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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 regulates the retromer dependent recycling of Jag1a. (A) Subcellular distribution of SNX17. It was detected at early endosomes (Rab5), late endosomes (Rab7), the retromer complex (Vps35) but not the recycling endosomes (Rab11). (B) The retromer pathway was required for the SNX17-stimulated recycling of Jag1a. Whole cell lysate or the isolated plasma membrane fraction was subject to western blot analysis using the indicated antibodies. Knockdown of Vps35 (35–1, 35–2) but not Rab11 (11–1, 11–2) blocked the SNX17-induced accumulation of Jag1a on the plasma membrane. On the other hand, the recycling of TFR was Rab11 dependent. (C) Knockdown of Vps35 but not Rab11 reduced the Notch reporter activity in 293 T cells. (D) Vps35 was required in the ligand-expressing cells for Notch signaling as determined using the cell co-culture system. Assays are performed as described in Figure 2D. Data represent mean ± SD from three independent assays.
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Fig5: SNX17 regulates the retromer dependent recycling of Jag1a. (A) Subcellular distribution of SNX17. It was detected at early endosomes (Rab5), late endosomes (Rab7), the retromer complex (Vps35) but not the recycling endosomes (Rab11). (B) The retromer pathway was required for the SNX17-stimulated recycling of Jag1a. Whole cell lysate or the isolated plasma membrane fraction was subject to western blot analysis using the indicated antibodies. Knockdown of Vps35 (35–1, 35–2) but not Rab11 (11–1, 11–2) blocked the SNX17-induced accumulation of Jag1a on the plasma membrane. On the other hand, the recycling of TFR was Rab11 dependent. (C) Knockdown of Vps35 but not Rab11 reduced the Notch reporter activity in 293 T cells. (D) Vps35 was required in the ligand-expressing cells for Notch signaling as determined using the cell co-culture system. Assays are performed as described in Figure 2D. Data represent mean ± SD from three independent assays.

Mentions: The endocytosed membrane proteins can be transported back to the plasma membrane through the recycling endosome pathway (Rab11-dependent) or the retromer pathway (Vps35-dependent). We first determined the subcellular distribution of SNX17 and found that it localized to the Rab5, Rab7 or Vps35 but not Rab11-positive vesicles (Figure 5A). This observation suggested that the retromer pathway could be involved in the SNX17-induced recycling of Jag1a. We tested this hypothesis by determining whether the recycling of Jag1a is affected when this pathway is blocked. As shown in Figure 5B, pretreatment of cells with siRNAs to Vps35 but not Rab11 blocked the SNX17-stimulated accumulation of Jag1a. On the other hand, the recycling of TFR which is recycling endosome dependent was clearly down-regulated by siRNAs to Rab11 but not Vps35. We concluded that Jag1a and TFR used different pathways for their recycling and SNX17 regulated the recycling of Jag1a through a Vps35 but not Rab11-dependent pathway. If the retromer pathway is essential for the maintenance of the homeostasis of Jag1a on the plasma membrane, it might be required for Notch signaling. We investigated this possibility by performing the luciferase reporter assay. As shown in Figure 5C, inhibition of Vps35 but not Rab11 reduced the reporter activity. We further found that, similar to SNX17, Vps35 was only required in the ligand-expressing cells for Notch signaling (Figure 5D). Taken together, these results demonstrate that the SNX17-retromer pathway is required for the homeostasis of Jag1a and subsequent Notch signaling.Figure 5


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 regulates the retromer dependent recycling of Jag1a. (A) Subcellular distribution of SNX17. It was detected at early endosomes (Rab5), late endosomes (Rab7), the retromer complex (Vps35) but not the recycling endosomes (Rab11). (B) The retromer pathway was required for the SNX17-stimulated recycling of Jag1a. Whole cell lysate or the isolated plasma membrane fraction was subject to western blot analysis using the indicated antibodies. Knockdown of Vps35 (35–1, 35–2) but not Rab11 (11–1, 11–2) blocked the SNX17-induced accumulation of Jag1a on the plasma membrane. On the other hand, the recycling of TFR was Rab11 dependent. (C) Knockdown of Vps35 but not Rab11 reduced the Notch reporter activity in 293 T cells. (D) Vps35 was required in the ligand-expressing cells for Notch signaling as determined using the cell co-culture system. Assays are performed as described in Figure 2D. Data represent mean ± SD from three independent assays.
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Related In: Results  -  Collection

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Fig5: SNX17 regulates the retromer dependent recycling of Jag1a. (A) Subcellular distribution of SNX17. It was detected at early endosomes (Rab5), late endosomes (Rab7), the retromer complex (Vps35) but not the recycling endosomes (Rab11). (B) The retromer pathway was required for the SNX17-stimulated recycling of Jag1a. Whole cell lysate or the isolated plasma membrane fraction was subject to western blot analysis using the indicated antibodies. Knockdown of Vps35 (35–1, 35–2) but not Rab11 (11–1, 11–2) blocked the SNX17-induced accumulation of Jag1a on the plasma membrane. On the other hand, the recycling of TFR was Rab11 dependent. (C) Knockdown of Vps35 but not Rab11 reduced the Notch reporter activity in 293 T cells. (D) Vps35 was required in the ligand-expressing cells for Notch signaling as determined using the cell co-culture system. Assays are performed as described in Figure 2D. Data represent mean ± SD from three independent assays.
Mentions: The endocytosed membrane proteins can be transported back to the plasma membrane through the recycling endosome pathway (Rab11-dependent) or the retromer pathway (Vps35-dependent). We first determined the subcellular distribution of SNX17 and found that it localized to the Rab5, Rab7 or Vps35 but not Rab11-positive vesicles (Figure 5A). This observation suggested that the retromer pathway could be involved in the SNX17-induced recycling of Jag1a. We tested this hypothesis by determining whether the recycling of Jag1a is affected when this pathway is blocked. As shown in Figure 5B, pretreatment of cells with siRNAs to Vps35 but not Rab11 blocked the SNX17-stimulated accumulation of Jag1a. On the other hand, the recycling of TFR which is recycling endosome dependent was clearly down-regulated by siRNAs to Rab11 but not Vps35. We concluded that Jag1a and TFR used different pathways for their recycling and SNX17 regulated the recycling of Jag1a through a Vps35 but not Rab11-dependent pathway. If the retromer pathway is essential for the maintenance of the homeostasis of Jag1a on the plasma membrane, it might be required for Notch signaling. We investigated this possibility by performing the luciferase reporter assay. As shown in Figure 5C, inhibition of Vps35 but not Rab11 reduced the reporter activity. We further found that, similar to SNX17, Vps35 was only required in the ligand-expressing cells for Notch signaling (Figure 5D). Taken together, these results demonstrate that the SNX17-retromer pathway is required for the homeostasis of Jag1a and subsequent Notch signaling.Figure 5

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