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SNX17 regulates Notch pathway and pancreas development through the retromer-dependent recycling of Jag1.

Yin W, Liu D, Liu N, Xu L, Li S, Lin S, Shu X, Pei D - Cell Regen (Lond) (2012)

Bottom Line: SNX17 is a sorting nexin family protein implicated in vesicular trafficking and we find it is specifically required in the ligand-expressing cells for Notch signaling.Mechanistically, SNX17 regulates the protein level of Jag1a on plasma membrane by binding to Jag1a and facilitating the retromer-dependent recycling of the ligand.In zebrafish, inhibition of this SNX17-mediated Notch signaling pathway results in defects in neurogenesis as well as pancreas development.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.

ABSTRACT

Background: Notch is one of the most important signaling pathways involved in cell fate determination. Activation of the Notch pathway requires the binding of a membrane-bound ligand to the Notch receptor in the adjacent cell which induces proteolytic cleavages and the activation of the receptor. A unique feature of the Notch signaling is that processes such as modification, endocytosis or recycling of the ligand have been reported to play critical roles during Notch signaling, however, the underlying molecular mechanism appears context-dependent and often controversial.

Results: Here we identified SNX17 as a novel regulator of the Notch pathway. SNX17 is a sorting nexin family protein implicated in vesicular trafficking and we find it is specifically required in the ligand-expressing cells for Notch signaling. Mechanistically, SNX17 regulates the protein level of Jag1a on plasma membrane by binding to Jag1a and facilitating the retromer-dependent recycling of the ligand. In zebrafish, inhibition of this SNX17-mediated Notch signaling pathway results in defects in neurogenesis as well as pancreas development.

Conclusions: Our results reveal that SNX17, by acting as a cargo-specific adaptor, promotes the retromer dependent recycling of Jag1a and Notch signaling and this pathway is involved in cell fate determination during zebrafish neurogenesis and pancreas development.

No MeSH data available.


Related in: MedlinePlus

SNX17 is required specifically in the ligand-expressing cells for Notch signaling. (A) Knockdown of SNX17 by siRNAs in 293 T cells down-regulated the Notch luciferase reporter activity while over-expression of SNX17 stimulated the reporter activity. (B) Over-expression of Jag and Delta family Notch ligands stimulated the reporter activity which was blocked by the knockdown of SNX17. (C) Down-regulation of SNX17 reduced the expression of endogenous Notch target genes hes1/7 and hey1 in 293 T cells as determined by real-time RT-PCR (p < 0.05 in all cases). hey2 was not affected in the same assay (P > 0.1). β-actin was the reference. (D) Co-culture of the Jag1-expressing 293 T cells with the Notch1-expressing NIH3T3 cells. Knockdown of SNX17 in the ligand-expressing cells reduced the Notch reporter activity while down-regulation of SNX17 in the receptor-expressing cells did not have inhibitory effect. All assays were repeated at least three times and data represent mean ± SD from three independent experiments.
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Fig2: SNX17 is required specifically in the ligand-expressing cells for Notch signaling. (A) Knockdown of SNX17 by siRNAs in 293 T cells down-regulated the Notch luciferase reporter activity while over-expression of SNX17 stimulated the reporter activity. (B) Over-expression of Jag and Delta family Notch ligands stimulated the reporter activity which was blocked by the knockdown of SNX17. (C) Down-regulation of SNX17 reduced the expression of endogenous Notch target genes hes1/7 and hey1 in 293 T cells as determined by real-time RT-PCR (p < 0.05 in all cases). hey2 was not affected in the same assay (P > 0.1). β-actin was the reference. (D) Co-culture of the Jag1-expressing 293 T cells with the Notch1-expressing NIH3T3 cells. Knockdown of SNX17 in the ligand-expressing cells reduced the Notch reporter activity while down-regulation of SNX17 in the receptor-expressing cells did not have inhibitory effect. All assays were repeated at least three times and data represent mean ± SD from three independent experiments.

Mentions: We used the Notch luciferase reporter system [29] in 293 T cells to further characterize the molecular mechanisms of SNX17 in Notch signaling. As shown in Figure 2A, over-expression of SNX17 stimulated the Notch reporter activity; on the other hand, siRNA-mediated knockdown of SNX17 reduced the reporter activity (the efficiencies of siRNAs were shown in Additional file 1: Figure S1). We investigated whether the SNX17 regulation of Notch was ligand specific. Jagged (Jag) and Delta family ligands were transfected into the 293 T cells and we found that Jag1 was the most potent one in stimulating the reporter. When SNX17 was knocked-down, the reporter activity was clearly inhibited in all of the ligand-transfected cells (Figure 2B). We further examined the expression levels of several Notch target genes (hes1/7 and hey1/2) by real-time RT-PCR. As shown in Figure 2C, inhibition of SNX17 decreased the expression of hes7hes1 and hey1. Thus, SNX17 regulated the expression of both the transiently transfected Notch reporter and the endogenous Notch target genes. As Notch signaling requires the direct contact of a ligand-expressing cell (signal-sending cell) with a Notch-expressing cell (signal-receiving cell), we used a cell co-culture system [30] to determine in which cell SNX17 functioned. As shown in Figure 1D, when SNX17 was knocked-down in the Jag1-expressing 293 T cells, these cells became less efficient to stimulate the luciferase reporter expressed in the Notch-expressing 3T3 cells. On the other hand, when SNX17 was down-regulated in the Notch-expressing 3T3 cells, luciferase reporter activity in these cells was still stimulated by the addition of Jag1-expressing 293 T cells. Taken together, these results demonstrate that SNX17 is specifically required in the ligand-expressing cells for Notch signaling.Figure 2


SNX17 regulates Notch pathway and pancreas development through the retromer-dependent recycling of Jag1.

Yin W, Liu D, Liu N, Xu L, Li S, Lin S, Shu X, Pei D - Cell Regen (Lond) (2012)

SNX17 is required specifically in the ligand-expressing cells for Notch signaling. (A) Knockdown of SNX17 by siRNAs in 293 T cells down-regulated the Notch luciferase reporter activity while over-expression of SNX17 stimulated the reporter activity. (B) Over-expression of Jag and Delta family Notch ligands stimulated the reporter activity which was blocked by the knockdown of SNX17. (C) Down-regulation of SNX17 reduced the expression of endogenous Notch target genes hes1/7 and hey1 in 293 T cells as determined by real-time RT-PCR (p < 0.05 in all cases). hey2 was not affected in the same assay (P > 0.1). β-actin was the reference. (D) Co-culture of the Jag1-expressing 293 T cells with the Notch1-expressing NIH3T3 cells. Knockdown of SNX17 in the ligand-expressing cells reduced the Notch reporter activity while down-regulation of SNX17 in the receptor-expressing cells did not have inhibitory effect. All assays were repeated at least three times and data represent mean ± SD from three independent experiments.
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Related In: Results  -  Collection

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Fig2: SNX17 is required specifically in the ligand-expressing cells for Notch signaling. (A) Knockdown of SNX17 by siRNAs in 293 T cells down-regulated the Notch luciferase reporter activity while over-expression of SNX17 stimulated the reporter activity. (B) Over-expression of Jag and Delta family Notch ligands stimulated the reporter activity which was blocked by the knockdown of SNX17. (C) Down-regulation of SNX17 reduced the expression of endogenous Notch target genes hes1/7 and hey1 in 293 T cells as determined by real-time RT-PCR (p < 0.05 in all cases). hey2 was not affected in the same assay (P > 0.1). β-actin was the reference. (D) Co-culture of the Jag1-expressing 293 T cells with the Notch1-expressing NIH3T3 cells. Knockdown of SNX17 in the ligand-expressing cells reduced the Notch reporter activity while down-regulation of SNX17 in the receptor-expressing cells did not have inhibitory effect. All assays were repeated at least three times and data represent mean ± SD from three independent experiments.
Mentions: We used the Notch luciferase reporter system [29] in 293 T cells to further characterize the molecular mechanisms of SNX17 in Notch signaling. As shown in Figure 2A, over-expression of SNX17 stimulated the Notch reporter activity; on the other hand, siRNA-mediated knockdown of SNX17 reduced the reporter activity (the efficiencies of siRNAs were shown in Additional file 1: Figure S1). We investigated whether the SNX17 regulation of Notch was ligand specific. Jagged (Jag) and Delta family ligands were transfected into the 293 T cells and we found that Jag1 was the most potent one in stimulating the reporter. When SNX17 was knocked-down, the reporter activity was clearly inhibited in all of the ligand-transfected cells (Figure 2B). We further examined the expression levels of several Notch target genes (hes1/7 and hey1/2) by real-time RT-PCR. As shown in Figure 2C, inhibition of SNX17 decreased the expression of hes7hes1 and hey1. Thus, SNX17 regulated the expression of both the transiently transfected Notch reporter and the endogenous Notch target genes. As Notch signaling requires the direct contact of a ligand-expressing cell (signal-sending cell) with a Notch-expressing cell (signal-receiving cell), we used a cell co-culture system [30] to determine in which cell SNX17 functioned. As shown in Figure 1D, when SNX17 was knocked-down in the Jag1-expressing 293 T cells, these cells became less efficient to stimulate the luciferase reporter expressed in the Notch-expressing 3T3 cells. On the other hand, when SNX17 was down-regulated in the Notch-expressing 3T3 cells, luciferase reporter activity in these cells was still stimulated by the addition of Jag1-expressing 293 T cells. Taken together, these results demonstrate that SNX17 is specifically required in the ligand-expressing cells for Notch signaling.Figure 2

Bottom Line: SNX17 is a sorting nexin family protein implicated in vesicular trafficking and we find it is specifically required in the ligand-expressing cells for Notch signaling.Mechanistically, SNX17 regulates the protein level of Jag1a on plasma membrane by binding to Jag1a and facilitating the retromer-dependent recycling of the ligand.In zebrafish, inhibition of this SNX17-mediated Notch signaling pathway results in defects in neurogenesis as well as pancreas development.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.

ABSTRACT

Background: Notch is one of the most important signaling pathways involved in cell fate determination. Activation of the Notch pathway requires the binding of a membrane-bound ligand to the Notch receptor in the adjacent cell which induces proteolytic cleavages and the activation of the receptor. A unique feature of the Notch signaling is that processes such as modification, endocytosis or recycling of the ligand have been reported to play critical roles during Notch signaling, however, the underlying molecular mechanism appears context-dependent and often controversial.

Results: Here we identified SNX17 as a novel regulator of the Notch pathway. SNX17 is a sorting nexin family protein implicated in vesicular trafficking and we find it is specifically required in the ligand-expressing cells for Notch signaling. Mechanistically, SNX17 regulates the protein level of Jag1a on plasma membrane by binding to Jag1a and facilitating the retromer-dependent recycling of the ligand. In zebrafish, inhibition of this SNX17-mediated Notch signaling pathway results in defects in neurogenesis as well as pancreas development.

Conclusions: Our results reveal that SNX17, by acting as a cargo-specific adaptor, promotes the retromer dependent recycling of Jag1a and Notch signaling and this pathway is involved in cell fate determination during zebrafish neurogenesis and pancreas development.

No MeSH data available.


Related in: MedlinePlus