Biology 2e
2nd Edition
ISBN:9781947172517
Author:Matthew Douglas, Jung Choi, Mary Ann Clark
Publisher:Matthew Douglas, Jung Choi, Mary Ann Clark
Chapter9: Cell Communication
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Transcribed Image Text:THE JOURNAL OF BIOLOGICAL CHEMISTRY
Ⓒ2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Cell Signaling Microdomain with Na,K-ATPase and Inositol
1,4,5-Trisphosphate Receptor Generates Calcium Oscillations*
Received for publication, May 22, 2003, and in revised form, August 20, 2003
Published, JBC Papers in Press, August 28, 2003, DOI 10.1074/jbc.M305378200
Ayako Miyakawa-Naito‡, Per Uhlén, Mark Lal, Oleg Aizman, Katsuhiko Mikoshibas,
Hjalmar Brismar, Sergey Zelenin, and Anita Aperia
From the Department of Woman and Child Health, Karolinska Institutet, Astrid Lindgren Children's Hospital, S-171 76
Stockholm, Sweden and the Department of Basic Medical Science, The Institute of Medical Science, The University of
Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
Recent studies indicate novel roles for the ubiquitous
ion pump, Na,K-ATPase, in addition to its function as a
key regulator of intracellular sodium and potassium
concentration. We have previously demonstrated that
ouabain, the endogenous ligand of Na,K-ATPase, can
trigger intracellular Ca²+ oscillations, versatile intra-
cellular signal controlling a diverse range of cellular
processes. Here we report that Na,K-ATPase and inosi-
tol 1,4,5-trisphosphate (InsP3) receptor (InsP,R) form a
cell signaling microdomain that, in the presence of oua-
bain, generates slow Ca²+ oscillations in renal cells. Us-
ing fluor
fluorescent
escent resonance energy transfer (FRET) meas-
urements, we detected a close spatial proximity between
Na,K-ATPase and InsP.R. Ouabain significantly en-
hanced FRET between Na,K-ATPase and InsP3R. The
FRET effect and ouabain-induced Ca²+ oscillations were
not observed following disruption of the actin cytoskel-
eton. Partial truncation of the NH₂ terminus of Na,K-
ATPase catalytic al-subunit abolished Ca²+ oscillations
and downstream activation of NF-kB. Ouabain-induced
Ca²+ oscillations occurred in cells expressing an InsP3
sponge and were hence independent of InsP3 generation.
Thus, we present a novel principle for a cell signaling
microdomain where an ion pump serves as a receptor.
my
Na,K-ATPase is an integral membrane protein expressed in
all eukaryotic cells, where it functions as a key regulator of
intracellular Na+ and K concentrations (1). Recent studies,
however, point to an additional role for Na,K-ATPase as a
signal transducer (2-5). Importantly, Na,K-ATPase has an en-
dogenous ligand, ouabain, a steroid hormone that t dose-depend-
ently inhibits the activity of Na,K-ATPase. The biological role
of ouabain is, despite extensive research, not well understood.
Ouabain belongs to the family of cardiac glycosides, which have
been used for centuries in the treatment of heart disease.
Recently, several investigators have noted that cardiac glyco-
sides may act as anticancer agents (6, 7).
Vol. 278, No. 50, Issue December 12, pp. 50355-50361, 2003
Printed in U.S.A.
*This work was supported by grants from the Swedish Research
Council, the Persson Family Foundation, the Märta and Gunnar V.
Philipson Foundation (to A. A. and H. B.), Japan Science and Technol-
ogy Corporation "Calcium Oscillation Project" (to K. M. and A. A.), and
the Ministry of Education, Science, Sports and Culture of Japan (to
A. M.-N.). 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.
Both authors contributed equally to this work.
To whom correspondence should be addressed: Dept. of Woman and
Child Health, Karolinska Institutet, Astrid Lindgren Children's Hospi-
tal, Q2:09, S-171 76 Stockholm, Sweden. Tel.: 46-8-51777326; Fax:
46-8-51777328; E-mail: anita.aperia@ks.se.
This paper is available on line at http://www.jbc.org
This is an Open Access article under the CC BY license.
We have described a new cell signaling pathway triggered by
ouabain (2). Using rat renal proximal tubule (RPT)¹ cells, we
showed that exposure to concentrations of ouabain that cause
only partial inhibition of Na,K-ATPase activity induces slow
intracellular Ca²+ oscillations and subsequent activation of the
transcription factors NF-kB and CAMP-response element-bind-
ing protein. Our results from that study indicated that Ca²+
oscillations occurred as an interplay between different Ca²+
transporters and that Ca²+ release via the inositol 1,4,5-
trisphosphate (InsP3) receptor (InsP,R) was involved in this
event. Na,K-ATPase does not possess the characteristics of a
G-protein-coupled receptor. Given the generality of Na,K-
ATPase expression and its significant role in cell homeostasis,
it is
s important to identify the molecular mechanisms by which
Na,K-ATPase can act as a signal transducer. Here we show
that the generation of Ca²+ oscillations by ouabain is depend-
ent on the physical association of Na,K-ATPase and InsP3R in
a signaling microdomain.
EXPERIMENTAL PROCEDURES
Expression Plasmids-A cDNA fragment encoding wild type rat
Na,K-ATPase al-subunit (NKAa1) was amplified by AmpliTaq Gold
(Applied Biosystems). The PCR product was digested by Apal/Xbal
restriction enzymes and cloned into pEGFP-C2 (Clontech) to obtain
pGFP-NKAa1. A mutant NKAa1 with truncation of the first 32 amino
acids (NKAa1.M32) was generated using PCR. The region of truncation
of the NH₂ terminus was decided on the basis of structure/function
analysis reported elsewhere (8). The sense primer sequence was 5'-
AAAGGGCCCATGGAAGTGTCTATGGACGAC-3', corresponding to
nucleotide positions 349-366 of NKAa1 (GenBank™ accession number
NM_012504) with an additional Apal site and ATG codon on the 5'-end
of the primer. The antisense primer was 5'-CTTGCCGTGGAGGAG-
GATAGAACT-3', corresponding to nucleotide positions 1792-1815 of
NKAa1. The PCR product and pGFP-NKAa1 were hydrolyzed by Apal/
AflII restriction enzymes and ligated for cloning PGFP-NKAa1.M32.
A fusion protein with NH₂-terminal glutathione S-transferase (GST)
and 95 amino acids of NKAa1 (GST-NKAa1.N95) was constructed
using Gateway Technology (Invitrogen). Briefly, a cDNA fragment en-
coding 95 amino acids of the Na,K-ATPase al-subunit NH₂ terminus
was amplified by AmpliTaq Gold (Applied Biosystems). The PCR prod-
uct was cloned in pENTR/D-TOPO vector using pENTR Directional
TOPO cloning kit (Invitrogen) and subcloned into pDEST-15 using
Gateway System (Invitrogen) to obtain pGST-NKAa1.N95. The nucle-
otide sequences of all constructs were confirmed by automated sequenc-
ing (KISEQ, Core Facilities of Karolinska Institutet, Stockholm, Swe-
den) and subsequent bioinformatics analysis using Lasergene software
(DNASTAR).
The abbreviations used are: RPT, renal proximal tubule; InsP™,
inositol 1,4,5-trisphosphate; InsP,R, inositol 1,4,5-trisphosphate recep-
tor; FRET, fluorescent resonance energy transfer; PLC, phospholipase
C; ER, endoplasmic reticulum; SERCA, sarco-endoplasmic reticulum
Ca²+ ATPase; CPA, cyclopiazonic acid; 2-APB, 2-aminoethoxydiphenyl
borate; CytD, cytochalasin D; AQP4, aquaporin-4; GST, glutathione
S-transferase; GFP, green fluorescent protein; EGFP, enhanced GFP.
50355
Transcribed Image Text:Na,K-ATPase and InsP, Receptor in a Signaling Microdomain
50356
For FRET control experiments, the cytosolic NH₂ terminus of aqua-
porin-4 (AQP4) was tagged with GFP to obtain pGFP-AQP4 (9). InsP,R
type 1 ligand binding protein (226-604 amino acids) with point muta-
tion (R441Q) encoding InsP, sponge was cloned into a pEF-BOS-MCS
vector (pEF-GSTm49-IRES-GFP) (10). pEGFP-actin was from
Clontech.
Cell Culture and Transfections-Three types of renal cells were used:
primary cultures of rat RPT cells prepared as described (11), COS-7
cells, a cell line derived from fetal monkey kidney, and LLC-PK, cells,
a cell line derived from pig kidney. GFP-NKAa1 was stably expressed in
COS-7 cells (12). pGFP-NKAal, pGFP-NKAa1.M32, pEF-GSTm49-
IRES-GFP, or pEGFP-actin was transiently transfected into RPT cells
on culture day 2 using CLONfectin (Clontech). pGFP-AQP4 was tran-
siently transfected into COS-7 cells on culture day 2 using CLONfectin
(Clontech).
Reagents-Reagents were used at the following concentrations: 20
μM cyclopiazonic acid (CPA), 5 μM cytochalasin D (CytD), 100 pM-250
μM ouabain, 5 μM 2-aminoethoxydiphenyl borate (2-APB), 0.5 μM bra-
dykinin, and 5 μM U73122. All reagents were from Sigma.
Intracellular Calcium and Sodium Measurements-Intracellular
Ca²+ and Nat measurements were performed using Fura-2/AM and
SBFI/AM (Molecular Probes), respectively, as described previously (2,
11). After baseline recording, cells were treated, and ratio images were
recorded every 30 s for 45-90 min. In each dish, 20-30 individual cells
from a single cluster of cells were analyzed. Results presented are
representative single cell traces obtained from a minimum of 12
experiments.
Immunoprecipitation Studies-Cells were solubilized in lysis buffer
(50 mM Tris/HCL (pH 7.4), 150 mm NaCl, 0.25% sodium deoxycholate,
1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, protease inhib-
itors (Roche Applied Science)) and left for 30 min on ice. Cell lysates
were sonicated (3 x 2 s at setting 2 using a Branson Sonifier 250,
Branson Ultrasonics) and centrifuged at 9,000 xg at 4 °C to obtain a
crude cell extract. 500 µg of supernatant protein/reaction were pre-
cleared with protein G-Sepharose for 1 h to reduce background that
may be caused by non-specific adsorption of cellular debris. After a low
speed centrifugation, the resultant supernatant was incubated for 1 h
at 4 °C with a mouse monoclonal anti-Na,K-ATPase al-subunit anti-
body (1:250; Upstate Biotechnology), a mouse monoclonal anti-InsP,R2
antibody (1:50; Santa Cruz Biotechnology), or a mouse monoclonal
antibody anti-InsP,R3 antibody (1:50; BD Biosciences). Immunocom-
plexes were incubated with protein G-Sepharose beads overnight at
4 °C. Beads were pelleted, washed, and incubated with 2x Laemmli
buffer, and supernatants were subjected to SDS gel electrophoresis
using 6% acrylamide gels. Membranes were incubated overnight with a
mouse monoclonal anti-InsP,R3 antibody (1 µg/ml; KM1082) and then
for 1 h using a horseradish peroxidase-conjugated secondary antibody
(1:5000) prior to detection using ECL plus (Amersham Biosciences). The
resultant protein bands were scanned digitally and densitometrically
analyzed by Bio-Rad QuantitativeOne software.
GST Pull-down Assay-GST-NKAa1.N95 was produced in the BL21
strain of Escherichia coli and purified with glutathione-Sepharose 4B
beads (Amersham Biosciences). Non-recombinant GST was used as a
control. Detergent-extracted RPT cell lysate (prepared as described
above for the co-immunoprecipitation protocol) was added to the beads
in a 5:1 ratio (v/v) and incubated overnight at 4 °C with gentle rotation.
Beads were washed and resuspended in 2x Laemmli buffer prior to
SDS gel electrophoresis (6% gel) and immunoblotting for InsP R3.
Immunocytochemistry and Confocal Microscopy-For co-immunolo-
calization and FRET studies, COS-7 cells stably expressing GFP-
NKAa1 were fixed with acetone for 3 min at room temperature and then
incubated with phosphate-buffered saline containing 5% (v/v) normal
goat serum and 3% (w/v) bovine serum albumin for 1 h. InsP,Rs were
probed with monoclonal mouse anti-rat InsP,R type 2 (KM1083) or type
3 (KM1082) antibodies (1 µg/ml) (13) overnight at 4 °C. Cy3-conjugated
goat anti-mouse IgG antibody served as secondary antibody (1:1000,
Jackson ImmunoResearch Laboratories). Cells were scanned with at
Leica TCS SP inverted confocal scanning laser microscope.
NF-kB activation was measured in RPT cells by immunocytochemi-
cal staining as described previously (2). RPT cells transiently trans-
fected with PGFP-NKAa1.M32 were treated with 250 μM ouabain for 30
In accordance with previous observations (2), ouabain (250
μM) induced highly regular intracellular Ca²+ oscillations with
a periodicity in the minute range in RPT cells (Fig. 1a). Typical
min and then fixed using 3% paraformaldehyde (10 min). Following Ca²+ oscillations were detected about 5-15 min after ouabain
blocking as described above, cells were incubated with NF-kB p65
antibody (1:200, Santa Cruz Biotechnology) for 1 h and then with Alexa
546 fluorescent secondary antibody (1:500, Molecular Probes) for 30
min. Slides were scanned using a Leica TCS SP inverted confocal
scanning laser microscope, and images of cells expressing the construct
were identified by GFP signal; NF-kB immunostaining of cells was
captured for the same field of view. NF-kB activation in individual cells
was semiquantitatively estimated by measuring the ratio between the
mean NF-kB immunosignal in a given comparable area in the nucleus
and cytoplasm in cells expressing GFP-NKAa1.M32 or those adjacent
cells not expressing the construct using ImageJ (Wayne Rasband, Na-
tional Institutes of Health).
exposure in approximately one-third of the cells and were gen-
erally initiated in one cell at the periphery of a cell cluster.
Quantitatively and qualitatively, COS-7 cells treated with oua-
bain showed a similar Ca²+ oscillatory response (Fig. 1b).
Spontaneous oscillations in cytosolic Ca²+ were never observed
in untreated cells. Na,K-ATPase activity is dose-dependently
inhibited by ouabain, and 250 μM ouabain causes ~50% inhi-
bition of rat Na,K-ATPase activity (2). Ouabain, 250 μm, ex-
ceeds circulating levels in rat, estimated to be in the pM-nM
range (17). When cells were exposed to physiological ouabain
doses (100 pm), Ca²+ oscillations were observed (Fig. 1c) in
-1% of cells. For subsequent experiments designed to explore
the mechanism by which Na,K-ATPase triggers Ca²+ oscilla-
tions, we used 250 μM ouabain.
To elucidate the source of the Ca²+ oscillatory response,
intracellular endoplasmic reticulum (ER) Ca2+ stores were de-
pleted by preincubation with a sarco-endoplasmic reticulum
2+ AMD (SERCA)
Ca²+ ATPase (SERCA) inhibitor, CPA (Fig. 1d). Ouabain did
ancillation in
De
not induce Ca²+ oscillations in CPA-pretreated cells. Regulated
AND
Ca²+ release from intracellular ER Ca²+ stores occurs mainly
intracentral
Ima Dam
via InsP Rs or via ryanodine receptors. InsP3Rs are abun-
dantly expressed in RPT cells, whereas ryanodine receptors do
not have any functional importance for ouabain-triggered Ca²+
oscillations in these cells (2). The membrane-permeable sub-
stance, 2-APB, was initially introduced as a specific inhibitor of
InsP3Rs (18). The IC50 for inhibition of InsP3R-evoked Ca²+
release was reported to be 1-20 µM. Since then, 2-APB has, in
addition to its inhibitory effect on InsP3-induced Ca²+ release,
been shown to block store-operated calcium-mediated cytosolic
Ca²+ influx (19). Store-operated calcium is generally fully in-
hibited by 50-100 μM 2-APB. Exposure of cells to concentra-
tions of 2-APB higher than 100 μM may also cause a pro-
FRET-Fluorescent resonance energy transfer (FRET) measure-
ments were performed on a Leica TCS SP inverted confocal scanning
laser microscope using a X40/1.4 NA objective. A detailed description of
the FRET technique can be found elsewhere (14, 15). The Förster
constant, Ro, for the donor-acceptor pair, GFP and Cy3, used in this
study was 6 nm (16). FRET occurs when the fluorophores are separated
by distances 0.5 R, <r < 2 Ro. Thus, it is possible to distinguish
proteins that are spatially co-localized within a 12-nm radius. To de-
termine FRET, we quantified the quenching of donor fluorescence by
performing acceptor photobleaching (14). COS-7 cells stably expressing
GFP-NKAa1 and stained with Cy3-labeled secondary goat anti-mouse
IgG secondary antibody (Jackson ImmunoResearch Laboratories) to
detect mouse monoclonal antibody to InsP,R2 (KM1083) and InsP,R3
(KM1082) were excited with 488 and 543 nm and collected separately.
The acceptor, Cy3, was then irreversibly photobleached in a selected
adequate region by continuous excitation with 543 and 633 nm lasers
for 30-90 s. Thereafter, the residual Cy3 and GFP image was obtained,
and identical regions, at the plasma membrane on individual cells, were
outlined in the photobleached area and processed using ImageJ (Wayne
Rasband, National Institutes of Health). Ratios between GFP intensi-
ties of the plasma membrane region, after and before photobleaching,
were calculated to quantify FRET. The FRET values presented are
corrected for erroneous intensity changes in a selected region outside
the bleached area. In a typical experiment, 10-15 cells were measured
for each sample.
Data Presentation and Analysis-Data are presented as means ±
S.E. of a minimum of 10 experiments, unless indicated otherwise.
Student's t test was used, and significance was accepted at p < 0.05.
RESULTS
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