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CCAAT/enhancer binding protein β induces motility and invasion of glioblastoma cells through transcriptional regulation of the calcium binding protein S100A4.

Aguilar-Morante D, Morales-Garcia JA, Santos A, Perez-Castillo A - Oncotarget (2015)

Bottom Line: In agreement with this, we showed that C/EBPβ depletion decreases the mRNA levels of different genes involved in metastasis and invasion.Our results show that C/EBPβ suppression significantly reduced the levels of S100A4 in murine GL261 and human T98G glioblastoma cells.By employing an S100A4-promoter reporter, we observed a significant induction in the transcriptional activation of the S100A4 gene by C/EBPβ.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones Biomédicas, (CSIC-UAM), Departamento Modelos Experimentales de Enfermedades Humanas, Arturo Duperier, Madrid, Spain.

ABSTRACT
We have previously shown that decreased expression of CCAAT/enhancer binding protein β (C/EBPβ) inhibits the growth of glioblastoma cells and diminishes their transformation capacity and migration. In agreement with this, we showed that C/EBPβ depletion decreases the mRNA levels of different genes involved in metastasis and invasion. Among these, we found S100 calcium binding protein A4 (S100A4) to be almost undetectable in glioblastoma cells deficient in C/EBPβ. Here, we have evaluated the possible role of S100A4 in the observed effects of C/EBPβ in glioblastoma cells and the mechanism through which S100A4 levels are controlled by C/EBPβ. Our results show that C/EBPβ suppression significantly reduced the levels of S100A4 in murine GL261 and human T98G glioblastoma cells. By employing an S100A4-promoter reporter, we observed a significant induction in the transcriptional activation of the S100A4 gene by C/EBPβ. Furthermore, overexpression of S100A4 in C/EBPβ-depleted glioblastoma cells reverses the enhanced migration and motility induced by this transcription factor. Our data also point to a role of S100A4 in glioblastoma cell invasion and suggest that the C/EBPβ gene controls the invasive potential of GL261 and T98G cells through direct regulation of S100A4. Finally, this study indicates a role of C/EBPβ on the maintenance of the stem cell population present in GL261 glioblastoma cells.

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Effect of C/EBPβ depletion on GL261 stem cells(A) Representative Western blot showing expression of Musashi-1 and Oct3/4 in adherent C1 and I4 cell lines and neurospheres cultures (GNSF) from PC1 and PI4 7 days after plating. (B) Representative Western blot showing expression of C/EBPβ and S100A4 in neurospheres cultures derived from C1 and I4 cells (PC1 and PI4, respectively). (C) Representative images of primary GNSF from PC1 and PI4 cells. (D) Primary GNSF were dissociated and grown during 14 days to analyze secondary GNSF formation. Quantification of secondary GNSF was performed counting 12 randomly selected fields in 3 different wells. Within each assay the experiments were performed in triplicates. Values represent the means ± SD. **p < 0.01 (E) Representative microphotographs showing self-renewal capacity of neurosphere cultures from PC1 and PI4 cells 7 and 14 days after plating. Quantification of new GNSF after 14 days growing was performed counting 12 randomly selected fields in 3 different wells. Scale bar 250 μm. **p < 0.01 (F) Transient transfection experiments in GNSF. The entire promoter fragment (pS100A4/1248), a 5′-deletion construct (pS100A4/298), and two constructs containing the mutated C/EBPβ (pS100A4/Mut-C/EBPβ) and the mutated C/EBPβ and AP1 binding sites (pS100A4//Mut-C/EBPβ-AP1) were created as indicated in Methods. Data are expressed relative to the basal values and represent the mean ± SD luciferase activity determined in triplicate in at least three independent experiments. ***p < 0.001.
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Figure 8: Effect of C/EBPβ depletion on GL261 stem cells(A) Representative Western blot showing expression of Musashi-1 and Oct3/4 in adherent C1 and I4 cell lines and neurospheres cultures (GNSF) from PC1 and PI4 7 days after plating. (B) Representative Western blot showing expression of C/EBPβ and S100A4 in neurospheres cultures derived from C1 and I4 cells (PC1 and PI4, respectively). (C) Representative images of primary GNSF from PC1 and PI4 cells. (D) Primary GNSF were dissociated and grown during 14 days to analyze secondary GNSF formation. Quantification of secondary GNSF was performed counting 12 randomly selected fields in 3 different wells. Within each assay the experiments were performed in triplicates. Values represent the means ± SD. **p < 0.01 (E) Representative microphotographs showing self-renewal capacity of neurosphere cultures from PC1 and PI4 cells 7 and 14 days after plating. Quantification of new GNSF after 14 days growing was performed counting 12 randomly selected fields in 3 different wells. Scale bar 250 μm. **p < 0.01 (F) Transient transfection experiments in GNSF. The entire promoter fragment (pS100A4/1248), a 5′-deletion construct (pS100A4/298), and two constructs containing the mutated C/EBPβ (pS100A4/Mut-C/EBPβ) and the mutated C/EBPβ and AP1 binding sites (pS100A4//Mut-C/EBPβ-AP1) were created as indicated in Methods. Data are expressed relative to the basal values and represent the mean ± SD luciferase activity determined in triplicate in at least three independent experiments. ***p < 0.001.

Mentions: To ensure that we were working with an enriched population in GNSF, we first analyzed by western blot the protein levels of Musashi-1 and Oct3/4, which are well-known stem cells markers [36]. As expected, adherent C1 and I4 cells did not have Mushashi-1 and Oct3/4 proteins, while GNSF populations of PC1 and PI4 presented detectable levels of these markers (Figure 8A). Also, we observed a decrease in the levels of Musashi-1 in PI4 cells, compared to PC1, suggesting that C/EBPβ can alter the population of stem cells present in these cultures.


CCAAT/enhancer binding protein β induces motility and invasion of glioblastoma cells through transcriptional regulation of the calcium binding protein S100A4.

Aguilar-Morante D, Morales-Garcia JA, Santos A, Perez-Castillo A - Oncotarget (2015)

Effect of C/EBPβ depletion on GL261 stem cells(A) Representative Western blot showing expression of Musashi-1 and Oct3/4 in adherent C1 and I4 cell lines and neurospheres cultures (GNSF) from PC1 and PI4 7 days after plating. (B) Representative Western blot showing expression of C/EBPβ and S100A4 in neurospheres cultures derived from C1 and I4 cells (PC1 and PI4, respectively). (C) Representative images of primary GNSF from PC1 and PI4 cells. (D) Primary GNSF were dissociated and grown during 14 days to analyze secondary GNSF formation. Quantification of secondary GNSF was performed counting 12 randomly selected fields in 3 different wells. Within each assay the experiments were performed in triplicates. Values represent the means ± SD. **p < 0.01 (E) Representative microphotographs showing self-renewal capacity of neurosphere cultures from PC1 and PI4 cells 7 and 14 days after plating. Quantification of new GNSF after 14 days growing was performed counting 12 randomly selected fields in 3 different wells. Scale bar 250 μm. **p < 0.01 (F) Transient transfection experiments in GNSF. The entire promoter fragment (pS100A4/1248), a 5′-deletion construct (pS100A4/298), and two constructs containing the mutated C/EBPβ (pS100A4/Mut-C/EBPβ) and the mutated C/EBPβ and AP1 binding sites (pS100A4//Mut-C/EBPβ-AP1) were created as indicated in Methods. Data are expressed relative to the basal values and represent the mean ± SD luciferase activity determined in triplicate in at least three independent experiments. ***p < 0.001.
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Related In: Results  -  Collection

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Show All Figures
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Figure 8: Effect of C/EBPβ depletion on GL261 stem cells(A) Representative Western blot showing expression of Musashi-1 and Oct3/4 in adherent C1 and I4 cell lines and neurospheres cultures (GNSF) from PC1 and PI4 7 days after plating. (B) Representative Western blot showing expression of C/EBPβ and S100A4 in neurospheres cultures derived from C1 and I4 cells (PC1 and PI4, respectively). (C) Representative images of primary GNSF from PC1 and PI4 cells. (D) Primary GNSF were dissociated and grown during 14 days to analyze secondary GNSF formation. Quantification of secondary GNSF was performed counting 12 randomly selected fields in 3 different wells. Within each assay the experiments were performed in triplicates. Values represent the means ± SD. **p < 0.01 (E) Representative microphotographs showing self-renewal capacity of neurosphere cultures from PC1 and PI4 cells 7 and 14 days after plating. Quantification of new GNSF after 14 days growing was performed counting 12 randomly selected fields in 3 different wells. Scale bar 250 μm. **p < 0.01 (F) Transient transfection experiments in GNSF. The entire promoter fragment (pS100A4/1248), a 5′-deletion construct (pS100A4/298), and two constructs containing the mutated C/EBPβ (pS100A4/Mut-C/EBPβ) and the mutated C/EBPβ and AP1 binding sites (pS100A4//Mut-C/EBPβ-AP1) were created as indicated in Methods. Data are expressed relative to the basal values and represent the mean ± SD luciferase activity determined in triplicate in at least three independent experiments. ***p < 0.001.
Mentions: To ensure that we were working with an enriched population in GNSF, we first analyzed by western blot the protein levels of Musashi-1 and Oct3/4, which are well-known stem cells markers [36]. As expected, adherent C1 and I4 cells did not have Mushashi-1 and Oct3/4 proteins, while GNSF populations of PC1 and PI4 presented detectable levels of these markers (Figure 8A). Also, we observed a decrease in the levels of Musashi-1 in PI4 cells, compared to PC1, suggesting that C/EBPβ can alter the population of stem cells present in these cultures.

Bottom Line: In agreement with this, we showed that C/EBPβ depletion decreases the mRNA levels of different genes involved in metastasis and invasion.Our results show that C/EBPβ suppression significantly reduced the levels of S100A4 in murine GL261 and human T98G glioblastoma cells.By employing an S100A4-promoter reporter, we observed a significant induction in the transcriptional activation of the S100A4 gene by C/EBPβ.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones Biomédicas, (CSIC-UAM), Departamento Modelos Experimentales de Enfermedades Humanas, Arturo Duperier, Madrid, Spain.

ABSTRACT
We have previously shown that decreased expression of CCAAT/enhancer binding protein β (C/EBPβ) inhibits the growth of glioblastoma cells and diminishes their transformation capacity and migration. In agreement with this, we showed that C/EBPβ depletion decreases the mRNA levels of different genes involved in metastasis and invasion. Among these, we found S100 calcium binding protein A4 (S100A4) to be almost undetectable in glioblastoma cells deficient in C/EBPβ. Here, we have evaluated the possible role of S100A4 in the observed effects of C/EBPβ in glioblastoma cells and the mechanism through which S100A4 levels are controlled by C/EBPβ. Our results show that C/EBPβ suppression significantly reduced the levels of S100A4 in murine GL261 and human T98G glioblastoma cells. By employing an S100A4-promoter reporter, we observed a significant induction in the transcriptional activation of the S100A4 gene by C/EBPβ. Furthermore, overexpression of S100A4 in C/EBPβ-depleted glioblastoma cells reverses the enhanced migration and motility induced by this transcription factor. Our data also point to a role of S100A4 in glioblastoma cell invasion and suggest that the C/EBPβ gene controls the invasive potential of GL261 and T98G cells through direct regulation of S100A4. Finally, this study indicates a role of C/EBPβ on the maintenance of the stem cell population present in GL261 glioblastoma cells.

Show MeSH
Related in: MedlinePlus