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. 2010 Aug 20;285(34):26417-30.
doi: 10.1074/jbc.M110.121830. Epub 2010 Jun 16.

Differential up-regulation of MAP kinase phosphatases MKP3/DUSP6 and DUSP5 by Ets2 and c-Jun converge in the control of the growth arrest versus proliferation response of MCF-7 breast cancer cells to phorbol ester

Caroline E Nunes-Xavier  1 , Céline Tárrega, Rocío Cejudo-Marín, Jeroen Frijhoff, Asa Sandin, Arne Ostman, Rafael Pulido
Affiliations

Differential up-regulation of MAP kinase phosphatases MKP3/DUSP6 and DUSP5 by Ets2 and c-Jun converge in the control of the growth arrest versus proliferation response of MCF-7 breast cancer cells to phorbol ester

Caroline E Nunes-Xavier et al. J Biol Chem. .

Abstract

Different levels of regulation account for the inactivation of MAP kinases by MAPK phosphatases (MKPs), in a cell type- and stimuli-dependent manner. MCF-7 human breast carcinoma cells treated with the phorbol 12-myristate 13-acetate (PMA) suffer growth arrest and show morphological alterations, which depend on the activation of the ERK1/2 MAP kinases. MKP3/DUSP6 and DUSP5 MAP kinase phosphatases, two negative regulators of ERK1/2, were specifically up-regulated in MCF-7 and SKBR3 cells in response to PMA. MKP3 and DUSP5 up-regulation required the prolonged activation of the ERK1/2 pathway, and correlated with the shutdown of this route. MKP3 induction relied on the activation of the Ets2 transcription factor, whereas DUSP5 induction depended on the activation of c-Jun. Diminishing the expression of MKP3 and DUSP5 raised the activation of ERK1/2, and accelerated growth arrest of PMA-treated MCF-7 cells. Conversely, MCF-7 cell lines expressing high levels of MKP3 or DUSP5 did not undergo PMA-triggered growth arrest, displayed a migratory phenotype, and formed colonies in soft agar. We propose that the differential up-regulation of MKP3 by Ets2 and of DUSP5 by c-Jun may converge in similar functional roles for these MAP kinase phosphatases in the growth arrest versus proliferation decisions of breast cancer cells.

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Figures

FIGURE 1.
FIGURE 1.
Activation of MAPKs on human breast carcinoma MCF-7 cells treated with PMA. Cells were incubated in the presence of PMA (50 ng/ml) during the indicated times, and the activation status of ERK1/2, p38, and JNK determined using phospho-specific anti-ERK1/2 (α-pERK1/2), anti-p38 (α-pp38), or anti-JNK (α-pJNK) antibodies. The total amount of ERK1/2, p38, and JNK is also shown. GAPDH expression is included as a loading control. The arrows in the α-pJNK and α-JNK panels indicate the migration of JNK isoforms. A representative experiment is shown of at least three different experiments.
FIGURE 2.
FIGURE 2.
Regulation of MKP expression in MCF-7 cells treated with PMA. A, DNA microarray analysis of the expression of the MKPs. mRNA levels of MKPs from MCF-7 cells untreated or treated with PMA for 4 days were analyzed by microarray hybridization. MKP gene expression was obtained from normalized data, and calculation of Log2 fold-change ±S.D. was performed dividing values from PMA treatment to control. B, semi-quantitative RT-PCR analysis of MKP expression in MCF-7 cells treated with PMA. mRNA levels of MKPs from cells untreated or treated with PMA for 4 days were measured by semi-quantitative RT-PCR, using primers specific for β-actin and the 10 different members of the MKP family. In the upper panel, samples were resolved on 1% agarose gels, and the amplified cDNA was stained with ethidium bromide. Size standards are shown at the left in base pairs (bp). In the lower panel (bar diagram), the average mRNA expression levels ±S.D. of three independent experiments is shown. Bands were quantified using the ImageQuant software package, and normalized with respect to the corresponding β-actin control bands. Note the increase in MKP3 and DUSP5 mRNA content. C, up-regulated mRNA expression of MKP3 and DUSP5 in MCF-7 cells treated with PMA. mRNA levels of MKPs from cells untreated or treated with PMA for 1–4 days were measured by qRT-PCR, and relative expression values ±S.D. are shown. D, coordination of ERK1/2 activation and MKP3 and DUSP5 expression during 4 days of PMA treatment. Data are normalized from C and Fig. 1. E, up-regulated protein expression of MKP3 and DUSP5 proteins in MCF-7 cells treated with PMA. Endogenous MKP3 and DUSP5 protein levels from cells untreated or treated with PMA for 1 to 4 days were determined by immunoblot using anti-MKP3 and anti-DUSP5 antibodies. In the upper panel, an anti-MKP3 immunoblot is shown; the migration of recombinant MKP3 (overexpressed in MCF-7 PMA-treated cells using pTREhyg-MKP3) is illustrated in lane 6. In the lower panel, an anti-DUSP5 immunoblot is shown; the migration of recombinant DUSP5 (overexpressed in MCF-7 cells using pTREhyg-DUSP5) is illustrated in lanes 6 (cells untreated) and 7 (PMA-treated cells). GAPDH expression is included as a loading control. The arrowheads indicate the migration of the different MKP3 and DUSP5 species in the presence and absence of PMA. F, mRNA expression of MKP3 and DUSP5 in human breast carcinoma cell lines. In the upper panel, semi-quantitative RT-PCR analysis was performed on the different cell lines grown under control conditions, as in Fig. 2B. Note the detection of both MKP3 and DUSP5 mRNAs on all analyzed cell lines. In the lower panel (bar diagram), qRT-PCR analysis was performed from untreated cells or from cells treated with 50 ng/ml of PMA for 3 days. Mean fold change ±S.D. is shown. In the case of SKBR3 cells, 1 ng/ml of PMA were used, because higher concentrations during long-term incubation triggered cell death. Note the increase in the mRNA content of both MKP3 and DUSP5 from MCF-7 and SKBR3 cells.
FIGURE 3.
FIGURE 3.
MKP3 and DUSP5 up-regulation by PMA required the prolonged activation of the ERK1/2 pathway. A, inhibition of PKC and ERK1/2 decreased the up-regulation of MKP3 and DUSP5 expression in MCF-7 cells upon PMA treatment. Cells were pre-treated with dimethyl sulfoxide (−), or with the indicated inhibitors prior to PMA treatment, and harvested after 24 h of PMA treatment. In the upper panel, the mRNA levels of MKP3, DUSP5, and p21Waf1/Cip1 in the presence of the inhibitors were measured by qRT-PCR, and relative expression values ±S.D. are shown. In the lower panel, pERK1/2, ERK1/2, pAKT, AKT, MKP3, and DUSP5 levels were analyzed for the effect of inhibitors; PD, MEK-1 inhibitor (PD98059); SB, p38 inhibitor (SB203580); SP, JNK inhibitor (SP600125); Wo, PI3K inhibitor (wortmannin); and GF, PKC inhibitor (GF109203X). GAPDH expression is included as a loading control. B, prolonged ERK1/2 activation is necessary for the continuous mRNA and protein induction of MKP3 and DUSP5. Cells were treated with PMA, and MEK-1 inhibitor (PD98059) was added at 3 and 24 h. Cells were harvested after 48 h of PMA treatment. In the upper panel, the mRNA levels of MKP3, DUSP5, and p21Waf1/Cip1 were measured by qRT-PCR, and relative expression values ±S.D. are shown. In the lower panel, pERK1/2, ERK1/2, MKP3, and DUSP5 levels were analyzed for the effect of PD98059 inhibitor. GAPDH expression is included as a loading control. In both panels, a representative immunoblot is shown of at least three different experiments.
FIGURE 4.
FIGURE 4.
Differential regulation of MKP3 and DUSP5 gene expression in MCF-7 cells in the response to PMA. A, Ets2 is up-regulated in PMA-treated MCF-7 and SKBR3 cells. In the upper left panel, data from semi-quantitative RT-PCR are shown, illustrating the expression of Ets1 and Ets2 mRNAs in MCF-7 cells grown in the presence and absence of PMA. Note the lack of Ets1 expression. In the left bottom panel, quantification of microarray hybridization analysis is provided, showing mRNA up-regulation of Ets2, but not of Ets1, in MCF-7 cells treated with PMA. In the middle panel, Ets2 mRNA up-regulation was measured by qRT-PCR in MCF-7 and SKBR3 cells upon PMA (50 ng/ml) cell treatment during several periods of time, and relative expression values are shown. In the right panel, Ets2 protein up-regulation and activation (phospho-Ets2, pEts2) in MCF-7 and SKBR3 cells treated with PMA was monitored by immunoblot. Note that Ets2 up-regulation in SKBR3 cells followed a faster kinetics. B, alignment of MKP3 and DUSP5 promoters showed putative Ets2 transcription factor binding sites. Aligned upstream MKP3 genomic sequence, NC_000012.11:c89746400–89746297; MKP3 genomic sequence, NC_000012.11:c89746296–89741837; aligned upstream DUSP5 genomic sequence, NC_000010.10:c112257572–112257624; DUSP5 genomic sequence, NC_000010.10:c112257625–112271302. Alignments were made using EMBOSS pairwise alignment algorithms (72). C, transcription factor Ets2 is responsible for up-regulation of MKP3 and p21Waf1/Cip1 in MCF-7 cells in the response to PMA. In the left panel, Ets2 protein expression and activation were monitored by immunoblot, from untreated (−) or PMA-treated (24 h) (+) MCF-7 cells. Transfection with negative control (siNS, nonspecific silencer) or with Ets1- or Ets2-specific (siEts1, siEts2) silencing oligonucleotides was performed 48 h before PMA stimulation. The phospho-Ets2, Ets2, pERK1/2, and ERK1/2 contents under the silencing conditions are shown. In the right panel, mRNA levels of MKP3, DUSP5, and p21Waf1/Cip1, from silenced control or Ets2-silenced MCF-7 cells, grown in the absence (−) or presence of PMA (3 h) (+), were measured by qRT-PCR, and relative expression values are shown. Results represent the mean ± S.D. (*, p < 0,005). D, transcription factor c-Jun is responsible for up-regulation of DUSP5 in MCF-7 cells in the response to PMA. Cells were silenced with nonspecific (siNS) or with c-Jun-specific (sic-Jun) oligonucleotides, as in C, and phospho-c-Jun (pc-Jun), c-Jun, pERK1/2, and ERK1/2 content was monitored by immunoblot (left panel). In the right panel, MKP3, DUSP5, and p21Waf1/Cip1 mRNAs were measured by qRT-PCR, as in C. Results represent the mean ± S.D. (*, p < 0,005). Data are representative of more than 3 different experiments. In all the panels, a representative immunoblot is shown of at least three different experiments.
FIGURE 5.
FIGURE 5.
Silencing of MKP3 and DUSP5 by RNA interference favors growth arrest of MCF-7 cells by PMA. A, silencing of MKP3 and DUSP5 mRNA expression upon RNA interference using siRNAs. Cells were transfected with nonspecific siRNA (siNS) or MKP3- or DUSP5-specific siRNAs (siMKP3, siDUSP5), and mRNA levels were determined by qRT-PCR after 48 h. B, morphological changes in MKP3- and DUSP5-silenced MCF-7 cells upon PMA treatment. 48 h after transfection with siRNA negative control (siNS), siMKP3, or siDUSP5, cells were kept untreated (−) or were treated with PMA (+), and photographed after 3 h. The arrows indicate cells with filopodia. Cultures silenced for MKP3 of DUSP5 showed more cells with filopodia. C, quantification of filopodia from silenced MCF-7 cells. Cells were treated with PMA for 3 or 24 h and filopodia were counted. Data are shown as the mean ± S.D. D, silencing of MKP3 and DUSP5 increased the activation of ERK1/2, caused a rapid decay of ERα, and a faster up-regulation of p21Waf1/Cip1 upon PMA treatment. 48 h after transfection with siRNA negative control (NS), siMKP3, or siDUSP5, cells were kept untreated or treated with PMA for 3 and 24 h, before harvesting. pERK1/2, ERK1/2, ERα, and p21Waf1/Cip1 levels were analyzed by immunoblot. Actin expression is included as a loading control. A representative immunoblot is shown of at least three different experiments.
FIGURE 6.
FIGURE 6.
Stable expression of MKP3 and DUSP5 in MCF-7 cells inhibits growth arrest upon PMA treatment. A, expression of MKP3 and DUSP5 in stable, Tet-On-inducible MCF-7 cell lines. Clones obtained after transfection with empty pTRE2hyg vector (EV) or with pTRE2hyg-MKP3 (wild type of C293S mutation) or pTRE2hyg-DUSP5 (wild type of C263S mutation), were analyzed by immunoblot for the expression of MKP3, DUSP5, and GAPDH (as a loading control), in the absence (−) or presence (+) of doxycyline (DOX) for 24 h. Note the leaking expression of MKP3 and DUSP5 in the absence of doxycycline. B and C, stable MCF-7 cell lines expressing MKP3 and DUSP5 abandon their mosaic-like appearance and do not display cell-growth arrest upon PMA treatment. In B, empty vector-control cells, and MKP3- and DUSP5-inducible cell lines, were kept untreated (−) or were treated (+) with PMA for 4 days, and pictures of the cultures were taken. In C, the distinct cell lines were grown in the presence of PMA from 1 to 4 days, and cell proliferation was measured by the 3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyltetrazolium bromide assay. Note the significant increase in cell growth in the presence of PMA of the MKP3 and DUSP5 cell lines. D, p21Waf1/Cip1 protein levels on stable MCF-7 cell lines expressing MKP3 and DUSP5. Cells were incubated in the presence of PMA during 24 h, and levels of p21Waf1Cip1 and GAPDH were determined by immunoblot. E, stable MCF-7 cell lines expressing MKP3 and DUSP5 do not display prolonged activation of ERK1/2 upon PMA treatment. Cells were incubated in the presence of PMA during the indicated times, and levels of MKP3, DUSP5, pERK1/2, and ERK1/2 were determined by immunoblot. In all experiments, results from one clone are shown, which are representative of results from at least two different clones. In A, D, and E, a representative immunoblot is shown of at least three different experiments.
FIGURE 7.
FIGURE 7.
Effect of MKP3 and DUSP5 expression on anchorage-independent colony formation, and wound repair and migratory properties of MCF-7 cell lines in the presence of PMA. A, stable MCF-7 cell lines expressing MKP3 and DUSP5 form colonies in soft agar in the presence of PMA. Stable cell lines expressing MKP3 and DUSP5 were pre-treated for 24 h without (−) or with (+) PMA before plating in soft agar for the formation of anchorage-independent colonies. Cells were grown for 2 weeks in soft agar, and photographs of representative plates were taken. In the bottom of the figure, quantification of the number of colonies, using ImageJ 1.40g, from triplicate plates, is shown. Results represent the mean values ± S.D. (*, p < 0.005). B, stable MCF-7 cell lines expressing MKP3 and DUSP5 display enhanced migration properties in the presence of PMA. Cells were plated and wounded with a pipette tip, followed by incubation for 24 h without (−) or with (+) PMA. Photographs of the cultures at the beginning of the assay, and after 24 h of PMA treatment, are shown. In the bottom of the figure, quantification of cell migration is shown. Results represent the mean ± S.D. (*, p < 0,005). In both panels, results from one clone are shown, which are representative of results from at least two different clones.
FIGURE 8.
FIGURE 8.
Scheme of the up-regulation mechanisms of MKP3 and DUSP5, and their role in the control of growth arrest versus proliferation response of MCF-7 cells to PMA. PMA triggers the activation of ERK1/2 in MCF-7 cells, which induces MKP3 and DUSP5 expression through the activity of transcription factors Ets2 and c-Jun, respectively. Low levels of MKP3 and DUSP5 results in ERK1/2 sustained activation and cell growth arrest. Overexpression of MKP3 and DUSP5 results in ERK1/2 transient activation and enhancement of proliferation and migration. In this model, the Ets2 and c-Jun pathways converge, through MKP3 and DUSP5, to produce a similar functional output in the regulation of cell growth of MCF-7 cells.
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