Role of IFITM2 in osteogenic differentiation of C3H10T1/2 mesenchymal stem cells

Abstract

Interferon-inducible transmembrane (IFITM) are a family of small proteins localized to plasma and endolysosomal membranes. Their functions beyond restricting viral entry and replication have been revealed in recent years. IFITM5 is involved in bone mineralization and is an osteogenic cell surface marker. IFITM1 and 3 interact with desmin and myosin, and are involved in myogenic differentiation. This study found upregulation of Ifitm2 during osteogenic differentiation of C3H10T1/2 cells. This positively correlated to the expression of osteogenic differentiation markers Col1a1, Alp, Runx2, and Ocn. Knockdown of Ifitm2 by siRNAs inhibited osteogenic differentiation, calcium deposition, and osteogenic marker expression of C3H10T1/2 cells. The osteoblast transcriptome revealed that knocking down Ifitm2 affected the expression Wnt signaling pathway-related genes, including Wnt family members, their receptors Lrp, Frizzled, and Lgr, and transmembrane molecule Rnf43 that suppresses the Wnt signaling pathway. Luciferase assays indicated enhancement of canonical Wnt signaling pathways by Ifitm2 overexpression. Furthermore, IFITM2 was colocalized in the metaphyseal bone and growth plate of the mouse tibial bone with SP7, a transcription factor essential for osteoblast differentiation and bone formation. These findings reveal a possible novel function and potential mechanisms of Ifitm2 in osteogenic differentiation.

Keywords: C3H10T1/2 cells; IFITM2; TOP/FOP assay; Wnt/β-catenin signaling pathway; osteogenic differentiation.

Figure 1.

Alizarin red S staining and osteogenic biomarker expression of C3H10T1/2 cells at various days of osteogenic induction. (A) Representative alizarin red S staining of cells in 24-well plates. (B) Quantification of alizarin red S staining showed an increase in calcium deposition after osteogenic induction (**P < 0.01 vs. day 0 group). (C) Expression levels of Col1a1, Alp, Runx2, and Ocn measured by RT-qPCR. (D) Representative bands of COL1A1, ALP, RUNX2, OCN, and GAPDH in western blots. Semi-quantification of the band intensity in western blots is shown. Protein levels were normalized to GAPDH. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. day 0 group.

Figure 2.

Increased expression of IFITM2 during osteoblastic differentiation of C3H10T1/2 cells. (A) Relative expression of Ifitm2 mRNA measured by RT-qPCR after osteogenic induction for 3, 5, and 7 days. (B) Representative bands of IFITM2 in western blots and densitometry after osteogenic induction for 3, 5, and 7 days. Protein levels were normalized to GAPDH. (C) Immunofluorescence staining of IFITM2 (green) in C3H10T1/2 cells without or with osteogenic induction (day 5). Scale bars, 50 μm. Nuclei were visualized by DAPI staining. Quantitative analysis of the relative fluorescence intensity by one-way ANOVA is shown (n = 3). **P < 0.01, and ***P < 0.001.

Figure 3.

Alizarin red S staining and osteogenic biomarker expression of C3H10T1/2 cells transfected with si-Ifitm2 after 5 days of osteogenic induction. (A) Alizarin red S staining indicated a decrease in calcium deposition in the si- Ifitm2 group compared with siRNA negative control (si- NC) and transection reagent only (mock) groups. (B) Quantification of alizarin red S staining indicated that si-Ifitm2 reduced the mineralization capacity from 49.19% to 41.89% compared with si-NC. (C) Representative bands of COL1A1, ALP, RUNX2, OCN, and GAPDH in western blots. (D) Quantification of the band intensities. Protein levels were normalized to GAPDH. Magnification: ×10. *P < 0.05, ***P < 0.001.

Figure 4.

Volcano plot, heat map, GO, and KEGG analyses of RNA-sequencing data. (A) Volcano plots of significantly differentially expressed genes (FDR < 0.05 and |FC| ≥ 1.5; red, upregulated; blue, downregulated). (B) Heat map showing 3912 significantly (FDR < 0.05) differentially expressed genes between si1-Ifitm2 and si-NC groups. Each row of the heat map represents the z-score transformed log₂(1+FPKM) values of one differentially expressed gene across all samples (blue, low expression; red, high expression). (C) Significantly differentially expressed genes related to the Wnt signaling pathway. (D-F) Biological process (top 30), cellular components (top 30), molecular functions (top 30), and (G) KEGG enrichment analysis (top 20) of differentially expressed genes, respectively. (H) TOP/FOP luciferase reporter assays in HEK293T cells. TOP/FOP luciferase reporter activity was enhanced by Ifitm2 overexpression.

Figure 5.

Expression of IFITM2 examined by histology in mice. (A-D) Representative immunofluorescence staining of IFITM2 (green) and SP7 (red) in the hind limb bone of 15-week-old mice. Scale bars, 200 μm. (E-H) Articular cartilage and (I-L) metaphyseal bone regions magnified to display positive cells. White arrow indicates IFITM2 and SP7 co-localization in endochondral ossification centers and articular cartilage. Scale bar: 50 μm. DAPI (blue) was used to counterstain nuclei.