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Regulation of MARCKS and MARCKS-related protein expression in BV-2 microglial cells in response to lipopolysaccharide
J R Sunohara1, N D Ridgway, H W Cook
1Atlantic Research Centre, Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada.
Abstract:
Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP) have been implicated in membrane-cytoskeletal events underlying cell adhesion, migration, secretion, and phagocytosis. In BV-2 microglial cells, lipopolysaccharide (LPS) elicited a dose-dependent increase in mRNA of both MRP (sixfold) and MARCKS (threefold) with corresponding increases in [3H]myristoylated and immunoreactive protein levels. LPS also produced significant increases in protein kinase C (PKC)-beta twofold and PKC-epsilon (1.5-fold). Pro-inflammatory cytokines produced by activated microglia (IL-1beta, IL-6, TNF-alpha) did not mimic LPS effects on MARCKS or MRP expression when added individually or in combination. LPS and IFN-gamma produced a synergistic induction of iNOS but not MARCKS or MRP. Induction of MARCKS and MRP by LPS was completely blocked by inhibitors of NF-kappaB (PDTC) and protein tyrosine kinases (herbimycin A), partially blocked by the p38 kinase inhibitor SB203580, and unaffected by the MEK inhibitor PD98059. LPS induction of iNOS was considerably more sensitive to all these inhibitors. The Src kinase inhibitor PP2 had no effect, while the closely related inhibitor PP1 actually increased LPS induction of MARCKS and MRP. Our results suggest that MARCKS and MRP may play an important role in LPS-activated microglia, but are not part of the neuroinflammatory response produced by cytokines.
Insights
Lipopolysaccharide (LPS) activates microglia, increasing Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP) expression. These proteins are involved in microglial activation but not the neuroinflammatory response to cytokines.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP) are involved in critical cellular functions.
- Microglia play a key role in the central nervous system's immune response.
Purpose of the Study:
- To investigate the role of MARCKS and MRP in lipopolysaccharide (LPS)-activated microglia.
- To determine the signaling pathways involved in LPS-induced MARCKS and MRP expression.
Main Methods:
- BV-2 microglial cells were treated with LPS.
- mRNA and protein levels of MARCKS, MRP, and various kinases were analyzed.
- Inhibitors of signaling pathways (NF-kappaB, protein tyrosine kinases, p38, MEK, Src) were used to elucidate mechanisms.
Main Results:
- LPS dose-dependently increased MARCKS and MRP mRNA and protein levels in microglia.
- LPS also increased protein kinase C (PKC)-beta and PKC-epsilon expression.
- LPS-induced MARCKS and MRP expression was dependent on NF-kappaB and protein tyrosine kinases, partially dependent on p38 kinase, but independent of MEK and Src kinases.
- Pro-inflammatory cytokines did not affect MARCKS or MRP levels.
Conclusions:
- MARCKS and MRP are upregulated in LPS-activated microglia and may play a significant role in this process.
- The induction of MARCKS and MRP by LPS involves specific signaling pathways distinct from those mediating the pro-inflammatory cytokine response.
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