S100A6 Regulates nucleus pulposus cell apoptosis via Wnt/β-catenin signaling pathway: an in vitro and in vivo study

Abstract

Background: Intervertebral disc degeneration (IDD) is a common musculoskeletal degenerative disease, which often leads to low back pain and even disability, resulting in loss of labor ability and decreased quality of life. Although many progresses have been made in the current research, the underlying mechanism of IDD remains unclear. The apoptosis of nucleus pulposus (NP) cells (NPCs) is an important pathological mechanism in intervertebral disc degeneration (IDD). This study evaluated the relationship between S100A6 and NPCs and its underlying mechanism.

Methods: Mass spectrometry, bioinformatics, and quantitative real-time polymerase chain reaction (qRT-PCR) analyses were used to screen and verify hub genes for IDD in human IVD specimens with different degeneration degrees. Western blotting, immunohistochemistry (IHC), and/or immunofluorescence (IF) were used to detect the expression level of S100A6 in human NP tissues and NPCs. The apoptotic phenotype of NPCs and Wnt/β-catenin signaling pathway were evaluated using flow cytometry, western blotting, and IF. S100A6 was overexpressed or knocked down in NPCs to determine its impact on apoptosis and Wnt/β-catenin signaling pathway activity. Moreover, we used the XAV-939 to inhibit and SKL2001 to activate the Wnt/β-catenin signaling pathway. The therapeutic effect of S100A6 inhibition on IDD was also evaluated.

Results: S100A6 expression increased in IDD. In vitro, increased S100A6 expression promoted apoptosis in interleukin (IL)-1β-induced NPCs. In contrast, the inhibition of S100A6 expression partially alleviated the progression of annulus fibrosus (AF) puncture-induced IDD in rats. Mechanistic studies revealed that S100A6 regulates NPC apoptosis via Wnt/β-catenin signaling pathway.

Conclusions: This study showed that S100A6 expression increased during IDD and promoted NPCs apoptosis by regulating the Wnt/β-catenin signaling pathway, suggesting that S100A6 is a promising new therapeutic target for IDD.

Keywords: Apoptosis; Intervertebral disc degeneration; S100A6; Wnt/β-catenin signaling pathway.

Fig. 1

Screening of hub genes in IDD. (A)Mass spectrometry analysis revealed the differentially expressed genes. The colors represent P-values and circles represent Log2FC. (B) Differentially expressed genes in IDD according to the GSE23130 dataset analysis. The colors represent P-values and circles represent Log2FC. (C) Venn analysis showing DEGs in GSE23130 dataset, CTD, and GeneCards (370 common genes were obtained). (D) Venn analysis showing the TOP100 proteins of DEPs in mass spectrometry and the result of (C) four co-expressed hub genes (COL6A2, FLOT1, S100A6, and SOD1). (E) Expression levels of MMP13, COL6A2, FLOT1, S100A6, and SOD1 in human IVDs of Pfirrmann Grade II and Grade IV detected using qRT-PCR. * P < 0.05, ** P < 0.01, and *** P < 0.001

Fig. 2

The expressions of S100A6, cleaved caspase 3, and β-catenin were increased in human degenerative NP tissues. (A) Human IVD MRI examination, HE, and Safranin-O/fast green staining with Pfirmmann grading Grade II and Grade IV. (B, C) Western blot and quantitative analysis of S100A6, cleaved caspase 3, β-catenin, MMP13, and Collagen II in Grade IV and Grade II groups. (D-J) IHC staining and quantitative analysis of S100A6, cleaved caspase 3, β-catenin, MMP13 and Collagen II were performed in Grade IV and Grade II groups. * P < 0.05, ** P < 0.01, and *** P < 0.001

Fig. 3

The expressions of S100A6, cleaved caspase 3, and β-catenin increased in the AF puncture-induced rat IDD model. (A, F) MRI examination and Pfirrmann score were performed 8 weeks after surgery in the AF puncture and normal groups. (B) HE and Safranin-O/fast green staining at 8 weeks after surgery in the AF puncture and normal groups. (C, D) Western blot and quantitative analysis of S100A6, cleaved caspase 3, β-catenin, MMP13, and Collagen II in the AF puncture and normal groups. (E, G-I) IHC staining and quantitative analysis of S100A6, cleaved caspase 3, and β-catenin in the AF puncture and normal groups. * P < 0.05, ** P < 0.01, and *** P < 0.001

Fig. 4

The expression of S100A6 and β-catenin and the apoptosis rate of human NPCs were concentration-dependent with IL-1β. (A-D) IF staining and quantitative analysis of S100A6 and β-catenin in human NPCs treated with different concentrations of IL-1β. (E-H) Western blot and quantitative analysis of S100A6, β-catenin, P16, cleaved caspase 3, caspase 9, BAX, and MMP13 in human NPCs treated with different concentrations of IL-1β. * P < 0.05, ** P < 0.01, and *** P < 0.001

Fig. 5

Targeted regulation of S100A6 can affect the apoptosis and the activity of Wnt/β-catenin signaling pathway in IL-1β-induced human NPCs. (A) The apoptosis rates of NPCs determined using flow cytometry. (B) Percentages of apoptotic cells. (C-F) IF staining and quantitative analysis of S100A6 and β-catenin. (G-M) Western blot and quantitative analysis of cleaved caspase 3, caspase 9, BAX, BCL-2, MMP13, and Collagen II. (N-Q) Western blot and quantitative analysis of Wnt3a and β-catenin. * P < 0.05, ** P < 0.01, and *** P < 0.001

Fig. 6

Inhibition of Wnt/β-catenin signaling pathway partially reversed IL-1β-induced human NPCs apoptosis caused by S100A6 overexpression. (A) The apoptosis rates of NPCs were determined using flow cytometry. (B) Percentages of apoptotic cells. (C-F) IF and quantitative analysis of S100A6 and β-catenin. (G-M) Western blot and quantitative analysis of cleaved caspase 3, caspase 9, BCL-2, BAX, Wnt3a, MMP13, β-catenin and Collagen II. * P < 0.05, ** P < 0.01, and *** P < 0.001

Fig. 7

Activation of Wnt/β-catenin signaling pathway could partially reverse the anti-apoptosis of IL-1β-induced human NPCs caused by S100A6 knockdown. (A) The apoptosis rates of NPCs were determined using flow cytometry. (B) Percentages of apoptotic cells. (C-F) IF and quantitative analysis of S100A6 and β-catenin. (G-M) Western blot and quantitative analysis of cleaved caspase 3, caspase 9, BCL-2, BAX, Wnt3a, MMP13, β-catenin, and Collagen II. * P < 0.05, ** P < 0.01, and *** P < 0.001

Fig. 8

S100A6 inhibition alleviated IDD progression in vivo. (A, B) MRI examination and Pfirrmann score at 8 weeks after surgery. (C) HE and Safranin-O/fast green staining at 8 weeks after surgery. (D-H) IHC staining and quantitative analysis of S100A6, cleaved caspase 3, β-catenin, and Collagen II at 8 weeks after operation. * P < 0.05, ** P < 0.01, and *** P < 0.001

Fig. 9

S100A6 mediates the apoptosis of human NPCs by regulating the activity of Wnt/β-catenin signaling pathway