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PROP1 triggers epithelial-mesenchymal transition-like process in pituitary stem cells

View Article: PubMed Central - PubMed

ABSTRACT

Mutations in PROP1 are the most common cause of hypopituitarism in humans; therefore, unraveling its mechanism of action is highly relevant from a therapeutic perspective. Our current understanding of the role of PROP1 in the pituitary gland is limited to the repression and activation of the pituitary transcription factor genes Hesx1 and Pou1f1, respectively. To elucidate the comprehensive PROP1-dependent gene regulatory network, we conducted genome-wide analysis of PROP1 DNA binding and effects on gene expression in mutant mice, mouse isolated stem cells and engineered mouse cell lines. We determined that PROP1 is essential for stimulating stem cells to undergo an epithelial to mesenchymal transition-like process necessary for cell migration and differentiation. Genomic profiling reveals that PROP1 binds to genes expressed in epithelial cells like Claudin 23, and to EMT inducer genes like Zeb2, Notch2 and Gli2. Zeb2 activation appears to be a key step in the EMT process. Our findings identify PROP1 as a central transcriptional component of pituitary stem cell differentiation.

Doi:: http://dx.doi.org/10.7554/eLife.14470.001

No MeSH data available.


Related in: MedlinePlus

Zeb2 knockdown resemble colonies derived from Prop1 mutant mice.(A) Zeb2, Gli2 and Cdh1 mRNA levels in colonies from Prop1df/+ and Prop1df/df pituitaries treated with Zeb2 siRNA and negative control siRNA (control). Samples were normalized to GAPDH. qPCRs were done using at least three technical replicates. (B) Pictures of the colonies from Prop1df/+ and Prop1df/df pituitaries fixed and stained, show the different morphology of colonies between control siRNA and treated with Zeb2 siRNA.DOI:http://dx.doi.org/10.7554/eLife.14470.010
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fig6: Zeb2 knockdown resemble colonies derived from Prop1 mutant mice.(A) Zeb2, Gli2 and Cdh1 mRNA levels in colonies from Prop1df/+ and Prop1df/df pituitaries treated with Zeb2 siRNA and negative control siRNA (control). Samples were normalized to GAPDH. qPCRs were done using at least three technical replicates. (B) Pictures of the colonies from Prop1df/+ and Prop1df/df pituitaries fixed and stained, show the different morphology of colonies between control siRNA and treated with Zeb2 siRNA.DOI:http://dx.doi.org/10.7554/eLife.14470.010

Mentions: We validated differential expression of selected genes in stem cell colonies from Prop1 mutant and wild-type pituitaries. A qRT-PCR analysis showed that Cdh1 and Claudin 23 were elevated, and Mmp2, Mmp3, Mmp16, Zeb2 and Gli2 were decreased in the Prop1 mutant colonies relative to colonies from wild-type littermates (Figure 5B). Expression of CDH1 was also analyzed by immunohistochemistry. Colonies from Prop1df/df pituitaries showed increased in CDH1-positive cells (Figure 5B). CyclinE was decreased in Prop1 mutant colonies, at mRNA and protein levels, consistent with our findings in pituitary tissue (Figure 5B). Because Zeb2 is an inducer of EMT in other tissues (Vandewalle et al., 2005), we tested whether reduction in Zeb2 expression was sufficient to alter colony morphology and gene expression. We achieved effective reduction of Zeb2 expression with siRNA designed to target Zeb2, but not with the siRNA negative control (Figure 6). Lowered Zeb2 expression was sufficient to reduce expression of Gli2 and increase expression of Cdh1. Treatment with Zeb2 siRNA appeared to block formation of tightly packed colonies typical of normal pituitary stem cell cultures (Figure 6A and B). Thus, in the absence of Prop1 or Zeb2 the colonies fail to initiate the EMT-like process and exhibit abnormal morphology and EMT marker gene expression. This supports the idea that Prop1 induces EMT-like process in the stem cell population.10.7554/eLife.14470.010Figure 6.Zeb2 knockdown resemble colonies derived from Prop1 mutant mice.


PROP1 triggers epithelial-mesenchymal transition-like process in pituitary stem cells
Zeb2 knockdown resemble colonies derived from Prop1 mutant mice.(A) Zeb2, Gli2 and Cdh1 mRNA levels in colonies from Prop1df/+ and Prop1df/df pituitaries treated with Zeb2 siRNA and negative control siRNA (control). Samples were normalized to GAPDH. qPCRs were done using at least three technical replicates. (B) Pictures of the colonies from Prop1df/+ and Prop1df/df pituitaries fixed and stained, show the different morphology of colonies between control siRNA and treated with Zeb2 siRNA.DOI:http://dx.doi.org/10.7554/eLife.14470.010
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Related In: Results  -  Collection

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fig6: Zeb2 knockdown resemble colonies derived from Prop1 mutant mice.(A) Zeb2, Gli2 and Cdh1 mRNA levels in colonies from Prop1df/+ and Prop1df/df pituitaries treated with Zeb2 siRNA and negative control siRNA (control). Samples were normalized to GAPDH. qPCRs were done using at least three technical replicates. (B) Pictures of the colonies from Prop1df/+ and Prop1df/df pituitaries fixed and stained, show the different morphology of colonies between control siRNA and treated with Zeb2 siRNA.DOI:http://dx.doi.org/10.7554/eLife.14470.010
Mentions: We validated differential expression of selected genes in stem cell colonies from Prop1 mutant and wild-type pituitaries. A qRT-PCR analysis showed that Cdh1 and Claudin 23 were elevated, and Mmp2, Mmp3, Mmp16, Zeb2 and Gli2 were decreased in the Prop1 mutant colonies relative to colonies from wild-type littermates (Figure 5B). Expression of CDH1 was also analyzed by immunohistochemistry. Colonies from Prop1df/df pituitaries showed increased in CDH1-positive cells (Figure 5B). CyclinE was decreased in Prop1 mutant colonies, at mRNA and protein levels, consistent with our findings in pituitary tissue (Figure 5B). Because Zeb2 is an inducer of EMT in other tissues (Vandewalle et al., 2005), we tested whether reduction in Zeb2 expression was sufficient to alter colony morphology and gene expression. We achieved effective reduction of Zeb2 expression with siRNA designed to target Zeb2, but not with the siRNA negative control (Figure 6). Lowered Zeb2 expression was sufficient to reduce expression of Gli2 and increase expression of Cdh1. Treatment with Zeb2 siRNA appeared to block formation of tightly packed colonies typical of normal pituitary stem cell cultures (Figure 6A and B). Thus, in the absence of Prop1 or Zeb2 the colonies fail to initiate the EMT-like process and exhibit abnormal morphology and EMT marker gene expression. This supports the idea that Prop1 induces EMT-like process in the stem cell population.10.7554/eLife.14470.010Figure 6.Zeb2 knockdown resemble colonies derived from Prop1 mutant mice.

View Article: PubMed Central - PubMed

ABSTRACT

Mutations in PROP1 are the most common cause of hypopituitarism in humans; therefore, unraveling its mechanism of action is highly relevant from a therapeutic perspective. Our current understanding of the role of PROP1 in the pituitary gland is limited to the repression and activation of the pituitary transcription factor genes Hesx1 and Pou1f1, respectively. To elucidate the comprehensive PROP1-dependent gene regulatory network, we conducted genome-wide analysis of PROP1 DNA binding and effects on gene expression in mutant mice, mouse isolated stem cells and engineered mouse cell lines. We determined that PROP1 is essential for stimulating stem cells to undergo an epithelial to mesenchymal transition-like process necessary for cell migration and differentiation. Genomic profiling reveals that PROP1 binds to genes expressed in epithelial cells like Claudin 23, and to EMT inducer genes like Zeb2, Notch2 and Gli2. Zeb2 activation appears to be a key step in the EMT process. Our findings identify PROP1 as a central transcriptional component of pituitary stem cell differentiation.

Doi:: http://dx.doi.org/10.7554/eLife.14470.001

No MeSH data available.


Related in: MedlinePlus