Neurotrophin 3 activation of TrkC induces Schwann cell migration through the c-Jun N-terminal kinase pathway

Abstract

During development and nerve injury, complex interactions between glial cells and neurons are essential for establishing proper nerve function. Neurotrophins play multiple roles in the developing nervous system, including cell survival, growth, and differentiation. Here we show that migration of Schwann cells, isolated from sciatic nerves, is significantly enhanced by neurotrophin 3, but not by nerve growth factor or brain-derived neurotrophic factor. The neurotrophin-3-induced cell migration was also observed in Schwann cells isolated from sciatic nerves of p75NTR-/- mice, indicating that neurotrophin 3 enhances cell migration through TrkC. This effect was blocked by K252a, an inhibitor of the Trk receptor family. Additionally, the neurotrophin-3-induced cell migration depended on Rho GTPases (Rac1 and Cdc42) and c-Jun N-terminal kinase. We obtained the same results with Cos-7 cells expressing TrkC. Taken together, these results suggest that neurotrophin 3 activation of TrkC induces Schwann cell migration through the c-Jun N-terminal kinase signaling pathway.

Fig. 1.

NT3 mediates migration of primary Schwann cells and Cos-7 cells through TrkC. Cell migration was measured in Schwann cells (A–D), isolated from sciatic nerves of rats (A–C) and mice (D), and Cos-7 cells transiently transfected with the plasmid encoding TrkC (E–G), by using Boyden chambers. Cells were pretreated with or without K252a (A, B, E, and F). After incubation with (+) or without (-) NT3, the migrating cells were stained and analyzed (A and E), and the number of stained cells was counted (B and F). (C) Rat Schwann cells were incubated with NT3, BDNF, or nerve growth factor, and the number of stained, migrating cells was counted. (D) Schwann cells isolated from sciatic nerves of mice (p75^NTR+/+ or p75^NTR-/-) were incubated with or without NT3, and the number of stained, migrating cells was counted. (G) Cos-7 cells were transfected with or without TrkC and were incubated with or without NT3. Data were evaluated by using Student's t test. *, P < 0.01.

Fig. 2.

The NT3-induced cell migration involves JNK. Rat Schwann cells (A) and Cos-7 cells transfected with TrkC (B) were pretreated with SP600125. After incubation with NT3, the migrating cells in the Boyden chambers were stained and counted.

Fig. 3.

JNK is activated after stimulation with NT3. JNK activity was measured in rat Schwann cells (A–C) and Cos-7 cells cotransfected with TrkC and HA-JNK (D–F). (A) Schwann cells were treated with NT3. Endogenous JNK was immunoprecipitated with anti-JNK antibody and blotted with antiphosphorylated JNK antibody or anti-JNK antibody. Asterisks indicate the heavy chain of IgGs. (B) Levels of JNK phosphorylation were quantified and normalized against the total immunoprecipitated JNK levels. As a positive control, anisomysin (10 μg/ml, 30 min)-induced JNK phosphorylation is also shown. (C) Schwann cells were pretreated with K252a, and the activity of JNK was measured 30 min after stimulation with NT3. (D) Cos-7 cells cotransfected with TrkC and HA-JNK were treated with NT3. Transfected HA-JNK was immunoprecipitated with anti-JNK antibody and blotted with antiphosphorylated JNK antibody or anti-JNK antibody. (E) Levels of HA-JNK phosphorylation were quantified and normalized against the total immunoprecipitated HA-JNK levels (•). Mock transfections are represented (○). As a positive control, anisomysin-induced HA-JNK phosphorylation is also shown (▪). (F) Cos-7 cells cotransfected with TrkC and HA-JNK were pretreated with K252a, and the activity of HA-JNK was measured 30 min after stimulation with NT3.

Fig. 4.

Involvement of Rho GTPases in NT3-induced cell migration and JNK activation. Rat Schwann cells (A and C) and Cos-7 cells transfected with TrkC (B) or TrkC and HA-JNK (D) were pretreated with Toxin B. (A and B) After incubation with NT3, the migrating cells in the Boyden chambers were stained and counted. (C and D) The activity of endogenous JNK or HA-JNK was measured 30 min after stimulation with NT3. Data were evaluated by using Student's t test. *, P < 0.01.

Fig. 5.

Rho GTPases Rac1 and Cdc42 are activated after stimulation with NT3. The activities of Rho GTPases were measured in rat Schwann cells (A–E) and Cos-7 cells cotransfected with RhoA (F), Rac1 (G and I), or Cdc42 (H and J), together with TrkC. (A–C) The activities of endogenous Rho GTPases were measured after the addition of NT3 with the pull-down assay by using recombinant mDia1-RBD or αPak-CRIB. (D and E) Cells were pretreated with K252a, and the activities of endogenous Rac1 and Cdc42 were measured after the addition of NT3. (F–H) The activities of transfected Rho GTPases were measured after the addition of NT3. (I and J) Cells were pretreated with K252a, and the activities of transfected Rac1 and Cdc42 were measured after the addition of NT3. The total Rho GTPases in the cell lysates were immunoblotted with anti-Rho GTPases antibodies.

Fig. 6.

Schematic model for the signaling pathway coupling NT3/TrkC to Schwann cell migration. Details are described in Discussion.