[Na+]i-induced c-Fos expression is not mediated by 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 Hamet1 and Sergei N Orlov1 ˆ ´ ´ Centre de recherche, Centre hospitalier de l’Universite de Montreal (CHUM) ) Technopole ANGUS, Montreal, PQ, Canada Department of Medicine, University of Chicago, Chicago, IL, USA Keywords c-Fos; expression; intracellular Na+; ouabain; 5¢-UTR Correspondence S N Orlov, Centre de recherche, ˆ CHUM ) Technopole 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 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 h of Na+ ⁄ K+-ATPase inhibition with ouabain that was abolished by actinomycin D, an inhibitor of RNA synthesis c-Fos protein accumulation in ouabain-treated cells correlated with a gain of Na+i and loss of K+i Augmented 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 attenuated under dissipation of the transmembrane Na+ gradient, but was preserved 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 Negative 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’ 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 medium, 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 by 3H-induced DNA damage via elevation of [Na+]i rather than attenuation of [K+]i [5] We also noted that in VSMCs, protection against apoptosis 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 FEBS Journal 274 (2007) 3557–3567 ª 2007 The Authors Journal compilation ª 2007 FEBS 3557 Intracellular Na+ and gene expression M Haloui et al SIE TCF STAT STAT was 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 triggered by elevation of the [Na+]i ⁄ [K+]i ratio [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 mortalin, 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, hepatocytes, and leukemia and melanoma cell lines, as well as in freshly isolated cardiomyocytes [9] In VSMCs, 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, respectively [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 accumulation was preserved in the presence of nicardipine SRF SRF AP-1 Ets CArG FAP NF-1 NF-1 SRE 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+-sensitive, Ca2+-independent transi i 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 cultured cells with different origins Figure 2A shows that h of inhibition of Na+ ⁄ K+-ATPase with ouabain led to an  10-fold elevation of intracellular exchangeable Na+ in all types of cells investigated In contrast, the action of ouabain on c-Fos mRNA was cell typespecific, 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 existence of a commercially available activation domain fusion cDNA library from HeLa cells that could be employed for the identification of NaRE-binding CREB/ ATF SP- SPGC-rich direct repeats/ RCE TATA CRE MLTF/USF CArG -650 -348 -335 -320 -313 -297 -312 wt-SRE -291 -176 -166 -97 -76 -67 -62 -59 -55 -31 +103 -303 CCATATTAGG mt-SRE GGATATTACC Fig 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 transcription 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 3558 FEBS Journal 274 (2007) 3557–3567 ª 2007 The Authors Journal compilation ª 2007 FEBS Intracellular Na+ and gene expression M Haloui et al control A Na+i, nmol/mg protein 600 600 600 600 600 400 400 400 400 400 200 200 200 200 200 Cell type Ouabain ouabain 0 HeLa C7-MDCK - + - - - + - 0 C11-MDCK H9C2 + - - + - - + - - - + NIH 3T3 + - - + - B Serum - - - + - - + Fig Effect of ouabain on intracellular exchangeable Na+ (A) and c-Fos mRNA (B) in different cell types Serum-deprived cells were incubated for h in DMEM ± 10 lM (HeLa, C7-MDCK and C11-MDCK cells) or mM (NIH 3T3 and H9C2 cells) ouabain In some of the experiments, 10% fetal bovine serum was added for 20 as a positive control The means ± SE from experiments performed in triplicate are shown protein(s) involved in [Na+]i sensing, engaging the yeast two-hybrid system A Ouabain-induced c-Fos accumulation is abolished by inhibition of RNA synthesis (hr) Figure 3A shows that c-Fos mRNA expression in quiescent HeLa cells was below the detection limit and was time-dependently increased after h of ouabain addition The inclusion of lgỈmL)1 of actinomycin D decreased RNA synthesis by 95–98% (data not presented), and completely abolished ouabain-induced c-Fos mRNA accumulation (Fig 3B) Neither ouabain nor actinomycin affected survival of HeLa cells, as indicated 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+]i elevation 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 0.5 12 B ouabain Act-D - + - + + + Fig c-Fos mRNA expression in HeLa cells (A) Kinetics of c-Fos mRNA accumulation in serum-deprived cells incubated in DMEM containing 10 lM ouabain (B) c-Fos mRNA in HeLa cells after h of incubation in DMEM ± 10 lM ouabain and lgỈmL)1 actinomycin D (Act-D) 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 shows that, similarly to the effect of ouabain, Na+ ⁄ K+-ATPase inhibition in K+-free media resulted in an approximately seven-fold elevation of intracellular Na+ content and a 12-fold increase of c-Fos protein Importantly, the effect of ouabain on c-Fos FEBS Journal 274 (2007) 3557–3567 ª 2007 The Authors Journal compilation ª 2007 FEBS 3559 Intracellular Na+ and gene expression M Haloui et al Table 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 h Total cell number and LDH content were taken as 100% The means ± SE from experiments performed in quadruplicate are shown Additions Number of trypan blue-positive cells (%) LDH release (%) None (control) Ouabain, 10 lM Monensin, lgỈmL)1 Monensin, 10 lgỈmL)1 Actinomycin D, lgỈmL)1 24 4 17 ± ± ± ± ± 8* ± ± ± ± ± 2 3* 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 collectively, these data strongly indicate that c-Fos expression 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 expression 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 accumulation in ouabain-treated cells positively and negatively correlated with the gain of Na+i and loss of K+i, respectively (Fig 5) Third, we treated cells with monensin, an ionophore providing electroneutral Na+ ⁄ H+ exchange At a concentration of lgỈmL)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 compared 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 concentration up to 10 lgỈmL)1 attenuated cell survival, as indicated by the accumulation of trypan blue-posit- The presence of CRE in the c-Fos promoter (Fig 1) suggests 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-aminophenoxy)ethane-N,N,N¢,N¢-tetraacetic acid (BAPTA) As predicted, the procedure almost completely abolished an increase of [Ca2+]i triggered by inhibition of endoplasmic 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 triggered by ouabain (Fig 6) In recent studies, we documented that addition of the extracellular Ca2+ chelator EGTA leads to a four-fold increase in Na+ influx, probably due to heightened passive permeability of the plasma membrane [16] This observation provides an control ouabain A 1000 B 1400 1200 c-Fos, % Na+i, % 800 600 400 1000 800 600 400 200 200 0 K+o, mM Na+ o, mM 5.4 140.1 145.5 5.4 30.4 5.4 140.1 145.5 5.4 30.4 Fig Effect of 10 lM ouabain on intracellular Na+ (A) and immunoreactive c-Fos (B) after 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 (mM) NaCl, 109.4; KCl, 5.4; CaCl2, 1.8; MgSO4, 0.8; NaHCO3, 29.8; NaH2PO4, 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 3560 FEBS Journal 274 (2007) 3557–3567 ª 2007 The Authors Journal compilation ª 2007 FEBS Intracellular Na+ and gene expression M Haloui et al A 0.03 0.1 control 0.3 ouabain, 10 monensin, g/ml M 1200 Na+i, c-Fos (%) 1000 c-Fos 800 100 K+i 80 Na+ i 600 60 400 40 200 K+i (%) B 20 0 0,1 10 ouabain, µM Fig (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 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 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 h The means ± SE from experiments performed in quadruplicate (ion content) or three independent experiments (c-Fos expression) are shown Immunoreactive c-Fos content in the absence of monensin was taken as 100% Control Na+i [nmolỈ(mg protein))1] K+i [nmolỈ(mg protein))1] c-Fos (%) Monensin, lgỈmL)1 67 ± 24 1873 ± 102 100 201 ± 33 1904 ± 211 263 ± 87 explanation for the modest rise of c-Fos seen in EGTAtreated 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 ⁄ 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 examined the effect of ouabain on phosphorylated ERK1 ⁄ content and the effect of the ERK kinase inhibitor PD98059 on c-Fos expression evoked by ouabain Figure shows that h of incubation of HeLa cells with lm ouabain did not affect ERK1 ⁄ 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 expression in ouabain-treated cells is mediated by NaRE within the 5¢-UTR containing all known transcriptional 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 thymidine kinase promoter as a negative control Figure shows that the addition of serum to F-luc- and R-luctransfected 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, 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 presence of this compound (Figs 2–5) The lack of significant 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 FEBS Journal 274 (2007) 3557–3567 ª 2007 The Authors Journal compilation ª 2007 FEBS 3561 Intracellular Na+ and gene expression M Haloui et al A A ouabain CaCl2 EGTA BAPTA B + - + + - + - + + - + + + + + 18 12 90 - + + - + + 120 (min) + - + + control PD98059 1000 c-Fos, % 800 600 400 200 Fig Effect of Ca2+ chelators on c-Fos accumulation in ouabaintreated HeLa cells (A) Representative western blot showing the effect of extracellular (EGTA) and intracellular (BAPTA) Ca2+ chelators on c-Fos expression in control and ouabain-treated cells (B) Immunoreactive c-Fos content in the absence and presence of ouabain, EGTA and BAPTA Cells were treated with 10 lM ouabain in control and Ca2+-free media for h In Ca2+-free medium, CaCl2 was substituted with 0.1 mM EGTA For complete composition of the DMEM-like medium, see legend to Fig BAPTA-AM was added in Ca2+-free medium at a concentration of 20 lM for 30 before ouabain c-Fos content in the absence of ouabain and Ca2+ chelators was taken as 1.0 The means ± SE from three independent experiments are shown + 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 immunoreactive c-Fos content after 4–6 h of incubation with mm ouabain [10] Role of SRE Previously, we demonstrated that Na+ ⁄ K+-ATPase inhibition in VSMCs did not affect luciferase expression driven by CRE, but evoked a two-fold decrease in luciferase expression driven by SRE [10] To investigate the impact of SRE on the negative results obtained in the study of F-luc expression in ouabaintreated cells, we explored F-luc expression driven by the c-Fos 5¢-UTR containing mutated SRE (Fig 1) Figure 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 3562 60 1400 1200 10 30 c-Fos C 14 15 B ouabain serum PD98059 16 c-Fos, relative units p44~P p42~P control ouabain serum Fig 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 lM ouabain for up to h (B, C) Effect of the ERK kinase inhibitor PD98059 on c-Fos expression in control cells and in cells treated for 30 with 5% fetal bovine serum or for h with lM ouabain PD98059, 20 lM, was added 20 before serum and ouabain c-Fos content in the absence of any compounds was taken as 100% The means ± SE from three independent experiments are shown only slightly affected by the combined addition of epidermal and insulin-like growth factors This observation is consistent with the sharp elevation 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 inhibition and occurs via elevation of [Na+]i This conclusion is supported by several observations: (a) similar to the effects of ouabain, massive c-Fos accumulation was seen under Na+ ⁄ K+-ATPase inhibition in K+-free medium (Fig 4); (b) by comparing dosedependencies of the effect of ouabain on [Na+]i, FEBS Journal 274 (2007) 3557–3567 ª 2007 The Authors Journal compilation ª 2007 FEBS Intracellular Na+ and gene expression M Haloui et al 10 control ouabain FBS p