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Activation of the p38 mitogen-activated protein kinase pathway arrests cell cycle progression and differentiation of immature thymocytes in vivo.

Diehl NL, Enslen H, Fortner KA, Merritt C, Stetson N, Charland C, Flavell RA, Davis RJ, Rincón M - J. Exp. Med. (2000)

Bottom Line: The development of T cells in the thymus is coordinated by cell-specific gene expression programs that involve multiple transcription factors and signaling pathways.Persistent activation of p38 MAP kinase blocks fetal thymocyte development at the CD25(+)CD44(-) stage in vivo, and results in the lack of T cells in the peripheral immune system of adult mice.The arrest of cell cycle in mitosis is partially responsible for the blockade of differentiation.

View Article: PubMed Central - PubMed

Affiliation: Immunobiology Program, Department of Medicine, University of Vermont, Burlington, Vermont 05405, USA.

ABSTRACT
The development of T cells in the thymus is coordinated by cell-specific gene expression programs that involve multiple transcription factors and signaling pathways. Here, we show that the p38 mitogen-activated protein (MAP) kinase signaling pathway is strictly regulated during the differentiation of CD4(-)CD8(-) thymocytes. Persistent activation of p38 MAP kinase blocks fetal thymocyte development at the CD25(+)CD44(-) stage in vivo, and results in the lack of T cells in the peripheral immune system of adult mice. Inactivation of p38 MAP kinase is required for further differentiation of these cells into CD4(+)CD8(+) thymocytes. The arrest of cell cycle in mitosis is partially responsible for the blockade of differentiation. Therefore, the p38 MAP kinase pathway is a critical regulatory element of differentiation and proliferation during the early stages of in vivo thymocyte development.

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Inactivation of p38 MAP kinase is required for CD25+CD44− cell cycle progression and differentiation. (A) Inactivation of p38 MAP kinase allows progression of mitosis in CD25+CD44− MKK6(Glu) thymocytes. Thymocytes from the MKK6(Glu) transgenic mice were incubated in the presence of the p38 MAP kinase inhibitor, SB203580, at the indicated dose. After 6 h, the cells were cytospun and stained with Giemsa. Two or three fields from the same slide are presented. (B) Decreased cyclin A expression in MKK6(Glu) thymocytes upon inactivation of p38 MAP kinase. Total thymocytes from the MKK6(Glu) transgenic mice were treated in the presence (red line) or absence (black line) of SB203580 (1 μM) for 12 h. Cyclin A expression was examined by intracellular staining. (C) Inactivation of p38 MAP kinase restores differentiation of CD25+CD44− MKK6(Glu) thymocytes. Total thymocytes from adult negative littermate control (NLC) or MKK6(Glu) transgenic (Tg+) mice were incubated for 2 d with the indicated concentrations of the p38 MAP kinase inhibitor, SB203580. The recovered cells were stained for CD4 and CD8 and analyzed by flow cytometry. (D) Inhibition of p38 MAP kinase in MKK6(Glu) thymocytes restores cell proliferation. Total thymocytes (106 cells) were treated as described in C. After 2 d, the number of live cells was determined by trypan blue staining.
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Figure 5: Inactivation of p38 MAP kinase is required for CD25+CD44− cell cycle progression and differentiation. (A) Inactivation of p38 MAP kinase allows progression of mitosis in CD25+CD44− MKK6(Glu) thymocytes. Thymocytes from the MKK6(Glu) transgenic mice were incubated in the presence of the p38 MAP kinase inhibitor, SB203580, at the indicated dose. After 6 h, the cells were cytospun and stained with Giemsa. Two or three fields from the same slide are presented. (B) Decreased cyclin A expression in MKK6(Glu) thymocytes upon inactivation of p38 MAP kinase. Total thymocytes from the MKK6(Glu) transgenic mice were treated in the presence (red line) or absence (black line) of SB203580 (1 μM) for 12 h. Cyclin A expression was examined by intracellular staining. (C) Inactivation of p38 MAP kinase restores differentiation of CD25+CD44− MKK6(Glu) thymocytes. Total thymocytes from adult negative littermate control (NLC) or MKK6(Glu) transgenic (Tg+) mice were incubated for 2 d with the indicated concentrations of the p38 MAP kinase inhibitor, SB203580. The recovered cells were stained for CD4 and CD8 and analyzed by flow cytometry. (D) Inhibition of p38 MAP kinase in MKK6(Glu) thymocytes restores cell proliferation. Total thymocytes (106 cells) were treated as described in C. After 2 d, the number of live cells was determined by trypan blue staining.

Mentions: Differentiation and proliferation are closely regulated during the development of T cells in the thymus. Differentiation of CD25−CD44+ thymocytes into CD25+CD44+ and CD25+CD44− thymocytes is associated with some proliferation, but the principal expansion of cells occurs during the differentiation of CD25+CD44− thymocytes into CD25− CD44− thymocytes (for a review, see reference 1). However, it is unclear which of these processes, cell cycle progression or differentiation, initiates the transition between two differentiation stages. Our results indicated that activation of p38 MAP kinase prevented cell cycle progression and differentiation of CD25+CD44− thymocytes. To address which of these two biological functions is the cause of the arrest of thymocyte development, we examined the effect of p38 MAP kinase inhibition on thymocytes from the MKK6(Glu) transgenic mice. Thymocytes from adult MKK6 (Glu) transgenic mice were incubated in vitro with medium alone or in the presence of different concentrations of SB203580, a pyridinyl imidazol drug that selectively inhibits p38 MAP kinase 18. After 6 h, the nuclear morphology of MKK6(Glu) thymocytes was examined by Giemsa staining. As shown above, in the absence of the inhibitor a large proportion of these cells remained in prophase (Fig. 5 A). However, after treatment with 1 μM SB203580, a significant number of thymocytes in late anaphase/early telophase was observed (Fig. 5 A). Cells in late telophase and small cells in interphase were also detected upon incubation with 5 μM SB203580 (Fig. 5 A). No SB203580 effect was observed in control thymocytes (data not shown). Downregulation of cyclin A expression was also observed in the MKK6(Glu) thymocytes treated with SB203580 (Fig. 5 B). These results indicated that inhibition of p38 MAP kinase is required for progression of mitosis in CD25+CD44− thymocytes.


Activation of the p38 mitogen-activated protein kinase pathway arrests cell cycle progression and differentiation of immature thymocytes in vivo.

Diehl NL, Enslen H, Fortner KA, Merritt C, Stetson N, Charland C, Flavell RA, Davis RJ, Rincón M - J. Exp. Med. (2000)

Inactivation of p38 MAP kinase is required for CD25+CD44− cell cycle progression and differentiation. (A) Inactivation of p38 MAP kinase allows progression of mitosis in CD25+CD44− MKK6(Glu) thymocytes. Thymocytes from the MKK6(Glu) transgenic mice were incubated in the presence of the p38 MAP kinase inhibitor, SB203580, at the indicated dose. After 6 h, the cells were cytospun and stained with Giemsa. Two or three fields from the same slide are presented. (B) Decreased cyclin A expression in MKK6(Glu) thymocytes upon inactivation of p38 MAP kinase. Total thymocytes from the MKK6(Glu) transgenic mice were treated in the presence (red line) or absence (black line) of SB203580 (1 μM) for 12 h. Cyclin A expression was examined by intracellular staining. (C) Inactivation of p38 MAP kinase restores differentiation of CD25+CD44− MKK6(Glu) thymocytes. Total thymocytes from adult negative littermate control (NLC) or MKK6(Glu) transgenic (Tg+) mice were incubated for 2 d with the indicated concentrations of the p38 MAP kinase inhibitor, SB203580. The recovered cells were stained for CD4 and CD8 and analyzed by flow cytometry. (D) Inhibition of p38 MAP kinase in MKK6(Glu) thymocytes restores cell proliferation. Total thymocytes (106 cells) were treated as described in C. After 2 d, the number of live cells was determined by trypan blue staining.
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Related In: Results  -  Collection

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Figure 5: Inactivation of p38 MAP kinase is required for CD25+CD44− cell cycle progression and differentiation. (A) Inactivation of p38 MAP kinase allows progression of mitosis in CD25+CD44− MKK6(Glu) thymocytes. Thymocytes from the MKK6(Glu) transgenic mice were incubated in the presence of the p38 MAP kinase inhibitor, SB203580, at the indicated dose. After 6 h, the cells were cytospun and stained with Giemsa. Two or three fields from the same slide are presented. (B) Decreased cyclin A expression in MKK6(Glu) thymocytes upon inactivation of p38 MAP kinase. Total thymocytes from the MKK6(Glu) transgenic mice were treated in the presence (red line) or absence (black line) of SB203580 (1 μM) for 12 h. Cyclin A expression was examined by intracellular staining. (C) Inactivation of p38 MAP kinase restores differentiation of CD25+CD44− MKK6(Glu) thymocytes. Total thymocytes from adult negative littermate control (NLC) or MKK6(Glu) transgenic (Tg+) mice were incubated for 2 d with the indicated concentrations of the p38 MAP kinase inhibitor, SB203580. The recovered cells were stained for CD4 and CD8 and analyzed by flow cytometry. (D) Inhibition of p38 MAP kinase in MKK6(Glu) thymocytes restores cell proliferation. Total thymocytes (106 cells) were treated as described in C. After 2 d, the number of live cells was determined by trypan blue staining.
Mentions: Differentiation and proliferation are closely regulated during the development of T cells in the thymus. Differentiation of CD25−CD44+ thymocytes into CD25+CD44+ and CD25+CD44− thymocytes is associated with some proliferation, but the principal expansion of cells occurs during the differentiation of CD25+CD44− thymocytes into CD25− CD44− thymocytes (for a review, see reference 1). However, it is unclear which of these processes, cell cycle progression or differentiation, initiates the transition between two differentiation stages. Our results indicated that activation of p38 MAP kinase prevented cell cycle progression and differentiation of CD25+CD44− thymocytes. To address which of these two biological functions is the cause of the arrest of thymocyte development, we examined the effect of p38 MAP kinase inhibition on thymocytes from the MKK6(Glu) transgenic mice. Thymocytes from adult MKK6 (Glu) transgenic mice were incubated in vitro with medium alone or in the presence of different concentrations of SB203580, a pyridinyl imidazol drug that selectively inhibits p38 MAP kinase 18. After 6 h, the nuclear morphology of MKK6(Glu) thymocytes was examined by Giemsa staining. As shown above, in the absence of the inhibitor a large proportion of these cells remained in prophase (Fig. 5 A). However, after treatment with 1 μM SB203580, a significant number of thymocytes in late anaphase/early telophase was observed (Fig. 5 A). Cells in late telophase and small cells in interphase were also detected upon incubation with 5 μM SB203580 (Fig. 5 A). No SB203580 effect was observed in control thymocytes (data not shown). Downregulation of cyclin A expression was also observed in the MKK6(Glu) thymocytes treated with SB203580 (Fig. 5 B). These results indicated that inhibition of p38 MAP kinase is required for progression of mitosis in CD25+CD44− thymocytes.

Bottom Line: The development of T cells in the thymus is coordinated by cell-specific gene expression programs that involve multiple transcription factors and signaling pathways.Persistent activation of p38 MAP kinase blocks fetal thymocyte development at the CD25(+)CD44(-) stage in vivo, and results in the lack of T cells in the peripheral immune system of adult mice.The arrest of cell cycle in mitosis is partially responsible for the blockade of differentiation.

View Article: PubMed Central - PubMed

Affiliation: Immunobiology Program, Department of Medicine, University of Vermont, Burlington, Vermont 05405, USA.

ABSTRACT
The development of T cells in the thymus is coordinated by cell-specific gene expression programs that involve multiple transcription factors and signaling pathways. Here, we show that the p38 mitogen-activated protein (MAP) kinase signaling pathway is strictly regulated during the differentiation of CD4(-)CD8(-) thymocytes. Persistent activation of p38 MAP kinase blocks fetal thymocyte development at the CD25(+)CD44(-) stage in vivo, and results in the lack of T cells in the peripheral immune system of adult mice. Inactivation of p38 MAP kinase is required for further differentiation of these cells into CD4(+)CD8(+) thymocytes. The arrest of cell cycle in mitosis is partially responsible for the blockade of differentiation. Therefore, the p38 MAP kinase pathway is a critical regulatory element of differentiation and proliferation during the early stages of in vivo thymocyte development.

Show MeSH
Related in: MedlinePlus