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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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RT-PCR analysis of Wnt pathway-related genes.A. Most Wnt genes are overexpressed in HB1.F3 whereas the expression of Wnt 10b is increased in F3.Olig2. Interestingly, Wnt7b is expressed only in F3.Olig2. B. Except FZD5, all Wnt receptor and co-receptor genes that are expressed in HB1.F3 are suppressed in F3.Olig2. C. Wnt pathway target genes are expressed only in HB1.F3. D. Dkk1, a soluble Wnt antagonist, is expressed only in F3.Olig2.
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pone-0003917-g003: RT-PCR analysis of Wnt pathway-related genes.A. Most Wnt genes are overexpressed in HB1.F3 whereas the expression of Wnt 10b is increased in F3.Olig2. Interestingly, Wnt7b is expressed only in F3.Olig2. B. Except FZD5, all Wnt receptor and co-receptor genes that are expressed in HB1.F3 are suppressed in F3.Olig2. C. Wnt pathway target genes are expressed only in HB1.F3. D. Dkk1, a soluble Wnt antagonist, is expressed only in F3.Olig2.

Mentions: As described above, both GSEA and the reporter assay revealed that Wnt pathway is active in HB1.F3 and downregulated in F3.Olig2. To further confirm these findings at the molecular level, we performed semiquantitative RT-PCR analysis of Wnt pathway components including 18 Wnt families, 4 Wnt receptors, 2 Wnt co-receptors, 2 antagonists of Wnt signaling, and 2 target genes of Wnt pathway. Most of Wnt genes which expressed in HB1.F3 are suppressed in F3.Olig2, whereas only Wnt 10b gene is increased in F3.Olig2 (Fig. 3A). Interestingly, Wnt7b is expressed only in F3.Olig2. Wnt receptors and co-receptor are all downregulated in F3.Olig2 and frizzled 5(FZD5) was not expressed in both HB1.F3 and F3.Olig2 (Fig. 3B). In accordance with GSEA results, Wnt pathway target genes such as Axin2 [24] and c-myc [25] are expressed only in HB1.F3 (Fig. 3C). On the other hand, Dickkopf-1 (Dkk1), a secreted Wnt antagonist [26], is expressed only in F3.Olig2 (Fig. 3D). Unlike other Wnt antagonists, the function of Dkk1 is independent of Frizzled, and inhibits canonical Wnt signaling by binding to LRP6 [27], [28], the only Wnt co-receptor expressed in F3.Olig2.


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)

RT-PCR analysis of Wnt pathway-related genes.A. Most Wnt genes are overexpressed in HB1.F3 whereas the expression of Wnt 10b is increased in F3.Olig2. Interestingly, Wnt7b is expressed only in F3.Olig2. B. Except FZD5, all Wnt receptor and co-receptor genes that are expressed in HB1.F3 are suppressed in F3.Olig2. C. Wnt pathway target genes are expressed only in HB1.F3. D. Dkk1, a soluble Wnt antagonist, is expressed only in F3.Olig2.
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Related In: Results  -  Collection

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pone-0003917-g003: RT-PCR analysis of Wnt pathway-related genes.A. Most Wnt genes are overexpressed in HB1.F3 whereas the expression of Wnt 10b is increased in F3.Olig2. Interestingly, Wnt7b is expressed only in F3.Olig2. B. Except FZD5, all Wnt receptor and co-receptor genes that are expressed in HB1.F3 are suppressed in F3.Olig2. C. Wnt pathway target genes are expressed only in HB1.F3. D. Dkk1, a soluble Wnt antagonist, is expressed only in F3.Olig2.
Mentions: As described above, both GSEA and the reporter assay revealed that Wnt pathway is active in HB1.F3 and downregulated in F3.Olig2. To further confirm these findings at the molecular level, we performed semiquantitative RT-PCR analysis of Wnt pathway components including 18 Wnt families, 4 Wnt receptors, 2 Wnt co-receptors, 2 antagonists of Wnt signaling, and 2 target genes of Wnt pathway. Most of Wnt genes which expressed in HB1.F3 are suppressed in F3.Olig2, whereas only Wnt 10b gene is increased in F3.Olig2 (Fig. 3A). Interestingly, Wnt7b is expressed only in F3.Olig2. Wnt receptors and co-receptor are all downregulated in F3.Olig2 and frizzled 5(FZD5) was not expressed in both HB1.F3 and F3.Olig2 (Fig. 3B). In accordance with GSEA results, Wnt pathway target genes such as Axin2 [24] and c-myc [25] are expressed only in HB1.F3 (Fig. 3C). On the other hand, Dickkopf-1 (Dkk1), a secreted Wnt antagonist [26], is expressed only in F3.Olig2 (Fig. 3D). Unlike other Wnt antagonists, the function of Dkk1 is independent of Frizzled, and inhibits canonical Wnt signaling by binding to LRP6 [27], [28], the only Wnt co-receptor expressed in F3.Olig2.

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