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Deficiency of the stress kinase p38alpha results in embryonic lethality: characterization of the kinase dependence of stress responses of enzyme-deficient embryonic stem cells.

Allen M, Svensson L, Roach M, Hambor J, McNeish J, Gabel CA - J. Exp. Med. (2000)

Bottom Line: Mice for the p38alpha allele die during embryonic development. p38alpha(1/)- embryonic stem (ES) cells grown in the presence of high neomycin concentrations demonstrated conversion of the wild-type allele to a targeted allele. p38alpha(-/)- ES cells lacked p38alpha protein and failed to activate MAP kinase-activated protein (MAPKAP) kinase 2 in response to chemical stress inducers.Comparison of chemical-induced apoptosis processes revealed no significant difference between the p38alpha(1/+) and p38alpha(-/)- ES cells.However, p38alpha does not serve an indispensable role in apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory, Allergy, Immunology, Inflammation, and Infectious Diseases, Central Research, Pfizer, Inc., Groton, CT 06340, USA.

ABSTRACT
The mitogen-activated protein (MAP) kinase p38 is a key component of stress response pathways and the target of cytokine-suppressing antiinflammatory drugs (CSAIDs). A genetic approach was employed to inactivate the gene encoding one p38 isoform, p38alpha. Mice for the p38alpha allele die during embryonic development. p38alpha(1/)- embryonic stem (ES) cells grown in the presence of high neomycin concentrations demonstrated conversion of the wild-type allele to a targeted allele. p38alpha(-/)- ES cells lacked p38alpha protein and failed to activate MAP kinase-activated protein (MAPKAP) kinase 2 in response to chemical stress inducers. In contrast, p38alpha(1/+) ES cells and primary embryonic fibroblasts responded to stress stimuli and phosphorylated p38alpha, and activated MAPKAP kinase 2. After in vitro differentiation, both wild-type and p38alpha(-/)- ES cells yielded cells that expressed the interleukin 1 receptor (IL-1R). p38alpha(1/+) but not p38alpha(-/)- IL-1R-positive cells responded to IL-1 activation to produce IL-6. Comparison of chemical-induced apoptosis processes revealed no significant difference between the p38alpha(1/+) and p38alpha(-/)- ES cells. Therefore, these studies demonstrate that p38alpha is a major upstream activator of MAPKAP kinase 2 and a key component of the IL-1 signaling pathway. However, p38alpha does not serve an indispensable role in apoptosis.

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Demonstration that p38α−/− ES cells lack immunodetectable p38α. Wild-type (ES+/+) and p38α-deficient ES cells were stimulated with sodium arsenite for 15 min and then solubilized by detergent extraction. Duplicate samples of each extract were loaded onto separate gels and processed for Western blotting. Antigen detected after staining these blots with an antibody against total (p38) or one selective for phosphospecific (phospho-p38) forms are indicated. Extracts of IL-1–stimulated PEFs and C6 cells are included as standards. Arrow indicates the migration of p38α, and the arrowhead marks the antigen detected by the phospho-specific antiserum in the ES cell samples that is not p38 dependent.
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Figure 6: Demonstration that p38α−/− ES cells lack immunodetectable p38α. Wild-type (ES+/+) and p38α-deficient ES cells were stimulated with sodium arsenite for 15 min and then solubilized by detergent extraction. Duplicate samples of each extract were loaded onto separate gels and processed for Western blotting. Antigen detected after staining these blots with an antibody against total (p38) or one selective for phosphospecific (phospho-p38) forms are indicated. Extracts of IL-1–stimulated PEFs and C6 cells are included as standards. Arrow indicates the migration of p38α, and the arrowhead marks the antigen detected by the phospho-specific antiserum in the ES cell samples that is not p38 dependent.

Mentions: p38α-deficient ES cells were subjected to a similar analysis. Extracts derived from anisomycin-treated p38α1/− and p38α1/+ ES cells contained p38α, and this enzyme was phosphorylated in response to the chemical stress inducer (Fig. 6). In contrast, extracts derived from aniosomycin-treated p38α−/− ES cells did not contain p38α or the phosphorylated p38α polypeptide species (Fig. 6).


Deficiency of the stress kinase p38alpha results in embryonic lethality: characterization of the kinase dependence of stress responses of enzyme-deficient embryonic stem cells.

Allen M, Svensson L, Roach M, Hambor J, McNeish J, Gabel CA - J. Exp. Med. (2000)

Demonstration that p38α−/− ES cells lack immunodetectable p38α. Wild-type (ES+/+) and p38α-deficient ES cells were stimulated with sodium arsenite for 15 min and then solubilized by detergent extraction. Duplicate samples of each extract were loaded onto separate gels and processed for Western blotting. Antigen detected after staining these blots with an antibody against total (p38) or one selective for phosphospecific (phospho-p38) forms are indicated. Extracts of IL-1–stimulated PEFs and C6 cells are included as standards. Arrow indicates the migration of p38α, and the arrowhead marks the antigen detected by the phospho-specific antiserum in the ES cell samples that is not p38 dependent.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195860&req=5

Figure 6: Demonstration that p38α−/− ES cells lack immunodetectable p38α. Wild-type (ES+/+) and p38α-deficient ES cells were stimulated with sodium arsenite for 15 min and then solubilized by detergent extraction. Duplicate samples of each extract were loaded onto separate gels and processed for Western blotting. Antigen detected after staining these blots with an antibody against total (p38) or one selective for phosphospecific (phospho-p38) forms are indicated. Extracts of IL-1–stimulated PEFs and C6 cells are included as standards. Arrow indicates the migration of p38α, and the arrowhead marks the antigen detected by the phospho-specific antiserum in the ES cell samples that is not p38 dependent.
Mentions: p38α-deficient ES cells were subjected to a similar analysis. Extracts derived from anisomycin-treated p38α1/− and p38α1/+ ES cells contained p38α, and this enzyme was phosphorylated in response to the chemical stress inducer (Fig. 6). In contrast, extracts derived from aniosomycin-treated p38α−/− ES cells did not contain p38α or the phosphorylated p38α polypeptide species (Fig. 6).

Bottom Line: Mice for the p38alpha allele die during embryonic development. p38alpha(1/)- embryonic stem (ES) cells grown in the presence of high neomycin concentrations demonstrated conversion of the wild-type allele to a targeted allele. p38alpha(-/)- ES cells lacked p38alpha protein and failed to activate MAP kinase-activated protein (MAPKAP) kinase 2 in response to chemical stress inducers.Comparison of chemical-induced apoptosis processes revealed no significant difference between the p38alpha(1/+) and p38alpha(-/)- ES cells.However, p38alpha does not serve an indispensable role in apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory, Allergy, Immunology, Inflammation, and Infectious Diseases, Central Research, Pfizer, Inc., Groton, CT 06340, USA.

ABSTRACT
The mitogen-activated protein (MAP) kinase p38 is a key component of stress response pathways and the target of cytokine-suppressing antiinflammatory drugs (CSAIDs). A genetic approach was employed to inactivate the gene encoding one p38 isoform, p38alpha. Mice for the p38alpha allele die during embryonic development. p38alpha(1/)- embryonic stem (ES) cells grown in the presence of high neomycin concentrations demonstrated conversion of the wild-type allele to a targeted allele. p38alpha(-/)- ES cells lacked p38alpha protein and failed to activate MAP kinase-activated protein (MAPKAP) kinase 2 in response to chemical stress inducers. In contrast, p38alpha(1/+) ES cells and primary embryonic fibroblasts responded to stress stimuli and phosphorylated p38alpha, and activated MAPKAP kinase 2. After in vitro differentiation, both wild-type and p38alpha(-/)- ES cells yielded cells that expressed the interleukin 1 receptor (IL-1R). p38alpha(1/+) but not p38alpha(-/)- IL-1R-positive cells responded to IL-1 activation to produce IL-6. Comparison of chemical-induced apoptosis processes revealed no significant difference between the p38alpha(1/+) and p38alpha(-/)- ES cells. Therefore, these studies demonstrate that p38alpha is a major upstream activator of MAPKAP kinase 2 and a key component of the IL-1 signaling pathway. However, p38alpha does not serve an indispensable role in apoptosis.

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