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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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Progressive enlargement of the thymus in the MKK6(Glu) transgenic mice. (A) p38 MAP kinase activity in thymocyte populations from wild-type mice. Whole cell extracts (4 × 105) of freshly isolated total thymocytes (T.T), DN, DP, and mature single CD4+ thymocytes from wild-type mice were assayed for p38 MAP kinase activity using the substrate GST-ATF2. (B) Expression of MKK6(Glu) in the MKK6(Glu) transgenic mice. The expression of endogenous p38 MAP kinase and the MKK6(Glu) transgene (MKK6) was analyzed in thymocytes and spleen cells from negative littermate control mice (NLC) and mice from three different MKK6(Glu) transgenic (Tg+) lines (lines 45, 3, and 6) by Western blot analysis. Blots were probed with either an anti-Flag antibody (MKK6) or an anti-p38 MAP kinase polyclonal antibody (p38). (C) Activation of p38 MAP kinase by the expression of MKK6(Glu) in thymocytes. Total thymocytes (5 × 105 cells) from MKK6(Glu) transgenic mice and negative littermate control mice were lysed, and whole extracts were assayed for p38 activity using the substrate GST-ATF2 or for JNK activity using the substrate GST-c-Jun. (D) Diminished thymocyte number in the MKK6(Glu) transgenic mice. Data represent the percentage of the total number of cells in the thymus from the MKK6(Glu) transgenic mice from different lines compared with the total number of thymocytes in negative littermate control mice. (E) Enlarged thymus in the MKK6(Glu) transgenic mice. Thymi from 3- and 5.5-mo-old (3 m and 5 m, respectively) negative littermate control and MKK6(Glu) transgenic (line 6) mice (left two panels). Thymus (thick arrow) and lungs (thin arrow) from 5.5-mo-old (5 m) negative littermate control and MKK6(Glu) transgenic mice (right two panels). (F) Weight of thymi from 2.5–3.5- and 4.5–5.5-mo-old (3 m and 5 m, respectively) negative littermate control and MKK6(Glu) transgenic mice. Values represent average weight (n = 4). (G) Progressive accumulation of thymocytes in the MKK6(Glu) transgenic mice. Values represent the average (n = 4) of the number of total thymocytes in 3- and 5-mo-old negative littermate control and MKK6(Glu) transgenic mice.
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Figure 1: Progressive enlargement of the thymus in the MKK6(Glu) transgenic mice. (A) p38 MAP kinase activity in thymocyte populations from wild-type mice. Whole cell extracts (4 × 105) of freshly isolated total thymocytes (T.T), DN, DP, and mature single CD4+ thymocytes from wild-type mice were assayed for p38 MAP kinase activity using the substrate GST-ATF2. (B) Expression of MKK6(Glu) in the MKK6(Glu) transgenic mice. The expression of endogenous p38 MAP kinase and the MKK6(Glu) transgene (MKK6) was analyzed in thymocytes and spleen cells from negative littermate control mice (NLC) and mice from three different MKK6(Glu) transgenic (Tg+) lines (lines 45, 3, and 6) by Western blot analysis. Blots were probed with either an anti-Flag antibody (MKK6) or an anti-p38 MAP kinase polyclonal antibody (p38). (C) Activation of p38 MAP kinase by the expression of MKK6(Glu) in thymocytes. Total thymocytes (5 × 105 cells) from MKK6(Glu) transgenic mice and negative littermate control mice were lysed, and whole extracts were assayed for p38 activity using the substrate GST-ATF2 or for JNK activity using the substrate GST-c-Jun. (D) Diminished thymocyte number in the MKK6(Glu) transgenic mice. Data represent the percentage of the total number of cells in the thymus from the MKK6(Glu) transgenic mice from different lines compared with the total number of thymocytes in negative littermate control mice. (E) Enlarged thymus in the MKK6(Glu) transgenic mice. Thymi from 3- and 5.5-mo-old (3 m and 5 m, respectively) negative littermate control and MKK6(Glu) transgenic (line 6) mice (left two panels). Thymus (thick arrow) and lungs (thin arrow) from 5.5-mo-old (5 m) negative littermate control and MKK6(Glu) transgenic mice (right two panels). (F) Weight of thymi from 2.5–3.5- and 4.5–5.5-mo-old (3 m and 5 m, respectively) negative littermate control and MKK6(Glu) transgenic mice. Values represent average weight (n = 4). (G) Progressive accumulation of thymocytes in the MKK6(Glu) transgenic mice. Values represent the average (n = 4) of the number of total thymocytes in 3- and 5-mo-old negative littermate control and MKK6(Glu) transgenic mice.

Mentions: To investigate the role of the p38 MAP kinase pathway in thymocyte development, we first examined p38 MAP kinase activity in freshly isolated DN, DP, and mature CD4+ thymocytes from wild-type mice. Consistent with a previous report 14, we found that p38 MAP kinase was highly activated in DN thymocytes (Fig. 1 A). The level of p38 MAP kinase activity in DP thymocytes was lower than the activity found in DN thymocytes. Only low levels of p38 MAP kinase activity were detected in mature CD4+ thymocytes (Fig. 1 A). These results suggested that inactivation of p38 MAP kinase may be important for thymocyte maturation. To address this possibility, we have developed a mouse model in which p38 MAP kinase is persistently activated during early thymocyte development.


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)

Progressive enlargement of the thymus in the MKK6(Glu) transgenic mice. (A) p38 MAP kinase activity in thymocyte populations from wild-type mice. Whole cell extracts (4 × 105) of freshly isolated total thymocytes (T.T), DN, DP, and mature single CD4+ thymocytes from wild-type mice were assayed for p38 MAP kinase activity using the substrate GST-ATF2. (B) Expression of MKK6(Glu) in the MKK6(Glu) transgenic mice. The expression of endogenous p38 MAP kinase and the MKK6(Glu) transgene (MKK6) was analyzed in thymocytes and spleen cells from negative littermate control mice (NLC) and mice from three different MKK6(Glu) transgenic (Tg+) lines (lines 45, 3, and 6) by Western blot analysis. Blots were probed with either an anti-Flag antibody (MKK6) or an anti-p38 MAP kinase polyclonal antibody (p38). (C) Activation of p38 MAP kinase by the expression of MKK6(Glu) in thymocytes. Total thymocytes (5 × 105 cells) from MKK6(Glu) transgenic mice and negative littermate control mice were lysed, and whole extracts were assayed for p38 activity using the substrate GST-ATF2 or for JNK activity using the substrate GST-c-Jun. (D) Diminished thymocyte number in the MKK6(Glu) transgenic mice. Data represent the percentage of the total number of cells in the thymus from the MKK6(Glu) transgenic mice from different lines compared with the total number of thymocytes in negative littermate control mice. (E) Enlarged thymus in the MKK6(Glu) transgenic mice. Thymi from 3- and 5.5-mo-old (3 m and 5 m, respectively) negative littermate control and MKK6(Glu) transgenic (line 6) mice (left two panels). Thymus (thick arrow) and lungs (thin arrow) from 5.5-mo-old (5 m) negative littermate control and MKK6(Glu) transgenic mice (right two panels). (F) Weight of thymi from 2.5–3.5- and 4.5–5.5-mo-old (3 m and 5 m, respectively) negative littermate control and MKK6(Glu) transgenic mice. Values represent average weight (n = 4). (G) Progressive accumulation of thymocytes in the MKK6(Glu) transgenic mice. Values represent the average (n = 4) of the number of total thymocytes in 3- and 5-mo-old negative littermate control and MKK6(Glu) transgenic mice.
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Related In: Results  -  Collection

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Figure 1: Progressive enlargement of the thymus in the MKK6(Glu) transgenic mice. (A) p38 MAP kinase activity in thymocyte populations from wild-type mice. Whole cell extracts (4 × 105) of freshly isolated total thymocytes (T.T), DN, DP, and mature single CD4+ thymocytes from wild-type mice were assayed for p38 MAP kinase activity using the substrate GST-ATF2. (B) Expression of MKK6(Glu) in the MKK6(Glu) transgenic mice. The expression of endogenous p38 MAP kinase and the MKK6(Glu) transgene (MKK6) was analyzed in thymocytes and spleen cells from negative littermate control mice (NLC) and mice from three different MKK6(Glu) transgenic (Tg+) lines (lines 45, 3, and 6) by Western blot analysis. Blots were probed with either an anti-Flag antibody (MKK6) or an anti-p38 MAP kinase polyclonal antibody (p38). (C) Activation of p38 MAP kinase by the expression of MKK6(Glu) in thymocytes. Total thymocytes (5 × 105 cells) from MKK6(Glu) transgenic mice and negative littermate control mice were lysed, and whole extracts were assayed for p38 activity using the substrate GST-ATF2 or for JNK activity using the substrate GST-c-Jun. (D) Diminished thymocyte number in the MKK6(Glu) transgenic mice. Data represent the percentage of the total number of cells in the thymus from the MKK6(Glu) transgenic mice from different lines compared with the total number of thymocytes in negative littermate control mice. (E) Enlarged thymus in the MKK6(Glu) transgenic mice. Thymi from 3- and 5.5-mo-old (3 m and 5 m, respectively) negative littermate control and MKK6(Glu) transgenic (line 6) mice (left two panels). Thymus (thick arrow) and lungs (thin arrow) from 5.5-mo-old (5 m) negative littermate control and MKK6(Glu) transgenic mice (right two panels). (F) Weight of thymi from 2.5–3.5- and 4.5–5.5-mo-old (3 m and 5 m, respectively) negative littermate control and MKK6(Glu) transgenic mice. Values represent average weight (n = 4). (G) Progressive accumulation of thymocytes in the MKK6(Glu) transgenic mice. Values represent the average (n = 4) of the number of total thymocytes in 3- and 5-mo-old negative littermate control and MKK6(Glu) transgenic mice.
Mentions: To investigate the role of the p38 MAP kinase pathway in thymocyte development, we first examined p38 MAP kinase activity in freshly isolated DN, DP, and mature CD4+ thymocytes from wild-type mice. Consistent with a previous report 14, we found that p38 MAP kinase was highly activated in DN thymocytes (Fig. 1 A). The level of p38 MAP kinase activity in DP thymocytes was lower than the activity found in DN thymocytes. Only low levels of p38 MAP kinase activity were detected in mature CD4+ thymocytes (Fig. 1 A). These results suggested that inactivation of p38 MAP kinase may be important for thymocyte maturation. To address this possibility, we have developed a mouse model in which p38 MAP kinase is persistently activated during early thymocyte development.

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