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MEKK3 is essential for lipopolysaccharide-induced interleukin-6 and granulocyte-macrophage colony-stimulating factor
Kihwan Kim1, Omar Duramad, Xiao-Feng Qin
1Department of Immunology, The University of Texas, M. D. Anderson Cancer Center, Houston, TX, USA.
Abstract:
Mitogen-activated protein/ERK kinase kinase 3 (MEKK3) is a Ser/Thr protein kinase belonging to the MEKK/STE11 subgroup of the MAP3K family. Recently, we found that MEKK3 plays a critical role in interleukin-1 (IL-1) receptor and Toll-like receptor 4 signalling using established primary mouse embryonic fibroblast (MEF) cell lines. However, the function of MEKK3 in immune cells has not been studied because germ-line MEKK3 knockout mice are embryonically lethal between embryonic days 10 and 11. In this study, we used small interference RNA to the mouse Mekk3 gene to specifically knock down MEKK3 expression in the macrophage line Raw264.7. We found that the lipopolysaccharide-induced IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) production was dramatically decreased in MEKK3 knockdown cells whereas the tumour necrosis factor-alpha and IL-1beta production were not affected. We also observed that the ERK1/2, p38 and JNK MAPK induction in MEKK3 knockdown cells were moderately inhibited within the first 60 min of stimulation, while the ERK and p38 were more severely inhibited after 2-4 hr of stimulation. Degradation of IkappaBalpha was also partially blocked in MEKK3 knockdown cells. Notably, the impairment in IL-6 and GM-CSF production in the MEKK3 knockdown cells was restored by reintroducing a human Mekk3 cDNA that could not be targeted by mouse Mekk3-siRNAs. In conclusion, this study showed that MEKK3 is a crucial and specific regulator of the proinflammatory cytokines IL-6 and GM-CSF in macrophages and provided a novel method for investigating MEKK3 function in other immune cells.
Insights
Mitogen-activated protein/ERK kinase kinase 3 (MEKK3) is vital for producing IL-6 and GM-CSF in macrophages. This study developed a method to investigate MEKK3's role in immune cells, overcoming embryonic lethality issues.
Area of Science:
- Immunology
- Molecular Biology
- Signal Transduction
Background:
- Mitogen-activated protein/ERK kinase kinase 3 (MEKK3) is a MAP3K family kinase involved in IL-1 receptor and Toll-like receptor 4 signaling.
- Previous studies were limited by embryonic lethality in germ-line MEKK3 knockout mice, preventing investigation in immune cells.
- Understanding MEKK3's function in macrophages is crucial for immune response regulation.
Purpose of the Study:
- To investigate the function of MEKK3 in immune cells, specifically macrophages.
- To determine the role of MEKK3 in lipopolysaccharide (LPS)-induced cytokine production.
- To elucidate the impact of MEKK3 knockdown on MAPK signaling pathways.
Main Methods:
- Utilized small interference RNA (siRNA) to specifically knock down MEKK3 expression in the Raw264.7 macrophage cell line.
- Assessed the production of cytokines (IL-6, GM-CSF, TNF-α, IL-1β) following LPS stimulation.
- Monitored the activation of MAPK pathways (ERK1/2, p38, JNK) and IκBα degradation.
Main Results:
- MEKK3 knockdown significantly decreased LPS-induced IL-6 and GM-CSF production, but not TNF-α or IL-1β.
- MEKK3 knockdown partially inhibited IκBα degradation and affected MAPK pathway induction at early and late time points.
- Reintroduction of human MEKK3 cDNA rescued the impaired IL-6 and GM-CSF production, confirming MEKK3 specificity.
Conclusions:
- MEKK3 is a critical and specific regulator of pro-inflammatory cytokines IL-6 and GM-CSF in macrophages.
- This study provides a novel siRNA-based approach to study MEKK3 function in immune cells, circumventing embryonic lethality.
- Findings highlight MEKK3's essential role in macrophage-mediated inflammatory responses.
