responses to ouabain and are both essential for survival of human neuroblastoma Larisa Karpova1,2, Alexander Eva1, Ulrike Kirch1, Alexander Boldyrev2, Georgios Scheiner-Bobis1 1 Institut
Trang 1responses to ouabain and are both essential for survival of human neuroblastoma
Larisa Karpova1,2, Alexander Eva1, Ulrike Kirch1, Alexander Boldyrev2, Georgios Scheiner-Bobis1
1 Institut fu¨r Biochemie und Endokrinologie, Fachbereich Veterina¨rmedizin, Justus-Liebig-Universita¨t Giessen, Germany
2 Department of Biochemistry, Lomonosov Moscow State University, Moscow, Russia
Introduction
The sodium pump (Na+,K+-ATPase; EC 3.6.1.37)
maintains the Na+gradient across plasma membranes
of animal cells [1] By hydrolyzing ATP, the enzyme
transports three Na+ ions out of the cell in exchange
for two K+ions that are brought into the cytosol This
activity can be interrupted by a group of substances
that are referred to as cardiotonic steroids (CTS),
a name linked to their clinical use for the treatment of
heart failure [2]
In recent years, numerous publications have
established that CTS not only inhibit the sodium pump
but also induce signaling cascades that may be
associ-ated with cell growth and proliferation as well as with
apoptotic cell death, depending upon the cell type or
CTS investigated CTS-induced signaling does not depend on sodium pump inhibition [3–8], as inactive sodium pump mutants can still transmit signals when CTS are added to the cell culture [9,10]
The sodium pump of animal cells is an oligomeric enzyme consisting of a and b subunits [1] In some tissues, a regulatory c subunit is associated with the a and b subunits [11] All three subunits have been co-crystallized in several conformational states of the enzyme [12–14] The a subunit, which is referred to as the catalytic subunit, has ten transmembrane domains, hydrolyzes ATP, transports the cations and is the phar-macological receptor for CTS The b subunit is a highly glycosylated protein with a single transmembrane span,
Keywords
cardiotonic steroids; Erk1⁄ 2;
Na + ,K + -ATPase; signaling; SK-N-AS cells
Correspondence
G Scheiner-Bobis, Institut fu¨r Biochemie
und Endokrinologie, Fachbereich
Veterina¨rmedizin, Justus-Liebig-Universita¨t
Giessen, Frankfurter Strasse 100, D-35392
Giessen, Germany
Fax: +49 641 9938179
Tel: +49 641 9938180
E-mail: georgios.scheiner-bobis@vetmed.
uni-giessen.de
(Received 9 December 2009, revised 11
January 2010, accepted 1 February 2010)
doi:10.1111/j.1742-4658.2010.07602.x
Using SK-N-AS human neuroblastoma cells, which co-express the a1 and a3 isoforms of the sodium pump a subunit, we selectively silenced either the a1 or a3 subunit by means of transfection with small interfering RNA, and investigated cell survival and the cellular response to ouabain We found that both of the a subunits are essential for cell survival, indicating that substitution of one subunit for the other is not sufficient In the pres-ence of both a subunits, ouabain causes sustained activation of extracellu-lar signal-regulated kinases 1 and 2 (Erk1⁄ 2) This activation is not affected when the a1 subunit is silenced However, when a3 expression is silenced, ouabain-induced activation of Erk1⁄ 2 does not occur, even at a high concentration of ouabain (1 lm) Thus, ouabain-induced Erk1⁄ 2 acti-vation is mediated in SK-N-AS cells by a3 only, and a1 does not partici-pate in this event This is a clear demonstration of selective involvement of
a specific sodium pump a subunit isoform in ouabain-induced signaling
Abbreviations
CTS, cardiotonic steroids; Erk1 ⁄ 2, extracellular signal-regulated kinases 1 and 2; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide.
Trang 2and appears to function as a molecular chaperone for
correct folding of the a subunit and its transportation
to the plasma membrane [15] The c subunit (also
termed FXYD2) is a member of the FXYD family of
proteins that includes phospholemman (FXYD1) and
corticosteroid hormone-induced factor (FXYD4)
These very hydrophobic proteins are characterized by a
single transmembrane span (except FXYD3, mammary
tumor marker Mat-8, which has two transmembrane
spans) and an FYXD motif near the transmembrane
domain, in the extracellular N-terminal part of the
pro-tein [16] Each of the subunits exists in various isoforms
[1] There are four a subunits (a1–a4), three b subunits
(b1–b3) and two splice variants of the c subunit
(FXYD2a and FXYD2b) Various investigations have
shown that at least five of the seven FXYD proteins
interact with the a and b subunits of Na+,K+-ATPase
and regulate functions of the enzyme [16] In several
cases, multiple isoforms of the a subunit are found in
the same cell type, thus raising questions about the
physiological significance of such co-existence
Given that CTS⁄ sodium pump interactions resemble
typical hormone⁄ receptor-mediated events in many
respects [7,17], it may be assumed that co-existing
subunits are involved in other signaling events in
addition to the ion pump function [6–8]; however, there
is no direct evidence to support this notion so far We investigated this question by using the human neuro-blastoma cell line SK-N-AS These neuroneuro-blastoma cells, which co-express the a1 and a3 subunits, were shown to interact with the CTS ouabain The results provide evidence for distinctive roles in signal media-tion for the two subunit isoforms of the sodium pump
Results
Expression of a subunit isoforms of
Na+,K+-ATPase in SK-N-AS cells before and after transfection with StealthTMRNAi to silence a1 and a3
SK-N-AS is a cancerous neuroblastoma cell line that, like other neuronal cells [18,19], expresses a1 and a3 subunit isoforms (Fig 1) As shown in Fig 1A, in control (untreated) SK-N-7AS cells, a1 and a3 subunit-specific cDNA bands are present in equivalent quanti-ties (left lanes) The same result is seen when the cells are transfected with control RNAi: expression of both a1-specific and a3-specific cDNA is like that of the con-trol cells (Fig 1A, middle lanes) However, transfection
Transfected with negative control RNAi
M
M
Control cells
Transfected with αα1 RNAi
500 bp
α1 α2 α3 α1 α2 α3 α1 α2 α3 GAPDH H 2 O M
Control cells Transfected with negative control RNAi
Transfected with α3 RNAi
500 bp
A
Control Lipofectamine Negative
Control RNAi
α1 isoform of
SP RNAi
Control Lipofectamine Negative
control RNAi
α3 isoform of
SP RNAi
**
**
0 25 50 75 100 125
0 25 50 75 100 125
B
Fig 1 Effect of transfection with various RNAi on expression of a subunit isoforms (A) Expression of a subunit isoforms in SK-N-AS cells without and with transfection with RNAi Like other neuronal cells, the neuroblastoma cell line SK-N-AS expresses the a1 and a3 isoforms of the sodium pump a subunit (left lanes, upper and lower panels) Transfection with a1- or a3-specific RNAi silences expression of the corre-sponding mRNA Detection of glyceraldehyde 3-phosphate dehydrogenase (lower panel, right) was used for normalization purposes (B) Expression of a subunit isoforms in SK-N-AS cells without and with transfection with RNAi (normalized) Transfection of SK-N-AS cells with either a1- or a3-specific RNAi significantly silences the expression of corresponding mRNA (n = 9; **P < 0.01) Lipofectamine alone and negative-control RNAi do not have any effect on the expression of a1 or a3 mRNA.
Trang 3of the SK-N-AS cells with either a1-specific RNAi
(Fig 1A, upper panel) or a3-specific RNAi (Fig 1A,
lower panel) leads to a reduction in the corresponding
mRNA⁄ cDNA (Fig 1A, right lanes) In both cases, the
reduction in expression of either a1- or a3-specific
mRNA is significant (Fig 1B) mRNA⁄ cDNA for
the a2 subunit was not detected For normalization of
data, glyceraldehyde 3-phosphate
dehydrogenase-specific mRNA⁄ cDNA from every probe was amplified
in parallel experiments (Fig 1A) Its expression was
not affected
Survival of SK-N-AS cells before and after
transfection with StealthTMRNAi to silence
a1 and a3
Experiments with knockout mice demonstrated that
either the a1 or a2 isoforms are essential for survival of
the animals [20] Nevertheless, it is not known whether
survival of cells that co-express multiple a subunits
depends on the simultaneous presence of the various
subunits or whether one subunit isoform can substitute
for the other To investigate this question, SK-N-AS
cells were treated with specific RNAi to silence the
expression of either the a1 or a3 subunit of the sodium
pump, and cells were cultured for several days
Untreated cells served as a control The MTT assay
was used to determine the number of living cells under
each condition Figure 2 shows that, for the first 48 h,
growth is the same for all cell types However, cells
lacking either a1 or a3 do not multiply further
thereaf-ter, and after 8 days (192 h), the number of living cells
was reduced by more than 25% compared to the
origi-nal number of cells, and by about 70% when compared
to the number of living cells expressing both a1 and a3 subunits It should be noted, however, that the cells expressing both subunits reached confluence after the 4th day of incubation and therefore did not multiply any further
Ouabain-induced signaling in SK-N-AS cells Ouabain and other CTS induce signaling cascades in a variety of cells and also in the neuroblastoma cell line SH-SY5Y [7,21,22] One of the first events seen upon exposure of various cell types to CTS is activation of extracellular signal-regulated kinases 1 and 2 (Erk1⁄ 2) [3,21,23–26] Therefore, we focused our attention on
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
α1 RNAi α3 RNAi
Hours after transfection
Fig 2 MTT assay of cell viability Proliferation of SK-N-AS cells treated with a1- (open circles) or a3- (open triangles) specific RNAi was assessed by the MTT assay over a period of 8 days Non-trea-ted cells (open squares) served as a control The proliferation of cells treated with either a1 or a3 RNAi declines after 48 h By the end of the observation time, RNAi-treated cells account for about 30% of the non-treated control (n = 6–12).
50 kDa
40 kDa
30 kDa
Phospho Erk 1/2
0 10 100 1000
0
M 10 100 1000 M
Total Erk 1/2
50 kDa
40 kDa
30 kDa
0 10 n M 100 n M 1000 n M
0 100 200
Ouabain
Phospho Erk 1/2
**
**
**
C
Fig 3 Activation of Erk1 ⁄ 2 by ouabain.
(A) Detection of total, non-phosphorylated
Erk1 ⁄ 2 was used for normalization
purposes (B) Incubation of SK-N-AS cells
for 30 min with low concentrations of
ouabain stimulates Erk1 ⁄ 2 activation
(phosphorylation), as detected by specific
antibody against the phosphorylated form of
Erk1 ⁄ 2 (C) Erk1 ⁄ 2 activation by ouabain is
highly significant at all ouabain
concentra-tions tested (n = 4; **P < 0.01) M,
molecular weight markers.
Trang 4possible stimulation (phosphorylation) of these kinases
in SK-N-AS cells As in other cell types, low concentra-tions of ouabain trigger significant activation of Erk1⁄ 2 within 30 min in SK-N-AS cells (Fig 3), thus raising the question of whether ouabain-induced activation of these kinases is mediated through the a1 or a3 subunit or through both subunit isoforms of the sodium pump
Ouabain-induced activation of Erk1⁄ 2 after silencing either a1 or a3 subunits
Cells expressing only the ubiquitous a1 subunit of the sodium pump respond to CTS by induction of a variety
of signaling cascades However, in the present study, cells lacking a1 clearly show concentration-dependent ouabain-induced activation of Erk1⁄ 2 (Fig 4) In con-trast, cells lacking a3 did not respond to ouabain, even
at the very high concentration of 1 lm (Fig 4)
Discussion
Previous experiments with knockout mice have demon-strated that the presence of either a1 or a2 subunits is critical for animal survival [20] However, as single cells can simultaneously express various a subunits, we addressed the question of whether survival of cells would be affected by loss of only one a subunit isoform, and, if so, which one is more essential for survival Using the same experimental set-up, we also determined whether a1 and a3 subunits respond to ouabain by induction of different signaling events or whether signaling cascades are isoform-independent When either the a1 or a3 subunits are silenced, cells proliferate over a period of 2 days in a manner similar
to the control, in which neither of the two subunits had been silenced (Fig 2) After that, the number of living cells starts declining, until, at day 8, the numbers
of cells that lack either a1 or a3 are only about 30%
of the number of control cells that express both subunit isoforms The data in Fig 2 indicate that cells lacking a1 show an earlier loss of viability than those lacking a3; however, the impact of this is not known Nevertheless, the results clearly show that both a1 and a3 subunits are essential for survival, and that the a1 and a3 isoforms have distinct roles in SK-N-AS cells and loss of one cannot be compensated by the other Based on these and previous findings, it is possible to speculate that these subunits are similarly essential in other cell types as well
What niche of cell biological functions do a1 or a3 subunits occupy that makes them essential for sur-vival? Are their functions identical, or do they differ in some respects? In the investigation presented here, the
α3/+ α3/- α3/+ α3/- α3/+ α3/- α3/+
α3/-Ouabain (n M )
α1/+ α1/- α1/+ α1/- α1/+ α1/- α1/+
α1/-Ouabain (n M )
0
50
100
150
200
250
0
50
100
150
200
250
Erk 1/2 activation (%) α3/+ α3/- α3/+ α3/- α3/+ α3/- α3/+
α3/-α1/+ α1/- α1/+ α1/- α1/+ α1/- α1/+
α1/-Ouabain (n M ) 0 10 100 1000
Ouabain (n M ) 0 10 100 1000
*
°
*
*
*
*
*
°
°
°
A
B
C
D
Fig 4 Activation of Erk1 ⁄ 2 by ouabain in cells lacking either a1 or
a3 subunits (A) When cells are transfected with a1-specific RNAi
(a1 ⁄ -), ouabain still induces activation (phosphorylation) of Erk1 ⁄ 2.
(B) When cells are transfected with a3-specific RNAi (a3 ⁄ -),
activa-tion of Erk1 ⁄ 2 is absent, even at the high concentration of 1 l M
ouabain, indicating involvement of the a3 subunit in the signaling
process (C) Statistical analysis of data similar to those shown in
(A) obtained from other western blot experiments, showing
stimu-lation of Erk1 ⁄ 2 by ouabain in cells with silenced a1 subunits
(asterisk indicates that Erk1 ⁄ 2 is significantly activated at all
oua-bain concentrations used in both RNAi-treated and untreated cells;
P < 0.05; n = 4) (D) Statistical analysis of data similar to those
shown in (B) obtained from other western blot experiments,
show-ing stimulation of Erk1 ⁄ 2 by ouabain in cells with silenced a3
subunits (asterisk indicates that Erk1 ⁄ 2 is significantly activated at
all ouabain concentrations used in cells not treated with RNAi;
P < 0.05; circle indicates significantly reduced Erk1 ⁄ 2 activation in
all cells with silenced a3; n = 4).
Trang 5latter seems to be the case; cells lacking the a1 subunit
respond to ouabain by activation of Erk1⁄ 2, indicating
that the a3 subunit can transmit CTS-induced
signal-ing (Fig 4A,C) However, silencsignal-ing the a3 subunit
abolishes the ability of the cells to respond to CTS
(Fig 4B), indicating that the presence of a3 is essential
for this signaling pathway in SK-N-AS cells
Activa-tion of Erk1⁄ 2 was not observed in these cells even at
the high concentration of 1 lm ouabain (Fig 4B),
indi-cating that inhibition of the sodium pump, which
occurs at this concentration, is not a requirement for
CTS-induced signaling, as shown previously [9,10]
Figure 4B,D additionally shows that, after silencing
the a3 subunit, the Erk1⁄ 2 activity is already reduced
compared to the activity seen in control cells,
indicat-ing that, even in the absence of externally added
oua-bain, part of the Erk1⁄ 2 basic activity is contributed
by the a3 subunit of the sodium pump
Here we demonstrate for the first time that a1 and
a3 have distinct functions in cell physiology, but this
leads to a fundamental question: why does a1 not
mediate CTS-induced activation of Erk1⁄ 2 in SK-N-AS
cells when it has been shown to be involved in
CTS-induced signaling in various other cells? This question
can be addressed by taking into consideration the
mechanisms by which CTS generate signals and the
structural differences between the a1 and a3 sodium
pump subunits CTS-induced signaling can be
explained by two different models In the first model, it
is assumed that cell signaling induced by CTS is due to
inhibition of the sodium pump and a local increase in
intracellular [Na+] followed by a subsequent increase
in [Ca2+] in the small space between the plasmalemma
and endoplasmic⁄ sarcoplasmic reticulum This space,
referred to as the plasmerosome, contains sodium
pump isoforms a2and a3but not a1 in smooth muscle
cells and astrocytes [6,8,27] In an alternative model,
the sodium pump is considered to be a member of a
caveolae-defined environment of proteins that are
capa-ble of communicating with each other This entity is
referred to as the signalosome [28] This model
pro-poses that it is not inhibition of the sodium
pump but rather conformational changes of the
CTS⁄ Na+, K+-ATPase complex that trigger the
signal-ing cascade This is supported by the fact that signalsignal-ing
cascades are activated when CTS interact with
non-pumping sodium pump mutants [9] Although the
models differ in their basic assumption, they have in
common the requirement that the sodium pump be
targeted to a defined environment
The results presented here show that the a3 isoform
can generate CTS-induced activation of Erk1⁄ 2,
indi-cating its localization in an environment different to
that of the a1 isoform The basis for these different environments is may be due to differences in the struc-ture of the two proteins The primary strucstruc-tures of the human a1 and a3 isoforms are 87% identical and display a similarity of 94% Nevertheless, at the level
of tertiary structure, they display significant differ-ences In a recently published comprehensive work, comparison of the tertiary structures of a1⁄ a2 and a1⁄ a3 subunits revealed that surface-exposed areas of the a2 or a3 isoforms were very different from the corresponding areas of the a1 isoform [29] These areas are found mainly within the N-domain but also within the A-domain of the proteins The membrane-spanning segments of the three isoforms are rather conserved [29] We assume that the clusters of isoform-specific differences in the surface-exposed regions might be important for isoform-specific interactions with other proteins These specific interactions, which may result
in either distinctive targeting of the isoforms to differ-ent areas of the plasma membrane or specific interac-tions with signaling molecules (or both), could be the reason for the differences found for ouabain-induced signaling through the a1 or a3 isoforms The recent demonstration that the a1 isoform is recruited to the plasma membrane via interaction of adaptor protein 1 with Tyr255 of this isoform supports this hypothesis, and demonstrates that even small differences in surface-exposed areas may have a big impact in target-ing of the proteins [30] Isoforms a2 or a3 lack this tyrosine residue, and we assume that their targeting to specific areas of the plasma membrane must be defined
by other parameters
Thus, based on our results and those of others discussed above, we suggest that the differences seen in the CTS-induced signaling through the a1 or a3 isoforms are associated with differences in the surface-exposed regions of the two proteins These might lead to specific targeting of each isoform to different micro-environments of the plasma membrane or to isoform-specific interactions with other proteins of the micro-environments Future work should help to verify this assumption
Experimental procedures
Cell culture
Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium (PromoCell, Heidelberg, Germany)
Trang 6medium was replaced twice per week Cells were harvested
by incubating with trypsin (0.25%; PromoCell) for 2 min at
Preparation of cell lysates
Cell lysates were prepared as described previously [21]
SDS/PAGE and western blotting of isolated
proteins
A total of 10–50 lg of protein was separated by SDS/PAGE
using 10% acrylamide and 0.3%
N,N¢-methylene-bis-acryla-mide gels Biotinylated molecular weight markers (Cell
Signaling Technology, Frankfurt am Main, Germany) were
run in parallel After SDS/PAGE, proteins were
electro-blotted onto nitrocellulose membranes (Schleicher & Schuell,
Dassel, Germany) at 500 mA for 30–40 min Detection of
proteins was performed as described by the manufacturers of
the antibodies (Cell Signaling Technology, Santa Cruz Inc
or Dianova, Hamburg, Germany) in combination with an
enhanced chemiluminescence (ECL) kit (GE HealthCare,
Munich, Germany) Chemiluminescence was visualized and
quantified using a molecular imager ChemiDoc XRS system
(Bio-Rad, Munich, Germany)
Detection of a1-, a2- and a3-specific mRNA in
SK-N-AS cells
Cells were grown to 75% confluence before isolation of
total mRNA using the RNeasy mini kit (Qiagen, Hilden,
Germany) RNase-free DNase I (Qiagen) was used to
elimi-nate potential contamination by DNA The concentration
and purity of total mRNA were determined by measuring
the absorbance at 260 and 280 nm
The OneStep RT-PCR kit (Qiagen) was used for reverse
transcription and PCR amplification of DNA In a total
dNTPs), 2 lL of a mixture of Omniscript and Sensiscript
reverse transcriptases and HotStar Taq DNA polymerase
were incubated in a MasterCycler gradient (Eppendorf,
for 15 min, followed by 40–45 cycles of denaturation at
10 min For specific amplification of a1, the forward
and reverse primers were 5¢-GTTGGGGCTCCGATGTGTT
GGGGT-3¢ and 5¢-CTGGCTGGAGGCTGTCATCTTCTT
CAT-3¢, respectively; for specific amplification of a2, the
forward and reverse primers were 5¢-CTGGCTGGAGGC
TGTCATCTTCTTCAT-3¢ and 5¢-GGCTCTTGGGGGCT
GTCTTCTCGCT-3¢, respectively; for specific amplification
of a3, the forward and reverse primers were 5¢-CTGGCTT GAGGCTGTCATCTTCTTCAT-3¢ and 5¢-ATCGGTTGT CGTTGGGGTCCTCGGT-3¢ respectively The correspond-ing fragment sizes are 560 bp (a1), 557 bp (a2) and 560 bp (a3) To control PCR efficiency and the quality of the cDNA,
and 5¢-TAAGCAGTTGGTGGTGCAGGAGGCA-3¢ were used to co-amplify a specific fragment of 469 bp coding for the housekeeping gene glyceraldehyde 3-phosphate dehydro-genase
The RT-PCR products were analyzed by electrophoresis
in a 1.7% agarose gel The correct identity of a1- or a3-spe-cific amplified sequences was further verified by digestion with Bpi, Eco47I, BspTI (all MBI Fermentas, St Leon-Rot, Germany) and subsequent agarose gel electrophoresis
Silencing a1 or a3 mRNA biosynthesis by siRNA transfection
transfected into SK-N-AS cells using Lipofectamine 2000
SK-N-AS cells were placed in each well of a six-well culture vessel in Dulbecco’s modified Eagle’s medium without antibiotics Cells were 30–50% confluent at the time of
Lipo-fectamine 2000 were diluted with Opti MEM I Reduced Serum Medium (Invitrogen) and incubated for 5 min at
diluted Lipofectamine 2000 were combined, mixed gently and added to the cells after 20 min The final concentration
trea-ted with Lipofectamine 2000 only In parallel, cells were
trans-fection kit (Invitrogen) according the manufacturer’s proto-col Total RNA was isolated from SK-N-AS cells, and the extracted RNA was subjected to RT-PCR to amplify a1-, a2-and a3-specific DNA fragments as described above
UGUGGUGCUAUCAGCCGUUGUA-3¢ and 5¢-UACAA CGGCUGAUAGCACCACACCC-3¢ (a1 subunit); 5¢-AC GACAACCGAUACCUGCUGGUGAU-3¢ and ‘5-AUCA CCAGCAGGUAUCGGUUGUCGU-3¢ (a3 subunit)
RNAi, cells were exposed to various ouabain concentrations for 3 h These cells were used to produce lysates that were subsequently used in western blot experiments
In a different set of experiments, the survival of cells
period of time using the MTT assay described below
Trang 7MTT assay
above After replacing the transfection medium by complete
growth medium, incubation was continued for various
times (1–8 days) Afterwards, the medium was aspirated
and replaced by 300 lL of fresh medium containing
addi-tional 4 h The medium containing MTT was removed by
inverting the plate, and the resulting formazan crystals were
solubilized by adding 200 lL dimethyl sulfoxide to each
well After 10 min of vigorous vortexing, the absorbance in
each well was read in a microplate reader at 540 nm
Statistical analysis
Data were analyzed by one-way anova and by applying
Dunnett’s comparison for evaluation of all data with respect
to control values Significance was accepted at P < 0.05
Acknowledgements
L.K was supported by a stipend granted through the
German Academic Exchange Service (Deutscher
Aka-demischer Austausch Dienst)
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