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. 2010 Mar 23;107(12):5375-80.
doi: 10.1073/pnas.0910015107. Epub 2010 Mar 8.

p38 MAPK/MK2-mediated induction of miR-34c following DNA damage prevents Myc-dependent DNA replication

Ian G Cannell  1 , Yi W Kong, Samantha J Johnston, Melissa L Chen, Hilary M Collins, Helen C Dobbyn, Androulla Elia, Theresia R Kress, Martin Dickens, Michael J Clemens, David M Heery, Matthias Gaestel, Martin Eilers, Anne E Willis, Martin Bushell
Affiliations

p38 MAPK/MK2-mediated induction of miR-34c following DNA damage prevents Myc-dependent DNA replication

Ian G Cannell et al. Proc Natl Acad Sci U S A. .

Abstract

The DNA damage response activates several pathways that stall the cell cycle and allow DNA repair. These consist of the well-characterized ATR (Ataxia telangiectasia and Rad-3 related)/CHK1 and ATM (Ataxia telangiectasia mutated)/CHK2 pathways in addition to a newly identified ATM/ATR/p38MAPK/MK2 checkpoint. Crucial to maintaining the integrity of the genome is the S-phase checkpoint that functions to prevent DNA replication until damaged DNA is repaired. Inappropriate expression of the proto-oncogene c-Myc is known to cause DNA damage. One mechanism by which c-Myc induces DNA damage is through binding directly to components of the prereplicative complex thereby promoting DNA synthesis, resulting in replication-associated DNA damage and checkpoint activation due to inappropriate origin firing. Here we show that following etoposide-induced DNA damage translation of c-Myc is repressed by miR-34c via a highly conserved target-site within the 3(') UTR. While miR-34c is induced by p53 following DNA damage, we show that in cells lacking p53 this is achieved by an alternative pathway which involves p38 MAPK signalling to MK2. The data presented here suggest that a major physiological target of miR-34c is c-Myc. Inhibition of miR-34c activity prevents S-phase arrest in response to DNA damage leading to increased DNA synthesis, DNA damage, and checkpoint activation in addition to that induced by etoposide alone, which are all reversed by subsequent c-Myc depletion. These data demonstrate that miR-34c is a critical regulator of the c-Myc expression following DNA damage acting downstream of p38 MAPK/MK2 and suggest that miR-34c serves to remove c-Myc to prevent inappropriate replication which may otherwise lead to genomic instability.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
miR-34c represses translation of c-Myc. (A) Sequestering miR-34c relieves repression of a reporter construct harboring the wild-type c-myc 3′ UTR in HeLa cells. HeLa cells were cotransfected with pLSVM3′ or pLSVM3′mut and either a control anti-miR or anti-miR-34c. Luciferase acitivity of the wild-type c-myc 3′ UTR (gray bars) construct are expressed as a percentage of luciferase activity of the seed-mutant (black bars) normalized to a LacZ transfection control. Values are mean ± SD (t-test, n = 3, compared to control anti-miR transfected cells). (B) Transfection of exogenous miR-34c represses translation of a reporter construct harboring the wild-type c-myc 3′ UTR. HEK293 cells were transfected with pLSVM3′ or pLSVM3′mut and either a control siRNA or a miR-34c mimic. Values are expressed and normalized as in (B). Values are mean ± SD (t-test, n = 3, compared to control siRNA cells transfected cells). (C) Myc is repressed at the posttranscriptional level following etoposide treatment. HEK293 cells were treated with etoposide at the indicated concentrations for 24 h. Parallel samples were taken for western and northern analysis as indicated. (D) The c-myc 3′ UTR reporter construct is repressed in response to etoposide in a miR-34c-dependent manner. HEK293 cells were transfected as indicated and 24 h later treated with 12.5 μM etoposide. Luciferase activity was normalized to a LacZ transfection control and expressed as a percentage of luciferase activity from DMSO treated cells (t-test, n = 3, compared to DMSO control anti-miR transfected cells). (E) Endogenous Myc is repressed in response to etoposide in a miR-34c-dependent manner. HEK293 cells were transfected with control anti-miR or anti-miR-34c and 24 h later treated with 12.5 μM etoposide. Percentages indicate relative translational efficiency compared to control.
Fig. 2.
Fig. 2.
miR-34c-mediated repression of Myc in response to DNA damage is maintained in p53-deficient cells. (A) miR-34c is induced following DNA damage in p53-/- MEFs. p53 -/- MEFs were treated with 25 μM etoposide for 24 h, RNA extracted and subjected to qPCR analysis for miR-34c. Data are expressed as fold-change relative to control. Values are mean ± SD (t-test, n = 3, compared to DMSO treated cells). (B) Myc is repressed in response to etoposide in a miR-34c-dependent manner in p53-/- MEFs. p53 -/- MEFs were transfected as indicated and 24 h later treated with 25 μM etoposide. Data shown are representative of three independent experiments.
Fig. 3.
Fig. 3.
The p38 MAPK/MK2 pathway controls miR-34c induction in response to DNA damage. (A) Inhibition of sensor kinases ATM, ATR, and DNA-PK prevents miR-34c induction and Myc repression in response to DNA damage. HEK293 cells were pretreated with LY294002 for 30 min prior to treatment with 12.5 μM etoposide for 24 h. Values are mean ± SD (t-test, n = 3, compared to DMSO treated cells). (B) Inhibition of p38 MAPK but not MEK1/2 prevents miR-34c induction and Myc repression in response to DNA damage. HEK293 cells were pretreated with SB20350 or PD098059 for 30 min prior to treatment with 12.5 μM etoposide for 24 h. Values are mean ± SD (t-test, n = 3, compared to DMSO treated cells). (C) MK2 influences miR-34c induction in response to DNA damage. HEK293 cells were transfected as indicated and 24 h later treated with etoposide. Values are mean ± SD (t-test, n = 3, compared to control DMSO treated cells). (D) Constitutively active MK2 can restore miR-34c induction and Myc repression following p38 MAPK inhibition. p53 -/- MEFs were transfected with empty vector or MK2 EE and treated with etoposide or pretreated with SB203580 for 30 min followed by etoposide for 24 h. Values are mean ± SD (t-test, n = 3, compared to DMSO treated cells).
Fig. 4.
Fig. 4.
miR-34c-mediated repression of Myc promotes S-phase arrest and prevents Myc-induced DNA damage. (A) Inhibition of miR-34c following DNA damage prevents S-phase arrest in a Myc-dependent manner. To determine the effect of miR-34c-mediated repression of Myc HEK293 cells were transfected with combinations of siRNA and anti-miRs for 24 h as indicated prior to treatment with 12.5 μM etoposide for 24 h. Data were analyzed as described in Materials and Methods and are shown as mean ± SD (t-test, n = 3, compared to control). (B) Inhibition of miR-34c increases DNA synthesis following DNA damage in a Myc-dependent manner. Quantifiation of EdU incorporation of cells from (A). Values are mean ± SD (t-test, n = 3, compared to DMSO treated, control anti-miR, control siRNA transfected cells). (C) Inhibition of miR-34c leads to Myc-dependent DNA damage and checkpoint activation following DNA damage. Western analysis of lysates generated from cells in Fig. 1A. Data shown are pooled samples from three independent experiments.
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