Irisin enhances chondrogenic differentiation of human mesenchymal stem cells via Rap1/PI3K/AKT axis

Abstract

Background: Human mesenchymal stem cells (hMSCs) have been proven to have inherent chondrogenic differentiation potential, which appears to be used in cartilage regeneration. Increasing evidence suggests that irisin enhances osteoblast differentiation of MSCs, but little is known about its potential on chondrogenic differentiation.

Methods: In the study, we investigated the effects of irisin on chondrogenic differentiation of hMSCs using a high-density pellet culture system. The cartilage pellets were evaluated by morphology, and the metabolism of cartilage matrix was detected by qPCR, western blot and immunohistochemistry. Next, RNA-seq was performed to explore the underlying mechanism. Furthermore, using the transduction of plasmid, miRNAs mimics and inhibitor, the activation of Rap1/PI3K/AKT axis, the expression level of SIPA1L2, and the functional verification of miR-125b-5p were detected on day 7 of chondrogenic differentiation of hMSCs.

Results: Compared with the controls, we found that irisin treatment could significantly enhance the chondrogenic differentiation of hMSCs, enlarge the induced-cartilage tissue and up-regulate the expression levels of cartilage markers. RNA-seq indicated that irisin activated the Rap1 and PI3K/AKT signaling pathway, and the lower expression level of SIPA1L2 and the higher expression level of miR-125b-5p were found in irisin-treated group. Further, we found that irisin treatment could up-regulate the expression level of miR-125b-5p, targeting SIPA1L2 and consequently activating the Rap1/PI3K/AKT axis on the process of chondrogenic differentiation of hMSCs.

Conclusions: Collectively, our study reveals that irisin can enhance chondrogenic differentiation of hMSCs via the Rap1/PI3K/AKT pathway, suggesting that irisin possesses prospects in cartilage regeneration.

Keywords: Chondrogenic differentiation; Human mesenchymal stem cells; Irisin; Rap1/PI3K/AKT signaling pathway; SIPA1L2; miR-125b-5p.

Fig. 1

Irisin enhances chondrogenic differentiation of hMSCs. A Macro-images of induced-cartilage tissues were taken on Day-7 and Day-14 (Left panel). (Magnification: × 15, scale bar: 2 mm). The relative diameter of cartilage tissues in controls and r-irisin-treated group (Right panel). B The expression of integrin αVβ5 receptor in hMSCs-induced cartilage tissue. (Magnification: × 400, scale bar: 50 μm). C The mRNA expression levels of COL2A1, ACAN and SOX9 were detected by qPCR in different groups on Day-7 and Day-14. D Protein expression levels of COL2A1, ACAN and SOX9 were detected by western blot in different groups on Day-7 and Day-14. E Immunohistochemistry for COL2A1 and ACAN on Day-7 and Day-14 in different groups. (Magnification: × 200, scale bar: 100 μm). F Safranin O and Alcian Blue staining for cartilage matrix metabolism on 7 and 14 days in different groups. (Magnification: × 200, scale bar: 100 μm). The levels were determined using the ImageJ software. The concentration of irisin used in this figure was 100 ng/ml. *P < 0.05, **P < 0.01 compared with the control-group

Fig. 2

Irisin activates the Rap1 and PI3K/AKT pathways. A The heatmap of DEGs, as detected by RNA-seq in hMSCs induced-cartilage tissue treated with and without irisin for 7 d (n = 3 biological replicates). B The volcano map of genes distribution. C GO terms with the most significant P values. BP: biological process, CC: cellular component, MF: molecular function. D GSEA of positively associated cartilage condensation process (Left panel) and cartilage condensation hallmark genes treated with irisin compared with vehicle (Right panel). E The top 20 of pathways enrichment by KEGG analysis. F The mRNA expression levels of key genes (RAP1A, RAP1B, RAP1GAP, SIPA1L2, RASGRP) in Rap1 signaling pathway. The concentration of irisin used in this figure was 100 ng/ml. *P < 0.05, **P < 0.01 compared to control

Fig. 3

Irisin activates the Rap1 signaling pathway by suppressing the expression of SIPA1L2. A Protein expression levels of SIPA1L2 were detected by western blot in control and irisin-induced group on 7 and 14 days. B Immunofluorescence for SIPA1L2 on 7 and 14 days in different groups. (Magnification: × 400, scale bar: 50 μm). C Transfection efficiency verification of SIPA1L2 by western blot. D The mRNA expressions of COL2A1, ACAN, and SOX9. E Immunofluorescence of COL2A1 and SOX9 of MSCs-induced cartilage tissue on 7 days. (Magnification: × 400, scale bar: 50 μm). F Protein expression levels of PI3K, p-PI3K, AKT, p-AKT, mTOR, p-mTOR, COL2A1, ACAN and SOX9 were detected by western blot in different groups. G Safranin O and Alcian Blue staining in different groups. (Magnification: × 200, scale bar: 100 μm). The concentration of irisin used in this figure was 100 ng/ml. ^*P < 0.05, ^**P < 0.01 compared with the control group. ^#P < 0.05, ^##P < 0.01 compared with the irisin + OE-NC group

Fig. 4

Irisin enhances chondrogenic differentiation of hMSCs by activating the PI3K/AKT pathway. A Protein expression levels of PI3K, p-PI3K, AKT and p-AKT were detected by western blot in controls and irisin-treated groups on 7 days. B Quantitative expression levels of PI3K, p-PI3K, AKT and p-AKT using Image J software. C The ratios of relative protein expression of p-PI3K to relative protein expression of PI3K (p-PI3K/PI3K), and p-AKT to relative protein expression of AKT (p-AKT/AKT). D The mRNA and protein expression levels of COL2A1, ACAN, and SOX9 were detected by qPCR and western blot in control, r-irisin, and r-irisin + 3MA-treated group. E The mRNA and protein expressions of COL2A1, ACAN, and SOX9 were detected by qPCR and western blot in control, irisin, and irisin + GSK690693-treated group. F Immunofluorescence of COL2A1 and SOX9 of hMSCs-induced cartilage tissues. (Magnification: × 400, scale bar: 50 μm). G Safranin O and Alcian Blue staining were performed after 7 days of differentiation in different group. (Magnification: × 200, scale bar: 100 μm). The concentration of irisin used in this figure was 100 ng/ml. *P < 0.05, **P < 0.01 compared with the control group. ^#P < 0.05, ^##P < 0.01 compared with the irisin-treated group

Fig. 5

Irisin-mediated up-regulation of miR-125b-5p targeting SIPA1L2 to promote chondrogenic differentiation of hMSCs. A Human MSCs was seeded in 24-wells plates via high-density micromass culture, and chondrogenic differentiation was induced in chondrogenic medium with or without irisin (100 ng/ml) treatment for 7 days. Differently miRNA expressions in control and irisin-treated group were shown. B Relative expressions of the indicated miRNAs in cartilage tissues detected by qPCR. C The mRNA expressions (Left panel) of COL2A1, ACAN, and SOX9, and the protein expressions (Right panel) of SIPA1L2, COL2A1, ACAN, and SOX9 in the control, NC, and miR-125b-5p mimics transduction groups. D The mRNA expressions (Left panel) of COL2A1, ACAN, and SOX9, and the protein expressions (Right panel) of SIPA1L2, COL2A1, ACAN, and SOX9 in the control, NC, and miR-125b-5p inhibitor transduction groups under irisin treatment. E, F The expressions of SIPA1L2, COL2A1, and SOX9 were detected by immunofluorescence combined with DAPI staining for the nuclei in different groups. (Magnification: × 400, scale bar: 50 μm). G, H Safranin O and Alcian Blue staining were performed in cartilage tissue paraffin sections with different treatments as shown. (Magnification: × 200, scale bar: 100 μm). The concentration of irisin used in this figure was 100 ng/ml. *P < 0.05, **P < 0.01 compared with the control group or NC group

Fig. 6

Schematic diagram highlighting the primary findings of this study. Irisin up-regulates miR-125b-5p, targeting SIPA1L2 and activates the Rap1/PI3K/AKT signaling axis, and consequently promoted the expression levels of chondrogenic differentiation genes (COL2A1, ACAN, and SOX9), enhancing chondrogenic differentiation in hMSCs via integrin receptor.