In HeLa cells, the highest increment of c-Fos mRNA content was noted after 6 h of Na+⁄ K+-ATPase inhibition with ouabain that was abolished by actino-mycin D, an inhibitor of RNA synthes
Trang 1by activation of the 5¢-promoter containing known
transcriptional elements
Mounsif Haloui1,*, Sebastien Taurin1,2,*, Olga A Akimova1, Deng-Fu Guo1, Johanne Tremblay1, Nickolai O Dulin2, Pavel Hamet1and Sergei N Orlov1
1 Centre de recherche, Centre hospitalier de l’Universite´ de Montre´al (CHUM) ) Technopoˆle ANGUS, Montreal, PQ, Canada
2 Department of Medicine, University of Chicago, Chicago, IL, USA
Previously, we reported that elevation of the
[Na+]i⁄ [K+]i ratio, caused by sustained inhibition of
Na+⁄ K+-ATPase with ouabain or in K+-free
med-ium, inhibits apoptosis in serum-deprived vascular
smooth muscle cells (VSMCs) [1] Subsequently, the
antiapoptotic action of Na+⁄ K+-ATPase inhibition
was also detected in cultured renal proximal tubule
cells [2], freshly isolated rat cerebellar granule cells [3], and endothelial cells from the human umbilical vein [4] and porcine aorta [5] With endothelial cells, we found that Na+⁄ K+-ATPase inhibition blocked apoptosis triggered by3H-induced DNA damage via elevation of [Na+]i rather than attenuation of [K+]i [5] We also noted that in VSMCs, protection against apoptosis
Keywords
c-Fos; expression; intracellular Na+; ouabain;
5¢-UTR
Correspondence
S N Orlov, Centre de recherche,
CHUM ) Technopoˆle ANGUS, 2901, Rachel
est, Montreal, Quebec H1W 4A4, Canada
Fax: +1 514 412 7638
Tel: +1 514 890 8000
E-mail: sergei.n.orlov@umontreal.ca
*These authors contributed equally to this
work
(Received 4 January 2007, revised 25 April
2007, accepted 16 May 2007)
doi:10.1111/j.1742-4658.2007.05885.x
In vascular smooth muscle cells and several other cell types, inhibition of
Na+⁄ K+-ATPase leads to the expression of early response genes, including c-Fos We designed this study to examine whether or not a putative
Na+i⁄ K+
i-sensitive element is located within the c-Fos 5¢-UTR from) 650
to + 103 containing all known response elements activated by ‘classic’ stimuli, such as growth factors and Ca2+
i-raising compounds In HeLa cells, the highest increment of c-Fos mRNA content was noted after 6 h of
Na+⁄ K+-ATPase inhibition with ouabain that was abolished by actino-mycin D, an inhibitor of RNA synthesis c-Fos protein accumulation in ouabain-treated cells correlated with a gain of Na+iand loss of K+i Aug-mented c-Fos expression was also observed under inhibition of Na+⁄ K+ -ATPase in K+-free medium and in the presence of the Na+ ionophore monensin The effect of ouabain on c-Fos expression was sharply attenu-ated under dissipation of the transmembrane Na+ gradient, but was pre-served in the presence of Ca2+ chelators and the extracellular regulated kinase inhibitor PD98059, thus indicating an Na+i-mediated, Ca2+i- and extracellular regulated kinase-independent mechanism of gene expression
In contrast to massive c-Fos expression, we failed to detect any effect of ouabain on accumulation of luciferase driven by the c-Fos 5¢-UTR Negat-ive results were also obtained in ouabain-treated vascular smooth muscle cells and C11 Madin–Darby canine kidney cells possessing augmented c-Fos expression Our results reveal that Na+i-induced c-Fos expression is not mediated by the 5¢-UTR containing transcriptional elements activated
by growth factors and other ‘classic stimuli’
Abbreviations
CRE, Ca2++ cAMP response element; ERK, extracellular regulated kinase; LDH, lactate dehydrogenase; MDCK, Madin–Darby canine kidney; NaRE, Na + response element; SRE, serum response element; VSMC, vascular smooth muscle cells.
Trang 2was almost completely abolished by inhibitors of RNA
and protein synthesis [6], suggesting the de novo
expression of genes involved in suppression of the cell
death machinery This hypothesis was confirmed by
proteomics-based analysis of soluble proteins from
control and ouabain-treated VSMCs, leading to the
identification of mortalin as an antiapoptotic gene
trig-gered by elevation of the [Na+]i⁄ [K+]iratio [7]
Keeping in mind the increase of total RNA synthesis
seen in ouabain-treated VSMCs [6], we proposed that
the expression of antiapoptotic genes, including
morta-lin, is at least partially mediated by early response
genes [8] This hypothesis was also consistent with
accumulation of early response gene mRNA detected
in cultured renal epithelial cells, fibroblasts,
hepato-cytes, and leukemia and melanoma cell lines, as well as
in freshly isolated cardiomyocytes [9] In VSMCs, 2 h
of incubation with ouabain or K+-free medium led to
a 10-fold increase of immunoreactive c-Fos protein
content, followed by c-Jun expression Importantly,
accumulation of c-Fos mRNA in ouabain-treated
VSMCs correlated with increased [Na+]i and preceded
the loss of K+i[10]
It is well documented that the c-Fos promoter
contains a serum response elements (SRE) and a
Ca2++ cAMP response element (CRE) activated by
[Ca2+] elevation in the cytoplasm and nucleus,
respect-ively [11,12] (Fig 1) These data suggest that c-Fos
expression in Na+i-loaded cells might be mediated by
activation of the Na+⁄ Ca2+exchanger and an increase
in [Ca2+]i However, neither [Ca2+]i nor total
exchangeable Ca2+ content in VSMCs was augmented
by ouabain Moreover, ouabain-induced c-Fos
accu-mulation was preserved in the presence of nicardipine
as well as extracellular and intracellular Ca2+chelators [10] These data allowed us to conclude that c-Fos expression in ouabain-treated VSMCs was mediated
by a novel Na+i-sensitive, Ca2+i-independent trans-cription factor We designed this study to localize the putative Na+response element (NaRE) within the 5¢-UTR containing all known elements involved in the regulation of c-Fos expression
Results The effect of ouabain on c-Fos expression
is cell type-specific Keeping in mind that VSMCs from the rat aorta used
in our previous study [10] possess low transfection efficacy [7], we compared the action of ouabain on cul-tured cells with different origins Figure 2A shows that
5 h of inhibition of Na+⁄ K+-ATPase with ouabain led to an 10-fold elevation of intracellular exchange-able Na+in all types of cells investigated In contrast, the action of ouabain on c-Fos mRNA was cell type-specific, with the highest accumulation in HeLa and C11 Madin-Darby canine kidney (MDCK) cells and a modest rise in HIH 3T3 fibroblasts and C7-MDCK cells; no effect of ouabain on c-Fos expression was observed in H9C2 cells (Fig 2B) To search for NaRE within the 5¢-UTR c-Fos promoter, we tested HeLa cells Side-by-side with the highest increment of c-Fos expression and perfect susceptibility to transfection, the choice of HeLa cells was motivated by the exist-ence of a commercially available activation domain fusion cDNA library from HeLa cells that could be employed for the identification of NaRE-binding
-650 -348 -335 -320 -313 -297 -291 -176 -166 -97 -76 -67 -62 -59 -55 -31 +103
CArG
GGATATTACC
CCATATTAGG
-303 -312
TATA CRE
MLTF/USF GC-rich
direct repeats/
RCE
FAP Ets CArG NF-1 SIE
SRF SRF
NF-1
SP-CREB/
ATF
STAT STAT
SRE
AP-1
wt-SRE
mt-SRE
Fig 1 Structure of c-Fos promoters containing wild-type (wt) and mutated (mt) SRE STAT, signal transducers and activators of transcription (transcription factor family); SIE, sis-inducible element; Ets, E 26 domain; TCF, ternary complex factor, including Elk-1, Net, and Sap-1 tran-scription factors; SRF, serum response factor; CArG, CC-A + T-rich-GG box; AP-1, activator protein-1; FAP, fos-AP-1 binding sequence; RCE, retinoblastoma (Rb) control element; CREB, CRE-binding protein; MLTF ⁄ USF, major late transcription factor ⁄ upstream stimulating factor.
Trang 3protein(s) involved in [Na+]i sensing, engaging the
yeast two-hybrid system
Ouabain-induced c-Fos accumulation is abolished
by inhibition of RNA synthesis
Figure 3A shows that c-Fos mRNA expression in
qui-escent HeLa cells was below the detection limit and
was time-dependently increased after 1 h of ouabain
addition The inclusion of 1 lgÆmL)1of actinomycin D
decreased RNA synthesis by 95–98% (data not
presen-ted), and completely abolished ouabain-induced c-Fos
mRNA accumulation (Fig 3B) Neither ouabain nor
actinomycin affected survival of HeLa cells, as
indica-ted by a low number of trypan blue-positive cells and
a lactate dehydrogenase (LDH) release assay (Table 1)
c-Fos expression in ouabain-treated HeLa cells
is mediated by [Na+]ielevation
Side-by-side with elevation of the [Na+]i⁄ [K+]i ratio,
interaction of ouabain and other cardiotonic steroids
with the Na+⁄ K+-ATPase a-subunit triggers diverse
Na+i-independent signals [8,13,14] Ouabain targets
other than Na+⁄ K+-ATPase also cannot be excluded
To examine these hypotheses, we adopted several approaches First, we compared the actions of ouabain
on c-Fos expression in control and K+-free media Figure 4 shows that, similarly to the effect of ouabain,
Na+⁄ K+-ATPase inhibition in K+-free media resulted
in an approximately seven-fold elevation of intracellu-lar Na+ content and a 12-fold increase of c-Fos protein Importantly, the effect of ouabain on c-Fos
-Serum - - + - - + - - + - - + - - +
A
B
0 200 400 600
0 200 400 600
0 200 400 600
0 200 400 600
0 200 400
600
ouabain control
+, nmol/mg protein
Fig 2 Effect of ouabain on intracellular exchangeable Na+(A) and c-Fos mRNA (B) in different cell types Serum-deprived cells were incuba-ted for 5 h in DMEM ± 10 l M (HeLa, C7-MDCK and C11-MDCK cells) or 3 m M (NIH 3T3 and H9C2 cells) ouabain In some of the experi-ments, 10% fetal bovine serum was added for 20 min as a positive control The means ± SE from experiments performed in triplicate are shown.
(hr) 0 0.5 1 3 6 12
A
B
Fig 3 c-Fos mRNA expression in HeLa cells (A) Kinetics of c-Fos mRNA accumulation in serum-deprived cells incubated in DMEM containing 10 l M ouabain (B) c-Fos mRNA in HeLa cells after 6 h
of incubation in DMEM ± 10 l M ouabain and 1 lgÆmL)1 actino-mycin D (Act-D).
Trang 4expression was abolished in K+-free medium Second,
we compared the dose-dependent action of ouabain on
intracellular monovalent cation content and c-Fos
expression This experiment revealed that c-Fos
accu-mulation in ouabain-treated cells positively and
negat-ively correlated with the gain of Na+iand loss of K+i,
respectively (Fig 5) Third, we treated cells with
monensin, an ionophore providing electroneutral
Na+⁄ H+exchange At a concentration of 1 lgÆmL)1,
this compound did not affect [K+]i but increased
[Na+]i and c-Fos content two- to three-fold (Table 2)
The modest impact of monensin on [Na+]i as
com-pared to ouabain was probably caused by the negative
resting membrane potential () 50 mV) [15], which
limits the accumulation of charge-balancing anions,
such as Cl– and HCO3 Elevation of monensin
con-centration up to 10 lgÆmL)1 attenuated cell survival,
as indicated by the accumulation of trypan
blue-posit-ive cells and augmented LDH release (Table 1) Fourth, as predicted, equimolar substitution of 80% of extracellular Na+ by N-methyl-d-glucamine sharply attenuated the action of ouabain on Na+i content (Fig 4A) In this medium, ouabain increased c-Fos content only two- to three-fold (Fig 4B) Viewed col-lectively, these data strongly indicate that c-Fos expres-sion in ouabain-treated HeLa cells is caused by inhibition of Na+⁄ K+-ATPase-mediated ion fluxes and elevation of [Na+]i
Evidence of Ca2- and extracellular regulated kinase (ERK)-independent signaling
The presence of CRE in the c-Fos promoter (Fig 1) sug-gests that in cells with highly active Na+⁄ Ca2+ exchange, ouabain and other [Na+]i-raising stimuli can trigger c-Fos expression via elevation of [Ca2+]i To test this hypothesis, we treated HeLa cells with ouabain in the absence of extracellular Ca2+ and in the presence
of the intracellular Ca2+ chelator 1,2-bis(O-amino-phenoxy)ethane-N,N,N¢,N¢-tetraacetic acid (BAPTA)
As predicted, the procedure almost completely abolished
an increase of [Ca2+]i triggered by inhibition of endo-plasmic reticulum Ca2+-ATPase with thapsigargin or
by activation of Ca2+ signaling with the P2Y agonist ATP (data not shown) However, neither Ca2+-free medium nor BAPTA abolished c-Fos expression trig-gered by ouabain (Fig 6) In recent studies, we docu-mented that addition of the extracellular Ca2+chelator EGTA leads to a four-fold increase in Na+influx, prob-ably due to heightened passive permeability of the plasma membrane [16] This observation provides an
Table 1 Effect of ouabain, monensin and actinomycin D on survival
of HeLa cells Cells were incubated in DMEM with compounds
indicated in the left column for 6 h Total cell number and LDH
con-tent were taken as 100% The means ± SE from experiments
per-formed in quadruplicate are shown.
Additions
Number of trypan blue-positive cells (%)
LDH release (%)
A
K +
o , mM
Na +
o , mM
5.4 140.1
0 145.5
5.4 30.4
0 200 400 600 800 1000
+ , %
control ouabain
0 200 400 600 800 1000 1200 1400
B
5.4 140.1
0 145.5
5.4 30.4
Fig 4 Effect of 10 l M ouabain on intracellular Na+(A) and immunoreactive c-Fos (B) after 6 h of incubation of HeLa cells in control, K+-free
or Na + -depleted medium Na +
i and immunoreactive c-Fos content in control medium without ouabain was taken as 100% Means ± SE obtained from experiments performed in quadruplicate are shown Control medium contained (m M ) NaCl, 109.4; KCl, 5.4; CaCl 2 , 1.8; MgSO 4 , 0.8; NaHCO 3 , 29.8; NaH 2 PO 4 , 0.9; Hepes, 8.4; glucose, 5; and vitamins and amino acids at concentrations indicated for DMEM recipes In
K + -free medium, KCl was substituted by NaCl; in Na + -depleted medium, NaCl was substituted by N-methyl- D -glucamine.
Trang 5explanation for the modest rise of c-Fos seen in
EGTA-treated cells in the absence of ouabain (Fig 6)
During the last decade, several research teams have
reported that, side-by-side with elevation of the
[Na+]i⁄ [K+]i ratio, exposure to ouabain results in the
transient activation of ERK1⁄ 2 via Ras ⁄ Raf-mediated
signaling [13,14,17] ERK-mediated signaling was also
demonstrated in studies of c-Fos expression triggered
by growth factors [11,12] Considering this, we
exam-ined the effect of ouabain on phosphorylated ERK1⁄ 2
content and the effect of the ERK kinase inhibitor
PD98059 on c-Fos expression evoked by ouabain
Figure 7 shows that 2 h of incubation of HeLa cells
with 3 lm ouabain did not affect ERK1⁄ 2
phosphory-lation As predicted, c-Fos expression evoked by serum was sharply attenuated in the presence of PD98059 However, this compound did not alter c-Fos expression
in ouabain-treated cells
Ouabain does not affect F-luc expression driven
by the c-Fos promoter
To examine whether or not augmented c-Fos expres-sion in ouabain-treated cells is mediated by NaRE within the 5¢-UTR containing all known transcrip-tional elements, we deployed the firefly luciferase gene (F-luc) as a reporter gene and the Renilla luciferase gene (R-luc) driven by the herpes simplex virus thymi-dine kinase promoter as a negative control Figure 8 shows that the addition of serum to F-luc- and R-luc-transfected HeLa cells augmented the F-luc⁄ R-luc ratio by three-fold, confirming the responsiveness of our construct to ‘classic stimuli’ In contrast to serum,
6 h of exposure to ouabain did not affect the F-luc⁄ R-luc ratio in HeLa cells or in C11-MDCK cells; a sharp increase of c-Fos expression was seen in the pres-ence of this compound (Figs 2–5) The lack of signifi-cant action of ouabain on the F-luc⁄ R-luc ratio was also documented in HeLa cells transfected with F-luc driven by the c-Fos promoter region from ) 1264 to
0.03 0.1 0.3 1 10
ouabain, M
A
B
+, c-Fos
20 40 60 80 100
0 200 400 600 800 1000 1200
ouabain, µM
c-Fos
Na+ i
K+ i
Fig 5 (A) Representative western blot showing the effects of ouabain and monensin on c-Fos content in HeLa cells (B) Dose-dependent effect of ouabain on intracellular Na + , K + and c-Fos content Cells were incubated for 6 h in DMEM with or without ouabain and monensin.
Na +
i , K +
i and c-Fos content in the absence of ouabain and monensin was taken as 100% The means ± SE from experiments performed in triplicate (Na+i , K+i ) or three independent experiments (c-Fos) are shown.
Table 2 Effect of monensin on intracellular Na + and K + content
and c-Fos expression in HeLa cells Cells were incubated in DMEM
with or without monensin for 6 h The means ± SE from
experi-ments performed in quadruplicate (ion content) or three
independ-ent experimindepend-ents (c-Fos expression) are shown Immunoreactive
c-Fos content in the absence of monensin was taken as 100%.
Control Monensin, 1 lgÆmL)1
Na +
i [nmolÆ(mg protein))1] 67 ± 24 201 ± 33
K+i [nmolÆ(mg protein))1] 1873 ± 102 1904 ± 211
Trang 6+ 103 (Table 3) Negative results were also obtained
with VSMCs from the rat aorta (Fig 8) expressing
Na+⁄ K+-ATPase with low affinity for cardiotonic
steroids and showing 10-fold elevation of
immuno-reactive c-Fos content after 4–6 h of incubation with
1 mm ouabain [10]
Role of SRE
Previously, we demonstrated that Na+⁄ K+-ATPase
inhibition in VSMCs did not affect luciferase
expres-sion driven by CRE, but evoked a two-fold decrease in
luciferase expression driven by SRE [10] To
investi-gate the impact of SRE on the negative results
obtained in the study of F-luc expression in
ouabain-treated cells, we explored F-luc expression driven by
the c-Fos 5¢-UTR containing mutated SRE (Fig 1)
Figure 9 shows that the F-luc⁄ R-luc ratio was
increased by three-fold in HeLa cells transfected with
F-luc driven by the mutated c-Fos promoter, and was
only slightly affected by the combined addition of epi-dermal and insulin-like growth factors This observa-tion is consistent with the sharp elevaobserva-tion of baseline expression of human c-Fos lacking SRE in stably transfected mouse NIH 3T3 cells that was insensitive
to the addition of serum [18] Similar to the situation with wild-type c-Fos, ouabain did not affect luciferase expression driven by the mutated c-Fos promoter in the absence or the presence of growth factors
Discussion Our results reveal that, similar to the situation in VSMCs [10], c-Fos accumulation in ouabain-treated HeLa cells is triggered by Na+⁄ K+-ATPase inhibi-tion and occurs via elevainhibi-tion of [Na+]i This conclu-sion is supported by several observations: (a) similar
to the effects of ouabain, massive c-Fos accumula-tion was seen under Na+⁄ K+-ATPase inhibition in
K+-free medium (Fig 4); (b) by comparing dose-dependencies of the effect of ouabain on [Na+]i,
p42~P p44~P
0 15 30 60 90 120 (min)
A
B
0 200 400 600 800 1000 1200
1400
C
control ouabain serum
control PD98059
c-Fos
-ouabain serum PD98059
-+
+
-+ -+
-+
-+ +
Fig 7 Ouabain-induced c-Fos expression in HeLa cells: role of ERK (A) ERK1 (p42) and ERK2 (p44) phosphorylation in control cells and in cells treated with 3 l M ouabain for up to 2 h (B, C) Effect of the ERK kinase inhibitor PD98059 on c-Fos expression in control cells and in cells treated for 30 min with 5% fetal bovine serum or for 6 h with 3 l M ouabain PD98059, 20 l M , was added 20 min before serum and ouabain c-Fos content in the absence of any compounds was taken as 100% The means ± SE from three inde-pendent experiments are shown.
-+
-+ +
-+
-+ -+
-+ +
+ -+ +
ouabain
CaCl2
EGTA
BAPTA
A
B
0
2
4
6
8
10
12
14
16
18
Fig 6 Effect of Ca 2+ chelators on c-Fos accumulation in
ouabain-treated HeLa cells (A) Representative western blot showing the
effect of extracellular (EGTA) and intracellular (BAPTA) Ca 2+
chela-tors on c-Fos expression in control and ouabain-treated cells (B)
Immunoreactive c-Fos content in the absence and presence of
oua-bain, EGTA and BAPTA Cells were treated with 10 l M ouabain in
control and Ca 2+ -free media for 6 h In Ca 2+ -free medium, CaCl2
was substituted with 0.1 m M EGTA For complete composition of
the DMEM-like medium, see legend to Fig 4 BAPTA-AM was
added in Ca 2+ -free medium at a concentration of 20 l M for 30 min
before ouabain c-Fos content in the absence of ouabain and Ca 2+
chelators was taken as 1.0 The means ± SE from three
independ-ent experimindepend-ents are shown.
Trang 7[K+]i and immunoreactive c-Fos content (Fig 5), we
demonstrated that c-Fos expression is correlated with
elevation of the [Na+]i⁄ [K+]i ratio; (c) both c-Fos
accumulation and Na+i elevation in ouabain-treated
cells were sharply attenuated in Na+-depleted medium
(Fig 4); and (d) c-Fos content was increased two- to
three-fold in monensin-treated HeLa cells manifesting a
three-fold elevation of [Na+]i (Table 2) Importantly,
about the same increase of [Na+]i seen in cells treated
with 0.1 lm ouabain resulted in a five- to six-fold
eleva-tion of c-Fos content (Fig 5B) It should be underlined,
however, that in contrast to the effects of ouabain, elevation of [Na+]i in monensin-treated cells was not accompanied by loss of [K+]i (Table 2) This finding allows us to speculate that high [K+]iprotects the signal transduction machinery from its activation by Na+i, probably by competing for the same binding sites within
Na+
i-sensing molecules
The presence of CRE in the c-Fos promoter (Fig 1) suggests that an increase of [Na+]i can evoke c-Fos expression via Na+⁄ Ca2+ exchange activation and [Ca2+]i elevation However, neither Ca2+-free medium nor the intracellular Ca2+ chelator BAPTA affected c-Fos expression in ouabain-treated cells (Fig 6) These results indicate that c-Fos expression is caused by activation of the Na+i-mediated, Ca2+i -independent signaling pathway It should be noted that the signaling pathways triggered by ouabain are concentration-dependent and cell type-specific Thus,
in rat cardiomyocytes, c-Fos expression triggered by a modest concentration of ouabain was completely abolished by BAPTA [19], which contrasts with the negative results obtained in BAPTA-loaded VSMCs [10] and HeLa cells (Fig 6) treated with high doses
of ouabain In rat cardiomyocytes [20–23], canine VSMCs [24] and human endothelial cells [25], ouabain
Table 3 Effect of ouabain and serum on the ratio of firefly
lucif-erase (F-luc) and Renilla luciflucif-erase (R-luc) luminescence in HeLa
cells The cells were transfected with R-luc and F-luc driven by the
c-Fos promoter region from ) 1264 to + 103 as indicated in
Experi-mental procedures Transfected cells were incubated for 6 h in
DMEM with or without 10 l M ouabain or 10% fetal bovine serum.
Means ± SE obtained from experiments performed in triplicate are
shown *P < 0.001 compared to control.
0 1 2 3 4 5
-+ + -+ +
-+ + -+ +
p<0.02 p<0.01
GF ouabain
Fig 9 Effect of serum and ouabain on the ratio of firefly luciferase (F-luc) and Renilla luciferase (R-luc) luminescence in HeLa cells Serum-starved cells, transfected with F-luc driven by the c-Fos pro-moter region from ) 650 to + 103 and containing wild-type (wt-c-Fos) (A) or mutated (mt-(wt-c-Fos) (B) SRE, were treated for 6 h
in control or K+-free medium with or without growth factors (GF, 40 ngÆmL)1 epidermal growth factor + 20 ngÆmL)1 insulin-like growth factor) and 10 l M ouabain For complete composition of the incubation medium, see the legend to Fig 3 Values of the F-luc ⁄ R-luc ratio in cells transfected with wt-c-Fos and incubated in con-trol medium without GF and ouabain were taken as 1.00 The means ± SE from experiments performed in triplicate are shown.
0
2
4
6
8
10
NS
NS
p<0.01
p<0.02
NS
p<0.01
Fig 8 Effect of ouabain and serum on the ratio of firefly luciferase
(F-luc) and Renilla luciferase (R-luc) luminescence in HeLa cells,
C11-MDCK cells and VSMCs The cells were transfected with R-luc
and F-luc driven by the c-Fos promoter region from ) 650 to + 103,
as indicated in Exeperimental procedures Transfected cells were
incubated for 6 h in DMEM with or without 10 l M (HeLa and
C11-MDCK cells) or 1000 l M (VSMCs) ouabain or 10% fetal bovine
serum (FBS) In the absence of the tested compounds, F-luc
lumin-escence in HeLa cells, C11-MDCK cells and VSMCs was
59 086 ± 5676, 9036 ± 843 and 1201 ± 148 c.p.m per well,
respectively; protein content of the cells varied in the range 120 to
142 lg per well Means ± SE obtained from experiments
per-formed in triplicate are shown NS, not significant.
Trang 8at low concentrations transiently phosphorylated
ERK1⁄ 2 This effect was absent in rat VSMCs [6]
and HeLa cells (Fig 7A) Moreover, in contrast to
serum, the increase of c-Fos content in
ouabain-trea-ted HeLa cells was insensitive to the presence of the
ERK inhibitor PD98059 (Fig 7C) These negative
results are consistent with the lack of activation by
ouabain of ERK-sensitive transcription factors, such
as Elk-1, SRF, CREB and AP-1, documented in
transfected VSMCs [10]
To further explore the role of CRE and other known
transcriptional elements within the c-Fos promoter, we
studied the expression of F-luc driven by the c-Fos
5¢-UTR, using, as a negative control, R-luc driven by
the herpes simplex virus thymidine kinase promoter
region The lack of effect of ouabain on the F-luc⁄ R-luc
ratio in HeLa cells as well as in C11-MDCK cells and
VSMCs (Fig 8) showing more than 10-fold c-Fos
accu-mulation (Fig 1) [10] shows that [Na+]ielevation does
not affect c-Fos 5¢-UTR promoter activity
Three hypotheses can be proposed to explain the
distinct action of increased [Na+]ion c-Fos expression
and c-Fos promoter activity First, [Na+]i elevation
decreases c-Fos mRNA or protein degradation rather
than triggering de novo gene expression The first
hypothesis contradicts our results, which showed
com-plete inhibition of c-Fos mRNA accumulation in cells
treated with ouabain in the presence of actinomycin D,
a potent inhibitor of RNA synthesis (Fig 3), as well
as data obtained by run-on assays demonstrating
heightened c-Fos expression in nuclei isolated from
ouabain-treated human diploid fibroblasts [18]
Sec-ond, an increase in [Na+]i evokes c-Fos expression by
activation of NaRE located upstream or downstream
of the 5¢-UTR tested in our study (Figs 1, 8 and 9) It
should be underlined, however, that extension of the
5¢-UTR from position ) 650 to ) 1264 did not lead
to significant elevation of the F-luc⁄ R-luc ratio in
ouabain-treated cells as compared to the controls
(Table 3) The role of introns has been proven in a
transcription study of several genes, including c-Fos
[26–28], and their implication in Na+i-induced c-Fos
expression deserves further investigation Third,
increasing evidence indicates that, side-by-side with
regulation of the 5¢-UTR by transcription factors, gene
activation or silencing is under the complex control of
three-dimensional positioning of genetic materials and
chromatin in the nuclear space These data suggest
that gene silencing results in decreased chromatin
mobility and, conversely, gene activation and sustained
transcription are associated with long-range movement
of chromatin [29] Nuclease digestion assays have
documented two hypersensitive sites of c-Fos
chroma-tin architecture at positions ) 350 and ) 1900 [28] Importantly, the chromatin-mediated mechanism of gene expression does not contribute to the regulation
of L-luc transcription in cells transiently transfected with plasmid encoding the gene and employed in our experiments Keeping this in mind, the negative results obtained for ouabain’s effect on L-luc expression dri-ven by the c-Fos 5¢-UTR suggest that the activation of c-Fos transcription in ouabain-treated cells is caused
by [Na+]i-mediated reorganization of the DNA– chromatin complex This working hypothesis should
be verified in forthcoming studies
Experimental procedures c-Fos promoter design and cloning The c-Fos promoter region from ) 650 to + 103, contain-ing the major known response elements (Fig 1), was ampli-fied by PCR from human HeLa cell genomic DNA, using TCGCTAGCGTCTGCTTCCACGCTTTGCACT and ACAGATCTGCTGTGGAGCAGAGCTGGGTA primers bearing NheI and BglII restriction sites, respectively c-Fos promoter fragments were cloned into pGL3 basic vector from Promega (Madison, WI, USA), with NheI and BglII restriction enzymes and the firefly luciferase gene (F-luc) as
a reporter gene Mutation within the SRE of the ) 312 to ) 303 region (Fig 1) was introduced with the Quickchange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA), following the recommendations of the manufacturer, with the sense primer CTCCCCCCTTACACAGGATG TGGATATTACCACATCTGCGTCAGC and the corres-ponding antisense primer In additional experiments, we used the pGL3 vector containing F-luc driven by the c-Fos promoter region from) 1264 to + 103
Cell cultures HeLa cells derived from human epitheloid carcinoma and NIH 3T3 mouse fibroblasts were purchased from the Ameri-can Type Culture Collection (Rockville, MA, USA) Embry-onic rat heart-derived H9C2 myoblasts were kindly provided
by P Isenring (Laval University, Quebec, PQ, Canada) C7-MDCK and C11-MDCK cells, resembling principal and intercalated cells from the collecting ducts, were provided by
M Gekle (University of Wu¨rzburg, Germany) VSMCs were obtained by previously described explant methods [30] from the aortae of 10- to 13-week-old male Brown Norway (BN.lx) rats under Brietal anesthesia (3 mgÆkg)1) in accord-ance with the procedures outlined in the Guide for the Care and Use of Experimental Animals endorsed by the Canadian Institutes of Health Research The cells were maintained in DMEM supplemented with 2.5 gÆL)1 sodium bicarbonate,
2 gÆL)1Hepes, 100 UÆmL)1penicillin, 100 lgÆmL)1
Trang 9strepto-mycin, 25 mm glucose and 10% fetal bovine serum with
minor modifications for C7-MDCK and C11-MDCK cells
as described elsewhere [31] They were plated in Petri dishes
or 24-well plates at a density of 2–3· 103cellsÆcm)2 To
establish quiescence, the cells were incubated for 48 h before
experiments in DMEM containing 0.2% serum RNA
synthesis was measured as RNA labeling in the presence of
2 lCiÆmL)1of [3H]uridine [6]
Cell viability
Cell viability was estimated by counting colored cells after
incubation of cell suspensions with 0.2% trypan blue and
LDH release To measure LDH activity, 0.5 mL of
incuba-tion medium was mixed with the same volume of 1%
Triton X-100, and attached cells were solubilized with 0.5%
Triton X-100 Then, 300 lL of samples were transferred
into 2 mL of buffer containing 50 mm Tris⁄ HCl (pH 7.4),
50 mm KCl, 0.075 mm NADH and 1 mm sodium pyruvate
The kinetics of NADH degradation were monitored with a
SPEX FluoroMax spectrofluorometer (Jobin Yvon Inc.,
Edison, NJ, USA) at kex¼ 349 nm and kem¼ 420 nm (slits
4 and 20 nm, respectively)
Transfection and luciferase assay
Transient transfections of cells with constructs were
per-formed using LipofectAMINE-PLUS reagent (Invitrogen,
Carlsbad, CA, USA) in accordance with the manufacturer’s
instructions Briefly, cells grown in 24-well plates ( 105
per well) were incubated with DNA–liposome complexes in
serum-free and antibiotic-free DMEM for 3 h in the
pres-ence of 100 ng per well of c-Fos-pGL3b firefly luciferase
(F-luc) reporter plasmid This medium also contained
100 ng per well of pRL-TK vector with the herpes simplex
virus thymidine kinase promoter region upstream of the
Renillaluciferase (R-luc) reporter gene (Promega) We
tes-ted the construct as a control for transfection efficacy and
the possible nonspecific impact of Na+⁄ K+-ATPase
inhibi-tion on the transcripinhibi-tion⁄ translation machinery The cells
were incubated overnight in serum-deprived DMEM
sup-plemented with glutamine 2 mm, Hepes 10 mm and 0.1%
BSA, and this was followed by 6 h of incubation with the
desired media, whose compositions are indicated in the
figure legends Then, the cells were washed twice with
NaCl⁄ Pi, and lysed in Mammalian Protein extraction
rea-gent (Pierce, Rockford, IL, USA), and the lysates were
assayed for F-luc and R-luc activity in the dual-luciferase
reporter assay system from Promega
Western blotting
Cells grown in petri dishes and treated as indicated in the
figure legends were scraped with a rubber policeman,
washed with ice-cold medium C, which contained 150 mm NaCl and 10 mm Hepes⁄ Tris (pH 7.4), and centrifuged (500 g, 5 min) The resultant cell pellet was washed twice with the same medium, and lysed with buffer containing
150 mm NaCl, 25 mm Hepes⁄ Tris (pH 7.5), 0.1% SDS, 0.25% sodium deoxycholate, 2 mm EGTA, 2 mm EDTA,
1 mm Na3VO4, 10 mm NaF, 200 lm phenylmethylsulpho-nyl fluoride, 1 lgÆmL)1 leupeptin and 1 lgÆmL)1 aprotinin Equal portions of cell lysate (20 lg per lane) were electro-phoresed on 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, washed with NaCl⁄ Piand incuba-ted overnight at 4C with antibodies to c-Fos or p-ERK (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in NaCl⁄ Pi containing 0.05% Tween-20 and 5% skimmed milk After incubation, the membranes were washed three times with NaCl⁄ Pi⁄ Tween and incubated for 1 h with horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology) The membranes were then washed with NaCl⁄ Pi⁄ Tween, and the protein bands were visualized with
an enhanced chemiluminescence detection kit (Santa Cruz Biotechnology) before exposure to X-ray film Relative pro-tein content was quantified with the nih image program
Northern blotting Total RNA from HeLa cells was isolated with TRIZOL reagent (Life Technologies, Burlington, ON, Canada) in accordance with the manufacturer’s instructions Ten micr-ograms of total RNA, measured by UV spectrophotometry, was subjected to gel electrophoresis, transferred to a Hybond N+ membrane (Amersham Pharmacia Biotech, Baie d’Urfe´, PQ, Canada), UV-immobilized and hybridized
to 32P-labeled probes c-Fos mRNA forward (AGG AATAAGATGGCTGCAGCCAAG) and reverse (GACTC TGGGGTGGTAGCCTCAG) primers, corresponding to the 569–838 region of rat c-Fos mRNA, were synthesized
by the primer-designed genefisher program The probes were purified with the Wizard PCR Preps DNA purification system from Promega, and quantified with the Low DNA Mass Ladder (Life Technologies) They were labeled with a Random primer DNA-labeling system kit (Life Technol-ogies), purified on Sephadex G-50, hybridized for 24 h, visualized on a Phosphor-Imager (Molecular Dynamics, San Diego, CA, USA) and quantified by imagequant (ver-sion 5.1) software from the same company
Intracellular exchangeable K+and Na+ These were measured as the steady-state distribution of extracellualr and intracellular 86Rb and 22Na, respectively,
as described previously in detail [1] Briefly, to establish iso-tope equilibrium, cells growing in 24-well plates were prein-cubated for 6 h in control or K+-free DMEM-like media with or without ouabain and containing 0.5 lCiÆmL)1
Trang 10RbCl or 2 lCiÆmL)1 22NaCl At the end of incubation,
the cells were transferred onto ice, washed four times with
2 mL of ice-cold medium W containing 100 mm MgCl2and
10 mm Hepes⁄ Tris buffer (pH 7.4), and lysed with 1%
SDS⁄ 4 mm EDTA The radioactivity of the incubation
medium and cell lysate was quantified, and intracellular
cation content was calculated as A⁄ am, where A was the
radioactivity of the samples (c.p.m.), a was the specific
radioactivity of 86Rb (K+) and 22Na in the medium
(c.p.m.Ænmol)1), and m was protein content
Chemicals
22
NaCl, 86RbCl, [3H]uridine and [32P]ATP[cP] were
pro-cured from Amersham Biosciences The remaining
chemi-cals were purchased from Gibco, Sigma (Oakville, ON,
Canada) and Anachemia Science (Montreal, PQ, Canada)
Statistics
The data were analyzed by Student’s t-test or the t-test for
dependent samples, as appropriate Significance was defined
as P < 0.05
Acknowledgements
This work was supported by grants from the Canadian
Institutes of Health Research and the Heart and
Stroke Foundation of Canada The technical assistance
of Monique Poirier and the editorial help of Ovid Da
Silva, Research Support Office, Research Centre and
CHUM are appreciated
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