Limits...
MK3 modulation affects BMI1-dependent and independent cell cycle check-points.

Prickaerts P, Niessen HE, Dahlmans VE, Spaapen F, Salvaing J, Vanhove J, Geijselaers C, Bartels SJ, Partouns I, Neumann D, Speel EJ, Takihara Y, Wouters BG, Voncken JW - PLoS ONE (2015)

Bottom Line: In the current study we show that MK3 overexpression results in reduced cellular EZH2 levels and concomitant loss of epigenetic H3K27me3-marking and PRC1/chromatin-occupation at the CDKN2A/INK4A locus.In contrast, BMI1 does not rescue the MK3 loss-of-function phenotype, suggesting the involvement of multiple different checkpoints in gain and loss of MK3 function.Taken together, our findings support a role for MK3 in control of proliferation and replicative life-span, in part through concerted action with BMI1, and suggest that the effect of MK3 modulation or mutation on M/SAPK signaling and, ultimately, proliferation, is cell context-dependent.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Maastricht University Medical Centre, Maastricht, the Netherlands.

ABSTRACT
Although the MK3 gene was originally found deleted in some cancers, it is highly expressed in others. The relevance of MK3 for oncogenesis is currently not clear. We recently reported that MK3 controls ERK activity via a negative feedback mechanism. This prompted us to investigate a potential role for MK3 in cell proliferation. We here show that overexpression of MK3 induces a proliferative arrest in normal diploid human fibroblasts, characterized by enhanced expression of replication stress- and senescence-associated markers. Surprisingly, MK3 depletion evokes similar senescence characteristics in the fibroblast model. We previously identified MK3 as a binding partner of Polycomb Repressive Complex 1 (PRC1) proteins. In the current study we show that MK3 overexpression results in reduced cellular EZH2 levels and concomitant loss of epigenetic H3K27me3-marking and PRC1/chromatin-occupation at the CDKN2A/INK4A locus. In agreement with this, the PRC1 oncoprotein BMI1, but not the PCR2 protein EZH2, bypasses MK3-induced senescence in fibroblasts and suppresses P16INK4A expression. In contrast, BMI1 does not rescue the MK3 loss-of-function phenotype, suggesting the involvement of multiple different checkpoints in gain and loss of MK3 function. Notably, MK3 ablation enhances proliferation in two different cancer cells. Finally, the fibroblast model was used to evaluate the effect of potential tumorigenic MK3 driver-mutations on cell proliferation and M/SAPK signaling imbalance. Taken together, our findings support a role for MK3 in control of proliferation and replicative life-span, in part through concerted action with BMI1, and suggest that the effect of MK3 modulation or mutation on M/SAPK signaling and, ultimately, proliferation, is cell context-dependent.

Show MeSH

Related in: MedlinePlus

Proliferative regulation by MK3 in cancer cell lines.(A) Proliferation curves (left) of U-2OS cells expressing a retroviral MK3 vector (MK3WTOE; filled circles) or an empty vector (con; open circles); overexpression of GST-MK3 (MK3WTOE) in U-2OS cells detected with and GST or a MK3-antiserum (right panel). Cell counts at t = 2 through t = 8 were normalized to cell counts at t = 0 for each transduced cell culture individually (see Methods section for details); statistical significance was determined by two-tailed Student’s t-test and is presented relative to the empty vector control (* p < 0.05). (B) Phase contrast images showing cell morphology in U-2OS/MK3WTOE cells and control cells. (C) Protein expression levels of the check-point regulator proteins TP53 and p21CIP1/WAF1 (P21) in U-2OS/MK3WTOE cells; loading control b-Actin (bAct). (D) DNA profile analysis of U-2OS/MK3OE versus control cells (4–6 days post-transduction; representative experiment). MK3WToverexpression elicits an intra S-phase arrest: table shows a substantially increased S-phase occupancy. (E) Immunohistochemical staining for phosphorylated H2A.X (γH2A.X) and phosphorylated KAP1pSer824 (pKAP1; arrows) to visualize DNA damage in U-2OS/MK3WTOE cultures; control (top panels) or MK3WTOE (bottom panels).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4390245&req=5

pone.0118840.g005: Proliferative regulation by MK3 in cancer cell lines.(A) Proliferation curves (left) of U-2OS cells expressing a retroviral MK3 vector (MK3WTOE; filled circles) or an empty vector (con; open circles); overexpression of GST-MK3 (MK3WTOE) in U-2OS cells detected with and GST or a MK3-antiserum (right panel). Cell counts at t = 2 through t = 8 were normalized to cell counts at t = 0 for each transduced cell culture individually (see Methods section for details); statistical significance was determined by two-tailed Student’s t-test and is presented relative to the empty vector control (* p < 0.05). (B) Phase contrast images showing cell morphology in U-2OS/MK3WTOE cells and control cells. (C) Protein expression levels of the check-point regulator proteins TP53 and p21CIP1/WAF1 (P21) in U-2OS/MK3WTOE cells; loading control b-Actin (bAct). (D) DNA profile analysis of U-2OS/MK3OE versus control cells (4–6 days post-transduction; representative experiment). MK3WToverexpression elicits an intra S-phase arrest: table shows a substantially increased S-phase occupancy. (E) Immunohistochemical staining for phosphorylated H2A.X (γH2A.X) and phosphorylated KAP1pSer824 (pKAP1; arrows) to visualize DNA damage in U-2OS/MK3WTOE cultures; control (top panels) or MK3WTOE (bottom panels).

Mentions: Given its possible dual involvement in tumorigenesis, we probed cancer cell lines for effects of gain or loss of MK3 function on cell proliferation. We here focused on U-2OS osteosarcoma cells and the HeLa cervical carcinoma cells. Of note, U-2OS/MK3WTOE cultures also showed an MK3WTOE-induced reduction of proliferation and enhanced flat cell morphology (9.6% ±1.8 MK3WTOE vs. 2.3% ±0.8 con, P<0.001) and expression of SA-bGal (Fig 5A, 5B and S6A Fig). HeLa/MK3OE cells also showed enhanced flat cell induction (data not shown). Consistent with a response resembling senescence, U-2OS/MK3WTOE cultures showed enhanced expression of TP53 and of its transcriptional target P21CIP1/WAF1 (Fig 5C). Consistent with the involvement of the TP53 pathway in DNA damage response (DDR), nuclei of flat U-2OS/MK3WTOE cells showed increased co-staining for TP53, P14ARF and P21CIP1/WAF1 (S6B Fig). Increased replication stress is known to induce a proliferative arrest which is often associated with double strand DNA breaks (DSB). The occurrence of replication stress-associated DSB is typically associated with an initial intraS-phase arrest, as part of a DDR [19,35,36]. U-2OS/MK3WTOE cells showed a ±1.5 fold increase of cells in S-phase within days after transduction (Fig 5D). To determine whether the flat cell phenotype in U-2OS/MK3OE cells was associated with DNA damage, we use immunofluorescence to study the occurrence of DSB by measuring phosphorylation of histone variant H2A.X (γH2A.X) and of KAP1 phosphorylation at serine 824 (pKAP1). Double-positive γH2A.X/pKAP1 cells were found in considerable numbers throughout U-2OS/MK3WTOE cultures; the nuclei of enlarged flat cells were prominently stained (Fig 5E).


MK3 modulation affects BMI1-dependent and independent cell cycle check-points.

Prickaerts P, Niessen HE, Dahlmans VE, Spaapen F, Salvaing J, Vanhove J, Geijselaers C, Bartels SJ, Partouns I, Neumann D, Speel EJ, Takihara Y, Wouters BG, Voncken JW - PLoS ONE (2015)

Proliferative regulation by MK3 in cancer cell lines.(A) Proliferation curves (left) of U-2OS cells expressing a retroviral MK3 vector (MK3WTOE; filled circles) or an empty vector (con; open circles); overexpression of GST-MK3 (MK3WTOE) in U-2OS cells detected with and GST or a MK3-antiserum (right panel). Cell counts at t = 2 through t = 8 were normalized to cell counts at t = 0 for each transduced cell culture individually (see Methods section for details); statistical significance was determined by two-tailed Student’s t-test and is presented relative to the empty vector control (* p < 0.05). (B) Phase contrast images showing cell morphology in U-2OS/MK3WTOE cells and control cells. (C) Protein expression levels of the check-point regulator proteins TP53 and p21CIP1/WAF1 (P21) in U-2OS/MK3WTOE cells; loading control b-Actin (bAct). (D) DNA profile analysis of U-2OS/MK3OE versus control cells (4–6 days post-transduction; representative experiment). MK3WToverexpression elicits an intra S-phase arrest: table shows a substantially increased S-phase occupancy. (E) Immunohistochemical staining for phosphorylated H2A.X (γH2A.X) and phosphorylated KAP1pSer824 (pKAP1; arrows) to visualize DNA damage in U-2OS/MK3WTOE cultures; control (top panels) or MK3WTOE (bottom panels).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390245&req=5

pone.0118840.g005: Proliferative regulation by MK3 in cancer cell lines.(A) Proliferation curves (left) of U-2OS cells expressing a retroviral MK3 vector (MK3WTOE; filled circles) or an empty vector (con; open circles); overexpression of GST-MK3 (MK3WTOE) in U-2OS cells detected with and GST or a MK3-antiserum (right panel). Cell counts at t = 2 through t = 8 were normalized to cell counts at t = 0 for each transduced cell culture individually (see Methods section for details); statistical significance was determined by two-tailed Student’s t-test and is presented relative to the empty vector control (* p < 0.05). (B) Phase contrast images showing cell morphology in U-2OS/MK3WTOE cells and control cells. (C) Protein expression levels of the check-point regulator proteins TP53 and p21CIP1/WAF1 (P21) in U-2OS/MK3WTOE cells; loading control b-Actin (bAct). (D) DNA profile analysis of U-2OS/MK3OE versus control cells (4–6 days post-transduction; representative experiment). MK3WToverexpression elicits an intra S-phase arrest: table shows a substantially increased S-phase occupancy. (E) Immunohistochemical staining for phosphorylated H2A.X (γH2A.X) and phosphorylated KAP1pSer824 (pKAP1; arrows) to visualize DNA damage in U-2OS/MK3WTOE cultures; control (top panels) or MK3WTOE (bottom panels).
Mentions: Given its possible dual involvement in tumorigenesis, we probed cancer cell lines for effects of gain or loss of MK3 function on cell proliferation. We here focused on U-2OS osteosarcoma cells and the HeLa cervical carcinoma cells. Of note, U-2OS/MK3WTOE cultures also showed an MK3WTOE-induced reduction of proliferation and enhanced flat cell morphology (9.6% ±1.8 MK3WTOE vs. 2.3% ±0.8 con, P<0.001) and expression of SA-bGal (Fig 5A, 5B and S6A Fig). HeLa/MK3OE cells also showed enhanced flat cell induction (data not shown). Consistent with a response resembling senescence, U-2OS/MK3WTOE cultures showed enhanced expression of TP53 and of its transcriptional target P21CIP1/WAF1 (Fig 5C). Consistent with the involvement of the TP53 pathway in DNA damage response (DDR), nuclei of flat U-2OS/MK3WTOE cells showed increased co-staining for TP53, P14ARF and P21CIP1/WAF1 (S6B Fig). Increased replication stress is known to induce a proliferative arrest which is often associated with double strand DNA breaks (DSB). The occurrence of replication stress-associated DSB is typically associated with an initial intraS-phase arrest, as part of a DDR [19,35,36]. U-2OS/MK3WTOE cells showed a ±1.5 fold increase of cells in S-phase within days after transduction (Fig 5D). To determine whether the flat cell phenotype in U-2OS/MK3OE cells was associated with DNA damage, we use immunofluorescence to study the occurrence of DSB by measuring phosphorylation of histone variant H2A.X (γH2A.X) and of KAP1 phosphorylation at serine 824 (pKAP1). Double-positive γH2A.X/pKAP1 cells were found in considerable numbers throughout U-2OS/MK3WTOE cultures; the nuclei of enlarged flat cells were prominently stained (Fig 5E).

Bottom Line: In the current study we show that MK3 overexpression results in reduced cellular EZH2 levels and concomitant loss of epigenetic H3K27me3-marking and PRC1/chromatin-occupation at the CDKN2A/INK4A locus.In contrast, BMI1 does not rescue the MK3 loss-of-function phenotype, suggesting the involvement of multiple different checkpoints in gain and loss of MK3 function.Taken together, our findings support a role for MK3 in control of proliferation and replicative life-span, in part through concerted action with BMI1, and suggest that the effect of MK3 modulation or mutation on M/SAPK signaling and, ultimately, proliferation, is cell context-dependent.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Maastricht University Medical Centre, Maastricht, the Netherlands.

ABSTRACT
Although the MK3 gene was originally found deleted in some cancers, it is highly expressed in others. The relevance of MK3 for oncogenesis is currently not clear. We recently reported that MK3 controls ERK activity via a negative feedback mechanism. This prompted us to investigate a potential role for MK3 in cell proliferation. We here show that overexpression of MK3 induces a proliferative arrest in normal diploid human fibroblasts, characterized by enhanced expression of replication stress- and senescence-associated markers. Surprisingly, MK3 depletion evokes similar senescence characteristics in the fibroblast model. We previously identified MK3 as a binding partner of Polycomb Repressive Complex 1 (PRC1) proteins. In the current study we show that MK3 overexpression results in reduced cellular EZH2 levels and concomitant loss of epigenetic H3K27me3-marking and PRC1/chromatin-occupation at the CDKN2A/INK4A locus. In agreement with this, the PRC1 oncoprotein BMI1, but not the PCR2 protein EZH2, bypasses MK3-induced senescence in fibroblasts and suppresses P16INK4A expression. In contrast, BMI1 does not rescue the MK3 loss-of-function phenotype, suggesting the involvement of multiple different checkpoints in gain and loss of MK3 function. Notably, MK3 ablation enhances proliferation in two different cancer cells. Finally, the fibroblast model was used to evaluate the effect of potential tumorigenic MK3 driver-mutations on cell proliferation and M/SAPK signaling imbalance. Taken together, our findings support a role for MK3 in control of proliferation and replicative life-span, in part through concerted action with BMI1, and suggest that the effect of MK3 modulation or mutation on M/SAPK signaling and, ultimately, proliferation, is cell context-dependent.

Show MeSH
Related in: MedlinePlus