Irisin Ameliorates Intervertebral Disc Degeneration by Activating LATS/YAP/CTGF Signaling

Abstract

Unbalanced metabolism of an extracellular matrix (ECM) in nucleus pulposus cells (NPCs) is widely acknowledged as the primary cause of intervertebral disc degeneration (IDD). Irisin, a novel myokine, is cleaved from fibronectin type III domain-containing 5 (FNDC5) and has recently been proven to regulate the metabolism of ECM. However, little is known about its potential on NPCs and the development of IDD. Therefore, this study sought to examine the protective effects and molecular mechanism of irisin on IDD in vivo and in vitro. Decreased expression levels of FNDC5 and anabolism markers (COL2A1 and ACAN) but increased levels of catabolism markers (ADAMTS4) were found in degenerative nucleus pulposus (NP) tissues. In a punctured-induced rat IDD model, irisin treatment was found to significantly slow the development of IDD, and in TNF-α-stimulated NPCs, irisin treatment partly reversed the disorder of ECM metabolism. In mechanism, RNA-seq results suggested that irisin treatment affected the Hippo signaling pathway. Further studies revealed that with irisin treatment, the phosphorylation levels of key factors (LATS and YAP) were downregulated, while the expression level of CTGF was upregulated. Moreover, CTGF knockdown partially eliminated the protective effects of irisin on the metabolism of ECM in NPCs, including inhibiting the anabolism and promoting the catabolism. Taken together, this study demonstrated that the expression levels of FNDC5 were decreased in degenerative NP tissues, while irisin treatment promoted the anabolism, inhibited the catabolism of the ECM in NPCs, and delayed the progression of IDD via LATS/YAP/CTGF signaling. These results shed light on the protective actions of irisin on NPCs, leading to the development of a novel therapeutic target for treating IDD.

Figure 1

Decreased expression of FNDC5 in degenerative human NP tissue. (a, b) IHC staining assay of ACAN, COL2A1, ADAMTS4, and TNF-α in control and degenerative human nucleus pulposus tissues. (c) The protein expression levels of COL2A1, ADAMTS4, and TNF-α were detected using western blotting in control and degenerative human NP tissues. (d) The quantitative analysis of the protein bands in (c) using ImageJ software. (e) The protein expression levels of FNDC5 were detected by western blot in the control and degenerative groups. (f) The quantitative analysis of the protein bands in (e) using ImageJ software. The scale bar of images in (a) and (b) was shown (magnification: ×200, scale bar: 100 μm; magnification: ×400, scale bar: 50 μm). ^∗P < 0.05, ^∗∗P < 0.01.

Figure 2

Irisin ameliorates the progression of IDD in a puncture-induced rat model in vivo. (a) The T2 phase images of micro-MRI scan of the caudal intervertebral disc in different groups: the white arrows indicated the control and affected discs. (b) The expression of the integrin αVβ5 receptor in the intervertebral disc tissues (magnification: ×400, scale bar: 50 μm). (c) HE staining of the affected intervertebral disc in different groups. Images (magnification: ×40, scale bar: 500 μm; magnification: ×100, scale bar: 200 μm). (d) IHC staining assay of COL2A1, ACAN, MMP9, and TNF-α in different groups. (e) The MRI Pfirrmann grade analysis of the intervertebral disc in different groups. (f) The histological score of the intervertebral disc in different groups. Images (magnification: ×400, scale bar: 50 μm). ^∗P < 0.05, ^∗∗P < 0.01.

Figure 3

Irisin treatment restored the metabolism of the ECM in TNF-α-induced NPCs. (a) CCK-8 assay was performed to detect the effects of irisin on the cells' viability at various concentrations (0, 25, 50, 100, 200, and 400 ng/ml) at 24 and 48 hours. (b) The relative expression levels of different genes (COL2A1, MMP9, ADAMTS4, and ADAMTS5) were detected using qPCR in different groups. (c) The protein expression levels of COL2A1, MMP9, MMP13, ADAMTS4, and ADAMTS5 were detected by western blot in different groups. (d) The expression levels of COL2A1 and ADAMTS4 were detected by immunofluorescence in different groups. DAPI was used to stain for the nuclei. Images (magnification: ×400, scale bar: 50 μm). In (b)–(d), irisin was used at the concentration of 100 ng/ml and TNF-α was used at the concentration of 10 ng/ml. ^∗P < 0.05, ^∗∗P < 0.01.

Figure 4

Irisin treatment affected the activation of the Hippo signaling pathway. (a) The heatmap of differentially expressed genes (DEGs), which were detected by RNA-seq in human nucleus pulposus cells between TNF-α-treated groups and TNF-α plus irisin-treated groups. (b) The volcano map of gene distribution (upregulation, downregulation, and none). (c) GO analysis terms with the most significant P values were determined, including three analyses (BP: biological process, CC: cellular component, and MF: molecular function). (d) KEGG pathway enrichment analysis showed the affected signaling pathways with P value less than 0.05. (e) The heatmap of genes' expression levels, including anabolic marker (COL2A1), catabolic markers (MMPs and ADAMTSs), and inflammation factors (IL-6 and NLRP3). In this figure, irisin was used at the concentration of 100 ng/ml and TNF-α was used at the concentration of 10 ng/ml.

Figure 5

LATS and YAP mediated the effects of irisin on the ECM metabolism in human NPCs. (a) The expression levels of YAP protein were detected by western blot between control and degenerative groups. (b) IHC staining was performed to detect the YAP expression level between control and puncture groups in rats' intervertebral disc. Images (magnification: ×400, scale bar: 50 μm). (c) Protein expression levels of the Hippo signaling pathway (LATS1, LATS2, p-LATS1/2, YAP, and p-YAP) were detected by western blot in different groups. (d) Quantitative analysis of the bands in (c) using ImageJ software. (e) The localization of YAP was assessed via immunofluorescence in different groups. Images (magnification: ×400, scale bar: 50 μm). (f) The protein expression levels of ECM metabolism markers (COL2A1, MMP13, ADAMTS4, and ADAMTS5) were assessed by western blot in human NPCs. (g) The expression levels of ADAMTS4, COL2A1, and CTGF were detected by immunofluorescence in human NPCs. Images (magnification: ×400, scale bar: 50 μm). In this figure, irisin was used at the concentration of 100 ng/ml, TNF-α was used at the concentration of 10 ng/ml, and verteporfin was used at the concentration of 5 μmol/l. ^∗P < 0.05, ^∗∗P < 0.01.

Figure 6

CTGF protein mediated the protective effect of irisin on the regulation of ECM metabolism in human NPCs. (a) The expression levels of CTGF protein were detected between control and degenerative groups. (b) Quantitative analyses of the bands in (a) using ImageJ software. (c) Immunofluorescence analysis was performed between control and puncture groups in rats' intervertebral disc. Images (magnification: ×400, scale bar: 50 μm). (d) The transfection efficiency verification of CTGF using western blot. (e) The mRNA expression levels of anabolic markers (COL2A1 and ACAN) and catabolic markers (MMP9, MMP13, ADAMTS4, and ADAMTS5) were detected by qPCR. (f) The protein levels of COL2A1, MMP9, ADAMTS4, and ADAMTS5 were assessed by western blot. In this figure, irisin was used at the concentration of 100 ng/ml and TNF-α was used at the concentration of 10 ng/ml. ^∗P < 0.05, ^∗∗P < 0.01 compared with the control group. ^#P < 0.05, ^##P < 0.01 compared with the TNF-α group. ^&P < 0.05, ^&&P < 0.01 compared with the TNF-α+irisin group.

Figure 7

Schematic diagram of irisin's effects on human NPCs. Irisin inhibited the phosphorylation of LATS and YAP proteins induced by TNF-α, promoting nucleus translocation of YAP and upregulating the expression level of CTGF, thus promoting the expression levels of matrix synthesis markers (COL2A1 and ACAN) and inhibiting the levels of matrix-degrading enzymes (MMPs and ADAMTSs), finally reversing the unbalanced metabolism of ECM in human NPCs.