MMP12-dependent myofibroblast formation contributes to nucleus pulposus fibrosis
Yi Sun, Wai-Kit Tam, Manyu Zhu, Qiuji Lu, Mengqi Yu, Yuching Hsu, Peng Chen, Peng Zhang, Minmin Lyu, Yongcan Huang, Zhaomin Zheng, Xintao Zhang, Victor Y. Leung
Yi Sun, Wai-Kit Tam, Manyu Zhu, Qiuji Lu, Mengqi Yu, Yuching Hsu, Peng Chen, Peng Zhang, Minmin Lyu, Yongcan Huang, Zhaomin Zheng, Xintao Zhang, Victor Y. Leung
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Research Article Bone biology Cell biology

MMP12-dependent myofibroblast formation contributes to nucleus pulposus fibrosis

Abstract

Intervertebral disc degeneration (IDD) is associated with low back pain, a leading cause of disability worldwide. Fibrosis of nucleus pulposus (NP) is a principal component of IDD, featuring an accumulation of myofibroblast-like cells. Previous study indicates that matrix metalloproteinase 12 (MMP12) expression is upregulated in IDD, but its role remains largely unexplored. We here showed that TGF-β1 could promote myofibroblast-like differentiation of human NP cells along with an induction of MMP12 expression. Intriguingly, MMP12 knockdown not only ameliorated the myofibroblastic phenotype but also increased chondrogenic marker expression. Transcriptome analysis revealed that the MMP12-mediated acquisition of myofibroblast phenotype was coupled to processes related to fibroblast activation and osteogenesis and to pathways mediated by MAPK and Wnt signaling. Injury induced mouse IDD showed NP fibrosis with marked increase of collagen deposition and αSMA-expressing cells. In contrast, MMP12-KO mice exhibited largely reduced collagen I and III but increased collagen II and aggrecan deposition, indicating an inhibition of NP fibrosis along with an enhanced cartilaginous matrix remodeling. Consistently, an increase of SOX9+ and CNMD+ but decrease of αSMA+ NP cells was found in the KO. Altogether, our findings suggest a pivotal role of MMP12 in myofibroblast generation, thereby regulating NP fibrosis in IDD.

Authors

Yi Sun, Wai-Kit Tam, Manyu Zhu, Qiuji Lu, Mengqi Yu, Yuching Hsu, Peng Chen, Peng Zhang, Minmin Lyu, Yongcan Huang, Zhaomin Zheng, Xintao Zhang, Victor Y. Leung

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Figure 5

Inhibition of NP fibrosis and myofibroblast formation in Mmp12–/– mice.

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Inhibition of NP fibrosis and myofibroblast formation in Mmp12–/– mice. ...
(A) Time course assessment of NP integrity and remodeling in punctured tail discs of WT and Mmp12-KO mice (Mmp12–/–) (n = 5 mice/group at each time point) by FAST staining, polarized microscopy of Picrosirius red staining (PSR), and immunofluorescence for collagen I (COL1), II (COL2), and III (COL3) as well as aggrecan proteoglycan. Expression of MMP12 and its substrate elastin were also examined. Cell nuclei are stained with DAPI. Bright red-yellow and green color in the PSR staining reflect collagen I and III containing fibers, respectively (39). NP regions are shown by dotted white line. Scale bar: 200 μm and 25 μm (insert). wpp, weeks postpuncture. (B) Disc height index calculated and represented as fold change from unoperated level. (C) Quantification of collagen fibers within the NP by CT-FIRE analysis based on the PSR staining signals: integrated optical density (IOD) of collagen I (red and yellow) and collagen III (green); collagen fiber density (× 100/mm2) and average fiber length (μm) (39). ROI, region of interest. (D) Detection of αSMA, SOX9, and CNMD expressing cells by immunofluorescence (n = 5 mice/group). NP regions are shown by dotted white line. Scale bar: 200 μm and 25 μm (insert). (E–G) Quantitative analysis of the αSMA– (E), SOX9– (F), and CNMD-expressing NPC (G) by counting of the positive cells and averaged to DAPI counts (indicative of total cell number) in the NP area (n = 5). *P < 0.01, **P < 0.01, ***P < 0.001 determined by 2-way ANOVA with Bonferroni post hoc test. Data are presented as the mean ± SD.