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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: 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.

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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PEFs, but not ES cells, produce IL-6 in response to inflammatory cytokine stimuli. (A) Cultures of PEFs or wild-type ES cells were incubated overnight in medium containing no effector (None) or 10 ng/ml of IL-1β or TNF-α, after which IL-6 released to the medium was measured by ELISA. The ELISA signal is indicated as a function of treatment (average of duplicate determinations). This experiment was repeated twice with comparable results. (B) PEFs were stimulated overnight with 10 ng/ml of IL-1 in the absence or presence of the indicated concentration of SB-203,580. IL-6 released to the medium was determined by ELISA (average of duplicate determinations). These data are representative of three separate experiments.
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Figure 4: PEFs, but not ES cells, produce IL-6 in response to inflammatory cytokine stimuli. (A) Cultures of PEFs or wild-type ES cells were incubated overnight in medium containing no effector (None) or 10 ng/ml of IL-1β or TNF-α, after which IL-6 released to the medium was measured by ELISA. The ELISA signal is indicated as a function of treatment (average of duplicate determinations). This experiment was repeated twice with comparable results. (B) PEFs were stimulated overnight with 10 ng/ml of IL-1 in the absence or presence of the indicated concentration of SB-203,580. IL-6 released to the medium was determined by ELISA (average of duplicate determinations). These data are representative of three separate experiments.

Mentions: Inflammatory cytokines such as IL-1 can promote IL-6 production by cell types that express the appropriate signaling receptors 3536. To determine whether inflammatory cytokine signaling cascades were operative in PEF and ES cells, individual cultures were treated with IL-1 or TNF-α, after which IL-6 released into the medium was determined by ELISA. In the absence of a cytokine effector, PEFs isolated from wild-type embryos produced minimal quantities of IL-6 (Fig. 4 A). However, in response to IL-1 stimulation, these fibroblasts generated large quantities of extracellular IL-6 (Fig. 4 A). Likewise, PEFs responded to TNF-α and generated IL-6, but cytokine levels produced in response to TNF-α were less than those elicited by an equivalent concentration of IL-1 (Fig. 4 A). Attempts to isolate p38α−/− PEFs from day 12 chimeric embryos were unsuccessful; fibroblast-like cells were recovered after G418 selection, but these did not survive during culture. Unstimulated ES cells did not produce IL-6, and addition of IL-1 or TNF-α to their medium did not result in an increase in IL-6 production (Fig. 4 A). This lack of responsiveness suggests that DBA/1 lacJ ES cells lack signaling receptors for IL-1 and TNF, as has previously been noted for ES cells derived from other mice strains 50.


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)

PEFs, but not ES cells, produce IL-6 in response to inflammatory cytokine stimuli. (A) Cultures of PEFs or wild-type ES cells were incubated overnight in medium containing no effector (None) or 10 ng/ml of IL-1β or TNF-α, after which IL-6 released to the medium was measured by ELISA. The ELISA signal is indicated as a function of treatment (average of duplicate determinations). This experiment was repeated twice with comparable results. (B) PEFs were stimulated overnight with 10 ng/ml of IL-1 in the absence or presence of the indicated concentration of SB-203,580. IL-6 released to the medium was determined by ELISA (average of duplicate determinations). These data are representative of three separate experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195860&req=5

Figure 4: PEFs, but not ES cells, produce IL-6 in response to inflammatory cytokine stimuli. (A) Cultures of PEFs or wild-type ES cells were incubated overnight in medium containing no effector (None) or 10 ng/ml of IL-1β or TNF-α, after which IL-6 released to the medium was measured by ELISA. The ELISA signal is indicated as a function of treatment (average of duplicate determinations). This experiment was repeated twice with comparable results. (B) PEFs were stimulated overnight with 10 ng/ml of IL-1 in the absence or presence of the indicated concentration of SB-203,580. IL-6 released to the medium was determined by ELISA (average of duplicate determinations). These data are representative of three separate experiments.
Mentions: Inflammatory cytokines such as IL-1 can promote IL-6 production by cell types that express the appropriate signaling receptors 3536. To determine whether inflammatory cytokine signaling cascades were operative in PEF and ES cells, individual cultures were treated with IL-1 or TNF-α, after which IL-6 released into the medium was determined by ELISA. In the absence of a cytokine effector, PEFs isolated from wild-type embryos produced minimal quantities of IL-6 (Fig. 4 A). However, in response to IL-1 stimulation, these fibroblasts generated large quantities of extracellular IL-6 (Fig. 4 A). Likewise, PEFs responded to TNF-α and generated IL-6, but cytokine levels produced in response to TNF-α were less than those elicited by an equivalent concentration of IL-1 (Fig. 4 A). Attempts to isolate p38α−/− PEFs from day 12 chimeric embryos were unsuccessful; fibroblast-like cells were recovered after G418 selection, but these did not survive during culture. Unstimulated ES cells did not produce IL-6, and addition of IL-1 or TNF-α to their medium did not result in an increase in IL-6 production (Fig. 4 A). This lack of responsiveness suggests that DBA/1 lacJ ES cells lack signaling receptors for IL-1 and TNF, as has previously been noted for ES cells derived from other mice strains 50.

Bottom Line: 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.

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.

Show MeSH
Related in: MedlinePlus