Limits...
Reciprocal roles for CCAAT/enhancer binding protein (C/EBP) and PU.1 transcription factors in Langerhans cell commitment.

Iwama A, Osawa M, Hirasawa R, Uchiyama N, Kaneko S, Onodera M, Shibuya K, Shibuya A, Vinson C, Tenen DG, Nakauchi H - J. Exp. Med. (2002)

Bottom Line: PU.1 and CCAAT/enhancing binding protein (C/EBP) family transcription factors have been characterized as key regulators for the development and function of the myeloid system.However, the roles of C/EBP transcription factors have not been fully identified because of functional redundancy among family members.Our findings establish reciprocal roles for C/EBP and PU.1 in LC development, and provide new insight into the molecular mechanism of LC development, which has not yet been well characterized.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Institute of Basic Medical Sciences, Core Research for Evolutional Science and Technology (CREST) Program of Japan Science and Technology (JST). aiwama@md.tsukuba.ac.jp

ABSTRACT
Myeloid progenitor cells give rise to a variety of progenies including dendritic cells. However, the mechanism controlling the diversification of myeloid progenitors into each progeny is largely unknown. PU.1 and CCAAT/enhancing binding protein (C/EBP) family transcription factors have been characterized as key regulators for the development and function of the myeloid system. However, the roles of C/EBP transcription factors have not been fully identified because of functional redundancy among family members. Using high titer--retroviral infection, we demonstrate that a dominant-negative C/EBP completely blocked the granulocyte--macrophage commitment of human myeloid progenitors. Alternatively, Langerhans cell (LC) commitment was markedly facilitated in the absence of tumor necrosis factor (TNF)alpha, a strong inducer of LC development, whereas expression of wild-type C/EBP in myeloid progenitors promoted granulocytic differentiation, and completely inhibited TNFalpha-dependent LC development. On the other hand, expression of wild-type PU.1 in myeloid progenitors triggered LC development in the absence of TNFalpha, and its instructive effect was canceled by coexpressed C/EBP. Our findings establish reciprocal roles for C/EBP and PU.1 in LC development, and provide new insight into the molecular mechanism of LC development, which has not yet been well characterized.

Show MeSH

Related in: MedlinePlus

Altered differentiation of CD34+ progenitors expressing a dominant-negative C/EBP. (A) The schematic representation of the retroviral vector, GCsam–A-C/EBP–IRES–EGFP, encoding A-C/EBP, a dominant-negative C/EBP, linked by an IRES to a cDNA encoding EGFP. The 3′ LTR of the vector is replaced with MSCV. ψ+, packaging signal; SD, splice donor; SA, splice acceptor. (B) The effect of A-C/EBP on the growth of transduced CD34+ cells. After transduction, EGFP-positive cells were selected. Then, cytokine-dependent cell growth was evaluated by CFU generated in the presence of indicated cytokines and by liquid culture in the presence of IL-5 or SCF+GM−CSF. To evaluate IL-5–dependent cell growth, cells were cultured in the presence of SCF, IL-3, and GM-CSF for the first 5 d to promote the development of eosinophil progenitors. Then, cytokines were replaced to IL-5 alone. Results are shown as mean ± SD of three representative experiments (CFU assay), or of triplicate cultures (liquid culture). (C) Flow cytometric profiles of transduced cells cultured for 8 d in the presence of SCF and GM-CSF. Mock represents the cells transduced with empty vector. Results represent repeated experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2193769&req=5

fig1: Altered differentiation of CD34+ progenitors expressing a dominant-negative C/EBP. (A) The schematic representation of the retroviral vector, GCsam–A-C/EBP–IRES–EGFP, encoding A-C/EBP, a dominant-negative C/EBP, linked by an IRES to a cDNA encoding EGFP. The 3′ LTR of the vector is replaced with MSCV. ψ+, packaging signal; SD, splice donor; SA, splice acceptor. (B) The effect of A-C/EBP on the growth of transduced CD34+ cells. After transduction, EGFP-positive cells were selected. Then, cytokine-dependent cell growth was evaluated by CFU generated in the presence of indicated cytokines and by liquid culture in the presence of IL-5 or SCF+GM−CSF. To evaluate IL-5–dependent cell growth, cells were cultured in the presence of SCF, IL-3, and GM-CSF for the first 5 d to promote the development of eosinophil progenitors. Then, cytokines were replaced to IL-5 alone. Results are shown as mean ± SD of three representative experiments (CFU assay), or of triplicate cultures (liquid culture). (C) Flow cytometric profiles of transduced cells cultured for 8 d in the presence of SCF and GM-CSF. Mock represents the cells transduced with empty vector. Results represent repeated experiments.

Mentions: By using a retrovirus-mediated gene transfer system, we expressed A-C/EBP in human cord blood CD34+ progenitor cells and analyzed its effects on myelopoiesis. We transduced cells with the retroviral vector GCsam–A-C/EBP–internal ribosome entry site (IRES)–EGFP (Fig. 1 A), which drives expression of both A-C/EBP and EGFP from a single bicistronic message. After transduction, EGFP-positive cells were selected by cell sorting and subjected to in vitro assay. Among various kinds of cytokines, CD34+ progenitors expressing A-C/EBP failed to respond to myeloid-specific cytokines, including G-CSF, M-CSF, and IL-5 (Fig. 1 B). On the other hand, they normally responded to erythropoietin, an erythroid-specific cytokine, and showed a distinct growth profile in response to GM-CSF as compared with the mock control (Fig. 1 B). GM-CSF is a cytokine that supports cell growth and the differentiation of all kinds of myeloid cells. In the presence of SCF and GM-CSF, the transduced cells showed comparable proliferation with the control at the beginning, but after 1 wk they stopped growing. Flow cytometric analysis revealed that although the transduced cells failed to develop CD15+ granulocytes and CD14+ macrophages, they unexpectedly gave rise to CD1a+ CD11b+ myeloid DCs (Fig. 1 C).


Reciprocal roles for CCAAT/enhancer binding protein (C/EBP) and PU.1 transcription factors in Langerhans cell commitment.

Iwama A, Osawa M, Hirasawa R, Uchiyama N, Kaneko S, Onodera M, Shibuya K, Shibuya A, Vinson C, Tenen DG, Nakauchi H - J. Exp. Med. (2002)

Altered differentiation of CD34+ progenitors expressing a dominant-negative C/EBP. (A) The schematic representation of the retroviral vector, GCsam–A-C/EBP–IRES–EGFP, encoding A-C/EBP, a dominant-negative C/EBP, linked by an IRES to a cDNA encoding EGFP. The 3′ LTR of the vector is replaced with MSCV. ψ+, packaging signal; SD, splice donor; SA, splice acceptor. (B) The effect of A-C/EBP on the growth of transduced CD34+ cells. After transduction, EGFP-positive cells were selected. Then, cytokine-dependent cell growth was evaluated by CFU generated in the presence of indicated cytokines and by liquid culture in the presence of IL-5 or SCF+GM−CSF. To evaluate IL-5–dependent cell growth, cells were cultured in the presence of SCF, IL-3, and GM-CSF for the first 5 d to promote the development of eosinophil progenitors. Then, cytokines were replaced to IL-5 alone. Results are shown as mean ± SD of three representative experiments (CFU assay), or of triplicate cultures (liquid culture). (C) Flow cytometric profiles of transduced cells cultured for 8 d in the presence of SCF and GM-CSF. Mock represents the cells transduced with empty vector. Results represent repeated experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2193769&req=5

fig1: Altered differentiation of CD34+ progenitors expressing a dominant-negative C/EBP. (A) The schematic representation of the retroviral vector, GCsam–A-C/EBP–IRES–EGFP, encoding A-C/EBP, a dominant-negative C/EBP, linked by an IRES to a cDNA encoding EGFP. The 3′ LTR of the vector is replaced with MSCV. ψ+, packaging signal; SD, splice donor; SA, splice acceptor. (B) The effect of A-C/EBP on the growth of transduced CD34+ cells. After transduction, EGFP-positive cells were selected. Then, cytokine-dependent cell growth was evaluated by CFU generated in the presence of indicated cytokines and by liquid culture in the presence of IL-5 or SCF+GM−CSF. To evaluate IL-5–dependent cell growth, cells were cultured in the presence of SCF, IL-3, and GM-CSF for the first 5 d to promote the development of eosinophil progenitors. Then, cytokines were replaced to IL-5 alone. Results are shown as mean ± SD of three representative experiments (CFU assay), or of triplicate cultures (liquid culture). (C) Flow cytometric profiles of transduced cells cultured for 8 d in the presence of SCF and GM-CSF. Mock represents the cells transduced with empty vector. Results represent repeated experiments.
Mentions: By using a retrovirus-mediated gene transfer system, we expressed A-C/EBP in human cord blood CD34+ progenitor cells and analyzed its effects on myelopoiesis. We transduced cells with the retroviral vector GCsam–A-C/EBP–internal ribosome entry site (IRES)–EGFP (Fig. 1 A), which drives expression of both A-C/EBP and EGFP from a single bicistronic message. After transduction, EGFP-positive cells were selected by cell sorting and subjected to in vitro assay. Among various kinds of cytokines, CD34+ progenitors expressing A-C/EBP failed to respond to myeloid-specific cytokines, including G-CSF, M-CSF, and IL-5 (Fig. 1 B). On the other hand, they normally responded to erythropoietin, an erythroid-specific cytokine, and showed a distinct growth profile in response to GM-CSF as compared with the mock control (Fig. 1 B). GM-CSF is a cytokine that supports cell growth and the differentiation of all kinds of myeloid cells. In the presence of SCF and GM-CSF, the transduced cells showed comparable proliferation with the control at the beginning, but after 1 wk they stopped growing. Flow cytometric analysis revealed that although the transduced cells failed to develop CD15+ granulocytes and CD14+ macrophages, they unexpectedly gave rise to CD1a+ CD11b+ myeloid DCs (Fig. 1 C).

Bottom Line: PU.1 and CCAAT/enhancing binding protein (C/EBP) family transcription factors have been characterized as key regulators for the development and function of the myeloid system.However, the roles of C/EBP transcription factors have not been fully identified because of functional redundancy among family members.Our findings establish reciprocal roles for C/EBP and PU.1 in LC development, and provide new insight into the molecular mechanism of LC development, which has not yet been well characterized.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Institute of Basic Medical Sciences, Core Research for Evolutional Science and Technology (CREST) Program of Japan Science and Technology (JST). aiwama@md.tsukuba.ac.jp

ABSTRACT
Myeloid progenitor cells give rise to a variety of progenies including dendritic cells. However, the mechanism controlling the diversification of myeloid progenitors into each progeny is largely unknown. PU.1 and CCAAT/enhancing binding protein (C/EBP) family transcription factors have been characterized as key regulators for the development and function of the myeloid system. However, the roles of C/EBP transcription factors have not been fully identified because of functional redundancy among family members. Using high titer--retroviral infection, we demonstrate that a dominant-negative C/EBP completely blocked the granulocyte--macrophage commitment of human myeloid progenitors. Alternatively, Langerhans cell (LC) commitment was markedly facilitated in the absence of tumor necrosis factor (TNF)alpha, a strong inducer of LC development, whereas expression of wild-type C/EBP in myeloid progenitors promoted granulocytic differentiation, and completely inhibited TNFalpha-dependent LC development. On the other hand, expression of wild-type PU.1 in myeloid progenitors triggered LC development in the absence of TNFalpha, and its instructive effect was canceled by coexpressed C/EBP. Our findings establish reciprocal roles for C/EBP and PU.1 in LC development, and provide new insight into the molecular mechanism of LC development, which has not yet been well characterized.

Show MeSH
Related in: MedlinePlus