Limits...
Mind bomb 1 in the lymphopoietic niches is essential for T and marginal zone B cell development.

Song R, Kim YW, Koo BK, Jeong HW, Yoon MJ, Yoon KJ, Jun DJ, Im SK, Shin J, Kong MP, Kim KT, Yoon K, Kong YY - J. Exp. Med. (2008)

Bottom Line: In addition, the endocytosis of Dll1 was impaired in the Mib1- microenvironment.Moreover, the block in T cell development and the failure of Dll1 endocytosis were also observed in coculture system by Mib1 knockdown.Our study reveals that Mib1 is the essential E3 ligase in T and MZB cell development, through the regulation of Notch ligands in the thymic and splenic microenvironments.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Pohang University of Science and Technology, Pohang, Kyungbuk 790-784, South Korea.

ABSTRACT
Notch signaling regulates lineage decisions at multiple stages of lymphocyte development, and Notch activation requires the endocytosis of Notch ligands in the signal-sending cells. Four E3 ubiquitin ligases, Mind bomb (Mib) 1, Mib2, Neuralized (Neur) 1, and Neur2, regulate the Notch ligands to activate Notch signaling, but their roles in lymphocyte development have not been defined. We show that Mib1 regulates T and marginal zone B (MZB) cell development in the lymphopoietic niches. Inactivation of the Mib1 gene, but not the other E3 ligases, Mib2, Neur1, and Neur2, abrogated T and MZB cell development. Reciprocal bone marrow (BM) transplantation experiments revealed that Mib1 in the thymic and splenic niches is essential for T and MZB cell development. Interestingly, when BM cells from transgenic Notch reporter mice were transplanted into Mib1- mice, the Notch signaling was abolished in the double-negative thymocytes. In addition, the endocytosis of Dll1 was impaired in the Mib1- microenvironment. Moreover, the block in T cell development and the failure of Dll1 endocytosis were also observed in coculture system by Mib1 knockdown. Our study reveals that Mib1 is the essential E3 ligase in T and MZB cell development, through the regulation of Notch ligands in the thymic and splenic microenvironments.

Show MeSH

Related in: MedlinePlus

Block in T cell development at DN1 stage in knockdown Mib1 in OP9-DL1 cells. (A) Immunoblot of Mib1 protein in OP9-DL1 cells 36 h after microporation with control (ctrl) or Mib1 siRNA. Dll1 expression was not affected. (B) Control siRNA/OP9-DL1 or Mib1 siRNA/OP9-DL1 cells were cocultured with C2C12-Notch1 cells transfected with 8× WT CBF-Luc vectors. 24 h after coculture, luciferase activities were measured. Data are mean ± SD from triplicate experiments. *, P < 0.001. (C) Lin−Sca-1+c-Kit+ fetal liver (FL-LSK) or BM (BM-LSK) cells were prepared and cultured on control siRNA/OP9-DL1 or Mib1 siRNA/OP9-DL1 cells. Growing cells collected on days 5 or 7, respectively, were stained for CD44 and CD25 gated on CD4−CD8− DN cells and analyzed by flow cytometry. A representative of three independent experiments is shown. (D) C2C12-Notch1 cells were cocultured on control siRNA/OP9-DL1 or Mib1 siRNA/OP9-DL1 cells. After 12 h, the cells were stained with Dll1 antibody. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2571928&req=5

fig8: Block in T cell development at DN1 stage in knockdown Mib1 in OP9-DL1 cells. (A) Immunoblot of Mib1 protein in OP9-DL1 cells 36 h after microporation with control (ctrl) or Mib1 siRNA. Dll1 expression was not affected. (B) Control siRNA/OP9-DL1 or Mib1 siRNA/OP9-DL1 cells were cocultured with C2C12-Notch1 cells transfected with 8× WT CBF-Luc vectors. 24 h after coculture, luciferase activities were measured. Data are mean ± SD from triplicate experiments. *, P < 0.001. (C) Lin−Sca-1+c-Kit+ fetal liver (FL-LSK) or BM (BM-LSK) cells were prepared and cultured on control siRNA/OP9-DL1 or Mib1 siRNA/OP9-DL1 cells. Growing cells collected on days 5 or 7, respectively, were stained for CD44 and CD25 gated on CD4−CD8− DN cells and analyzed by flow cytometry. A representative of three independent experiments is shown. (D) C2C12-Notch1 cells were cocultured on control siRNA/OP9-DL1 or Mib1 siRNA/OP9-DL1 cells. After 12 h, the cells were stained with Dll1 antibody. Bars, 10 μm.

Mentions: It has been demonstrated that the culture of HSCs on OP9-DL1 cells facilitates T cell development from HSCs (31), whereas progression through the DN stages of T cell development is impaired in the presence of the Notch signaling inhibitor (54). To further examine whether the defect in T cell development in the MMTV-Cre;Mib1f/f mice is caused by inability of Mib1-disrupted stromal cells to activate Notch signaling in hematopoietic cells, we transfected Mib1 small interfering RNA (siRNA) duplexes into the OP9-DL1 cells. Mib1 protein was significantly reduced 36 h after microporation in the Mib1 siRNA-treated OP9-DL1 cells (Mib1 siRNA/OP9-DL1; Fig. 8 A). In addition, when C2C12-Notch1 cells transfected with a CBF-Luc vector carrying RBP-Jκ binding sites were cocultured with the Mib1 siRNA/OP9-DL1 cells, CBF-luciferase reporter activity was markedly reduced compared with that of control siRNA-treated cells (control siRNA/OP9-DL1), suggesting that Mib1 is required for Notch signaling through regulating Dll1 function (Fig. 8 B [reference 56]).


Mind bomb 1 in the lymphopoietic niches is essential for T and marginal zone B cell development.

Song R, Kim YW, Koo BK, Jeong HW, Yoon MJ, Yoon KJ, Jun DJ, Im SK, Shin J, Kong MP, Kim KT, Yoon K, Kong YY - J. Exp. Med. (2008)

Block in T cell development at DN1 stage in knockdown Mib1 in OP9-DL1 cells. (A) Immunoblot of Mib1 protein in OP9-DL1 cells 36 h after microporation with control (ctrl) or Mib1 siRNA. Dll1 expression was not affected. (B) Control siRNA/OP9-DL1 or Mib1 siRNA/OP9-DL1 cells were cocultured with C2C12-Notch1 cells transfected with 8× WT CBF-Luc vectors. 24 h after coculture, luciferase activities were measured. Data are mean ± SD from triplicate experiments. *, P < 0.001. (C) Lin−Sca-1+c-Kit+ fetal liver (FL-LSK) or BM (BM-LSK) cells were prepared and cultured on control siRNA/OP9-DL1 or Mib1 siRNA/OP9-DL1 cells. Growing cells collected on days 5 or 7, respectively, were stained for CD44 and CD25 gated on CD4−CD8− DN cells and analyzed by flow cytometry. A representative of three independent experiments is shown. (D) C2C12-Notch1 cells were cocultured on control siRNA/OP9-DL1 or Mib1 siRNA/OP9-DL1 cells. After 12 h, the cells were stained with Dll1 antibody. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2571928&req=5

fig8: Block in T cell development at DN1 stage in knockdown Mib1 in OP9-DL1 cells. (A) Immunoblot of Mib1 protein in OP9-DL1 cells 36 h after microporation with control (ctrl) or Mib1 siRNA. Dll1 expression was not affected. (B) Control siRNA/OP9-DL1 or Mib1 siRNA/OP9-DL1 cells were cocultured with C2C12-Notch1 cells transfected with 8× WT CBF-Luc vectors. 24 h after coculture, luciferase activities were measured. Data are mean ± SD from triplicate experiments. *, P < 0.001. (C) Lin−Sca-1+c-Kit+ fetal liver (FL-LSK) or BM (BM-LSK) cells were prepared and cultured on control siRNA/OP9-DL1 or Mib1 siRNA/OP9-DL1 cells. Growing cells collected on days 5 or 7, respectively, were stained for CD44 and CD25 gated on CD4−CD8− DN cells and analyzed by flow cytometry. A representative of three independent experiments is shown. (D) C2C12-Notch1 cells were cocultured on control siRNA/OP9-DL1 or Mib1 siRNA/OP9-DL1 cells. After 12 h, the cells were stained with Dll1 antibody. Bars, 10 μm.
Mentions: It has been demonstrated that the culture of HSCs on OP9-DL1 cells facilitates T cell development from HSCs (31), whereas progression through the DN stages of T cell development is impaired in the presence of the Notch signaling inhibitor (54). To further examine whether the defect in T cell development in the MMTV-Cre;Mib1f/f mice is caused by inability of Mib1-disrupted stromal cells to activate Notch signaling in hematopoietic cells, we transfected Mib1 small interfering RNA (siRNA) duplexes into the OP9-DL1 cells. Mib1 protein was significantly reduced 36 h after microporation in the Mib1 siRNA-treated OP9-DL1 cells (Mib1 siRNA/OP9-DL1; Fig. 8 A). In addition, when C2C12-Notch1 cells transfected with a CBF-Luc vector carrying RBP-Jκ binding sites were cocultured with the Mib1 siRNA/OP9-DL1 cells, CBF-luciferase reporter activity was markedly reduced compared with that of control siRNA-treated cells (control siRNA/OP9-DL1), suggesting that Mib1 is required for Notch signaling through regulating Dll1 function (Fig. 8 B [reference 56]).

Bottom Line: In addition, the endocytosis of Dll1 was impaired in the Mib1- microenvironment.Moreover, the block in T cell development and the failure of Dll1 endocytosis were also observed in coculture system by Mib1 knockdown.Our study reveals that Mib1 is the essential E3 ligase in T and MZB cell development, through the regulation of Notch ligands in the thymic and splenic microenvironments.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Pohang University of Science and Technology, Pohang, Kyungbuk 790-784, South Korea.

ABSTRACT
Notch signaling regulates lineage decisions at multiple stages of lymphocyte development, and Notch activation requires the endocytosis of Notch ligands in the signal-sending cells. Four E3 ubiquitin ligases, Mind bomb (Mib) 1, Mib2, Neuralized (Neur) 1, and Neur2, regulate the Notch ligands to activate Notch signaling, but their roles in lymphocyte development have not been defined. We show that Mib1 regulates T and marginal zone B (MZB) cell development in the lymphopoietic niches. Inactivation of the Mib1 gene, but not the other E3 ligases, Mib2, Neur1, and Neur2, abrogated T and MZB cell development. Reciprocal bone marrow (BM) transplantation experiments revealed that Mib1 in the thymic and splenic niches is essential for T and MZB cell development. Interestingly, when BM cells from transgenic Notch reporter mice were transplanted into Mib1- mice, the Notch signaling was abolished in the double-negative thymocytes. In addition, the endocytosis of Dll1 was impaired in the Mib1- microenvironment. Moreover, the block in T cell development and the failure of Dll1 endocytosis were also observed in coculture system by Mib1 knockdown. Our study reveals that Mib1 is the essential E3 ligase in T and MZB cell development, through the regulation of Notch ligands in the thymic and splenic microenvironments.

Show MeSH
Related in: MedlinePlus