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Olig2-induced neural stem cell differentiation involves downregulation of Wnt signaling and induction of Dickkopf-1 expression.

Ahn SM, Byun K, Kim D, Lee K, Yoo JS, Kim SU, Jho EH, Simpson RJ, Lee B - PLoS ONE (2008)

Bottom Line: Furthermore, we found that Olig2-induced differentiation induces the expression of Dickkopf-1(Dkk1), a potent antagonist of Wnt signaling.Dkk1 treatment blocked Wnt signaling in HB1.F3 in a dosage-dependent manner, and induced differentiation into astrocytes, oligodendrocytes, and neurons.In our proposed model, Dkk1 may play an important role in downregulating self-renewal and proliferation pathway of stem cells at the late stage of differentiation, and its failure may lead to carcinogenesis.

View Article: PubMed Central - PubMed

Affiliation: Center for Genomics and Proteomics, Lee Gil Ya Cancer and Diabetes Institute, Gachon University of Medicine and Science, Incheon, Korea.

ABSTRACT
Understanding stem cell-differentiation at the molecular level is important for clinical applications of stem cells and for finding new therapeutic approaches in the context of cancer stem cells. To investigate genome-wide changes involved in differentiation, we have used immortalized neural stem cell (NSC) line (HB1.F3) and Olig2-induced NSC differentiation model (F3.Olig2). Using microarray analysis, we revealed that Olig2-induced NSC differentiation involves downregulation of Wnt pathway, which was further confirmed by TOPflash/FOPflash reporter assay, RT-PCR analysis, immunoblots, and immunocytochemistry. Furthermore, we found that Olig2-induced differentiation induces the expression of Dickkopf-1(Dkk1), a potent antagonist of Wnt signaling. Dkk1 treatment blocked Wnt signaling in HB1.F3 in a dosage-dependent manner, and induced differentiation into astrocytes, oligodendrocytes, and neurons. Our results support cancer stem cell hypothesis which implies that signaling pathway for self-renewal and proliferation of stem cells is maintained till the late stage of differentiation. In our proposed model, Dkk1 may play an important role in downregulating self-renewal and proliferation pathway of stem cells at the late stage of differentiation, and its failure may lead to carcinogenesis.

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Downregulation of Wnt pathway in F3.Olig2.GSEA shows that Wnt pathway genes (A), Wnt-responsive genes (B), and Wnt pathway target genes (C) are enriched in HB1.F3. TOPFLASH/FOPflash reporter analysis (D) confirms that Wnt pathway activity in HB1.F3 is significantly higher than that in F3.Olig2. The reporter activity in HB1.F3 was inhibited to the level of F3.Olig2 when HB1.F3 was co-transfected with ‘dominant negative’ (DN) TCF constructs. Data are means±S.D. of triplicate samples. A.U., Activity unit
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pone-0003917-g002: Downregulation of Wnt pathway in F3.Olig2.GSEA shows that Wnt pathway genes (A), Wnt-responsive genes (B), and Wnt pathway target genes (C) are enriched in HB1.F3. TOPFLASH/FOPflash reporter analysis (D) confirms that Wnt pathway activity in HB1.F3 is significantly higher than that in F3.Olig2. The reporter activity in HB1.F3 was inhibited to the level of F3.Olig2 when HB1.F3 was co-transfected with ‘dominant negative’ (DN) TCF constructs. Data are means±S.D. of triplicate samples. A.U., Activity unit

Mentions: Microarray analysis revealed global gene expression changes between HB1.F3 and F3.Olig2; more than 60% of genes that are present in HB1.F3 are absent in F3.Olig2. Since the global gene expression changes violate basic assumptions of statistical analysis of microarray data that most genes are not differentially expressed [18], we have employed the knowledge-based Gene Set Enrichment Analysis (GSEA) (Materials and Methods), instead of using conventional statistical analysis such as t-test, to investigate expression changes in functional groups of genes. Since the Wnt pathway is known to be involved in neural stem cell-differentiation in contra-acting ways (i.e., maintain stemness versus inducing differentiation [19]–[21], the investigation of the microarray data was focused on Wnt pathway-related gene sets. Using this method, we identified significant enrichment of Wnt pathway genes, genes upregulated by Wnt [22], and Wnt pathway target genes in HB1.F3, an immortalized neural stem cell line (Fig. 2A–C) (see Table S1, S2, S3 for detailed information). To obtain further evidence that Wnt pathway is active in HB1.F3 and suppressed in F3.Olig2, we transfected a transcription factor (TCF) reporter gene (TOPflash) containing five optimal TCF-binding sites or the mutant control plasmid (FOPflash) into HB1.F3 and F3.Olig2. pRL-TK was included to normalize data for transfection efficiency [23]. As shown in Fig. 2D, the reporter activity of Wnt pathway is more than three times higher in HB1.F3 than in F3.Olig2. The addition of dominant negative TCF plasmids (dnTCF) decreased the reporter activity in HB1.F3 close to the level of F3.Olig2, indicating that the Wnt activity in F3.Olig2 is at the background level.


Olig2-induced neural stem cell differentiation involves downregulation of Wnt signaling and induction of Dickkopf-1 expression.

Ahn SM, Byun K, Kim D, Lee K, Yoo JS, Kim SU, Jho EH, Simpson RJ, Lee B - PLoS ONE (2008)

Downregulation of Wnt pathway in F3.Olig2.GSEA shows that Wnt pathway genes (A), Wnt-responsive genes (B), and Wnt pathway target genes (C) are enriched in HB1.F3. TOPFLASH/FOPflash reporter analysis (D) confirms that Wnt pathway activity in HB1.F3 is significantly higher than that in F3.Olig2. The reporter activity in HB1.F3 was inhibited to the level of F3.Olig2 when HB1.F3 was co-transfected with ‘dominant negative’ (DN) TCF constructs. Data are means±S.D. of triplicate samples. A.U., Activity unit
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2602983&req=5

pone-0003917-g002: Downregulation of Wnt pathway in F3.Olig2.GSEA shows that Wnt pathway genes (A), Wnt-responsive genes (B), and Wnt pathway target genes (C) are enriched in HB1.F3. TOPFLASH/FOPflash reporter analysis (D) confirms that Wnt pathway activity in HB1.F3 is significantly higher than that in F3.Olig2. The reporter activity in HB1.F3 was inhibited to the level of F3.Olig2 when HB1.F3 was co-transfected with ‘dominant negative’ (DN) TCF constructs. Data are means±S.D. of triplicate samples. A.U., Activity unit
Mentions: Microarray analysis revealed global gene expression changes between HB1.F3 and F3.Olig2; more than 60% of genes that are present in HB1.F3 are absent in F3.Olig2. Since the global gene expression changes violate basic assumptions of statistical analysis of microarray data that most genes are not differentially expressed [18], we have employed the knowledge-based Gene Set Enrichment Analysis (GSEA) (Materials and Methods), instead of using conventional statistical analysis such as t-test, to investigate expression changes in functional groups of genes. Since the Wnt pathway is known to be involved in neural stem cell-differentiation in contra-acting ways (i.e., maintain stemness versus inducing differentiation [19]–[21], the investigation of the microarray data was focused on Wnt pathway-related gene sets. Using this method, we identified significant enrichment of Wnt pathway genes, genes upregulated by Wnt [22], and Wnt pathway target genes in HB1.F3, an immortalized neural stem cell line (Fig. 2A–C) (see Table S1, S2, S3 for detailed information). To obtain further evidence that Wnt pathway is active in HB1.F3 and suppressed in F3.Olig2, we transfected a transcription factor (TCF) reporter gene (TOPflash) containing five optimal TCF-binding sites or the mutant control plasmid (FOPflash) into HB1.F3 and F3.Olig2. pRL-TK was included to normalize data for transfection efficiency [23]. As shown in Fig. 2D, the reporter activity of Wnt pathway is more than three times higher in HB1.F3 than in F3.Olig2. The addition of dominant negative TCF plasmids (dnTCF) decreased the reporter activity in HB1.F3 close to the level of F3.Olig2, indicating that the Wnt activity in F3.Olig2 is at the background level.

Bottom Line: Furthermore, we found that Olig2-induced differentiation induces the expression of Dickkopf-1(Dkk1), a potent antagonist of Wnt signaling.Dkk1 treatment blocked Wnt signaling in HB1.F3 in a dosage-dependent manner, and induced differentiation into astrocytes, oligodendrocytes, and neurons.In our proposed model, Dkk1 may play an important role in downregulating self-renewal and proliferation pathway of stem cells at the late stage of differentiation, and its failure may lead to carcinogenesis.

View Article: PubMed Central - PubMed

Affiliation: Center for Genomics and Proteomics, Lee Gil Ya Cancer and Diabetes Institute, Gachon University of Medicine and Science, Incheon, Korea.

ABSTRACT
Understanding stem cell-differentiation at the molecular level is important for clinical applications of stem cells and for finding new therapeutic approaches in the context of cancer stem cells. To investigate genome-wide changes involved in differentiation, we have used immortalized neural stem cell (NSC) line (HB1.F3) and Olig2-induced NSC differentiation model (F3.Olig2). Using microarray analysis, we revealed that Olig2-induced NSC differentiation involves downregulation of Wnt pathway, which was further confirmed by TOPflash/FOPflash reporter assay, RT-PCR analysis, immunoblots, and immunocytochemistry. Furthermore, we found that Olig2-induced differentiation induces the expression of Dickkopf-1(Dkk1), a potent antagonist of Wnt signaling. Dkk1 treatment blocked Wnt signaling in HB1.F3 in a dosage-dependent manner, and induced differentiation into astrocytes, oligodendrocytes, and neurons. Our results support cancer stem cell hypothesis which implies that signaling pathway for self-renewal and proliferation of stem cells is maintained till the late stage of differentiation. In our proposed model, Dkk1 may play an important role in downregulating self-renewal and proliferation pathway of stem cells at the late stage of differentiation, and its failure may lead to carcinogenesis.

Show MeSH
Related in: MedlinePlus