Schwann Cells Accelerate Osteogenesis via the Mif/CD74/FOXO1 Signaling Pathway In Vitro

Abstract

Schwann cells have been found to promote osteogenesis by an unclear molecular mechanism. To better understand how Schwann cells accelerate osteogenesis, RNA-Seq and LC-MS/MS were utilized to explore the transcriptomic and metabolic response of MC3T3-E1 to Schwann cells. Osteogenic differentiation was determined by ALP staining. Lentiviruses were constructed to alter the expression of Mif (macrophage migration inhibitory factor) in Schwann cells. Western blot (WB) analysis was employed to detect the protein expression. The results of this study show that Mif is essential for Schwann cells to promote osteogenesis, and its downstream CD74/FOXO1 is also involved in the promotion of Schwann cells on osteogenesis. Further, Schwann cells regulate amino acid metabolism and lipid metabolism in preosteoblasts. These findings unveil the mechanism for Schwann cells to promote osteogenesis where Mif is a key factor.

Figure 1

(a) Diagram of the Schwann cells and MC3T3-E1 cell coculture model; (b) representative images showing ALP staining; (c) ALP activity was measured with an ALP assay kit, n = 6, p = 0.0108; l; (d) representative images showing Alizarin red staining; l; (e) real-time RT-PCR (qPCR) analyses. ^∗P < 0.05, Student's t-test. N = 4; (f–i) transcriptome differences between the coculture group and the control group in MC3T3-E1 cells; (f) the boxplots represent the distribution of expression levels; the ordinate is gene expression level; (g) volcano plot of differential gene expression analysis; the dotted blue line indicates the threshold of the differential genes selected; (h) cluster heatmap of differentially expressed genes; (i) the KEGG enrichment scatter plot. The size of the black spots represents the gene number; the gradual color represents the P value.

Figure 2

(a) OPLS-DA score scatter plot. Each point represents a sample; (b) representative chromatograms in positive (POS) and negative (NEG) ion modes; (c) volcano plot of differential metabolite analysis. The dotted line indicates the threshold of the differential metabolites selected; (d) cluster analysis of differential metabolites; (e) pathway analysis.

Figure 3

KEGG pathway classification: metabolites detected and annotated; red/blue dots represent the differentially expressed compounds; color depth represents the P value.

Figure 4

Heatmap indicating the correlation between the differential transcripts and metabolites. Red (corr = 1), blue (corr = −1), and white (corr = 0); ^∗P < 0.05 for Spearman correlation.

Figure 5

Schwann cells promote osteogenesis by Mif/CD74/FOXO1 signaling. (a) Mif concentration in the medium released from Schwann cells; (b) representative images showing ALP staining; (c) ALP activity was measured with an ALP assay kit, n = 6; (d–h) representative WB images for steroid biosynthesis (d), glycolysis/gluconeogenesis (e), and signaling pathway (f, g). WB analysis was also quantified by ImageJ.