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The stress kinase mitogen-activated protein kinase kinase (MKK)7 is a negative regulator of antigen receptor and growth factor receptor-induced proliferation in hematopoietic cells.

Sasaki T, Wada T, Kishimoto H, Irie-Sasaki J, Matsumoto G, Goto T, Yao Z, Wakeham A, Mak TW, Suzuki A, Cho SK, Zuniga-Pflucker JC, Oliveira-dos-Santos AJ, Katada T, Nishina H, Penninger JM - J. Exp. Med. (2001)

Bottom Line: Mutation of mkk7 in mast cells resulted in hyperproliferation in response to the cytokines interleukin (IL)-3 and stem cell factor (SCF).Loss of MKK7 did not affect activation of extracellular signal-regulated kinase (ERK)1/2 or p38 MAPK. mkk7(-/-) mast cells display reduced expression of JunB and the cell cycle inhibitor p16INK4a and upregulation of cyclinD1.These results indicate that the MKK7-regulated stress signaling pathway can function as negative regulator of cell growth in multiple hematopoietic lineages.

View Article: PubMed Central - PubMed

Affiliation: Amgen Institute, Ontario Cancer Institute, Canada.

ABSTRACT
The dual specificity kinases mitogen-activated protein kinase (MAPK) kinase (MKK)7 and MKK4 are the only molecules known to directly activate the stress kinases stress-activated protein kinases (SAPKs)/c-Jun N-terminal kinases (JNKs) in response to environmental or mitogenic stimuli. To examine the physiological role of MKK7 in hematopoietic cells, we used a gene targeting strategy to mutate MKK7 in murine T and B cells and non-lymphoid mast cells. Loss of MKK7 in thymocytes and mature B cells results in hyperproliferation in response to growth factor and antigen receptor stimulation and increased thymic cellularity. Mutation of mkk7 in mast cells resulted in hyperproliferation in response to the cytokines interleukin (IL)-3 and stem cell factor (SCF). SAPK/JNK activation was completely abolished in the absence of MKK7, even though expression of MKK4 was strongly upregulated in mkk7(-/-) mast cell lines, and phosphorylation of MKK4 occurred normally in response to multiple stress stimuli. Loss of MKK7 did not affect activation of extracellular signal-regulated kinase (ERK)1/2 or p38 MAPK. mkk7(-/-) mast cells display reduced expression of JunB and the cell cycle inhibitor p16INK4a and upregulation of cyclinD1. Reexpression of p16INK4a in mkk7(-/-) mast cells abrogates the hyperproliferative response. Apoptotic responses to a variety of stimuli were not affected. Thus, MKK7 is an essential and specific regulator of stress-induced SAPK/JNK activation in mast cells and MKK7 negatively regulates growth factor and antigen receptor-driven proliferation in hematopoietic cells. These results indicate that the MKK7-regulated stress signaling pathway can function as negative regulator of cell growth in multiple hematopoietic lineages.

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MKK7 is essential for SAPK/JNK activation in BMMCs. (A) Western blot analysis of expression levels of total p54 and p46 SAPK/JNK, p38 MAPK, ERK1, PKB/Akt, JunB, p16INK4a, p27, and cyclinD1 in mkk7+/+ (+/+) and mkk7−/− (−/−) BMMCs. Actin is shown as a loading control. Total cell lysate proteins (30 μg/lane) were separated by SDS-PAGE and incubated with Abs specific for the indicated molecules. (B) Western blot analysis of expression levels of p16INK4a and actin in mkk7+/+ (+/+) and mkk7−/− (−/−) B220+IgM+ B cells. B cells were differentiated in vitro from mkk7+/+ and mkk7−/− ES cells as described (reference 29). Total cell lysates of 106 cells were separated by SDS-PAGE and incubated with Abs specific for the indicated molecules. (C and D) SAPK/JNK activity in mkk7+/+ (+/+) and mkk7−/− (−/−) BMMCs. BMMCs were left untreated (None) or activated with anti-DNP IgE (7 μg/ml) plus DNP (50 ng/ml, 15 min), IL-3 (30 ng/ml, 20 min), or anisomycin (Aniso; 10 μg/ml, 20 min), heat shock (heat; 45°C, 30 min), UV-irradiation (500 mJ, 15 min), NaCl (0.5 M, 10 min), cycloheximide (CHX; 50 μg/ml, 20 min), TNFα (100 ng/ml, 20 min), or TNFα (100 ng/ml) plus CHX (50 μg/ml) for 20 min. Total SAPK/JNK was immunoprecipitated and assayed for in vitro kinase activity using GST-c-Jun as the substrate. In C, the levels of immunoprecipitated p46 and p54 SAPK/JNK are shown as a loading control. One result representative of four independent experiments is shown. (E) Expression and phosphorylation (Thr223) of MKK4 in mkk7+/+ (+/+) and mkk7−/− (−/−) BMMCs. BMMCs were stimulated with the indicated agents as in B. Proteins were separated by SDS-PAGE and detected using an Ab reactive to total MKK4 or an Ab specific for phospho-MKK4 (Thr223). Thr223 phosphorylation is indicative of activated MKK4. Actin levels are shown as a loading control.
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Figure 6: MKK7 is essential for SAPK/JNK activation in BMMCs. (A) Western blot analysis of expression levels of total p54 and p46 SAPK/JNK, p38 MAPK, ERK1, PKB/Akt, JunB, p16INK4a, p27, and cyclinD1 in mkk7+/+ (+/+) and mkk7−/− (−/−) BMMCs. Actin is shown as a loading control. Total cell lysate proteins (30 μg/lane) were separated by SDS-PAGE and incubated with Abs specific for the indicated molecules. (B) Western blot analysis of expression levels of p16INK4a and actin in mkk7+/+ (+/+) and mkk7−/− (−/−) B220+IgM+ B cells. B cells were differentiated in vitro from mkk7+/+ and mkk7−/− ES cells as described (reference 29). Total cell lysates of 106 cells were separated by SDS-PAGE and incubated with Abs specific for the indicated molecules. (C and D) SAPK/JNK activity in mkk7+/+ (+/+) and mkk7−/− (−/−) BMMCs. BMMCs were left untreated (None) or activated with anti-DNP IgE (7 μg/ml) plus DNP (50 ng/ml, 15 min), IL-3 (30 ng/ml, 20 min), or anisomycin (Aniso; 10 μg/ml, 20 min), heat shock (heat; 45°C, 30 min), UV-irradiation (500 mJ, 15 min), NaCl (0.5 M, 10 min), cycloheximide (CHX; 50 μg/ml, 20 min), TNFα (100 ng/ml, 20 min), or TNFα (100 ng/ml) plus CHX (50 μg/ml) for 20 min. Total SAPK/JNK was immunoprecipitated and assayed for in vitro kinase activity using GST-c-Jun as the substrate. In C, the levels of immunoprecipitated p46 and p54 SAPK/JNK are shown as a loading control. One result representative of four independent experiments is shown. (E) Expression and phosphorylation (Thr223) of MKK4 in mkk7+/+ (+/+) and mkk7−/− (−/−) BMMCs. BMMCs were stimulated with the indicated agents as in B. Proteins were separated by SDS-PAGE and detected using an Ab reactive to total MKK4 or an Ab specific for phospho-MKK4 (Thr223). Thr223 phosphorylation is indicative of activated MKK4. Actin levels are shown as a loading control.

Mentions: At the molecular level, immunoblot analyses of proteins in BMMCs revealed that expression levels of p46 and p56 SAPK/JNK isoforms, p38 MAPK, ERK1/ERK2, PKB/Akt, and actin were comparable in mkk7+/+ and mkk7−/− BMMCs (Fig. 6 A). However, expression of the cell cycle inhibitory molecules, p16INK4a, which act principally on cyclinD1 and CDK4-6, was completely abrogated in mkk7−/− BMMCs, leading to a concomitant increase in cyclinD1 expression (Fig. 6 A). Interestingly, whereas expression of c-Jun was comparable between mkk7+/+ and mkk7−/− BMMCs, expression of JunB, which has been shown to upregulate p16INK4a expression 38, was markedly decreased in mkk7−/− BMMCs. Expression of p27, another cell cycle inhibitor, was not affected by the absence of MKK7 (Fig. 6 A). However, it should be noted that loss of MKK7 does not always result in downregulation of p16INK4a expression, as p16INK4a expression appeared normal in B220+IgM+ B cells after in vitro differentiation of mkk7+/+ and mkk7−/− ES cells into B cells (Fig. 6 B, and data not shown). Thus, mutation of mkk7 in BMMCs, but not in B cells, results in loss of expression of JunB and the cell cycle inhibitor p16INK4a and upregulation of cyclinD1.


The stress kinase mitogen-activated protein kinase kinase (MKK)7 is a negative regulator of antigen receptor and growth factor receptor-induced proliferation in hematopoietic cells.

Sasaki T, Wada T, Kishimoto H, Irie-Sasaki J, Matsumoto G, Goto T, Yao Z, Wakeham A, Mak TW, Suzuki A, Cho SK, Zuniga-Pflucker JC, Oliveira-dos-Santos AJ, Katada T, Nishina H, Penninger JM - J. Exp. Med. (2001)

MKK7 is essential for SAPK/JNK activation in BMMCs. (A) Western blot analysis of expression levels of total p54 and p46 SAPK/JNK, p38 MAPK, ERK1, PKB/Akt, JunB, p16INK4a, p27, and cyclinD1 in mkk7+/+ (+/+) and mkk7−/− (−/−) BMMCs. Actin is shown as a loading control. Total cell lysate proteins (30 μg/lane) were separated by SDS-PAGE and incubated with Abs specific for the indicated molecules. (B) Western blot analysis of expression levels of p16INK4a and actin in mkk7+/+ (+/+) and mkk7−/− (−/−) B220+IgM+ B cells. B cells were differentiated in vitro from mkk7+/+ and mkk7−/− ES cells as described (reference 29). Total cell lysates of 106 cells were separated by SDS-PAGE and incubated with Abs specific for the indicated molecules. (C and D) SAPK/JNK activity in mkk7+/+ (+/+) and mkk7−/− (−/−) BMMCs. BMMCs were left untreated (None) or activated with anti-DNP IgE (7 μg/ml) plus DNP (50 ng/ml, 15 min), IL-3 (30 ng/ml, 20 min), or anisomycin (Aniso; 10 μg/ml, 20 min), heat shock (heat; 45°C, 30 min), UV-irradiation (500 mJ, 15 min), NaCl (0.5 M, 10 min), cycloheximide (CHX; 50 μg/ml, 20 min), TNFα (100 ng/ml, 20 min), or TNFα (100 ng/ml) plus CHX (50 μg/ml) for 20 min. Total SAPK/JNK was immunoprecipitated and assayed for in vitro kinase activity using GST-c-Jun as the substrate. In C, the levels of immunoprecipitated p46 and p54 SAPK/JNK are shown as a loading control. One result representative of four independent experiments is shown. (E) Expression and phosphorylation (Thr223) of MKK4 in mkk7+/+ (+/+) and mkk7−/− (−/−) BMMCs. BMMCs were stimulated with the indicated agents as in B. Proteins were separated by SDS-PAGE and detected using an Ab reactive to total MKK4 or an Ab specific for phospho-MKK4 (Thr223). Thr223 phosphorylation is indicative of activated MKK4. Actin levels are shown as a loading control.
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Figure 6: MKK7 is essential for SAPK/JNK activation in BMMCs. (A) Western blot analysis of expression levels of total p54 and p46 SAPK/JNK, p38 MAPK, ERK1, PKB/Akt, JunB, p16INK4a, p27, and cyclinD1 in mkk7+/+ (+/+) and mkk7−/− (−/−) BMMCs. Actin is shown as a loading control. Total cell lysate proteins (30 μg/lane) were separated by SDS-PAGE and incubated with Abs specific for the indicated molecules. (B) Western blot analysis of expression levels of p16INK4a and actin in mkk7+/+ (+/+) and mkk7−/− (−/−) B220+IgM+ B cells. B cells were differentiated in vitro from mkk7+/+ and mkk7−/− ES cells as described (reference 29). Total cell lysates of 106 cells were separated by SDS-PAGE and incubated with Abs specific for the indicated molecules. (C and D) SAPK/JNK activity in mkk7+/+ (+/+) and mkk7−/− (−/−) BMMCs. BMMCs were left untreated (None) or activated with anti-DNP IgE (7 μg/ml) plus DNP (50 ng/ml, 15 min), IL-3 (30 ng/ml, 20 min), or anisomycin (Aniso; 10 μg/ml, 20 min), heat shock (heat; 45°C, 30 min), UV-irradiation (500 mJ, 15 min), NaCl (0.5 M, 10 min), cycloheximide (CHX; 50 μg/ml, 20 min), TNFα (100 ng/ml, 20 min), or TNFα (100 ng/ml) plus CHX (50 μg/ml) for 20 min. Total SAPK/JNK was immunoprecipitated and assayed for in vitro kinase activity using GST-c-Jun as the substrate. In C, the levels of immunoprecipitated p46 and p54 SAPK/JNK are shown as a loading control. One result representative of four independent experiments is shown. (E) Expression and phosphorylation (Thr223) of MKK4 in mkk7+/+ (+/+) and mkk7−/− (−/−) BMMCs. BMMCs were stimulated with the indicated agents as in B. Proteins were separated by SDS-PAGE and detected using an Ab reactive to total MKK4 or an Ab specific for phospho-MKK4 (Thr223). Thr223 phosphorylation is indicative of activated MKK4. Actin levels are shown as a loading control.
Mentions: At the molecular level, immunoblot analyses of proteins in BMMCs revealed that expression levels of p46 and p56 SAPK/JNK isoforms, p38 MAPK, ERK1/ERK2, PKB/Akt, and actin were comparable in mkk7+/+ and mkk7−/− BMMCs (Fig. 6 A). However, expression of the cell cycle inhibitory molecules, p16INK4a, which act principally on cyclinD1 and CDK4-6, was completely abrogated in mkk7−/− BMMCs, leading to a concomitant increase in cyclinD1 expression (Fig. 6 A). Interestingly, whereas expression of c-Jun was comparable between mkk7+/+ and mkk7−/− BMMCs, expression of JunB, which has been shown to upregulate p16INK4a expression 38, was markedly decreased in mkk7−/− BMMCs. Expression of p27, another cell cycle inhibitor, was not affected by the absence of MKK7 (Fig. 6 A). However, it should be noted that loss of MKK7 does not always result in downregulation of p16INK4a expression, as p16INK4a expression appeared normal in B220+IgM+ B cells after in vitro differentiation of mkk7+/+ and mkk7−/− ES cells into B cells (Fig. 6 B, and data not shown). Thus, mutation of mkk7 in BMMCs, but not in B cells, results in loss of expression of JunB and the cell cycle inhibitor p16INK4a and upregulation of cyclinD1.

Bottom Line: Mutation of mkk7 in mast cells resulted in hyperproliferation in response to the cytokines interleukin (IL)-3 and stem cell factor (SCF).Loss of MKK7 did not affect activation of extracellular signal-regulated kinase (ERK)1/2 or p38 MAPK. mkk7(-/-) mast cells display reduced expression of JunB and the cell cycle inhibitor p16INK4a and upregulation of cyclinD1.These results indicate that the MKK7-regulated stress signaling pathway can function as negative regulator of cell growth in multiple hematopoietic lineages.

View Article: PubMed Central - PubMed

Affiliation: Amgen Institute, Ontario Cancer Institute, Canada.

ABSTRACT
The dual specificity kinases mitogen-activated protein kinase (MAPK) kinase (MKK)7 and MKK4 are the only molecules known to directly activate the stress kinases stress-activated protein kinases (SAPKs)/c-Jun N-terminal kinases (JNKs) in response to environmental or mitogenic stimuli. To examine the physiological role of MKK7 in hematopoietic cells, we used a gene targeting strategy to mutate MKK7 in murine T and B cells and non-lymphoid mast cells. Loss of MKK7 in thymocytes and mature B cells results in hyperproliferation in response to growth factor and antigen receptor stimulation and increased thymic cellularity. Mutation of mkk7 in mast cells resulted in hyperproliferation in response to the cytokines interleukin (IL)-3 and stem cell factor (SCF). SAPK/JNK activation was completely abolished in the absence of MKK7, even though expression of MKK4 was strongly upregulated in mkk7(-/-) mast cell lines, and phosphorylation of MKK4 occurred normally in response to multiple stress stimuli. Loss of MKK7 did not affect activation of extracellular signal-regulated kinase (ERK)1/2 or p38 MAPK. mkk7(-/-) mast cells display reduced expression of JunB and the cell cycle inhibitor p16INK4a and upregulation of cyclinD1. Reexpression of p16INK4a in mkk7(-/-) mast cells abrogates the hyperproliferative response. Apoptotic responses to a variety of stimuli were not affected. Thus, MKK7 is an essential and specific regulator of stress-induced SAPK/JNK activation in mast cells and MKK7 negatively regulates growth factor and antigen receptor-driven proliferation in hematopoietic cells. These results indicate that the MKK7-regulated stress signaling pathway can function as negative regulator of cell growth in multiple hematopoietic lineages.

Show MeSH
Related in: MedlinePlus