Involvement of the neurotensin receptor-3 in the neurotensin-induced migration of human microglia

Abstract

Microglia motility plays a crucial role in response to lesion or exocytotoxic damage of the cerebral tissue. We used two in vitro assays, a wound-healing model and a chemotaxis assay, to show that the neuropeptide neurotensin elicited the migration of the human microglial cell line C13NJ by a mechanism dependent on both phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein (MAP) kinase pathways. The effect of neurotensin on cell migration was blocked by the neurotensin receptor-3 propeptide, a selective ligand of this receptor. We demonstrate, by using RT-PCR, photoaffinity labeling, and Western blot analysis, that the type I neurotensin receptor-3 was the only known neurotensin receptor expressed in these microglial cells and that its activation led to the phosphorylation of both extracellular signal-regulating kinases 1/2 and Akt. Furthermore, the effect of neurotensin on cell migration was preceded by a profound modification of the F-actin cytoskeleton, particularly by the rapid formation of numerous cell filopodia. Both the motility and the filopodia appearance induced by neurotensin were totally blocked by selective inhibitors of MAP kinases or PI 3-kinase pathways. This demonstrates that the neurotensin receptor-3 is functional and mediates the migratory actions of neurotensin.

Fig. 1.

Expression of neurotensin receptor-3 by microglial cells. A, RNA from cultured C13NJ cells was reverse transcribed, and cDNA was amplified by PCR using sense and antisense NTR-specific primers (lane 1). Lane 2, H₂O with both sense and antisense primers; lane 3, RNA with both primers; lane 4, RT product with sense primers alone; lane 5, RT product with antisense primers alone; lane 6, PCR with plasmids controls. B, Whole cells were photolabeled using α-azidobenzoyl-¹²⁵I-Tyr₃-NT(2–13) (0.5 nm) as described in Materials and Methods in the absence (lane 1) or presence (lane 2) of 1 μm unlabeled NT. Proteins from cell homogenates were then separated by SDS-PAGE (8% acrylamide); the gel was fixed and air dried before phosphorimager analysis. Lane 3, Western blot analysis of cell homogenate using the NTR3 antibody. C, D, andC/D show that the fluorescence immunolabeling of NTR3 (C) and syntaxin-6 (D) in C13NJ cells is colocalized primarily (C/D) in the trans-Golgi compartment and processes. Scale bar, 10 μm.

Fig. 2.

Effect of NT on the phosphorylation of MAP kinases ERK1/2 in C13NJ cells. Microglial cells were stimulated either with 10 nm NT for various times (A) or with increasing concentrations of NT for 2.5 min at 37°C (B). The phosphorylation of MAP kinases (p-Erk) was determined by immunoblotting using an antibody directed against the phosphorylated active form of ERK. Immunoblots shown in A and B are representative of typical experiments. C,D, Data were standardized from three different experiments using the labeling obtained on the same blot with the anti-NTR3 antibody and the total ERK antibody and expressed as means ± SEM. PD98059 is the specific MAP kinase inhibitor.

Fig. 3.

Effect of NT on the phosphorylation of Akt in microglial cells. Cells were stimulated either with 10 nmNT for various times (A) or with increasing concentrations of the peptide for 30 min at 37°C (B). The phosphorylation of Akt was determined by immunoblotting using an antibody directed against the phosphorylated active form of Akt. A, B, Immunoblots representative of typical experiments. C,D, Data were standardized using the labeling obtained on the same blot with the anti-NTR3 antibody and expressed as means ± SEM from three independent experiments. Wortmannin and LY294002 are PI 3-kinase inhibitors.

Fig. 4.

Microglia migration into a cell-free area.A, The monolayer of microglia was scratched. Images were taken at 0 (Day 0) and 48 hr (Day 2) after wounding. Cells were incubated in the absence (No stimulation, a, a′) or presence (Control, b, b′) of 10% serum and compared with cells grown in the absence of serum and 10 nm NT (c, c′) or 10 nm NT and 1 μm propeptide (d,d′). B, Four representative fields were counted from normalized areas corresponding to each condition and expressed as the percentage ± SEM of cells that migrated in the presence of 10% serum. ***p < 0.001 forn = 6 (number of wells) when compared with nonstimulated cells. Scale bar, 50 μm.

Fig. 5.

Microglia migration in modified Boyden chamber. Microglia migration in response to various experimental conditions was tested in the modified Boyden chamber. The average number of migrating cells was expressed as the percentage of migrating cells counted in the presence of 10% serum. A, The number of cells migrating across the membrane was determined after incubation with either 10 nm NT or 1% bovine serum albumin (1% BSA) or 10 nm NT after preincubation with 24 μmPD98059 or 50 μm LY294002 or 1 μmwortmannin. B, The number of cells migrating across the membrane was determined after incubation with either 10 nmNT alone or with 10 nm NT in the presence of indicated concentrations of propeptide. ***p < 0.001 forn = 6 (number of wells) when compared with nonstimulated cells.

Fig. 6.

NT induces rapid formation of membrane filopodia. Human microglial cells were grown on glass coverslips, pretreated (c, c′) or not (a,b,a′,b′) with 1 μm wortmannin for 5 hr, and incubated in the absence (a,a′) or presence (b,b′) of 10 nm NT for 10 min at 37°C. Cells were labeled with Texas-Red phalloidin to detect F-actin. Control cells contain actin stress fibers throughout the cells body and processes. In the presence of NT, a significant increase of filopodia formation was observed (arrowheads); this formation was blocked in the presence of wortmannin (c,c′). a′, b′, andc′ are enlargements of a,b, and c images. Scale bar, 2 μm.