this transmembrane protein appears to be CO(2)-permeable and is highly expressed in cells with rapid O(2) turnover (erythrocytes and microvessel endothelium). Here we show that in mammalian cells overexpressing AQP-1 and exposed to hypoxia, the loss of cytosolic O(2), as well as stabilization of the O(2)-dependent hypoxia-inducible transcription factor and expression of its target genes, is accelerated. In normoxic endothelial cells, knocking down AQP-1 produces induction of hypoxia-inducible genes. Moreover, lung AQP-1 is markedly up-regulated in animals exposed to hypoxia. These data suggest that AQP-1 has O(2) permeability and thus could facilitate O(2) diffusion across the cell membrane.

Full-text

Available from: Miriam Echevarría, Oct 19, 2015
Development of Cytosolic Hypoxia and Hypoxia-inducible
Factor Stabilization Are Facilitated by Aquaporin-1 Expression
*
S
Received for publication,March 27, 2007, and in revised form, July 31, 2007 Published, JBC Papers in Press, August 2, 2007, DOI 10.1074/jbc.M702639200
Miriam Echevarrı´a
1
, Ana M. Mun˜ oz-Cabello
1,2
, Rocı´o Sa´nchez-Silva, Juan J. Toledo-Aral, and Jose´ Lo´ pez-Barneo
3
From the Laboratorio de Investigaciones Biome´dicas, Departamento de Fisiologı´a Me´dica y Biofı´sica, Hospital Universitario Virgen
del Rocı´o, Universidad de Sevilla, Sevilla, 41013 Spain
O
2
is essential for aerobic life, and the classic view is that it dif-
fuses freely across the plasma membrane.However, measurements
of O
2
permeability of lipid bilayers have indicated that it is much
lower than previously thought, and therefore, the existence of
membrane O
2
channels has been suggested.We hypothesized that,
besides its role as a water channel, aquaporin-1 (AQP-1) could also
work as an O
2
transporter, because this transmembrane protein
appears to be CO
2
-permeable and is highly expressed in cells with
rapid O
2
turnover (erythrocytes and microvessel endothelium).
Here we show that in mammalian cells overexpressing AQP-1 and
exposed to hypoxia, the loss of cytosolic O
2
, as well as stabilization
of the O
2
-dependent hypoxia-inducible transcription factor and
expression of its target genes, is accelerated. In normoxic endothe-
lial cells, knocking down AQP-1 produces induction of hypoxia-
inducible genes. Moreover, lung AQP-1 is markedly up-regulated
in animals exposed to hypoxia. These data suggest that AQP-1 has
O
2
permeability and thus could facilitate O
2
diffusion across the
cell membrane.
Oxygen (O
2
) is necessary for aerobic life because of its central
role in mitochondrial ATP synthesis by oxidative phosphoryl-
ation. Traditionally, O
2
is considered to diffuse freely across the
plasma membrane (1, 2); however, recent studies have shown
that O
2
permeability of lipid bilayers is some orders of magni-
tude lower than previously thought (3). Therefore, it has been
suggested that there exist yet unknown plasmalemmal O
2
chan-
nels to ensure the fluxes required for O
2
uptake in conditions
of high demand or limited O
2
availability. Good candidates are
aquaporins, widely distributed intrinsic membrane proteins
that form water-permeable complexes (3, 4). Mammalian aqua-
porin-1 (AQP-1)
4
is highly expressed in c ells wit h rap id gas
(O
2
/CO
2
) turnover such as ery throcytes (5) and microvessel
endothelium (6, 7), and experiments performed with recom-
binant AQP-1 expressed in Xenopus oocytes have sugge sted
that i t confers upon the cells increased membrane CO
2
per-
meability (8 –10). In addi tion, it has been shown that the
AQP-1 tobacco plant homolog Nt-AQP -1 facilitates CO
2
transport, particularly in conditions of small transmem-
brane CO
2
gradient, and has a significant fun ction in photo -
synthesis and in stom atal opening (11). Against a possible
role of AQP-1 as a gas chan nel is that AQP-1 null mice do not
show any obvious sign of respiratory distress or alteration of
lung or erythrocyte CO
2
transport (12, 13). This observation
could, however, be explained if other aquaporins can compen-
sate, at least partially, for the lack of AQP-1. In fact, AQP-1
functions as a well established water channel, but AQP-1 null
humans (Colton-null blood group) (14) and AQP-1-deficient
mice (12) hav e only subtle changes of erythrocyte wat er dif-
fusion o r renal urine concentr ation. A recent study shows
that after subcutaneous or intracranial malignant cell
implantation, AQP-1 null animals present impaired tumor
growth and vascularity (15), alterations that are compatible
with reduced O
2
uptake in extreme conditions by tumor
cells. We performed experiments designed to test the
hypothesis that AQP -1 regul ates tra nsmembrane O
2
trans-
port. Her e, we show that in mam malian cells stably trans -
fected with AQP-1 and exposed to hypoxia, the los s of intra-
cellular O
2
is accelerated, thus leading to faster stabilization
of the hypoxia-inducible transcription factor (HIF) and up-
regulation of HIF-dependent genes. Redistribution of intra-
cellular O
2
in hypo xia by mitochondrial inhibitors (16) is
blunted in cells with high AQP-1 content. Moreover, inhibi-
tion of native AQP-1 expression with a small interfering
RNA (siRNA) leads to up-regula tion of hypoxia-inducible
genes. Finally, we report that AQP-1 gene expression in lung
in vivo is markedly induced by hypoxia. These data indicate
that AQP-1 might have a role in O
2
homeostasis by facilitat-
ing O
2
diffusion acros s the cell membrane.
EXPERIMENTAL PROCEDURES
Cell Culture, Transfect ions, siRNA, and in Vitro Hypoxic
TreatmentsRat cDNAs for AQP-1 and AQP-3 were cloned
into pcDNA3 (Invitrogen). PC12 cells were cultu red in Dul-
* This work was funded in part by Ayuda a la Investigacio´ n 2000 of the Juan
March Foundation and by grants from the Instituto de Salud Carlos III PI
030296 and Consejerı´a de Salud, Junta de Andalucı´a, 22/02. The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Fig. 1.
1
Both authors contributed equally to this work.
2
Supported by a predoctoral fellowship from the Spanish Ministries of Health
and of Education.
3
To whom correspondence should be addressed: Laboratorio de Investiga-
ciones Biome´dicas, Edificio de Laboratorios, 2 planta, Hospital Universita-
rio Virgen del Rocı´o, Avenida Manuel Siurot s/n, 41013 Sevilla, Spain. Tel.:
34-955-012648; Fax: 34-954-617301; E-mail: lbarneo@us.es.
4
The abbreviations used are: AQP, aquaporin; HIF, hypoxia inducible factor;
siRNA, small interfering RNA; TH, tyrosine hydroxylase; PGK1, phospho-
glycerate kinase 1; VEGF, vascular endothelial growth factor; DETA-NO,
(Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-dio-
late; HAQP, high AQP-1 expression PC12 clone; LAQP, low AQP-1 expres-
sion PC12 clone.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 41, pp. 30207–30215, October 12, 2007
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
OCTOBER 12, 2007 VOLUME 282 NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 30207
at FAC BIOLOGIA/BIBLIOTECA on October 10, 2007 www.jbc.orgDownloaded from
http://www.jbc.org/cgi/content/full/M702639200/DC1
Supplemental Material can be found at:
Page 1
becco’s modified E agle’s mediu m (Invitrogen) supplemented
with 5% fetal bovine serum, 10% horse serum, and 1% peni-
cillin/streptomycin (Invitrogen) in a CO
2
(10%) incubator at
37 °C. To obtain stable su bclones, PC12 cells were trans-
fected with 20
!
g of pcDNA3-AQP1 or pcDNA3-A QP3.
Screening of positive clones was done by Northern blot and
in situ hybridization analysis. From 40 clones analyzed, 20
were positive for either AQP-1 or AQP-3 wit h vari able levels
of expression.
siRNA experiments were done in an endothelial murine cell
line derived from a mixed hemangioendothelioma (EOMA
cells, ATCC catalog No. CRL-2586). Transfections were per-
formed using Lipofectamine 2000 and an AQP-1-designed oli-
gonucleotide (100 n
M; ID 48361, Ambion, Austin, TX), with a
scrambled oligonucleotide as a negative control. After 48 h of
inhibition, levels of distinct mRNAs were analyzed by real-time
reverse transcription and polymerase chain reaction.
Hypoxia experiments performed to analyze mRNA levels of
tyrosine hydroxylase (TH), phosphoglycerate kinase 1 (PGK1),
or vascular endothelial growth factor (VEGF) were done on a
cell incubator with 1–10% O
2
(CO
2
10%) (17). Experiments
designed to measure stabilization of HIF-2
"
protein were done
in a sealed glove box anaerobic workstation (Coy Laboratory
Products Inc., Grass Lake, MI) with O
2
maintained at 1–2%.
Mitochondrial respiratory inhibitor myxothiazol (Sigma-Al-
drich) and the NO donor DETA-NO (Alexis Biochemicals, San
Diego, CA) were prepared fresh and added to the culture
medium.
In Vivo Hypoxic Treatments—Rats were maintained either in
normal conditions or in a hypoxia incubator (Coy Laboratory
Products) for 24 48 h at 10% O
2
. Animals were anesthetized by
injection of 350 mg/kg chloral hydrate and then sacrificed fol-
lowing the animal care protocols approved by our institution.
Tissues were removed either to extract RNA or for immunohis-
tochemistry studies.
Pimonidazole Staining of Hypoxic PC12 Clones—PC12 cells
were grown on coverslips 24 h prior to the hypoxia treatment.
Cells were then transferred to hypoxia (3% O
2
) for 0.5, 1, or 2 h,
or let in normoxia, and 200
!
M pimonidazole was added to the
medium for 30 min. After washing with PBS, cells were fixed
with paraformaldehyde (3%) for 10 min at room temperature
and subsequently washed again overni ght with PBS. After
blocking with bovine serum albumin, coverslips were incu-
bated for 30 min with the fluorescein isothiocyanate-labeled
Hypoxyprobe
TM
-1 monoclonal antibody 1 (mAb1, 1:100
dilution) from the Hypoxyprobe
TM
-1 Plus kit (Chemicon
International, Temecula, CA). As a secondary anti body, the
anti- fluorescein isothiocyanate monoclonal antibody con ju-
gated with horseradish peroxidase provided with th e kit was
used. Coverslips were mounted and immunostained cells
were examined on an Olympus Provis (Tokyo) microscope.
Densitometric measurements of photograp hs taken under
Normaski opti cs were performed using the NIH Image
software.
ImmunohistochemistryAfter removal from the animal,
tissues were kept overnight in 10% phormol and included on
paraffin. Five-
!
m slices were cut with a micro tome and
mounted on microscope slides (Superfrost/plus, Fisher Sci -
entific). Rabbit polyclonal anti-AQP-1 (1:500 dilution,
Abcam, Cambridge, UK) a nd biotin peroxidase-conjugated
secondary antibody (1:200, Pierce) were us ed. Sections were
developed in diaminobenzidine and photographed usi ng a
BX61-Olympus microscope. For cytochemistry, cells w ere
fixed for 10 min with 3% paraformaldehyde at room temper-
ature. A rabbit polyclonal anti-AQP-1 antibody (1:100 dilu-
tion, Chemicon) and a fluorescent polyclonal goa t a nti-rab-
bit antibody (Alexa Fluor 568, Molecular Probes, Invitrogen)
were used. Immunofluorescence was then analyzed with a
Leica DM IRBE co nfocal microscope.
RNA Analysis—Extraction of RNA from PC12 cells was done
following the method described by Cathala et al. (18). To
extract RNA from tissues, the TRIzol reagent (Invitrogen) was
used as indicated by the manufacturer. For Northern analysis,
10
!
g of total RNA were resolved in agarose/formaldehyde
gel and transferred to Hybond-N! nylon membrane. The
Ultrahyb solution (Ambion) was used for hybridization with
the specific
32
P probes (19, 20). Results were visualiz ed using
a PhosphorImager (Typhoon 9400, Amersham Biosciences).
The reverse transcription reaction was performed immediately
after the mRNA isolation using SuperScript II RNase H
"
reverse transcriptase (Invitrogen).
Real-time PCR analysis was performed in an ABI Prism 7500
Sequence Detection System (Applied Biosystems, Warrington,
UK) using SYBR Green PCR Master mix (Applied Biosystems)
and the thermocycler conditions recommended by the manu-
facturer. Amplification of 18 S ribosomal or cyclophilin RNA
was done to normalize for RNA input amounts. Primers were
designed using the Primer Express software (Applied Biosys-
tems). For AQP-1, the primers were: forward (F), 5#-CCA TTG
ACC ACT GGC ATA GTA CA-3# and reverse (R), 5#-AGT
GTC CTG ACC TGT GAA GTG AGT A-3#; for TH: F, 5#-TCG
GAA GCT GAT TGC AGA GA-3# and R, 5#-TTC CGC TGT
GTA TTC CAC ATG-3#; for VEGF: F, 5#-CGC AAG AAA TCC
CGG TTT AA-3# and R, 5#-CAA ATG CTT TCT CCG CTC
TGA-3#; for 18 S ribosomal RNA: F, 5#-AAC GAG ACT CTG
GCA TGC TAA CTA-3# and R, 5#-GCC ACT TGT CCC TCT
AAG AAG T-3#; for PGK1: F, 5#-AGA GCC CAC AGT TCC
ATG GT-3# and R, 5#-GCA AAG TAG TTC AGC T CC TTC
TTC A-3#; for
#
-actin: F, 5#-GGC CCA GAG CAA GAG
AGG TA-3# and R, 5#-CATGTCGTCCCAGTTGGTAACA-3#;
and for cyclophilin: F, 5#-GCA CTG GTG GCA AGT CCA T-3#
and R, 5#-GCC AGG ACC TGT ATG CTT CAG-3#. Melting
curve analysis showed a single sharp peak with the expected T
m
for all samples.
Western Blotting—Cells were washed with cold PBS, scrap-
collected in 1 ml of cold PBS, and centrifuged at 165 $ g for 5
min at 4 °C. For whole-cell protein extract, the pellet was lysed
in 150–300
!
l of homogenization buffer: 50 mM Hepes (pH
7.3), 5 m
M EDTA, 250 m M NaCl, 5 mM dithiothreitol, 0.2% (v/v)
Nonidet P-40 (Sigma-Aldrich), and 1% (v/v) of complete prote-
ase inhibitors mixture (Sigma-Aldrich). The resuspended pellet
was left on ice for 5 min, vortexed, and then centrifuged at
16,000 $ g for 15 min at 4 °C. For cell-membrane protein
extract, cells were scraped and collected in 1 ml of homogeni-
zation buffer containing 320 m
M sucrose, 5 mM Hepes, and 1%
(v/v) of complete protease inhibitors mixture. Samples were
Aquaporin-1 and Membrane O
2
Transport
30208 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 NUMBER 41 OCTOBER 12, 2007
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homogenized with a Polytron and centrifuged at 2000 $ g for 5
min at 4 °C, and then supernatant was centrifuged at 30,000 $ g
for 30 min at 4 °C. The pellet was resuspended in homogeniza-
tion buffer plus 1% SDS. Protein concentration was analyzed
with the Bradford method (Bio-Rad Protein Assay) for whole-
cell extract and with the Lowry method (Bio-Rad DC Protein
Assay) fo r cell mem brane pro tein extraction, and k ept at
"20 °C until Western blot assay (21). Twenty
!
g of whole-
cell extracts were resolved by SDS-PAGE (6%) for HIF2
"
. For
AQP-1, 40
!
g of cell membrane
proteins were resolved in 10%
SDS-PAGE. After electrophoresis,
proteins were transferred into
polyvinylidene difluoride mem-
branes (Hybond-P, Amersham
Biosciences) using a Novex appa-
ratus (Novel Experimental Tech-
nology, San Diego, CA). Mem-
branes were probed with 1:1000
anti-HIF2
"
(Abcam), 1:2000 anti
HIF1
#
(Abcam), 1:1000 anti-
AQP-1 (Chemicon), and 1:10,000
anti-
#
-tubulin (Sigma). Immuno-
reactive bands were developed
with the E CL system (Amers ham
Biosciences) and visualized using a
PhosphorImager (Typho on 940 0,
Amersham Biosci ences).
Water Permeability Measure-
ments—Cells grown on coverslips
were loaded for 5 min with 1
!
M cal-
cein acetoxymethyl ester (Molecu-
lar Probes) and mounted in a small
(%250
!
l) perfusion chamber that
allowed rapid exchange of a solution
with 300 (isosmotic) to another with
150 (hyposmotic) mmol/kg. The
rate of change in calcein fluores-
cence was monitored as described
(22). The isosmotic solution used
for these experiments contained in
m
M: 140 NaCl, 4.5 KCl, 2.5 CaCl
2
, 1
MgCl
2
, 10 Hepes, and 10 glucose
(pH: 7.4). The hyposmotic solution
was obtained by water dilution.
Statistical Analysis—Data were
presented as mean & S.E. and were
analyzed with either paired Stu-
dent’s t test or the one-way analysis
of variance followed by Tukey’s test.
RESULTS
Responsiveness to Hypoxia Is
Accelerated in Cells with High Levels
of AQP-1—To investigate the role of
AQP-1 in plasmalemmal O
2
trans-
port, we generated several sub-
clones of PC12 cells stably trans-
fected with either AQP-1 or AQP-3, representative members of
the two major classes of aquaporins (23, 24). Because direct
measurement of O
2
fluxes at the cellular level is not technically
feasible, in our initial experiments the expression of HIF-de-
pendent genes was monitored as a readout of cytosolic O
2
con-
centration (Fig. 1A). HIF, a master regulator of the responses of
tissues to low O
2
tension (25–27), is composed of
"
- and
#
-sub-
units; the stability of the
"
-subunit is regulated by a family of
O
2
-dependent prolyl hydroxylases (26, 28). At low O
2
tensions,
FIGURE 1. AQP-1 expression facilitates the hypoxia-dependent induction of TH gene. A, schematic illus-
tration of cytosolic O
2
concentration resulting from the equilibrium between transmembrane influx (1) and
uptake by mitochondria (2). HIF destabilization by O
2
and gene expression in hypoxia are represented. B, PC12
cell lines stably transfected with AQP-1 and AQP-3 and effect on the hypoxia-inducible TH gene. Top, Northern
blot analysis of TH mRNA in AQP-1 (clones 1) and AQP-3 (clones 3) expressing cell clones and wild type PC12 cells
exposed to normoxia (N, 21% O
2
) or hypoxia (H, 6% O
2
) for 12 h. Cyclophilin (Cp) mRNA is used as a load control.
Bottom, bar diagram representing average values (mean & S.E.) from 7–9 separate experiments. C, data from
the bar diagram in B (hypoxic mRNA TH induction) expressed as a function of the amount of AQP-1 and AQP-3
mRNA expressed in the various PC12 cell clones permanently transfected with either AQP-1 or AQP-3 gene. The
black symbol indicates the value in wild type PC-12 cells, which do not express either AQP-1 or AQP-3. Note that
for AQP-1-expressing clones, TH mRNA induction increases linearly (r
2
' 0.82) with the level of AQP-1 expres-
sion. D, hypoxic (6% O
2
for 12 h) TH mRNA expression as a function of the amount of AQP-1 protein detected by
Western blot in the different cell clones. TH mRNA induction increases linearly (r
2
' 0.92) with the level of
AQP-1 protein expression. The black symbol indicates the value in wild type PC-12 cells. The cell clones selected
for further studies (HAQP and LAQP) are indicated in the figure.
Aquaporin-1 and Membrane O
2
Transport
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cellular hydroxylase activity is inhibited, and HIF
"
accumulates
to heterodimerize with HIF
#
. The heterodimeric HIF activates
the expression of numerous genes. In AQP-1 or AQP-3-trans-
fected clones we tested the hypoxic induction of TH, an O
2
-de-
pendent gene up-regulated by even mild hypoxia in PC12 cells
(29). The rationale behind these experiments was that if AQP-1
is O
2
-permeant, development of cytosolic hypoxia and subse-
quent cumulative induction of TH mRNA should be faster in
cells expressing high levels of AQP-1 than in wild type PC12
cells. Hypoxic TH mRNA induction in the seven clones studied
besides wild type PC12 cells is shown in Fig. 1B (top). Although
the basal TH mRNA levels varied among the different clones
and in separate experiments on a given cell clone, we compared
for each cell type the relation of TH mRNA in hypoxia versus
the values in normoxia. A quantitative summary of TH mRNA
induction after 12 h of hypoxic (6% O
2
) exposure in several
clones of AQP-1- and AQP-3-transfected cells is shown in Fig.
1B (bottom) and C. Although for AQP-1-expressing clones
there was a correlation between hypoxic TH mRNA induction
and the level of AQP-1 expression, the response of AQP-3
clones (either with high or low expression levels) was indistin-
guishable from that of wild type PC12 cells. Differences
between AQP-1-expressing clones were also seen with shorter
(6 h) hypoxic (6%) exposure (supplemental Fig. 1A). In the case
of AQP-1 clones there was a lineal correlation between TH
mRNA expression and the level of AQP-1 in the various cell
types as determined by Western blot (Fig. 1D). As result of this
initial screening, two AQP-1-transfected clones, one with high
level of AQP-1 and highly responsive to hypoxia (clone HAQP)
and another with low level of AQP-1 and low responsiveness to
hypoxia (clone LAQP) similar to wild type cells (Fig. 1D), were
selected for further analysis. The high expression of AQP-1 in
HAQP in comparison with LAQP or wild type cells was further
confirmed by immunocytochemistry (Fig. 2A). In addition, we
showed that HAQP cells responded more rapidly to an hypos-
motic challenge than LAQP cells (Fig. 2B), thus indicating that
recombinant AQP-1 expressed in the PC12 cell membrane was
functional as a water-permeable channel. We also used in some
experiments a highly expressing AQP-3 clone (clone 3.3 in Fig.
1, B and C), which behaves similar to the wild type PC12 cells
(supplemental Fig. 1, B–D), to make comparisons with the
highly expressing AQP-1 cells.
The differential behavior of HAQP and LAQP cells that was
clear when they were assayed in mild hypoxia (6% O
2
) (Fig. 3A,
left) became even more apparent in conditions of extreme
hypoxia. After 12 h in 1% O
2
, TH mRNA accumulation in
HAQP was %3– 4-fold higher than in LAQP cells (Fig. 3A,
right), indicating that the loss of cytosolic O
2
was accelerated by
AQP-1. Contrarily, TH mRNA induction by cobalt, an agent
that stabilizes HIF independent of O
2
tension (25, 27), was the
same in the two cell clones (Fig. 3B). Similar to TH, the hypoxic
mRNA up-regulation of phosphoglycerate kinase 1 (PGK1) and
vascular endothelial growth factor (VEGF), t wo other O
2
-sen-
sitive genes (25, 27), was higher in HAQP than in LAQP or
wild type cells ( Fig. 3, C and D). Cobalt TH induction and
hypoxic induction of PGK1 in the AQP-3 clone was similar to
the values obtained in PC12 cells (supplemental Fig. 1, C and
D). Altogethe r these d ata supported the hypothesis that
AQP-1 expression confers increased O
2
permeability upon
PC12 cell membrane.
HIF-2
"
Stabilization i n Hypoxia Depends on the Level of
AQP-1 Expre ssion—Because the expression of the O
2
-sensi-
tive genes depends on the stabilization of HIF, we investi-
gated whether the regulation of the two com ponents of this
heterodimeric transcription factor (HIF
"
and HIF
#
) was
influenced by the presence of functional AQP-1. In fair
agreement with the res ults on the O
2
-dependent genes stud-
ied (TH, PGK1, and VEGF) , the accumulation of HIF-2
"
(the
most abundant O
2
-dependent isoform in PC12 cells (21, 30))
during the first few hours after exposure t o hypoxia was sig-
nificantly greater in HAQP than in LAQP or wild type cells.
As an internal control, the levels of HIF-1
#
(a cons titutive
protein that dimerizes with all HIF isoforms (25) ) were unal-
tered by hypoxia (Fig. 4 , A and B). To check w hether the
presence of AQP-1 has a bid irectional impact on transmem-
brane O
2
transport (i.e. it also facilitates O
2
influx), we
induced HIF-2
"
accumulation in hypoxia, and after rapidly
switching to norm oxia, HIF-2
"
degradation time course was
monitored. As proteasomal degradation of a ccumulated
HIF-2
"
could be nonspecific ally influenced by the amount of
proteins existing in the cells, we compared in the se exp eri-
ments clones with high levels of aquaporin expression
(clones 1.22 and 3.3, Fig. 1 and s upplemental Fig. 1B). Deg-
radation of HIF-2
"
in normoxia was clearly faster in cells
expressing A QP-1 (HAQP1) than in cells with AQP-3
(HAQP3), thus supporting the view that AQP-1 increases
transmembrane O
2
transport (Fig. 4, C and D).
To further analyze the alterations in membrane O
2
transport
caused by AQP-1 expression, we studied the differential
response of HAQP and LAQP cells to redistribution of intra-
FIGURE 2. A (top), confocal images illustrating the immunocytochemical local-
ization of AQP-1 in the indicated cell types. Bottom, Western blot analysis of
AQP-1 protein expression in the cells studied in comparison with kidney
homogenates. Note the high level of AQP-1 expression in HAQP cells.
"
-Tu-
bulin is used as a load control. B, change of volume of individual HAQP and
LAQP cells in response to a hyposmotic shock measured by calcein fluores-
cence (a.u., arbitrary units). Switching to solutions of different osmolality is
indicated by downward (150 mmol/kg) and upward (300 mmol/kg) arrows. wt,
wild type.
Aquaporin-1 and Membrane O
2
Transport
30210 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 NUMBER 41 OCTOBER 12, 2007
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cellular O
2
by mitochondrial inhibition. As reported before (16,
31), inhibition of mitochondrial electron transport in wild type
cells with myxothiazol (a complex III blocker) or DETA-NO (an
NO donor that inhibits cytochrome c oxidase) lead to destabi-
lization of HIF-2
"
in hypoxic (1% O
2
) cells (Fig. 5). It has been
proposed that HIF destabilization upon inhibition of mito-
chondrial respiration in hypoxic cells (16, 31) is caused, among
other mechanisms (32, 33), by intra-
cellular O
2
redistribution toward
nonrespiratory O
2
-dependent tar-
gets such as prolyl hydroxylases, so
that they do not register hypoxia
(16). Quite interestingly, HIF-2
"
destabilization produced by myx-
othiazol and by DETA-NO was less
pronounced in HAQP in compari-
son with LAQP or wild type cells
(Fig. 5). These observations further
support the view that AQP-1
increases O
2
permeability in HAQP
cells, thereby facilitating the trans-
membrane efflux of intracellular O
2
spared by inhibition of respiration.
Thus, upon mitochondrial inhibi-
tion hypoxic HAQP cells can main-
tain a more severe cytosolic hypoxia
and higher HIF-2
"
content than
LAQP or wild type cells.
Cytosolic Loss of O
2
in Hypoxia Is
Facilitated by AQP-1 Expression—To
obtain more direct evidence that
AQP-1 facilitates the development
of cytosolic hypoxia, and to rule out
that overexpression of AQP-1 acti-
vates HIF through an oxygen-inde-
pendent signal transduction path-
way, we studied the transmembrane
fluxes of O
2
in HAQP and LAQP
cells using pimonidazole staining, a
broadly used hypoxia marker that
forms intracellular protein adducts
at low O
2
tension (31, 34, 35).
With this technique, the loss of
cytosolic O
2
upon exposure to
hypoxia was clearly faster in
HAQP than in LAQP cells (Fig. 6).
These differen ces estimated with
the Hypoxyprobe method were
similar to those inferred from the
HIF-2
"
experiments (Fig. 4B),
thus suggesting that the distinct
time courses of HIF-2
"
stabiliza-
tion observed in HAQP and LAQP
cells reflect, indeed, differences in
membrane O
2
transport between
the two cell typ es.
Knocking Down Native AQP-1
with siRNA Results in Up-regulation
of Hypoxia-inducible Genes—Besides the studies on PC12 cells
overexpressing AQP-1, we also performed loss of function
experiments using a nonmetastatic murine hemangioendothe-
lioma cell line (EOMA cells), which behave in vitro in a manner
similar to microvascular endothelial cells (36, 37). First, we
investigated whether, as in microvessel endothelium (6, 7),
EOMA cells also express AQP-1 as well as hypoxia-dependent
FIGURE 3. Induction of oxygen-sensitive genes in AQP-1-expressing cells. A (left), Northern blot analysis of
TH mRNA expression in HAQP, LAQP, and wild type (wt) PC12 cells exposed to normoxia (N, 21% O
2
) or hypoxia
(H, 6% O
2
for 12 h). Cyclophilin (Cp) mRNA is used as a load control. The bar diagram represents values (mean &
S.E.) from 7–9 separate experiments. Right, similar experiment using lower O
2
tension (H, 1% O
2
for 12 h).
Average values from three separate experiments. B, Northern blot analysis of TH mRNA in normoxic HAQP,
LAQP, and wild type cells treated with 0.1 m
M cobalt chloride (Co) for 12 h (C, control). Average values from
three separate experiments. C, Northern blot analysis of phosphoglycerate kinase 1 (PGK1) mRNA expression in
AQP-1-expressing cell clones HAQP, LAQP, and wild type PC12 cells exposed to normoxia (N) or hypoxia (H, 6%
O
2
for 12 h). Cyclophilin (Cp) mRNA is used as a load control. The bar diagram represents values (mean & S.E.)
from 5 separate experiments. D, TH and VEGF mRNA analysis by real time PCR in the cell clones indicated
exposed to normoxia (N) or hypoxia (H, 6% O
2
for 12 h). Note that the level of hypoxic TH mRNA induction is
similar to that estimated by Northern blot (panel A, left). Mean & S.E. of 5 experiments. *, p ( 0.05; ***, p ( 0.001.
Aquaporin-1 and Membrane O
2
Transport
OCTOBER 12, 2007 VOLUME 282 NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 30211
at FAC BIOLOGIA/BIBLIOTECA on October 10, 2007 www.jbc.orgDownloaded from
Page 5
genes such as PGK1 or VEGF (Fig. 7A). In these cells the level of
PGK1 mRNA expression was quite insensitive to moderate
hypoxia; however, VEGF mRNA induction was clearly modu-
lated by moderate levels of hypoxia in an O
2
tension-dependent
manner (Fig. 7A). In normoxic EOMA cells treated with spe-
cific AQP-1 siRNA, the level of AQP-1 expression decreased to
%5060% of the value in control. In parallel with the decrease
of AQP-1 expression we observed an almost 2-fold increase of
VEGF mRNA (Fig. 7B). Knocking down AQP-1 with siRNA had
little effect on the levels of either
#
-actin (a gene independent of
O
2
tension) or PGK1, a gene that, as shown above, is less sensi-
tive to hypoxia than VEGF in EOMA cells (Fig. 7). These data
further suggest that the decrease of AQP-1 expression in endo-
thelial cells diminishes the transmembrane O
2
fluxes, which
results in reduced cytosolic O
2
tension and induction of HIF-
dependent genes.
AQP-1 Gene Expression Is Up-regulated by Hypoxia in Vivo
Because AQP-1 is expressed in microvessels (6, 7), we searched
its up-regulation in lungs of animals subjected to hypoxia. A
priori, we presumed that this could be an advantageous adapt-
ive response if AQP-1 plays any role in membrane O
2
transport.
Indeed, AQP-1 was expressed in
lung endothelial cells (inset in Fig.
8), and the AQP-1 gene was also
highly O
2
-sensitive in comparison
with the more classically studied
PGK1 gene. In animals exposed to
mild hypoxia (10% O
2
) for 24 h, the
level of PGK1 mRNA was barely
altered, but we observed a robust
%3-fold AQP-1 mRNA induction in
lung tissue (Fig. 8).
DISCUSSION
The results in this study provide
evidence that AQP-1 can accelerate
the establishment of cytosolic
hypoxia possibly through facilita-
tion of O
2
transport across the
plasma membrane. We have also
shown that AQP-1 gene expression
is regulated exquisitely by O
2
ten-
sion. Although we did not directly
measure transmembrane O
2
fluxes
in PC12 cells because it is not tech-
nically feasible, our conclusions are
supported by several independent
observations. (i) In cells overex-
pressing AQP-1 (HAQP cells) and
exposed to a hypoxic environment,
the levels of HIF
"
- and O
2
-depend-
ent genes (TH, PGK1, and VEGF)
were higher than in LAQP cells,
suggesting that the loss of cytosolic
O
2
was accelerated by the presence
of AQP-1. It must be noted that this
experimental protocol estimates the
cumulative stabilization of HIF (and
mRNA induction of HIF-dependent genes) during the develop-
ment of hypoxia rather than the level of cytosolic O
2
in steady
state conditions. (ii) HIF-2
"
degradation elicited by reoxygen-
ation of hypoxic cells was faster in cells expressing AQP-1 than
in cells highly expressing AQP-3. (iii) The differential behavior
exhibited by HAQP and LAQP cells during exposure to low O
2
tension was confirmed when the development of cytosolic
hypoxia was estimated by the cumulative staining of pimonida-
zole, an HIF-independent hypoxia marker (34, 35). (iv) In
hypoxic PC12 cells, inhibition of mitochondrial respiration
produced HIF-2
"
destabilization (16), which was less pro-
nounced in HAQP than in LAQP or wild type cells. This finding
is compatible with an increased O
2
permeability in HAQP cells,
thereby facilitating the transmembrane efflux of intracellular
O
2
spared by inhibition of respiration. (v) In normoxic endo-
thelial cells, lowering AQP-1 expression with a siRNA blunted
aquaporin mRNA expression by %50 60% and selectively up-
regulated VEGF, a gene induced in these cells by even moderate
levels of hypoxia. Altogether, these data strongly support the
view that AQP-1 is involved in O
2
homeostasis and facilitates
transmembrane O
2
transport.
FIGURE 4. Regulation of HIF in AQP-expressing cells. A, Western blot analysis of HIF2
"
and HIF1
#
in the cell
clones studied exposed to normoxia (N, 21% O
2
) or hypoxia (H, 1% O
2
) for the time indicated. B, summary of
HIF2
"
induction by hypoxia in HAQP (red), LAQP (blue), and wild type (black) cell clones. Data points are mean &
S.E. values of 5–13 different experiments. After 2 h in hypoxia, HIF2
"
value in HAQP cells is statistically different
(p ( 0.05) with respect to that in normoxia. For LAQP or wild type cells, 4 h in hypoxia are needed to reach HIF2
"
values statistically different from those in normoxia. C, representative Western blot analysis of HIF2
"
in cell
clones overexpressing AQP-1 (HAQP1) or AQP-3 (HAQP3) exposed to hypoxia (H, 1% O
2
) for 4 h and then
transferred back to normoxia (N, 21 O
2
%) for the time indicated. D, time course of HIF2
"
degradation during
reoxygenation (21% O
2
). Levels of HIF2
"
were normalized in each clone to the value obtained at the end of the
hypoxic treatment. Data points are mean & S.E. values of 3 different experiments. Degradation of HIF2
"
in
HAQP1 expressing cells was significantly higher (***, p ( 0.001) than in high AQP-3 expression PC12 clone after
20 and 30 min of reoxygenation.
Aquaporin-1 and Membrane O
2
Transport
30212 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 NUMBER 41 OCTOBER 12, 2007
at FAC BIOLOGIA/BIBLIOTECA on October 10, 2007 www.jbc.orgDownloaded from
Page 6
Our proposal that AQP-1 could function as an O
2
channel is
in fair agreement with previous reports suggesting that AQP-1
increases membrane CO
2
permeability (8–11). Recently, it has
been proposed that AQP-1 also transports NO across endothe-
lial cells and contributes to the regulation of vascular tone (38,
39). Although our experiments suggest that AQP-3 is not
O
2
-permeable, there are several other aquaporins that overlap
AQP-1 distribution and could partially compensate for the
AQP-1 deficiency (24). For example, in alveolar epithelial type I
FIGURE 5. Differential response by AQP-1-expressing clones to mitochon-
drial inhibition. A, induction of HIF2
"
in hypoxic (1% O
2
for 4 h) cells and its
degradation after treatment with myxothiazol (myxo, 1
!
M) or DETA-NO (100
!
M) for the indicated time periods. A representative example from 7–8 sepa-
rate experiments with
"
-tubulin used as a load control. B, summary of residual
HIF2
"
levels after treatment with mitochondrial inhibitors. Values (mean &
S.E. from 7– 8 different experiments) in the bar diagrams are expressed as the
percentage of the maximum level (100%) of HIF2
"
reached after 4 h of
hypoxia. Destabilization of HIF2
"
after treatment with myxothiazol (20 min)
and DETA-NO (60 min) was significantly less (*, p ( 0.05) in HAQP cells than in
LAQP or wild type (wt) cells.
FIGURE 6. Pimonidazole staining of hypoxic PC12 cell clones. A, micropho-
tographs of pimonidazole staining in the cell clones studied exposed to nor-
moxia (N, 21% O
2
) or hypoxia (H, 3% O
2
) for the time indicated. Pimonidazole
(200
!
M) was added to the cells for the last 30 min of incubation, and after-
ward the immunostaining was performed. B, summary of the densitometric
measurements of the pimonidazole staining in HAQP (red) and LAQP (blue)
cells. Data points are mean & S.E. values of seven different experiments. After
0.5 h in hypoxia,theincrease inpimonidazolestaininginHAQPcells isstatistically
different(p( 0.05)withrespecttothatin normoxia.In contrast, forLAQP cells1h
in hypoxia is needed to reach a value statistically different from the one in nor-
moxia (a.u., arbitrary units). *, p ( 0.05; **, p ( 0.01; ***, p ( 0.001.
FIGURE 7. Knocking down the level of native AQP-1 in EOMA cells modu-
lates the expression of endogenous O
2
-sensitive genes. A, levels of mRNA
expression of VEGF and PGK1 analyzed by quantitative real time PCR after
24 h of incubation of the cells in different hypoxic conditions (H, 10, 6, and 3%
O
2
) normalized to values in normoxia (N, 21% O
2
). Bars represent mean & S.E.
values of 6 –9 different experiments. Note that whereas the expression of
both genes was induced by hypoxia, the induction of VEGF was clearly higher
to that of PGK1. The inset shows a Western blot analysis confirming the
expression of AQP-1 protein in endothelial EOMA cells. B, effect of blunting
AQP-1 expression by siRNA on the mRNA expression of O
2
-sensitive genes
(VEGF, PGK1, and
#
-actin). Note that inhibition of AQP-1 expression by about
50% produced an almost 2-fold induction of the highly O
2
-sensitive gene
VEGF, whereas the others genes remained unaffected. Bars represent mean &
S.E. values of seven different experiments. For endogenous normalization of
the PCR amplifications, cyclophilin levels were used as load control. mRNA
levels are normalized to values obtained with scramble siRNA. Statistical anal-
ysis is referred to levels of
#
-actin expression. *, p ( 0.05; **, p ( 0.01; ***, p (
0.001.
Aquaporin-1 and Membrane O
2
Transport
OCTOBER 12, 2007 VOLUME 282 NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 30213
at FAC BIOLOGIA/BIBLIOTECA on October 10, 2007 www.jbc.orgDownloaded from
Page 7
cells the AQP-5 gene is up-regulated by HIF through interac-
tion with hypoxia-responsive elements in the promoter (40),
and AQP-4 mRNA has also been shown to be up-regulated by
hypoxia in astrocytes (41). This could explain why humans (14)
and mice (12, 13) lacking AQP-1 do not exhibit major gross
physiological alterations related with either water or O
2
metab-
olism. In addition, besides aquaporins, other membrane pro-
teins could also contribute to membrane O
2
transport (3, 10).
The novel role of AQP-1 in O
2
homeostasis proposed here is
compatible with its expression in erythrocytes (5) or vascular
endothelium (6, 7). AQP-1 is also overexpressed in cells of pro-
liferating microvessels (8), as well as in aberrant cells of human
and rat tumors (42– 44), situations in which a limited O
2
avail-
ability could have induced AQP-1 expression to facilitate O
2
uptake by the cells. In fact, AQP-1 null mice present impaired
tumor growth after subcutaneous or intracranial malignant cell
implantation, with reduced tumor vascularity and extensive
necrosis (15). Migration of AQP-1-deficient aortic endothelial
cells in vitro is greatly decreased, and it has been suggested that
the lack of AQP-1 alters water fluxes required for rapid turn-
over of cell membrane protrusions at the leading edge of
migrating cells (15). The presence of AQP-1 could also be crit-
ical for aerobic ATP synthesis, particularly in membranes of
low gas permeability (4) or when transmembrane O
2
gradients
are small, as it occurs in cells exposed to environments with
extremely low O
2
concentrations or in multilayered diffusion
barriers such as that existing between the lung alveoli and
blood. In conclusion, our observations support a new physio-
logic role for aquaporins as O
2
transporters (4) besides their
canonical function as water-permeable channels. AQP-1 phar-
macology could be of potential use in conditions of altered O
2
exchange or to control O
2
-dependent angiogenesis and tumor
cell growth.
Acknowledgments—We thank Dr. Carmen Sa´ez and Javier Villadiego
for their technical help with immunohistochemistry studies.
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Aquaporin-1 and Membrane O
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  • Source
    • "AZA decreased the expression of AQP5, cell proliferation and migration[21]Colon cancer Acetazolamide decreased AQP1 expression and the tumor growth.[6]AQP1 induced HT20 cell migration[50]AQP1 is required for angiogenesis[3]hEGF induced AQP3 expression, proliferation and migration. AQP3 is correlated lymph node differentiation and metastasis in CRC. "
    [Show abstract] [Hide abstract] ABSTRACT: Aquaporins (AQPs) are small (~30kDa monomers) integral membrane water transport proteins that allow water to flow through cell membranes in reaction to osmotic gradients in cells. In mammals, the family of AQPs has thirteen (AQP0-12) unique members that mediate critical biological functions. Since AQPs can impact cell proliferation, migration and angiogenesis, their role in various human cancers is well established. Recently, AQPs have been explored as potential diagnostic and therapeutic targets in gastrointestinal (GI) cancers. GI cancers encompass multiple sites including the colon, esophagus, stomach and pancreas. Research in the last three decades has revealed biological aspects and signaling pathways critical for the development of GI cancers. Since the majority of these cancers is very aggressive and rapidly metastasizes, identifying effective targets is crucial for treatment. Preclinical studies have utilized inhibitors of specific AQPs and knock down of AQP expression using siRNA. Although several studies have explored the role of AQPs in colorectal, esophageal, gastric, hepatocellular and pancreatic cancers, there is no comprehensive review compiling the available information on GI cancers as has been published for other malignancies such as ovarian cancer. Due to the similarities and association of various sites of GI cancers, it is helpful to consider these results collectively in order to better understand the role of specific AQPs in critical GI cancers. This review summarizes the current knowledge of the role of AQPs in GI malignancies with particular focus on diagnosis and therapeutic applications.
    Full-text · Article · Jan 2016 · Cancer letters
  • Source
    • "The resuspended pellet was left on ice for 5 min, vortexed, and then centrifuged at 16000 × g for 15 min at 4°C, and extracted proteins remain in the supernatant. Protein concentration was analyzed with the Bradford method (BioRad Protein Assay, BioRad) and kept at -20°C until western blot analysis [16]. For all proteins, 20 μg of whole-cell extracts were resolved by SDS-PAGE on 10% gels. "
    [Show abstract] [Hide abstract] ABSTRACT: Abnormal AQP3 overexpression in tumor cells of different origins has been reported and a role for this enhanced AQP3 expression in cell proliferation and tumor processess has been indicated. To further understand the role AQP3 plays in cell proliferation we explore the effect that stable over expression of AQP3 produces over the proliferation rate and cell cycle of mammalian cells. The cell cycle was analyzed by flow cytometry with propidium iodide (PI) and the cell proliferation rate measured through cell counting and BrdU staining. Cells with overexpression of AQP3 (AQP3-o) showed higher proliferation rate and larger percentage of cells in phases S and G2/M, than wild type cells (wt). Evaluation of the cell response against arresting the cell cycle with Nocodazole showed that AQP3-o exhibited a less modified cell cycle pattern and lower Annexin V specific staining than wt, consistently with a higher resistance to apoptosis of AQP3-overexpressing cells. The cell volume and complexity were also larger in AQP3-o compared to wt cells. After transcriptomic analysis, RT-qPCR was performed to highlight key molecules implicated in cell proliferation which expression may be altered by overexpression of AQP3 and the comparative analysis between both type of cells showed significant changes in the expression of Zeb2, Jun, JunB, NF-kβ, Cxcl9, Cxcl10, TNF, and TNF receptors. We conclude that the role of AQP3 in cell proliferation seems to be connected to increments in the cell cycle turnover and changes in the expression levels of relevant genes for this process. Larger expression of AQP3 may confer to the cell a more tumor like phenotype and contributes to explain the presence of this protein in many different tumors.
    Full-text · Article · Sep 2015 · PLoS ONE
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    • "Biomechanical function of the intervertebral disc is intricately linked to NP osmotic properties and the ability of matrix to bind water. Since AQPs are crucial in maintaining cellular water homeostasis under dynamic osmotic conditions and have been shown to co-transport many important metabolites such as CO 2 and O 2 [37, 38], it was important to investigate if their expression is sensitive to disc degeneration. Our results clearly show that levels of both AQPs are lower in NP tissues with progressive degeneration. "
    [Show abstract] [Hide abstract] ABSTRACT: Objectives of this study were to investigate whether AQP1 and AQP5 expression is altered during intervertebral disc degeneration and if hypoxia and HIF-1 regulate their expression in NP cells. AQP expression was measured in human tissues from different degenerative grades; regulation by hypoxia and HIF-1 was studied using promoter analysis and gain- and loss-of-function experiments. We show that both AQPs are expressed in the disc and that mRNA and protein levels decline with human disease severity. Bioinformatic analyses of AQP promoters showed multiple evolutionarily conserved HREs. Surprisingly, hypoxia failed to induce promoter activity or expression of either AQP. While genomic chromatin immunoprecipitation showed limited binding of HIF-1α to conserved HREs, their mutation did not suppress promoter activities. Stable HIF-1α suppression significantly decreased mRNA and protein levels of both AQPs, but HIF-1α failed to induce AQP levels following accumulation. Together, our results demonstrate that AQP1 and AQP5 expression is sensitive to human disc degeneration and that HIF-1α uniquely maintains basal expression of both AQPs in NP cells, independent of oxemic tension and HIF-1 binding to promoter HREs. Diminished HIF-1 activity during degeneration may suppress AQP levels in NP cells, compromising their ability to respond to extracellular osmolarity changes.
    Full-text · Article · Mar 2015 · Oncotarget
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