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Article |
Fast regulation of AP-1 activity through interaction of lamin A/C, ERK1/2, and c-Fos at the nuclear envelope
Correspondence to Vicente Andrés: vandres{at}ibv.csic.es
Sequestration of c-Fos at the nuclear envelope (NE) through interaction with A-type lamins suppresses AP-1–dependent transcription. We show here that c-Fos accumulation within the extraction-resistant nuclear fraction (ERNF) and its interaction with lamin A are reduced and enhanced by gain-of and loss-of ERK1/2 activity, respectively. Moreover, hindering ERK1/2-dependent phosphorylation of c-Fos attenuates its release from the ERNF induced by serum and promotes its interaction with lamin A. Accordingly, serum stimulation rapidly releases preexisting c-Fos from the NE via ERK1/2-dependent phosphorylation, leading to a fast activation of AP-1 before de novo c-Fos synthesis. Moreover, lamin A–null cells exhibit increased AP-1 activity and reduced levels of c-Fos phosphorylation. We also find that active ERK1/2 interacts with lamin A and colocalizes with c-Fos and A-type lamins at the NE. Thus, NE-bound ERK1/2 functions as a molecular switch for rapid mitogen-dependent AP-1 activation through phosphorylation-induced release of preexisting c-Fos from its inhibitory interaction with lamin A/C.
© 2008 González et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
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In addition to fulfilling structural functions at the nuclear envelope (NE), A-type lamins (lamin A and C) play important roles in the control of gene expression via their interaction with histones, transcription factors (e.g., SREBP1, MOK2, BAF, GCL, Mel18, and c-Fos), and cell cycle regulators (e.g., the retinoblastoma protein [pRb] and cyclin D3) (Taniura et al., 1995; Gruenbaum et al., 2005; Mariappan and Parnaik, 2005; Broers et al., 2006; Heessen and Fornerod, 2007; Vlcek and Foisner, 2007). We have recently shown that lamin A/C–dependent sequestration of c-Fos at the NE reduces AP-1 DNA-binding activity and cellular proliferation (Ivorra et al., 2006). However, the molecular mechanisms controlling this interaction remain unknown. Here we tested the hypothesis that ERK1/2 is a critical regulator of the interaction between lamin A/C and c-Fos. We show that active ERK1/2 directly interacts with lamin A and colocalizes with c-Fos at the NE. Therein, mitogen-induced ERK1/2-mediated phosphorylation of c-Fos releases it from the inhibitory interaction with lamin A/C before de novo synthesis of c-Fos, thus allowing a rapid induction of AP-1 activity.
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We then focused our attention on ERK1/2-dependent signaling, the main transduction pathway implicated in the rapid and transient phosphorylation and subsequent activation of c-Fos elicited by mitogens (Monje et al. 2003). As expected, Western blot analysis of the SNF of NIH-3T3 cells revealed the activation (phosphorylation) of ERK1/2 after serum stimulation (Fig. 1 E). Compared with serum-starved cells, the level of c-Fos protein was up-regulated in cultures stimulated for 90 min, which was markedly reduced after 180 min of serum stimulation (Fig. 1 E, lanes 1–3). Moreover, treatment with the proteasome inhibitor MG132 abrogated c-Fos down-regulation at 180 min after serum stimulation (Fig. 1 E, lane 5 vs. 3). These findings are in agreement with the concept that mitogen stimulation leads to de novo c-Fos expression, which then undergoes proteasome-dependent degradation (Bossis et al., 2003; Ito et al., 2005). In line with previous studies (Monje et al., 2003), we found that preincubation of serum-starved cells with the ERK1/2 inhibitor U0126 greatly reduces c-Fos level in cells stimulated with serum for 90 min (Fig. 1 F). Therefore, to circumvent the possible influence of ERK1/2 inhibitors on c-Fos synthesis and/or degradation, the effects of U0126 and PD98059 on c-Fos expression and subnuclear localization was investigated using the protocol schematized in Fig. 2 A. In brief, mitogen-depleted cells were stimulated with serum for 150 min in the presence of MG132 to avoid proteasome-dependent degradation of c-Fos. To prevent further protein synthesis after reaching a high level of c-Fos expression, cycloheximide was added at 90 min. Finally, U0126 or PD98059 was added at 100 min after serum stimulation. In agreement with the results of Fig. 1 C, the ERNF and SNF of NIH-3T3 cells manipulated as described above predominantly contained the fastest migrating hypophosphorylated and the two migrating forms of c-Fos, respectively (Fig. 2 B, ERNF: lanes 1 and 3; SNF: lanes 5 and 7). Treatment with either PD98059 or U0126 inhibited ERK1/2 activation, as revealed by the reduced amount of phosphorylated ERK1/2 (pERK1/2) in the SNF (Fig. 2 B, PD98059: lane 5 vs. 6; U0126: lane 7 vs. 8), and caused the accumulation of hypophosphorylated c-Fos both in the ERNF (Fig. 2 B, PD98059: lane 1 vs. 2; U0126: lane 3 vs. 4) and SNF (Fig. 2 B, PD98059: lane 5 vs. 6; U0126: lane 7 vs. 8). The pattern of expression of Sp1 and nucleoporin-50 + Npap60 (NUP50) suggested the absence of cross-contamination in these studies (Fig. 2 B). The results presented thus far indicated that the amount of c-Fos in the ERNF and perinuclear rim is regulated by the level of lamin A/C expression. They also suggested that inhibition of ERK1/2 activity reduces the extent of c-Fos phosphorylation and promotes its accumulation within the ERNF.
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To directly examine whether ERK1/2-dependent phosphorylation of c-Fos affects its affinity for lamin A, we performed in vitro pull-down assays using a GST-lamin A fusion protein containing amino acids 37–244 of rat lamin A, which interacts with recombinant c-Fos (Ivorra et al., 2006). GST-lamin A specifically interacted with the endogenous c-Fos protein present in extracts from serum-stimulated NIH-3T3 cells (Fig. 5 A, lane 3 vs. 4). Moreover, the amount of GST-lamin A–bound c-Fos was greatly increased upon treatment of cells with PD98059 (Fig. 5 B, lane 3 vs. 4). We also compared the binding of GST-lamin A to c-Fos-wt and c-Fos-m, which were ectopically expressed in serum-stimulated U2OS cells. Examination of input samples suggested the presence of phosphorylation activity in these cultures because c-Fos-wt exhibited slower electrophoretic mobility than c-Fos-m (Fig. 5 C, lane 2 vs. 3). Moreover, GST-lamin A interacted more efficiently with c-Fos-m compared with c-Fos-wt (Fig. 5 C, lane 5 vs. 6). These results suggested that the interaction of c-Fos with lamin A is facilitated by inhibiting ERK1/2-mediated phosphorylation of c-Fos, either by mutational blockade of ERK1/2 phosphorylation sites in c-Fos or by pharmacological inhibition of the ERK1/2 pathway.
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241-272) (Whitehurst et al., 2004) and point mutants within this region, Y261N and S264Y, were impaired in their association with lamin A/C (Fig. 8 D).
Lamin A/C, ERK1/2, and c-Fos colocalize at the mammalian NE
Double confocal immunofluorescence microscopy revealed areas of colocalization between endogenous ERK2 and lamin A/C at the perinuclear rim in COS7, U2OS, and HEK293 cells (Fig. 9 A, and unpublished data). Perinuclear colocalization of lamin A/C–ERK2 was also manifest after in situ extraction plus DNase treatment (Fig. 9 B). In contrast, perinuclear localization of Sp1 was negligible under the same experimental conditions (Fig. 9 B).
We next examined the possible colocalization of c-Fos and ERK1/2 at the NE. Lamin A/C entirely colocalized with lamin B at the perinuclear rim (Fig. 9 C); therefore, NE-associated lamin A/C could be unequivocally recognized by localizing lamin B expression. Analysis of asynchronously growing U2OS cells co-immunostained for c-Fos, ERK2, and lamin B revealed partial colocalization of these proteins in the perinuclear rim (Fig. 9 D).
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It is generally accepted that mitogen-dependent activation of ERK1/2 in fibroblasts is a biphasic process consisting of a rapid and strong burst of kinase activity peaking at 5–15 min, followed by a second wave of lower activity that persists throughout the G1 phase of the cell cycle (Kahan et al., 1992; Meloche et al., 1992; Meloche, 1995). c-Fos is expressed minimally in most nongrowing cells as well as during the initial phase of mitogen-induced ERK activation, with a peak of de novo c-Fos protein synthesis and AP-1 activation occurring between 1–2 h after stimulation (Kruijer et al., 1984; Kerr et al., 1988; Monje et al., 2003; Ivorra et al., 2006; Murphy and Blenis, 2006). In line with this notion, we observed maximum c-Fos protein expression at 60–90 min after serum stimulation (Fig. 1 E, Fig. 6 A), coinciding with a peak of AP-1 DNA-binding activity that could be attenuated by inhibitors of either protein synthesis or ERK1/2 activity (Fig. 6 D). Our novel observation is the induction of AP-1 DNA-binding activity as early as 15 min after serum addition concomitantly with maximum ERK1/2 activity and the release of c-Fos from the ERNF to the nucleoplasm in a manner dependent on ERK1/2 activation but independent of de novo protein synthesis (Fig. 6). Based on these findings and the observations that gain-of and loss-of ERK1/2 activity reduces and enhances, respectively, c-Fos accumulation within the ERNF and its interaction with lamin A, and that regulation of these processes is impaired by hindering ERK1/2-dependent phosphorylation of c-Fos, we propose the model depicted in Fig. 10. In serum-deprived cells, sequestration of c-Fos at the NE via its interaction with lamin A/C limits transcription of AP-1 target genes. Subsequent to mitogen stimulation, lamin A–bound active ERK1/2 phosphorylates c-Fos and releases it from the NE, thus allowing the rapid transcriptional activation of AP-1–responsive genes before de novo c-Fos protein synthesis. This mechanism permits the continuous existence in the cell of a certain level of inactive c-Fos (e.g., in quiescent cells) that can be rapidly activated by mitogen stimulation via ERK1/2-dependent phosphorylation without requiring de novo protein synthesis.
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The Insert and the CD domains of ERK1/2 are involved in protein–protein interactions. ERK1/2 substrate docking can be selectively dissociated in vitro by single point mutations without perturbing ERK activation or its intrinsic catalytic activity (Dimitri et al., 2005). Indeed, mutations affecting the Insert region (241-272, Y261N, and S264P) or the CD domain (D316,319A) alter the interaction of ERK2 with some substrates (Tanoue et al., 2000; Whitehurst et al., 2004; Casar et al., 2007). We have shown here that the ERK2–lamin A/C interaction requires the Insert domain of ERK2 and the coil 2 of lamin A/C. Other proteins such as PEA15 (Whitehurst et al., 2004) and Mxi2 (Casar et al., 2007) also interact with ERK through the Insert region. The determinant mediating in this interaction is a "reverse D domain", with a consensus sequence R/K-
a-X3/4-b (where are Leu, Ile, or Val) (Callaway et al., 2005). Notably, the R/K-a-X3/4-b motif is present in the region of A-type lamins, which we found to intervene in their interaction with ERK1/2 (RIRISL at residues 296–301).
In this study, we have focused our attention on how mitogenic signals regulate the interaction between c-Fos and lamin A/C. Further research is required to assess whether additional pathophysiological conditions regulate this interaction and the underlying molecular mechanisms. Of notable interest in this regard are the inherited diseases termed laminopathies, which are caused by either mutations in the LMNA gene (which encodes for A-type lamins) or defective posttranslational processing of prelamin A (Worman and Bonne, 2007). Notably, lamin A/C and lamin-associated polypeptides can physically interact with histones, chromatin, and transcription factors (e.g., c-Fos, SREBP1, MOK2, BAF, GCL, Mel18), suggesting that altered gene expression contributes to the pathogenesis of laminopathies (Taniura et al., 1995; Gruenbaum et al., 2005; Broers et al., 2006; Heessen and Fornerod, 2007; Vlcek and Foisner, 2007). Indeed, microarray studies using fibroblast from Hutchinson-Gilford progeria syndrome patients revealed differential transcription factor expression (Ly et al., 2000; Csoka et al., 2004). Moreover, certain pathogenic lamin A mutations cause alterations in the transcription factors MOK2 (Dreuillet et al., 2008) and SREBP1 (Lloyd et al., 2002; Hubner et al., 2006) and in the cell cycle regulator pRb (Hubner et al., 2006). Interestingly, transgenic mice overexpressing either wild-type MEK5 or constitutively active MEK1 exhibit excessive ERK1/2-dependent signaling and dilated cardiomyopathy (Bueno et al., 2000; Nicol et al., 2001), a clinical manifestation of several laminopathies. Moreover, expression of the Emery-Dreifuss muscular dystrophy–causing H222P–lamin A mutant protein in homozygous lmnaH222P/H222P knock-in mice causes in the heart aberrant ERK1/2 activity and myopathy, and leads to activation of several MAPKs—including ERK1/2—and downstream target genes in cultured cells (Muchir et al., 2007). It is also noteworthy that MEK/ERK pathway inhibition improves defective myogenic factor expression and differentiation of C2C12 myoblasts expressing the Emery-Dreifuss muscular dystrophy–causing R453W–lamin A mutant (Favreau et al., 2004, 2008). In the light of the aforementioned studies and the findings reported herein, it will be of interest to assess whether pathogenic lamin A/C mutants alter the interaction of c-Fos and/or ERK1/2 at the NE, and if so, whether they affect the regulation and function of AP-1 target genes.
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-tubulin (sc-8035), anti-FLAG, and HRP-coupled secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. Anti-NUP50 and anti-GFP were from Abcam (ab4005) and Invitrogen (A6455), respectively.
Plasmids
pECFP-YFP was constructed by inserting YFP into pECFP-C1 (gift from J. López-Giménez, University of Glasgow, Scotland). The following plasmids are described elsewhere: pGEX-lamin A (37–244), pGEX-lamin A (247–355), and pGEX-lamin A (356–571) (gift from T. Ozaki, Chiba Cancer Center Research Institute, Japan) (Ozaki et al., 1994); pEYFP-c-Fos and pECFP-lamin A (Ivorra et al., 2006); pmycCMV-ERK2-MEK1 (Robinson et al., 1998); pCDNAI-HA-ERK2 (Crespo et al., 1994); pCEFL-MEKE (Sanz-Moreno et al., 2003); pCMV-FLAG-ERK2; pCMV-FLAG-ERK2-Y261N; pCMV-FLAG-ERK2-241-272; pCMV-FLAG-ERK2-S264P and pCMV-FLAG-ERK2-D316A-D319A (Robinson et al., 2002); pCEFL-AU5-c-Fos (c-Fos-wt); and pCEFL-AU5-c-Fos-mut (c-Fos-m, which contains the following mutations: T232A, T325A, T331A, and S374A; gift from S. Gutkind, National Institutes of Health, MD; see Fig. 4 B) (Monje et al., 2003).
Cell culture
NIH-3T3, COS-7, HEK293, U20S, and HeLa cells were obtained from the American Type Culture Collection. Lmna-null and littermate wild-type MEFs are described elsewhere (Sullivan et al., 1999). Cells maintained in DME supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2 mmol/L L-glutamine (Invitrogen) and 10% FBS (or 10% NBS for NIH-3T3 cells) were incubated at 37°C in a 5% CO2/95% O2 atmosphere. Cultures were serum starved for 24 h and then stimulated with either 10% FBS, 10% NBS, PDGF-BB (20 ng/ml; Sigma-Aldrich), or EGF (100 ng/ml; Sigma-Aldrich). For ERK1/2 inhibition, PD98059 (20 µM; Tocris) or U0126 (10 µM; Promega) were added before serum stimulation. MG132 and cycloheximide were from Sigma-Aldrich. When indicated, cells were treated as schematized in Fig. 2 A.
In vitro pull-down assays
GST proteins were purified using glutathione-Sepharose 4B (GE Healthcare) and eluted with 50 mM Tris-Cl (pH 8.0). For the experiments of Fig. 5, GST proteins and cell lysates from NIH-3T3 cells were incubated in 20 mM Hepes (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF supplemented with complete protease inhibitor cocktail (Roche). After 16 h of incubation at 4°C, glutathione-Sepharose 4B was added to a final concentration of 10% and agitated at 4°C for 45 min. The beads were collected by centrifugation and washed three times with 20 mM Hepes (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF. For the experiments of Fig. 8 C, whole cell lysates from HEK293 cells were incubated with GST-lamin A bound to glutathione-Sepharose 4B beads. After 4 h of incubation at 4°C, the beads were collected by centrifugation and washed twice with 1% NP-40/PBS. In all cases, pellets were air-dried, resuspended in 2x Laemmli buffer, boiled for 5 min, and separated onto 12% SDS–polyacrylamide gels (SDS-PAGE).
Subcellular fractionation, immunoprecipitation, and immunoblot experiments
Immunoprecipitation and Western blot analysis were performed as previously described (Ivorra et al., 2006; Casar et al., 2007). Subcellular fractionation was performed as described by Schreiber et al. (1989) with minor modifications (Ivorra et al., 2006). In brief, cells were washed with PBS and scraped into TEN buffer (150 mM NaCl, 1 mM EDTA, and 40 mM TrisCl at pH 7.4). Cells were collected by brief centrifugation in microfuge tubes and resuspended in 10 mM Hepes (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF. After 15 min on ice, Nonidet NP-40 (Fluka) was added to a final concentration of 10%, and tubes were vortexed. Lysates were centrifuged at 4°C in a microfuge set at maximum speed to obtain the soluble cytoplasmic fraction (supernatant) and the nuclear pellet, which was resuspended in ice-cold 20 mM Hepes (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF, and agitated at 4°C for 15 min. The nuclear lysate was centrifuged for 45 min at 4°C to obtain the SNF and the pellet containing the ERNF.
In situ nuclear matrix isolation and indirect immunofluorescence analysis
In situ nuclear matrix isolation was performed as described previously (Fey et al., 1984). In brief, cells grown on coverslips were washed in PBS and extracted twice in cytoskeleton buffer (CSK: 100 mM NaCl, 300 mM sucrose, 10 mM PIPES (pH 6.8), 3 mM MgCl2, 0.5% Triton X-100, and 1.2 mM PMSF) for 10 min at 0°C. The resulting soluble fraction was removed. Extraction buffer (250 mM ammonium sulfate, 300 mM sucrose, 10 mM PIPES (pH 6.8), 3 mM MgCl2, 1.2 mM PMSF, and 0.5% Triton X-100) was added to the Triton X-100 insoluble structures for 10 min at 0°C and the cytoskeleton fraction was removed. DNase digestion was performed twice in digestion buffer (CSK buffer containing 100 µg/ml DNase I and 50 mM NaCl) followed by extraction in digestion buffer containing 0.25 M (NH4)2SO4. In situ– extracted and control cells were fixed in 4% formaldehyde/PBS and permeabilized with 0.5% Triton X-100. All samples were blocked for 5 min with 10 mM glycine (pH 8.5) and 1 h with 5% dry milk in 10% FBS, 0.5% BSA, 0.1% Triton X-100, and PBS, followed by an overnight incubation at 4°C with anti-c-Fos (1:100), anti-ERK2 (1:100), or anti-Sp1 (1:100) antibodies. Samples were then incubated with species-appropriate FITC-conjugated secondary antibody. After washes and incubation with anti-lamin A/C (1:100; sc-7292) for 1 h at room temperature, specimens were washed and incubated with an anti–mouse secondary antibody conjugated to Alexa 633 (1:300).
Phosphatase treatment
NIH-3T3 cells were rapidly washed with cold PBS and collected for subcellular fractionation as described above. When indicated, fractionation was performed in the absence of phosphatase inhibitors, DTT and PMSF. Aliquots of the SNF were incubated in the absence or presence of 1 U of alkaline phosphatase (Roche) for 1 h at 37°C. Reactions were stopped by adding SDS sample buffer and processed for immunoblotting.
EMSA
Double-stranded oligonucleotides containing the AP-1 (5'-CGCTTGATGAGTCAG-3'; AP-1 site underlined) and the Sp1 (5'-ATTCGATCGGGGCGGGGCGAGC-3'; Sp1 site underlined) consensus sites were labeled with 
[32P]dATP using polynucleotide kinase (New England Biolabs, Inc.) and purified on a Sephadex G-50 column. EMSA was performed using the SNF of NIH-3T3 cells (5 µg total protein) and wild-type and lamin A/C–null mice MEFs (15 µg total protein) as previously described (Ivorra et al., 2006).
Confocal microscopy
Images were acquired on a laser confocal microscope (TCS/SP2; Leica) with a 63x oil immersion objective (NA 1.4). For single lamin A/C (Fig. 7 C) and lamin B (Fig. 7 C), double ERK2–lamin A/C (Fig. 9 A) and lamin B–lamin A/C (Fig. 9 C), and triple ERK2–c-Fos–lamin B immunofluorescence (Fig. 9 D): cells were fixed with 4% PFA/PBS at room temperature (RT) on glass coverslips and permeabilized using 0.5% Triton X-100. Samples were blocked for 5 min with 10 mM glycine (pH 8.5) and 1 h with 5% dry milk in 10% FBS, 0.5% BSA, 0.1% Triton X-100, and PBS before an overnight incubation at 4°C with anti-lamin A/C (1:100; sc-7292) or anti-lamin B antibody (1:100). In double immunofluorescence experiments, samples were incubated with anti-ERK2 (1:100) or anti-lamin B (1:100) antibodies for 1 h at room temperature after anti-lamin A/C antibody incubation. For triple immunofluorescence, samples were incubated simultaneously with anti-c-Fos (1:100) and anti-ERK2 (1:100; sc-1647) antibodies after anti-lamin B antibody incubation. Finally, secondary antibodies conjugated to Alexa 488, Texas red, and Alexa 633 (Molecular Probes, Inc.) were used (1:300). Image quantification was done using MetaMorph software (MDS Analytical Technologies).
FRET
NIH-3T3 cells were cotransfected with pECFP-lamin A + pEYFP-c-Fos or pECFP-Lamin A + EYFP as a negative control (1 µg each plasmid) using Lipofectamine (Invitrogen). Cotransfection of pECFP-YFP + pcDNA3 (0.5 µg each) was used as positive control to calibrate the system. Images were acquired on a confocal microscope (TCS/SP2; Leica) with a 63x oil immersion objective (NA 1.4). An argon laser line of 458 nm was used to excite CFP (PMT window 465–505 nm) and a 514-nm line (20% laser intensity for acquisition and 65% for photobleaching) to excite YFP (PMT window: 525–600 nm). Studies were performed in 4% paraformaldehyde-fixed cells using the acceptor-photobleaching method (Kenworthy, 2001) as previously described (Ivorra et al., 2006), in which FRET is calculated as the relative increase in donor fluorescence as a result of the reduction or elimination of energy transfer when the acceptor is photobleached. Specifically, we used the following equation: FRET = (Cafter – Cbefore)/Cafter x 100, where Cbefore and Cafter are the total fluorescence intensity of the CFP channel before and after photobleaching, respectively. For negative values, this parameter was considered 0.
Luciferase gene reporter assays
Wild-type and lamin A/C–null MEFs were transiently transfected with 5 µg of AP-1–dependent coll 73-luciferase reporter plasmid and pGL4-Renilla luciferase using the calcium phosphate method. U2OS were transfected with 5 µg of coll73-luciferase plus either a plasmid encoding for a miRNA-control/GFP or miRNA-LMNA/GFP (BLOCK-IT; Invitrogen). After 48 h in 10% FBS, cells were harvested and luciferase activity was measured following the manufacturer's instructions (dual luciferase reporter assay system; Promega). Luciferase activity and GFP expression were measured in a luminometer (Victor). miRNA-transfected cells were also fixed in 4% PFA and studied by immunofluorescence confocal microscopy, or lysed for Western blot analysis.
Statistical analysis
Results are reported as mean ± SE. In experiments with two groups, differences were evaluated using a two-tail, unpaired Student's t test. One-way ANOVA and Bonferroni's post hoc test was used for experiments involving more than two groups.
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Acknowledgments |
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P. Crespo's laboratory is supported by grants BFU2005-00777 and GEN2003-20239-C06-03 (MICINN), the EU Sixth Framework Program under the GROWTHSTOP (LSHC CT-2006-037731) and SIMAP (IST-2004-027265) projects, and RD06/0020/0105 (Red Temática de Investigación Cooperativa en Cáncer, ISCIII). J.M. González received salary support from ISCIII and a research grant from Generalitat Valenciana (GVPRE/2008/163).
Submitted: 9 May 2008
Accepted: 20 October 2008
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90 min), protein synthesis was inhibited with cycloheximide (10 µg/ml). When indicated, the ERK1/2 inhibitors PD98059 (20 µM) and U0126 (10 µM) were added for the last 50 min (B) Western blot analysis of the SNF and ERNF of NIH-3T3 cells treated as described above. Numbers on the side of the blots indicate molecular weight (in kD). Arrows point to c-Fos species with different degree of phosphorylation.
