Dual Specificity Phosphatase 5, a Specific Negative Regulator of ERK Signaling, Is Induced by Serum Response Factor and Elk-1 Transcription Factor.

Serum stimulation of mammalian cells induces, via the MAPK pathway, the nuclear protein DUSP5 (dual-specificity phosphatase 5), which specifically interacts with and inactivates the ERK1/2 MAP kinases. However, molecular mechanisms underlying DUSP5 induction are not well known. Here, we found that the DUSP5 mRNA induction depends on a transcriptional regulation by the MAPK pathway, without any modification of the mRNA stability. Two contiguous CArG boxes that bind serum response factor (SRF) were found in a 1 Kb promoter region, as well as several E twenty-six transcription factor family binding sites (EBS). These sites potentially bind Elk-1, a transcription factor activated by ERK1/2. Using wild type or mutated DUSP5 promoter reporters, we demonstrated that SRF plays a crucial role in serum induction of DUSP5 promoter activity, the proximal CArG box being important for SRF binding in vitro and in living cells. Moreover, in vitro and in vivo binding data of Elk-1 to the same promoter region further demonstrate a role for Elk-1 in the transcriptional regulation of DUSP5. SRF and Elk-1 form a ternary complex (Elk-1-SRF-DNA) on DUSP5 promoter, consequently providing a link to an important negative feedback tightly regulating phosphorylated ERK levels

Proximal promoter region of <i>DUSP5</i> contains two functional CArG boxes.

<p>(A) Probes used in Electrophoretic Mobility Shift Assay (EMSA) containing CArG boxes of the proximal promoter region of <i>DUSP5</i> and an unrelated probe are shown. (B) EMSA was performed with the end labeled CArG boxes probe using 4 μg of nuclear extracts isolated from NIH/3T3 cells stimulated or not with 20% FCS for 30 minutes. For competition experiments, different unlabeled oligonucleotides (lane 10 to 14) were used. The indicated antibodies (lane 15 to 17) were used for supershift analysis.</p

<i>DUSP5</i> expression is regulated at the transcriptional level.

<p>(A) NIH/3T3 cells were treated with FCS 20% alone or in combination with the transcriptional inhibitor actinomycin D (5 μg/ml) or the protein synthesis inhibitor cycloheximide (100 μg/ml) for the indicated times. <i>DUSP5</i> mRNA levels were measured by RT-qPCR and normalized for cyclophilin mRNA levels. <i>DUSP5</i> mRNA levels at baseline were set at 1 and values at subsequent time points are indicated as fold induction compared to baseline. * <i>P</i> < 0.05, ** <i>P</i> = non-significant. (B) NIH/3T3 cells were stimulated with 20% FCS for one hour and then were treated with 5 μg/ml of actinomycin D with or without 20μM of UO126 (MEK inhibitor) for the indicated times. <i>DUSP5</i> mRNA levels, before actinomycin D treatment, were taken as 100%. <i>DUSP5</i> mRNA levels were measured at different times after UO126 treatment. <i>DUSP5</i> mRNA half-life (t ½) is indicated in cells with and without (control) UO126 treatment.</p

Proximal promoter region of <i>DUSP5</i> contains a functional serum responsive element.

<p>(A) DNA sequences of <i>DUSP5</i> promoter region containing proximal CArG box (CArG2) with proximal EBS and unrelated probe used in Electrophoretic Mobility Shift Assays (EMSA). (B) EMSA was performed with the end labelled probe containing wild type CArG Boxe and EBS using 5 μg of nuclear extracts isolated from NIH/3T3 transfected with hemagglutinin (HA) tagged Elk-1expression plasmid. For competition experiments, different unlabelled oligonucleotides (lane 3 to 7) were used. The indicated antibodies (lane 8 to 10) were used for supershift analysis. (C) EMSA was performed with the end labelled probe containing wild type CArG Boxe and mutated EBS using 5 μg of the same nuclear extracts. The indicated antibodies (lane 3 to 5) were used for supershift analysis.</p

SRF and Elk-1 binding to the endogenous <i>DUSP5</i> promoter proximal region.

<p>ChIP assay was performed in NIH/3T3 cells stimulated for 30 minutes with 20% FCS after 24 hours of serum starvation, using an unrelated control antibody (IgG) and specific antibodies (A) for SRF or (B) Elk-1. Binding of SRF or Elk-1 to an intronic part of the <i>DUSP5</i> gene (negative control), and to the proximal part of the <i>DUSP5</i> promoter was measured by qPCR and corrected for background measured in IgG immunoprecipitates. Graphs show the mean and standard deviation of 3 independent experiments. * <i>P</i> < 0.05.</p

Depletion of endogenous SRF or Elk-1 decreases DUSP5 mRNA induction by serum stimulation.

<p>NIH/3T3 cells were transfected with small interfering RNA directed against SRF or Elk-1 or control siRNA. Cells were serum deprived for 12 hours and then stimulated or not with 20% FCS for one hour. To quantify the gene-silencing efficiency by siRNA, SRF (A) and Elk-1 (B) mRNA levels were measured by RT-qPCR and normalized for cyclophilin mRNA levels. (C) DUSP5 mRNA relative levels were compared between the situations with control siRNA and SRF or Elk-1 specific siRNA. The results represent the mean of at least two independent experiments each performed in triplicate ± standard error. * <i>P</i> = 0.04; ° <i>P</i> = 0.8 °° <i>P</i> = 0.05.</p

<i>DUSP5</i> is an early response gene induced by ERK signalling.

<p>NIH/3T3 cells were stimulated by FCS 20% and treated either with 20 μM UO126 (MEK inhibitor) or 80nM wortmannin (PI3K inhibitor) for the indicated time. <i>DUSP5</i> mRNA levels were measured by RT-qPCR and normalized for cyclophilin mRNA levels. <i>DUSP5</i> mRNA levels at baseline were set at 1 and values at subsequent time points are indicated as fold induction compared to baseline. Protein expression levels were assayed by immunoblot for phosphorylated ERK (p-ERK), total ERK (t-ERK), phosphorylated (p-AKT) and total AKT (t-AKT). The effect of inhibitors on p-ERK and p-AKT levels is shown. * <i>P</i> < 0.05; ** <i>P</i> = non-significant.</p

Elk-VP16(L158P) mutant is defective in <i>DUSP5</i> transcriptional activation.

<p>Serum starved (0.25%) NIH/3T3 cells were transiently transfected with 250 ng of <i>DUSP5</i> proximal promoter reporter and with 200 ng of the plasmids Elk-VP16 or Elk-VP16(L158P) before assessment of luciferase activity. Luciferase assays were performed in triplicate and mean values ± S.D. are shown. * <i>P</i> < 0.05, ** <i>P</i>: statistically insignificant.</p

The proximal region of <i>DUSP5</i> promoter is sufficient for its serum induction.

<p>(A) Reporter vectors harboring full-length or various truncations of the promoter region of <i>DUSP5</i> (250 ng) were transfected in NIH/3T3 cells with (black boxes) or without (white boxes) stimulation by FCS 20% for nine hours (Luciferase activity was normalized to Renilla activity). Basal luciferase activities were related to that of full-length construct. Induced luciferase activities of each vector were reported to their own basal activity. * <i>P</i> < 0.05. (B) The sequence of the putative proximal promoter region of <i>DUSP5</i> gene is shown. Putative transcription factor binding sites are indicated: two contiguous CArG boxes potentially implicated in binding of SRF, two EBS sequences GGA(A/C) potentially implicated in binding of Elk-1, one putative Lef-TCF binding site (Wnt/ β-catenin pathway), and one cAMP response element (CRE).</p