Overexpression of LMP-1 Decreases Apoptosis in Human Nucleus Pulposus Cells via Suppressing the NF- κ B Signaling Pathway

Abstract

Intervertebral disc degeneration (IDD) is a prevalent disease characterized by low back pain. Increasing extracellular matrix (ECM) synthesis and decreasing nucleus pulposus cell (NPC) apoptosis are promising strategies to recover degenerated NP. LIM mineralization protein- (LMP-) 1 has anti-inflammatory potential and is a promising gene target for the treatment of NP degeneration. In this study, we measured the expression of LMP-1 in the NP of patients. Then, we constructed LMP-1-overexpressing NPCs using lentiviral vectors and investigated the effects of LMP-1 on cell proliferation, apoptosis, and ECM synthesis in NPCs. The results showed that LMP-1 was highly expressed in the NP of patients. LMP-1 overexpression significantly increased proliferation and decreased apoptosis in NPCs. The expression of collagen II and sulfated glycosaminoglycan (sGAG) in NPCs was also upregulated after LMP-1 was overexpressed. Moreover, we demonstrated that LMP-1 decreased apoptosis of NPCs by inhibiting NF-κB signaling activation. These findings suggest that LMP-1 plays an essential role in mediating apoptosis in NPCs by regulating NF-κB signaling and can be used as a gene target for the treatment of IDD.

Figure 1

Low LMP-1 expression in NP of patients with disc degeneration. (a) Representative T2 signal MRI images of each group. (b) Gene expression levels of LMP-1 in the NP of IDD patients were measured and normalized to 18 s and to the normal group. (c) The protein expression of LMP-1 in each group was measured by western blotting analysis and quantified. (d) CD45, CD73, and CD90 of the isolated cells were detected by flow cytometry. (e) The expression of collagen II and aggrecan of the isolated cells was measured by immunofluorescence staining. The degenerative-1 group represented patients aged from 20 to 30 years old, and the degenerative-2 group represented patients aged from 50 to 60 years old. Data represent mean ± SD; ^∗∗p < 0.01 vs. the normal group.

Figure 2

The construction of LMP-1 overexpression and lenti-control NPCs. (a) NPCs after lentiviral transfection and puromycin screening were observed under a normal microscope and a fluorescence microscope. (b) Cell viability was measured by CCK-8 on days 1 and 3. (c) The gene expression level of LMP-1 was determined by RT-qPCR and normalized to 18 s. (d) The protein expression of LMP-1 in each group was determined by western blotting analysis. (e) The protein expression of LMP-1 was quantified. Data represent mean ± SD; ^∗p < 0.05, ^∗∗p < 0.01 vs. the mock treated group. Scale bar = 200 μm.

Figure 3

LMP-1 overexpression mediated proliferation, ECM synthesis, and apoptosis of NPCs. (a) Cell proliferation of the LV-control and LV-LMP-1 groups was measured on days 1, 3, and 7. (b) Gene expression levels of Acan, SOX9, Col2, and Col1 of NPCs in each group were measured on day 14 and normalized to 18 s. (c) sGAG synthesis by NPCs was observed by Alcian blue staining on days 14. (d) Measurement of intracellular ROS generation in the LV-control and LV-LMP-1 groups using a DCFH-DA probe by fluorometry. (e) The protein expression levels of aggrecan, SOX9, collagen II, and collagen I of NPCs in each group were measured and (f) quantified on day 14. (g) The protein expression levels of caspase-3, cleaved-caspased-3, Bcl-2, and Bax of NPCs in each group were measured and (h) quantified on day 3. (i) Cell apoptosis of each group was detected by PI/Annexin V assays. Data represent mean ± SD; ^∗∗p < 0.01 vs. the LV-control group. Scale bar = 200 μm.

Figure 4

The activation of NF-κB signaling pathway was mediated by LMP-1. (a) The heat map showed differentially expressed miRNAs between the LV-control and LV-LMP-1 groups. (b) Pearson correlation between each sample was expressed by heat map. (c) GO terms with significant p values for biological processes, molecular function, and cellular component. (d) KEGG terms with significant p values were analyzed.

Figure 5

LMP-1 overexpression inhibited the activation of NF-κB signaling pathway. (a) Protein expression levels of pho-p65, p65, pho-IκBα, and IκBα of NPCs in each group were measured by western blotting analysis on day 3. (b) The protein expression was quantified according to signal intensity, and the ratio of pho-p65/p65 and pho-IκBα/IκBα in each group was calculated. siRNA for LMP-1 was transfected into NPCs, and the protein expression levels of pho-p65, p65, pho-IκBα, and IκBα of NPCs in each group were (c) measured and (d) quantified on day 3. Data represent mean ± SD; ^∗p < 0.05, ^∗∗p < 0.01.

Figure 6

LMP-1 silencing increased apoptosis of NPCs by activating the NF-κB signaling pathway. (a) TUNEL method was performed to measure apoptosis of NPCs (red), and the results were observed by fluorescence. Nuclei (blue) were stained by DAPI. (b) Cell apoptosis was quantified according to the results of TUNEL. ^∗∗p < 0.01. (c) The protein expression levels of caspase-3, cleaved-caspased-3, Bcl-2, and Bax of NPCs were measured by western blotting analysis on day 3. (d) The protein expression levels of caspase-3, cleaved-caspased-3, Bcl-2, and Bax of NPCs were quantified. ^∗∗p < 0.01. (e) Cell proliferation on each group was measured by CCK-8 assay on days 1, 3, and 7. ^∗∗p < 0.01 vs. the control group. (f) The protein expression levels of aggrecan, SOX9, and collagen II of NPCs in each group were measured on day 14 by western blotting analysis. (g) The protein expression levels of aggrecan, SOX9, and collagen II of NPCs were quantified. ^∗p < 0.05, ^∗∗p < 0.01. BAY11-7082 was used as a specific inhibitor of the NF-κB signaling pathway. Data represent mean ± SD; scale bar = 200 μm.

Figure 7

LMP-1 overexpression prevented the degeneration of IVDs. (a) Representative H&E and Safranin O staining of discs from different groups were observed. All samples were harvested at 4 weeks after injection. The contents of (b) sGAG and (c) hydroxyproline in each group at 4 weeks after injection were quantified. Data represent mean ± SEM; ^∗∗p < 0.01. Scale bar = 500 μm.