Down-regulation of insulin-like growth factor binding protein 5 is involved in intervertebral disc degeneration via the ERK signalling pathway

Abstract

It is obvious that epigenetic processes influence the evolution of intervertebral disc degeneration (IDD). However, its molecular mechanisms are poorly understood. Therefore, we tested the hypothesis that IGFBP5, a potential regulator of IDD, modulates IDD via the ERK signalling pathway. We showed that IGFBP5 mRNA was significantly down-regulated in degenerative nucleus pulposus (NP) tissues. IGFBP5 was shown to significantly promote NP cell proliferation and inhibit apoptosis in vitro, which was confirmed by MTT, flow cytometry and colony formation assays. Furthermore, IGFBP5 was shown to exert its effects by inhibiting the ERK signalling pathway. The effects induced by IGFBP5 overexpression on NP cells were similar to those induced by treatment with an ERK pathway inhibitor (PD98059). Moreover, qRT-PCR and Western blot analyses were performed to examine the levels of apoptosis-related factors, including Bax, caspase-3 and Bcl2. The silencing of IGFBP5 up-regulated the levels of Bax and caspase-3 and down-regulated the level of Bcl2, thereby contributing to the development of human IDD. Furthermore, these results were confirmed in vivo using an IDD rat model, which showed that the induction of Igfbp5 mRNA expression abrogated the effects of IGFBP5 silencing on intervertebral discs. Overall, our findings elucidate the role of IGFBP5 in the pathogenesis of IDD and provide a potential novel therapeutic target for IDD.

Keywords: ERK; IGFBP5; intervertebral disc degeneration; nucleus pulposus.

Figure 1

A, A heat map was generated by unsupervised clustering analyses with 23 significantly dysregulated mRNAs in patients with IDD. Hierarchical clustering was performed with average linkage and uncentered correlation. The mRNA expression profile effectively segregated patients with IDD from NCs. B, The expression level of IGFBP5 mRNA in NP cells was measured in 129 patients and 112 NCs (in the training and validation sets) using qRT‐PCR assays (***P < 0.001). C, The IGFBP5 mRNA expression levels were inversely correlated with the Pfirrmann scores (r = −0.72, P < 0.0001). D, The expression of IGFBP5 in degenerative NP tissues was significantly lower than that in normal NP samples, as assessed by Western blot analysis (P < 0.05). IDD, intervertebral disc degeneration; NCs, normal controls; NP, nucleus pulposus

Figure 2

Analysis of cell proliferation and apoptosis. A, An MTT assay showed that IGFBP5 overexpression increased cellular proliferation at day 10; **P < 0.01 and ***P < 0.001 compared with the normal control. B‐D, Flow cytometry and colony formation assays demonstrated that IGFBP5 overexpression could effectively promote NP cell proliferation and inhibit NP cell apoptosis. In contrast, IGFBP5‐shRNA could suppress NP cell proliferation and promote NP cell apoptosis; ***P < 0.001 compared with the normal control. NP, nucleus pulposus

Figure 3

IGFBP5 exerts its effects by inhibiting the ERK signalling pathway. A, The NP cells in each group were transfected and showed high transfection efficiency; ***P < 0.001 compared with the normal control. B and C, The overexpression of IGFBP5 inhibited ERK signalling pathways in NP cells. qRT‐PCR and Western blot analyses demonstrated that IGFBP5 overexpression led to decreases in ERK, pERK, Bax and caspase‐3; ***P < 0.001 compared with the normal control. In NP cells treated with IGFBP5‐shRNA, the expression levels of ERK, pERK, Bax and caspase‐3 were significantly increased, while the level of Bcl2 was decreased; ***P < 0.001 compared with the normal control. The values are presented as the mean ± SD. NP, nucleus pulposus

Figure 4

Effect of IGFBP5 in IDD rats. A and B, Severe intervertebral disc degeneration was induced in the rats in the IDD group. Igfbp5 mRNA expression treatment abrogated the effects on the intervertebral disc tissues. C and D, The expression level of IGFBP5 in NP cells was detected by immunofluorescence staining and qRT‐PCR; ***P < 0.001 compared with the normal control. D and E, The mRNA expression levels of Erk1/2, Bax, caspase‐3 and Bcl2 were measured by qRT‐PCR. The protein expression levels of ERK, pERK, Bax, caspase‐3 and Bcl2 were measured by Western blotting; ***P < 0.001 compared with the normal control. Data are shown as the mean ± SD of five rats in each group. NC = normal control group, IDD = IDD model group, IDD + Igfbp5=IDD model rats treated with an Igfbp5 mRNA‐expressing lentivirus