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MARCKS Is an Essential Regulator of Reactive Oxygen Species Production in the Monocytic Cell Type
René Huber1, Mareike Diekmann1, Leonie Hoffmeister1
1Institute of Clinical Chemistry, Hannover Medical School, 30625 Hannover, Germany.
Abstract:
Myristoylated alanine-rich C-kinase substrate (MARCKS) is a ubiquitous protein mediating versatile effects in a variety of cell types, including actin crosslinking, signal transduction, and intracellular transport processes. MARCKS's functional role in monocyte/macrophages, however, has not yet been adequately addressed. Thus, the aim of this study was to further elucidate the impact of MARCKS on central cellular functions of monocytic cells. To address this topic, we generated monocytic THP-1 (Tohoku Hospital Pediatrics-1)-derived MARCKS wildtype and knockout (KO) cells using the CRISPR/Cas9 technique. Remarkably, in the absence of MARCKS, both total and intracellular reactive oxygen species (ROS) production were strongly suppressed but restored following transient MARCKS re-transfection. In contrast, proliferation, differentiation, cytokine expression, and phagocytosis remained unaltered. A complete inhibition of ROS production could also be achieved in THP-1-derived PKCβ KO cells or in PKC inhibitor Staurosporine-treated primary human monocytes. MARCKS deficiency also involved reduced basal Akt phosphorylation and delayed re-phosphorylation. Further analyses indicated that long-term TNF pre-incubation strongly enhances monocytic ROS production, which was completely blocked in MARCKS and PKCβ KO cells. Collectively, our study demonstrates that MARCKS is an essential molecule enabling ROS production by monocytic cells and suggests that MARCKS is part of a signal cascade involved in ROS formation.
Insights
Myristoylated alanine-rich C-kinase substrate (MARCKS) is essential for reactive oxygen species (ROS) production in monocytic cells. MARCKS deficiency suppresses ROS, highlighting its role in monocyte signaling pathways.
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- Myristoylated alanine-rich C-kinase substrate (MARCKS) is a key protein involved in various cellular processes.
- The specific function of MARCKS in monocytes and macrophages remains incompletely understood.
- This study investigates the role of MARCKS in regulating monocytic cell functions.
Purpose of the Study:
- To elucidate the impact of MARCKS on critical cellular functions in monocytic cells.
- To determine the role of MARCKS in reactive oxygen species (ROS) production in monocytes.
- To explore the signaling pathways involving MARCKS in monocytic cells.
Main Methods:
- Generation of MARCKS wildtype and knockout (KO) monocytic THP-1 cells using CRISPR/Cas9.
- Assessment of reactive oxygen species (ROS) production, proliferation, differentiation, cytokine expression, and phagocytosis.
- Analysis of protein phosphorylation (Akt) and effects of PKC inhibition.
Main Results:
- MARCKS deficiency significantly suppressed both total and intracellular ROS production in monocytic cells.
- ROS production was restored upon transient MARCKS re-transfection.
- Proliferation, differentiation, cytokine expression, and phagocytosis were not significantly altered by MARCKS deficiency.
- MARCKS deficiency led to reduced basal Akt phosphorylation and delayed re-phosphorylation.
- ROS production, enhanced by TNF pre-incubation, was completely blocked in MARCKS and PKCβ KO cells.
Conclusions:
- MARCKS is an essential molecule for enabling ROS production in monocytic cells.
- MARCKS appears to be a component of a signaling cascade regulating ROS formation in monocytes.
- The findings suggest a novel role for MARCKS in innate immune responses involving ROS.
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