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Activation of p38mapk, MKK3, and MKK4 by TNF-alpha in mouse bone marrow-derived macrophages
B W Winston1, E D Chan, G L Johnson
1Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO 80206, USA.
Abstract:
TNF-alpha regulates the expression of many proinflammatory and profibrogenic gene products in macrophages, and hence plays a vital role in controlling the inflammatory response. We have shown previously that exposure of macrophages to TNF-alpha stimulates the activation of members of the mitogen-activated protein kinase (MAPK) family. In this study, we have investigated the mechanism of activation of the p38mapk by TNF-alpha in mouse bone marrow-derived macrophages. Exposure to TNF-alpha resulted in the activation of p38mapk, as measured by 1) the trans-phosphorylation of recombinant activating transcription factor-2 substrate by immunoprecipitated p38mapk and 2) specific tyrosine phosphorylation of immunoprecipitated p38mapk. In addition, selective ligation of the TNF-alpha receptor CD120a (p55) with human TNF-alpha was sufficient to induce p38mapk activation. Using an in vitro kinase assay with recombinant kinase-inactive p38mapk as substrate in the presence of [gamma-32P]ATP, the upstream kinases MKK3 (mitogen-activated protein kinase kinase 3) and MKK4 were found to be activated in response to TNF-alpha. These findings suggest that TNF-alpha transiently phosphorylates and activates the three members of the MAPK family, namely p42(mapk/erk2), p46 c-Jun amino-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38mapk following cross-linking of CD120a (p55), and that MKK3 and MKK4 are capable of phosphorylating p38mapk.
Insights
Tumor Necrosis Factor-alpha (TNF-alpha) activates the p38 mitogen-activated protein kinase (MAPK) pathway in macrophages. This activation involves upstream kinases MKK3 and MKK4, crucial for inflammatory responses.
Area of Science:
- Immunology
- Cell Signaling
- Molecular Biology
Background:
- Tumor Necrosis Factor-alpha (TNF-alpha) is a key regulator of inflammatory and profibrogenic gene expression in macrophages.
- TNF-alpha signaling activates mitogen-activated protein kinase (MAPK) family members in macrophages.
- Understanding the precise mechanisms of TNF-alpha-induced MAPK activation is critical for controlling inflammatory responses.
Purpose of the Study:
- To elucidate the mechanism by which TNF-alpha activates p38 MAPK in mouse bone marrow-derived macrophages.
- To identify the upstream kinases responsible for p38 MAPK activation by TNF-alpha.
Main Methods:
- Assessed p38 MAPK activation via trans-phosphorylation of activating transcription factor-2 and specific tyrosine phosphorylation.
- Utilized selective ligation of the TNF-alpha receptor CD120a (p55) to study receptor-specific activation.
- Employed in vitro kinase assays with recombinant proteins to identify upstream kinases (MKK3, MKK4).
Main Results:
- TNF-alpha exposure led to significant p38 MAPK activation in macrophages.
- Cross-linking of the CD120a (p55) receptor by TNF-alpha was sufficient to trigger p38 MAPK activation.
- Upstream kinases MKK3 and MKK4 were found to be activated by TNF-alpha and capable of phosphorylating p38 MAPK.
Conclusions:
- TNF-alpha activates p38 MAPK, along with ERK2 and JNK/SAPK, following CD120a receptor cross-linking.
- MKK3 and MKK4 act as crucial upstream activators of p38 MAPK in the TNF-alpha signaling pathway.
- These findings provide insight into the molecular mechanisms governing TNF-alpha-mediated inflammatory signaling.