Directed migration of mouse macrophages in vitro involves myristoylated alanine-rich C-kinase substrate (MARCKS) protein

Abstract

A role for MARCKS protein in directed migration of macrophages toward a chemoattractant was investigated. A peptide identical to the N-terminus of MARCKS (the MANS peptide), shown previously to inhibit the function of MARCKS in various cell types, was used. We investigated whether this MARCKS-related peptide could affect migration of macrophages, using the mouse macrophage-like J774A.1 cell line and primary murine macrophages. Both of these cell types migrated in response to the chemoattractants macrophage/MCPs, MCP-1 (25-100 ng/ml) or C5a (5-20 ng/ml). Cells were preincubated (15 min) with MANS or a mis-sense control peptide (RNS), both at 50 μM, and effects on migration determined 3 h after addition of chemoattractants. The movement and interactions of MARCKS and actin also were followed visually via confocal microscopy using a fluorescently labeled antibody to MARCKS and fluorescently tagged phalloidin to identify actin. MANS, but not RNS, attenuated migration of J774A.1 cells and primary macrophages in response to MCP-1 or C5a, implicating MARCKS in the cellular mechanism of directed migration. Exposure of cells to MCP-1 resulted in rapid phosphorylation and translocation of MARCKS from plasma membrane to cytosol, whereas actin appeared to spread through the cell and into cell protrusions; there was visual and biochemical evidence of a transient interaction between MARCKS and actin during the process of migration. These results suggest that MARCKS is involved in directed migration of macrophages via a process involving its phosphorylation, cytoplasmic translocation, and interaction with actin.

Figures

Figure 1.. J774A.1 cells migrate in response to: (A) MCP-1 (25, 50, or 100 ng/ml), (B) PMA (10, 50, or 100 nM), and (C) C5a (5, 10, or 20 ng/ml).

Cells were exposed for 3 h, at which time, migrating cells were quantified. Data represent mean ± sem; n = 3; **significantly different from media control, P < 0.01.

Figure 2.. MCP-1 acts as a chemoattractant for J774A.1 cells.

The MANS, but not the RNS, peptide inhibits cell migration. Cells were untreated or treated with 50 μM peptide prior to adding MCP-1 in a Transwell migration assay. Cells that migrated through the pores to the lower side of the insert membrane, were fixed and counted at 40× magnification. That number is given on each panel. Contrast was enhanced for this figure after the panels were assembled to clearly define the migrated cells from the visible membrane pores.

Figure 3.. Inhibitory effect of MANS peptide on MCP-1 induced migration.

J774A.1 cells were pretreated with 50 μM MANS or RNS. Migration was measured in response to: (A) MCP-1 (50 ng/ml); (B) MCP-1 (100 ng/ml); or (C) C5a (10 ng/ml). Cells were exposed for 3 h, at which time, migrating cells were quantified. Data represent mean ± sem; n = 3; **significantly different from media control; †significantly different from stimulated/media control; P < 0.05.

Figure 4.. Western blot analysis of expression of phosphorylated MARCKS and total MARCKS protein.

J774A.1 cells were exposed to: medium only; PMA (100 nM); MCP-1 (100 ng/ml); or C5a (10 ng/ml). Cells were phosphorylated (P) rapidly in response to all three stimuli compared with medium control. Blots are representative of three replicate experiments.

Figure 5.. MARCKS and actin localization and intracellular movement in response to MCP-1 (100 ng/ml) in J774A.1 cells.

(A) Cells were stained for MARCKS (red), actin (green), or nucleus (blue) and examined by confocal microscopy as described. Regions in which MARCKS and actin overlap appear yellow. In response to chemokine stimulation, MARCKS translocates to the cytoplasm within the cells, where actin appears as well at 30 s. Representative fields are shown. Original magnification, 600×. (B) Western blot probed for actin after immunoprecipitation (IP) with MARCKS antibody. The time-points correspond to those in A and show a decrease in interaction between the two proteins at 180 s. IB, Immunoblot.

Figure 6.. Inhibitory effect of MANS peptide on MCP-1-induced migration in primary mouse macrophages.

Isolated cells were pretreated with: (A) 25 μM or (B) 50 μM MANS or RNS peptide for 15 min. Migration was measured in response to MCP-1 (100 ng/ml). Cells were exposed for 3 h, at which time, migrating cells were quantified. Data represent mean ± sem; n = 3; **significantly different from media control; †significantly different from MCP-1/media control; P < 0.05.

Figure 7.. Western blots of phosphorylated MARCKS protein expression.

Primary murine macrophages were exposed to medium only, MCP-1 (100 ng/ml), or C5a (10 ng/ml). The cells were phosphorylated rapidly (within 30–60 s) upon exposure to either chemmoattractant. Blots are representative of five separate experiments.