Activin A Secreted From Peripheral Nerve Fibroblasts Promotes Proliferation and Migration of Schwann Cells

Abstract

The peripheral nervous system has remarkable regenerative capabilities. Schwann cells and fibroblasts are known to play crucial roles in these processes. In this study, we delineated the differential effects of peripheral nerve fibroblasts and cardiac fibroblasts on Schwann cells. We found that peripheral nerve fibroblasts significantly promoted Schwann cell proliferation and migration compared with cardiac fibroblasts. The cytokine array results identified 32 of 67 proteins that were considered differentially expressed in peripheral nerve fibroblasts versus cardiac fibroblasts. Among them, 25 were significantly upregulated in peripheral nerve fibroblasts compared with cardiac fibroblasts. Activin A, the protein with the greatest differential expression, clearly co-localized with fibroblasts in the in vivo sciatic never injury rat model. In vitro experiments proved that activin A secreted from nerve fibroblasts is the key factor responsible for boosting proliferation and migration of Schwann cells through ALK4, ALK5, and ALK7. Overall, these findings suggest that peripheral nerve fibroblasts and cardiac fibroblasts exhibit different patterns of cytokine secretion and activin A secreted from peripheral nerve fibroblasts can promote the proliferation and migration of Schwann cells.

Keywords: Schwann cells; activin A; cytokine array; fibroblasts; migration; peripheral nervous system; proliferation.

FIGURE 1

Cell characterization and purification. Phase-contrast micrographs of live primary cultured N-Fbs (A), cardiac fibroblasts (B), Schwann cells (C). Scale bar, 100 μm. Immunofluorescence for fibroblast marker CD90 in purified N-Fbs (D), C-Fbs (E), and SC marker GFAP in purified SCs (F). Scale bar: (D,E) 75 μm; (F) 100 μm. Representative figures of flow cytometry analysis of purified N-Fbs (G), C-Fbs (H), SCs (I). The upper graphics are control groups, and the lower graphics are experimental groups. M1/M2 stood for the area under the curve in upper/lower graphics; the percentage of M2 relative to M1 + M2 was the percentage of IgG 488-positive cells. (J) The purity of N-Fbs, C-Fbs, and SCs (three independent experiments).

FIGURE 2

N-Fbs facilitates Schwann cells migration and proliferation in vitro. (A) Phase-contrast micrographs of crystal violet-labeled migrated SCs co-cultured with blank control, C-Fbs, and N-Fbs in transwell for 6 h, 12 h, and 24 h. Scale bar, 50 μm. (B) Migrating SCs quantification (three independent experiments). Statistical significance is indicated as *p < 0.05 N-Fb-12h vs. Control-12h, ^**p < 0.01 C-Fb-24h vs. Control-24h, ^***p < 0.001 N-Fb-24h vs. C-Fb-24h, ^****p < 0.0001 N-Fb-24h vs. Control-24h (two-way analysis of variance followed by Tukey’s post hoc test). (C) EdU⁺ SCs (red) were labeled after 24-h co-culture with N-Fbs and C-Fbs, separately. Scale bar, 75 μm. (D) Quantification of the proportion of EdU⁺ SCs (three independent experiments). Statistical significance is indicated as ^**p < 0.01 (one-way analysis of variance followed by Tukey’s post hoc test).

FIGURE 3

Differentially expressed proteins in N-Fb CM vs. C-Fb CM. (A) Scatter plot of 32 differentially expressed proteins in N-Fb vs. C-Fb-conditioned medium with Log2-foldchange greater than 1.2 or less than 0.83. Red represents the upregulation, while blue represents the downregulation, and gray resembles no difference. Protein function annotation Gene Ontology (GO) and KEGG pathway were analyzed. GO analysis included three subtypes: (B) BP, (C) MF, and (D) CC. (E) KEGG pathway results reveal associated gene functions, linking genomic information with higher-order functional information. (F) q-PCR results of top ten differentially expressed proteins in N-Fb vs. C-Fb (three independent experiments). Statistical significance is indicated as *p < 0.05, ^**p < 0.01, ^****p < 0.0001 (unpaired Student’s t-test). ELISA results of activin A (G) (five independent experiments), Eotaxin (H) (three independent experiments), and Csf2 (I) (four independent experiments). Statistical significance is indicated as *p < 0.05, ^***p < 0.001 (unpaired Student’s t-test).

FIGURE 4

Localization and expression of activin A in N-Fbs in vitro and rat sciatic nerve injury model in vivo. (A) Immunofluorescence for activin A and fibroblast marker CD90 in purified N-Fbs. Scale bar, 100 μm. (B) Immunofluorescence for activin A and SC marker S100 in purified SCs. Scale bar, 100 μm. Expression of activin A in rat sciatic nerve injury model in vivo. (C–E) Immunofluorescence images of activin A (D) and CD90 (E) in sciatic nerve, proximal to distal. Scale bar, 1000 μm. (F–H) Magnified images of the regions demarcated by white boxes in (C). Scale bar, 100 μm.

FIGURE 5

Activin A facilitates Schwann cells proliferation and migration in vitro. (A) EdU⁺ SCs (red) were labeled after 24 h cultured with N-Fb CM and activin A (10-40 ng/ml), separately. Scale bar, 100 μm. (B) Quantification of the proportion of EdU⁺ SCs (4 independent experiments). Statistical significance is indicated as *p < 0.05, ^**p < 0.01, ^****p < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). (C) Phase-contrast micrographs of SCs in the cell-free gap at 0 h and 8 h (after 12 h pretreatment) with blank control, activin A (10-40 ng/ml), separately. Scale bar, 200 μm. (D) Quantification of the SC migrating area percentage relative to the cell-free area at 0 h (three independent experiments). Statistical significance is indicated as ^**p < 0.01, ^****p < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test).

FIGURE 6

SB-431542 inhibits the proliferation and migration promotion effects of N-Fb CM and activin A on Schwann cells in vitro. (A) EdU⁺ SCs (red) were labeled after 24-h culture. Scale bar, 100 μm. (B) Quantification of the proportion of EdU⁺ SCs. Statistical significance is indicated as *p < 0.05, ^***p < 0.001, ^****p < 0.0001 (four independent experiments, one-way analysis of variance followed by Tukey’s post hoc test). (C) Phase-contrast micrographs of SCs in the cell-free gap at 0 h and 8 h (after 12 h pretreatment). Scale bar, 200 μm. (D) Quantification of the SC migrating area percentage relative to the cell-free area at 0 h (three independent experiments). Statistical significance is indicated as ^****p < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test).

FIGURE 7

Activin A-neutralizing antibody inhibits the proliferation and migration promotion effects of N-Fb CM and activin A on Schwann cells in vitro. (A) EdU⁺ SCs (red) were labeled after 24-h culture. Scale bar, 100 μm. (B) Quantification of the proportion of EdU⁺ SCs. Statistical significance is indicated as ^**p < 0.01, ^****p < 0.0001 (three independent experiments, one-way analysis of variance followed by Tukey’s post hoc test). (C) Phase-contrast micrographs of SCs in the cell-free gap at 0 h and 8 h (after 12 h pretreatment). Scale bar, 200 μm. (D) Quantification of the SC migrating area percentage relative to the cell-free area at 0 h (three independent experiments). Statistical significance is indicated as ^****p < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test).