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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.

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MK3WT overexpression induces a proliferative arrest in normal human fibroblasts.(A) Retroviral expression systems were applied to enhance (MK3WTOE) or remove (shMK3) MK3 expression. Western blot shows MK3 proteins: endogenous (MK3endo) and overexpressed (GST:MK3). (B) Proliferation curves of TIG3 cells transduced with: a retroviral MK3 expression vector (MK3WTOE; filled circles), an empty vector (con; open circles) or a murine Bmi1 expression vector (open triangles); proliferation was determined at 1 week (top panel) or ±4 weeks (bottom panel) after retroviral transduction of TIG3 cells. 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). (C) Quantification of DNA profiles (BrdU pulse-labeling and S-phase quantification by FACS) in TIG3/MK3WTOE cells at approximately 1 week post-transduction. RasV12-transduced cells were used as a positive control. (D) MK3WTOE reduces de novo DNA synthesis in TIG3/MK3WTOE cells; cell counts: control (con) 699 BrdU-positive cells/10 fields; MK3WTOE: 442 BrdU-positive cells/9 fields.
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pone.0118840.g001: MK3WT overexpression induces a proliferative arrest in normal human fibroblasts.(A) Retroviral expression systems were applied to enhance (MK3WTOE) or remove (shMK3) MK3 expression. Western blot shows MK3 proteins: endogenous (MK3endo) and overexpressed (GST:MK3). (B) Proliferation curves of TIG3 cells transduced with: a retroviral MK3 expression vector (MK3WTOE; filled circles), an empty vector (con; open circles) or a murine Bmi1 expression vector (open triangles); proliferation was determined at 1 week (top panel) or ±4 weeks (bottom panel) after retroviral transduction of TIG3 cells. 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). (C) Quantification of DNA profiles (BrdU pulse-labeling and S-phase quantification by FACS) in TIG3/MK3WTOE cells at approximately 1 week post-transduction. RasV12-transduced cells were used as a positive control. (D) MK3WTOE reduces de novo DNA synthesis in TIG3/MK3WTOE cells; cell counts: control (con) 699 BrdU-positive cells/10 fields; MK3WTOE: 442 BrdU-positive cells/9 fields.

Mentions: To study the role of MK3 in cellular processes related to cell proliferation, we modulated cellular MK3 levels in different cell models using retroviral expression systems and measured the effects thereof. We initially focused on normal diploid human TIG3 fibroblasts to determine the effect of forced overexpression of wild type (non-mutated) MK3 (MK3WTOE) on cell proliferation (Fig 1A). Transduced TIG3 cells were expanded under selection pressure; proliferative capacity of these cultures was assessed at ±1 week and ±4 weeks after transduction. TIG3/MK3WTOE displayed clearly decreased proliferative capacity relative to empty vector control cells at the early time point and had altogether stopped dividing at 4 weeks post-transduction (Fig 1B). By means of reference, TIG3/Bmi1OE maintained proliferative capacity throughout the duration of the experiment, whereas primary empty vector-transduced TIG3 (con) progressively lost proliferative capacity due to limited proliferative lifespan in vitro (Fig 1B). DNA-profiling at late time points post-transduction revealed an increased number of cells in G0/G1 at the expense of cells in S-phase upon MK3-overexpression (Fig 1C); overexpression of the RASV12 oncogene reduced S-phase cell numbers as anticipated as RASV12 is known to evoke oncogene-induced senescence (OIS) [16], Fig 1C). Reduced de novo DNA synthesis in TIG3/MK3WTOE cells was confirmed by a markedly lower number of BrdU-positive cells in TIG3/MK3WTOE cultures and (Figs 1D and S1A); equal relative distribution of cells throughout S-phase suggested that TIG3/MK3WTOE cultures had undergone an intraS-phase arrest, a feature known to occur in the context of oncogene-induced senescence (OIS) (S1B Fig).


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)

MK3WT overexpression induces a proliferative arrest in normal human fibroblasts.(A) Retroviral expression systems were applied to enhance (MK3WTOE) or remove (shMK3) MK3 expression. Western blot shows MK3 proteins: endogenous (MK3endo) and overexpressed (GST:MK3). (B) Proliferation curves of TIG3 cells transduced with: a retroviral MK3 expression vector (MK3WTOE; filled circles), an empty vector (con; open circles) or a murine Bmi1 expression vector (open triangles); proliferation was determined at 1 week (top panel) or ±4 weeks (bottom panel) after retroviral transduction of TIG3 cells. 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). (C) Quantification of DNA profiles (BrdU pulse-labeling and S-phase quantification by FACS) in TIG3/MK3WTOE cells at approximately 1 week post-transduction. RasV12-transduced cells were used as a positive control. (D) MK3WTOE reduces de novo DNA synthesis in TIG3/MK3WTOE cells; cell counts: control (con) 699 BrdU-positive cells/10 fields; MK3WTOE: 442 BrdU-positive cells/9 fields.
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Related In: Results  -  Collection

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pone.0118840.g001: MK3WT overexpression induces a proliferative arrest in normal human fibroblasts.(A) Retroviral expression systems were applied to enhance (MK3WTOE) or remove (shMK3) MK3 expression. Western blot shows MK3 proteins: endogenous (MK3endo) and overexpressed (GST:MK3). (B) Proliferation curves of TIG3 cells transduced with: a retroviral MK3 expression vector (MK3WTOE; filled circles), an empty vector (con; open circles) or a murine Bmi1 expression vector (open triangles); proliferation was determined at 1 week (top panel) or ±4 weeks (bottom panel) after retroviral transduction of TIG3 cells. 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). (C) Quantification of DNA profiles (BrdU pulse-labeling and S-phase quantification by FACS) in TIG3/MK3WTOE cells at approximately 1 week post-transduction. RasV12-transduced cells were used as a positive control. (D) MK3WTOE reduces de novo DNA synthesis in TIG3/MK3WTOE cells; cell counts: control (con) 699 BrdU-positive cells/10 fields; MK3WTOE: 442 BrdU-positive cells/9 fields.
Mentions: To study the role of MK3 in cellular processes related to cell proliferation, we modulated cellular MK3 levels in different cell models using retroviral expression systems and measured the effects thereof. We initially focused on normal diploid human TIG3 fibroblasts to determine the effect of forced overexpression of wild type (non-mutated) MK3 (MK3WTOE) on cell proliferation (Fig 1A). Transduced TIG3 cells were expanded under selection pressure; proliferative capacity of these cultures was assessed at ±1 week and ±4 weeks after transduction. TIG3/MK3WTOE displayed clearly decreased proliferative capacity relative to empty vector control cells at the early time point and had altogether stopped dividing at 4 weeks post-transduction (Fig 1B). By means of reference, TIG3/Bmi1OE maintained proliferative capacity throughout the duration of the experiment, whereas primary empty vector-transduced TIG3 (con) progressively lost proliferative capacity due to limited proliferative lifespan in vitro (Fig 1B). DNA-profiling at late time points post-transduction revealed an increased number of cells in G0/G1 at the expense of cells in S-phase upon MK3-overexpression (Fig 1C); overexpression of the RASV12 oncogene reduced S-phase cell numbers as anticipated as RASV12 is known to evoke oncogene-induced senescence (OIS) [16], Fig 1C). Reduced de novo DNA synthesis in TIG3/MK3WTOE cells was confirmed by a markedly lower number of BrdU-positive cells in TIG3/MK3WTOE cultures and (Figs 1D and S1A); equal relative distribution of cells throughout S-phase suggested that TIG3/MK3WTOE cultures had undergone an intraS-phase arrest, a feature known to occur in the context of oncogene-induced senescence (OIS) (S1B Fig).

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