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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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The MK3WTOE-induced proliferative block in normal cells correlates with induction of known senescence markers.(A) Cell morphology in TIG3/MK3WTOE cells (phase contrast; right upper panel); senescence-associated beta-Galactosidase (SA-bGal) staining in TIG3/MK3WTOE cells (right lower panel). (B) Protein expression levels of MK3, TP53, P21CIP1/WAF1 (P21) and P16INK4A (P16) in TIG3/MK3WTOE (MK3WTOE) versus control (con) cell lysates; loading control: b-Actin (bAct). TIG3/MK3WTOE cells were cultured for approximately 3–4 weeks prior to extraction. (C) Nuclear TP53 accumulation in senescent TIG3/MK3WTOE cells; counterstaining: DAPI. (D) Phase contrast images showing cello morphology in TIG3/shMK3 cells (shMK3, lower panel; phase contrast) versus control cells (shcon; upper panel). Retrovirally transduced TIG3 cells (shcon and shMK3) were submitted to puromycin selection for 3–4 days, and plated at equal density (20–25% confluency) at the onset of the experiment; pictures were taken when shcon cells reached confluency. (E) Analysis of protein expression in TIG3/shMK3 and control (shcon) cell lysates: TP53, P21CIP1/WAF1 (P21) and P16INK4A (P16); loading control: b-Actin (bAct).
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pone.0118840.g002: The MK3WTOE-induced proliferative block in normal cells correlates with induction of known senescence markers.(A) Cell morphology in TIG3/MK3WTOE cells (phase contrast; right upper panel); senescence-associated beta-Galactosidase (SA-bGal) staining in TIG3/MK3WTOE cells (right lower panel). (B) Protein expression levels of MK3, TP53, P21CIP1/WAF1 (P21) and P16INK4A (P16) in TIG3/MK3WTOE (MK3WTOE) versus control (con) cell lysates; loading control: b-Actin (bAct). TIG3/MK3WTOE cells were cultured for approximately 3–4 weeks prior to extraction. (C) Nuclear TP53 accumulation in senescent TIG3/MK3WTOE cells; counterstaining: DAPI. (D) Phase contrast images showing cello morphology in TIG3/shMK3 cells (shMK3, lower panel; phase contrast) versus control cells (shcon; upper panel). Retrovirally transduced TIG3 cells (shcon and shMK3) were submitted to puromycin selection for 3–4 days, and plated at equal density (20–25% confluency) at the onset of the experiment; pictures were taken when shcon cells reached confluency. (E) Analysis of protein expression in TIG3/shMK3 and control (shcon) cell lysates: TP53, P21CIP1/WAF1 (P21) and P16INK4A (P16); loading control: b-Actin (bAct).

Mentions: In further support of activation of a senescence-associated response by MK3, TIG3/MK3WTOE cells displayed enlarged, flat-cell morphology (Fig 2A). The occurrence of senescence was further corroborated by expression of the senescence-associated beta-Galactosidase (SA-bGal) marker protein in large flat cells (Fig 2A). MK3WT-overexpression also negatively affected cell division in immortal TIG3hTERT cells, indicating that MK3 acts downstream or independent of hTERT in proliferative control (S1C Fig). TP53 is a senescence marker, activated downstream of e.g. increased mitogenic signaling through RAS/ERK [17–20]. Consistent with the observed MK3-mediated senescence response, global expression of TP53 was elevated in TIG3/MK3WTOE cells (Fig 2B). Increased staining of TP53 was readily detectable in senescent TIG3/MK3WTOE nuclei (Fig 2C); likewise expression of P21CIP1/WAF1, a direct transcriptional target of TP53, was increased (Fig 2B). The increased expression of P16INK4a in relation to sustained MK3WTOE was consistent with its crucial role in establishing irreversible senescence (Fig 2B) [17,21,22]. We next tested whether the adverse effect of MK3WTOE was dependent on its kinase activity. Surprisingly, the early effect of MK3WTOE on cell proliferation occurred independent of MK3 kinase activity, as expression of a kinase-dead mutant (MK3KMOE) and a constitutively active mutant (MK3CAOE) did not affect cell proliferation at 1 week post-transduction (S2A Fig). In agreement with this, cellular TP53 levels were only moderately increased in the TIG3/MK3OE cells, not in TIG3/MK3KMOE or TIG3/MK3CAOE cells. P16INK4A levels were substantially increased at 1 week post-transduction in both TIG3/MK3WTOE and TIG3/MK3CAOE cultures, despite the differences in proliferation rate (S2A and S2B Fig). P16INK4A was also induced by MK3KMOE, however, at a lower level than in both other conditions. Despite the initial absence of adverse effects on proliferation, all three MK3 variants reduced proliferation rate at 4 weeks post-transduction (S2A and S2B Fig). The absence of correlation between TP53 and P16INK4A levels and proliferation rate at 4 weeks suggests a potential contribution of additional molecular targets and mechanisms to the observed effects on proliferation. Combined this data shows that overexpression of MK3WT or two MK3 kinase mutants in human diploid fibroblasts reduces their proliferative capacity.


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)

The MK3WTOE-induced proliferative block in normal cells correlates with induction of known senescence markers.(A) Cell morphology in TIG3/MK3WTOE cells (phase contrast; right upper panel); senescence-associated beta-Galactosidase (SA-bGal) staining in TIG3/MK3WTOE cells (right lower panel). (B) Protein expression levels of MK3, TP53, P21CIP1/WAF1 (P21) and P16INK4A (P16) in TIG3/MK3WTOE (MK3WTOE) versus control (con) cell lysates; loading control: b-Actin (bAct). TIG3/MK3WTOE cells were cultured for approximately 3–4 weeks prior to extraction. (C) Nuclear TP53 accumulation in senescent TIG3/MK3WTOE cells; counterstaining: DAPI. (D) Phase contrast images showing cello morphology in TIG3/shMK3 cells (shMK3, lower panel; phase contrast) versus control cells (shcon; upper panel). Retrovirally transduced TIG3 cells (shcon and shMK3) were submitted to puromycin selection for 3–4 days, and plated at equal density (20–25% confluency) at the onset of the experiment; pictures were taken when shcon cells reached confluency. (E) Analysis of protein expression in TIG3/shMK3 and control (shcon) cell lysates: TP53, P21CIP1/WAF1 (P21) and P16INK4A (P16); loading control: b-Actin (bAct).
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pone.0118840.g002: The MK3WTOE-induced proliferative block in normal cells correlates with induction of known senescence markers.(A) Cell morphology in TIG3/MK3WTOE cells (phase contrast; right upper panel); senescence-associated beta-Galactosidase (SA-bGal) staining in TIG3/MK3WTOE cells (right lower panel). (B) Protein expression levels of MK3, TP53, P21CIP1/WAF1 (P21) and P16INK4A (P16) in TIG3/MK3WTOE (MK3WTOE) versus control (con) cell lysates; loading control: b-Actin (bAct). TIG3/MK3WTOE cells were cultured for approximately 3–4 weeks prior to extraction. (C) Nuclear TP53 accumulation in senescent TIG3/MK3WTOE cells; counterstaining: DAPI. (D) Phase contrast images showing cello morphology in TIG3/shMK3 cells (shMK3, lower panel; phase contrast) versus control cells (shcon; upper panel). Retrovirally transduced TIG3 cells (shcon and shMK3) were submitted to puromycin selection for 3–4 days, and plated at equal density (20–25% confluency) at the onset of the experiment; pictures were taken when shcon cells reached confluency. (E) Analysis of protein expression in TIG3/shMK3 and control (shcon) cell lysates: TP53, P21CIP1/WAF1 (P21) and P16INK4A (P16); loading control: b-Actin (bAct).
Mentions: In further support of activation of a senescence-associated response by MK3, TIG3/MK3WTOE cells displayed enlarged, flat-cell morphology (Fig 2A). The occurrence of senescence was further corroborated by expression of the senescence-associated beta-Galactosidase (SA-bGal) marker protein in large flat cells (Fig 2A). MK3WT-overexpression also negatively affected cell division in immortal TIG3hTERT cells, indicating that MK3 acts downstream or independent of hTERT in proliferative control (S1C Fig). TP53 is a senescence marker, activated downstream of e.g. increased mitogenic signaling through RAS/ERK [17–20]. Consistent with the observed MK3-mediated senescence response, global expression of TP53 was elevated in TIG3/MK3WTOE cells (Fig 2B). Increased staining of TP53 was readily detectable in senescent TIG3/MK3WTOE nuclei (Fig 2C); likewise expression of P21CIP1/WAF1, a direct transcriptional target of TP53, was increased (Fig 2B). The increased expression of P16INK4a in relation to sustained MK3WTOE was consistent with its crucial role in establishing irreversible senescence (Fig 2B) [17,21,22]. We next tested whether the adverse effect of MK3WTOE was dependent on its kinase activity. Surprisingly, the early effect of MK3WTOE on cell proliferation occurred independent of MK3 kinase activity, as expression of a kinase-dead mutant (MK3KMOE) and a constitutively active mutant (MK3CAOE) did not affect cell proliferation at 1 week post-transduction (S2A Fig). In agreement with this, cellular TP53 levels were only moderately increased in the TIG3/MK3OE cells, not in TIG3/MK3KMOE or TIG3/MK3CAOE cells. P16INK4A levels were substantially increased at 1 week post-transduction in both TIG3/MK3WTOE and TIG3/MK3CAOE cultures, despite the differences in proliferation rate (S2A and S2B Fig). P16INK4A was also induced by MK3KMOE, however, at a lower level than in both other conditions. Despite the initial absence of adverse effects on proliferation, all three MK3 variants reduced proliferation rate at 4 weeks post-transduction (S2A and S2B Fig). The absence of correlation between TP53 and P16INK4A levels and proliferation rate at 4 weeks suggests a potential contribution of additional molecular targets and mechanisms to the observed effects on proliferation. Combined this data shows that overexpression of MK3WT or two MK3 kinase mutants in human diploid fibroblasts reduces their proliferative capacity.

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