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Tumor suppressor Nf2/merlin drives Schwann cell changes following electromagnetic field exposure through Hippo-dependent mechanisms

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ABSTRACT

Previous evidence showed mutations of the neurofibromin type 2 gene (Nf2), encoding the tumor suppressor protein merlin, in sporadic and vestibular schwannomas affecting Schwann cells (SCs). Accordingly, efforts have been addressed to identify possible factors, even environmental, that may regulate neurofibromas growth. In this context, we investigated the exposure of SC to an electromagnetic field (EMF), which is an environmental issue modulating biological processes. Here, we show that SC exposed to 50 Hz EMFs changes their morphology, proliferation, migration and myelinating capability. In these cells, merlin is downregulated, leading to activation of two intracellular signaling pathways, ERK/AKT and Hippo. Interestingly, SC changes their phenotype toward a proliferative/migrating state, which in principle may be pathologically relevant for schwannoma development.

No MeSH data available.


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SCs change morphology and proliferation following EMF exposure. (a) Merge image of SC characterized by immunopositivity for P0 and S100 markers, respectively (anti-P0-594, in red; anti-s100-488, in green), showing a cell purity more than 98%. Nuclei were stained with DAPI, in blue. Scale bar 10 μm. (b) Scheme of the experimental model used. SCs were exposed to EMF of 50 Hz, 0.1 T, for 10 min, then cells were assayed for proliferation, migration, vitality, chemoresponsivity, morphology, western blot, qRT-PCR and RT2 profiler PCR. (c) EMF exposure induces SCs morphologic rearrangements in actin cytoskeleton, as assessed by immunopositivity for f-actin (phalloidin-FICT, in green). SCs turned from a spindle-shaped to an enlarged phenotype, suggesting alterations in the differentiation program. Nuclei were stained with DAPI, in blue. Scale bar 10 μm. (d) SCs proliferation was assessed at 6, 24, 48 and 72 h, following a single (dashed line) or double (dot line) EMF exposure. EMFs produced a significant (*P<0.05) increase in cell proliferation. Experiments were repeated at least three times and data expressed as cell number (×103). Two-way ANOVA using Bonferroni’s post hoc test was used for statistical analysis. (e) Percentage of SCs vitality was assessed at 6, 24, 48 and 72 h, following a single (white columns) or double (gray columns) EMF exposure, but no significant changes in SCs vitality were observed. Controls (CONTR, black columns). The values are means±S.D. (N=3).
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fig1: SCs change morphology and proliferation following EMF exposure. (a) Merge image of SC characterized by immunopositivity for P0 and S100 markers, respectively (anti-P0-594, in red; anti-s100-488, in green), showing a cell purity more than 98%. Nuclei were stained with DAPI, in blue. Scale bar 10 μm. (b) Scheme of the experimental model used. SCs were exposed to EMF of 50 Hz, 0.1 T, for 10 min, then cells were assayed for proliferation, migration, vitality, chemoresponsivity, morphology, western blot, qRT-PCR and RT2 profiler PCR. (c) EMF exposure induces SCs morphologic rearrangements in actin cytoskeleton, as assessed by immunopositivity for f-actin (phalloidin-FICT, in green). SCs turned from a spindle-shaped to an enlarged phenotype, suggesting alterations in the differentiation program. Nuclei were stained with DAPI, in blue. Scale bar 10 μm. (d) SCs proliferation was assessed at 6, 24, 48 and 72 h, following a single (dashed line) or double (dot line) EMF exposure. EMFs produced a significant (*P<0.05) increase in cell proliferation. Experiments were repeated at least three times and data expressed as cell number (×103). Two-way ANOVA using Bonferroni’s post hoc test was used for statistical analysis. (e) Percentage of SCs vitality was assessed at 6, 24, 48 and 72 h, following a single (white columns) or double (gray columns) EMF exposure, but no significant changes in SCs vitality were observed. Controls (CONTR, black columns). The values are means±S.D. (N=3).

Mentions: SCs primary cultures from 3-day-old rats were used for the experiments. These cells showed the characteristic spindle-shape morphology in vitro. Cell purity, more than 98%, was assessed performing immunolabeling for the typical markers myelin protein zero (P0) and protein S100 (Figure 1a). To test the effects of EMF on SC biological features, we applied an EMF intensity of 50 Hz, 0.1T for 10 min (Figure 1b). EMF exposure induced morphologic rearrangements in actin cytoskeleton, which might be critical for SCs differentiation and myelination. SCs turn from a spindle-shaped to an enlarged phenotype, indicating a dysregulation in the differentiation program; in fact, cells exposed to the treatment resemble to the undifferentiated phenotype (Figure 1c).


Tumor suppressor Nf2/merlin drives Schwann cell changes following electromagnetic field exposure through Hippo-dependent mechanisms
SCs change morphology and proliferation following EMF exposure. (a) Merge image of SC characterized by immunopositivity for P0 and S100 markers, respectively (anti-P0-594, in red; anti-s100-488, in green), showing a cell purity more than 98%. Nuclei were stained with DAPI, in blue. Scale bar 10 μm. (b) Scheme of the experimental model used. SCs were exposed to EMF of 50 Hz, 0.1 T, for 10 min, then cells were assayed for proliferation, migration, vitality, chemoresponsivity, morphology, western blot, qRT-PCR and RT2 profiler PCR. (c) EMF exposure induces SCs morphologic rearrangements in actin cytoskeleton, as assessed by immunopositivity for f-actin (phalloidin-FICT, in green). SCs turned from a spindle-shaped to an enlarged phenotype, suggesting alterations in the differentiation program. Nuclei were stained with DAPI, in blue. Scale bar 10 μm. (d) SCs proliferation was assessed at 6, 24, 48 and 72 h, following a single (dashed line) or double (dot line) EMF exposure. EMFs produced a significant (*P<0.05) increase in cell proliferation. Experiments were repeated at least three times and data expressed as cell number (×103). Two-way ANOVA using Bonferroni’s post hoc test was used for statistical analysis. (e) Percentage of SCs vitality was assessed at 6, 24, 48 and 72 h, following a single (white columns) or double (gray columns) EMF exposure, but no significant changes in SCs vitality were observed. Controls (CONTR, black columns). The values are means±S.D. (N=3).
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fig1: SCs change morphology and proliferation following EMF exposure. (a) Merge image of SC characterized by immunopositivity for P0 and S100 markers, respectively (anti-P0-594, in red; anti-s100-488, in green), showing a cell purity more than 98%. Nuclei were stained with DAPI, in blue. Scale bar 10 μm. (b) Scheme of the experimental model used. SCs were exposed to EMF of 50 Hz, 0.1 T, for 10 min, then cells were assayed for proliferation, migration, vitality, chemoresponsivity, morphology, western blot, qRT-PCR and RT2 profiler PCR. (c) EMF exposure induces SCs morphologic rearrangements in actin cytoskeleton, as assessed by immunopositivity for f-actin (phalloidin-FICT, in green). SCs turned from a spindle-shaped to an enlarged phenotype, suggesting alterations in the differentiation program. Nuclei were stained with DAPI, in blue. Scale bar 10 μm. (d) SCs proliferation was assessed at 6, 24, 48 and 72 h, following a single (dashed line) or double (dot line) EMF exposure. EMFs produced a significant (*P<0.05) increase in cell proliferation. Experiments were repeated at least three times and data expressed as cell number (×103). Two-way ANOVA using Bonferroni’s post hoc test was used for statistical analysis. (e) Percentage of SCs vitality was assessed at 6, 24, 48 and 72 h, following a single (white columns) or double (gray columns) EMF exposure, but no significant changes in SCs vitality were observed. Controls (CONTR, black columns). The values are means±S.D. (N=3).
Mentions: SCs primary cultures from 3-day-old rats were used for the experiments. These cells showed the characteristic spindle-shape morphology in vitro. Cell purity, more than 98%, was assessed performing immunolabeling for the typical markers myelin protein zero (P0) and protein S100 (Figure 1a). To test the effects of EMF on SC biological features, we applied an EMF intensity of 50 Hz, 0.1T for 10 min (Figure 1b). EMF exposure induced morphologic rearrangements in actin cytoskeleton, which might be critical for SCs differentiation and myelination. SCs turn from a spindle-shaped to an enlarged phenotype, indicating a dysregulation in the differentiation program; in fact, cells exposed to the treatment resemble to the undifferentiated phenotype (Figure 1c).

View Article: PubMed Central - PubMed

ABSTRACT

Previous evidence showed mutations of the neurofibromin type 2 gene (Nf2), encoding the tumor suppressor protein merlin, in sporadic and vestibular schwannomas affecting Schwann cells (SCs). Accordingly, efforts have been addressed to identify possible factors, even environmental, that may regulate neurofibromas growth. In this context, we investigated the exposure of SC to an electromagnetic field (EMF), which is an environmental issue modulating biological processes. Here, we show that SC exposed to 50&thinsp;Hz EMFs changes their morphology, proliferation, migration and myelinating capability. In these cells, merlin is downregulated, leading to activation of two intracellular signaling pathways, ERK/AKT and Hippo. Interestingly, SC changes their phenotype toward a proliferative/migrating state, which in principle may be pathologically relevant for schwannoma development.

No MeSH data available.


Related in: MedlinePlus