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MKK3 deletion improves mitochondrial quality
Anup Srivastava1, John McGinniss1, Yao Wong1
1Pulmonary, Critical Care and Sleep Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520-8057, USA.
Abstract:
Sepsis, a severe response to infection, leads to excessive inflammation and is the major cause of mortality in intensive care units. Mitochondria have been shown to influence the outcome of septic injury. We have previously shown that MAP kinase kinase 3 (MKK3)(-/-) mice are resistant to septic injury and MKK3(-/-) macrophages have improved mitochondrial function. In this study we examined processes that lead to improved mitochondrial quality in MKK3(-/-) mouse embryonic fibroblasts (MEFs) and specifically the role of mitophagy in mitochondrial health. MKK3(-/-) MEFs had lower inflammatory cytokine release and oxidant production after lipopolysaccharide (LPS) stimulation, confirming our earlier observations. MKK3(-/-) MEFs had better mitochondrial function as measured by mitochondrial membrane potential (MMP) and ATP, even after LPS treatment. We observed higher mitophagy in MKK3(-/-) MEFs compared to wild type (WT). Transmission electron microscopy studies showed longer and larger mitochondria in MKK3(-/-) MEFs, indicative of healthier mitochondria. We performed a SILAC (stable isotope labeling by/with amino acids in cell culture) study to assess differences in mitochondrial proteome between WT and MKK3(-/-) MEFs and observed increased expression of tricarboxylic acid (TCA) cycle enzymes and respiratory complex subunits. Further, inhibition of mitophagy by Mdivi1 led to loss in MMP and increased cytokine secretion after LPS treatment in MKK3(-/-) MEFs. In conclusion, this study demonstrates that MKK3 influences mitochondrial quality by affecting the expression of mitochondrial proteins, including TCA cycle enzymes, and mitophagy, which consequently regulates the inflammatory response. Based on our results, MKK3 could be a potential therapeutic target for inflammatory diseases like sepsis.
Insights
Mice lacking MAP kinase kinase 3 (MKK3) show improved mitochondrial function and reduced inflammation during sepsis. This suggests MKK3 is a potential therapeutic target for sepsis and other inflammatory diseases.
Area of Science:
- Cell Biology
- Immunology
- Mitochondrial Biology
Background:
- Sepsis is a life-threatening condition characterized by excessive inflammation and high mortality in intensive care units.
- Mitochondrial dysfunction is implicated in sepsis outcomes.
- Previous studies showed MAP kinase kinase 3 (MKK3) knockout mice are resistant to septic injury.
Purpose of the Study:
- To investigate the mechanisms underlying improved mitochondrial quality in MKK3 knockout mouse embryonic fibroblasts (MEFs).
- To determine the specific role of mitophagy in maintaining mitochondrial health in the absence of MKK3.
Main Methods:
- Stimulation of MKK3(-/-) and wild-type (WT) MEFs with lipopolysaccharide (LPS).
- Measurement of mitochondrial membrane potential (MMP), ATP levels, cytokine release, and oxidant production.
- Transmission electron microscopy (TEM) for mitochondrial morphology assessment.
- Stable isotope labeling by/with amino acids in cell culture (SILAC) for proteomic analysis.
- Inhibition of mitophagy using Mdivi-1.
Main Results:
- MKK3(-/-) MEFs exhibited lower inflammatory cytokine release and oxidant production post-LPS stimulation.
- MKK3(-/-) MEFs maintained higher mitochondrial membrane potential (MMP) and ATP levels compared to WT MEFs, even after LPS treatment.
- Increased mitophagy and longer, larger mitochondria were observed in MKK3(-/-) MEFs.
- Proteomic analysis revealed increased expression of tricarboxylic acid (TCA) cycle enzymes and respiratory complex subunits in MKK3(-/-) MEFs.
- Inhibition of mitophagy in MKK3(-/-) MEFs led to decreased MMP and increased cytokine secretion after LPS treatment.
Conclusions:
- MKK3 plays a critical role in regulating mitochondrial quality and inflammatory responses.
- MKK3 influences mitochondrial protein expression, including TCA cycle enzymes, and mitophagy.
- MKK3 deficiency enhances mitochondrial health and confers resistance to inflammatory injury, suggesting MKK3 as a potential therapeutic target for sepsis and inflammatory diseases.
