Grem1 accelerates nucleus pulposus cell apoptosis and intervertebral disc degeneration by inhibiting TGF-β-mediated Smad2/3 phosphorylation

Abstract

Intervertebral disc degeneration (IVDD) is a main cause of low back pain, and inflammatory factors play key roles in its pathogenesis. Gremlin-1 (Grem1) was reported to induce an inflammatory response in other fields. This study aimed to investigate the mechanisms of Grem1 in the degenerative process of intervertebral discs. Dysregulated genes were determined by analyzing microarray profiles. The expression of Grem1 in 17 human disc samples (male:female = 9:8) and rat models (n = 5 each group) was measured by western blotting (WB), real-time quantitative PCR (RT-qPCR), and immunohistochemistry (IHC). The regulatory effects of Grem1 on apoptosis were examined using siRNAs, flow cytometry, immunofluorescence (IF), and WB. The therapeutic effect was evaluated by locally injecting specific Grem1 siRNA into IVDD rats. The expression of Grem1 was significantly increased in human degenerative intervertebral discs; furthermore, the expression of Grem1 positively correlated with the level of intervertebral disc degeneration. Grem1 was significantly overexpressed in tumor necrosis factor (TNF)-α-induced degenerative NP cells. Apoptosis in degenerative NP cells transfected with siRNA targeting Grem1 was significantly lower than that in the control group. Specific Grem1 siRNA markedly repressed the development of IVDD in surgery-induced IVDD rats. These results indicated that the expression of Grem1 was positively correlated with the severity of intervertebral disc degeneration, and Grem1 siRNA could inhibit Grem1-induced apoptosis and extracellular matrix alterations by mediating the TGF-β/Smad signaling pathway. This study may provide a therapeutic strategy for alleviating inflammation-induced apoptosis associated with intervertebral disc degeneration.

Fig. 1. Identification of abnormally expressed genes associated with IVDD in the GSE70362 dataset.

a Heatmap depicting all abnormally expressed genes in degenerative NP and nondegenerative NP tissues. b, c Circos plot showing differentially expressed genes. The left column represents differentially expressed genes, and the right column is different biological processes. d Volcano plot showing dysregulated genes.

Fig. 2. Upregulated expression of Grem1 in human degenerative and rat model intervertebral discs.

a Representative MRI images showing the different grades of degeneration in human tissues: mild (Grades I and II) and severe (Grades III–V). b, c Grem1 was expressed in human NP tissues in the control (MDD) and SDD groups, as determined by immunohistochemistry, and quantitative analysis was performed. d, e Western blot analysis of Grem1 in disc tissues was performed. ImageJ or Image Lab software was used to determine the band intensities, which were normalized to GAPDH. f RT-qPCR analysis of Grem1 in MDD and SDD disc tissues. g, j Representative MRI images are shown for the control and IVDD rat models, and quantitative analysis based on the Pfirrmann grade was performed. h, k Representative images of HE staining, safranin-O staining, and histological scores of rat disc tissues are shown. i, l Immunohistochemical staining of Grem1 and qualification analysis. m, n Grem1 was expressed in rat model disc tissues, and quantitative analysis was performed. o RT-qPCR analysis of Grem1 in rat model disc tissues. The data are expressed as the mean ± SD (n = 3); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. MDD mildly degenerated intervertebral discs, SDD severely degenerated intervertebral discs.

Fig. 3. TNF-α induced Grem1 expression in NP cells.

a, h Representative images of western blots showing changes in Grem1, apoptosis-related proteins (cleaved Caspase3, Bax, and Bcl2), Collagen II and Aggrecan in a dose-dependent (0, 20, 50, 100 ng/ml, 24 h) and time-dependent (50 ng/ml, 0, 12, 24, 48 h) manner in NP cells. b–g, i–n ImageJ or Image Lab software was used to determine the band intensities, which were normalized to β-actin. o Representative immunofluorescence images are shown to visualize the expression of Grem1 in NP cells (magnification: ×400). The data are expressed as the mean ± SD (n = 3); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 4. Grem1 siRNA prevented TNF-α-induced apoptosis-related gene expression in NP cells.

a The protein expression of cleaved Caspase-3, Bax, and Bcl2 in the different treatment groups, as determined by western blotting. b–d ImageJ or Image Lab software was used to determine the band intensities, which were normalized to β-actin. e, g Representative images of flow cytometry and TUNEL staining (magnification: ×400) are shown in groups after the different treatments. f, h Quantitative analysis was performed. The data are expressed as the mean ± SD (n = 3); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 5. Identification of Smad-2/3 as a target gene for Grem1.

a p-Smad2 and p-Smad3 levels after rh-Grem1 (50 ng/mL, 30 min) or TGF-β1 (20 ng/mL, 30 min) treatment, as determined by western blotting. b, c ImageJ or Image Lab software was used to determine the band intensities, which were normalized to β-actin. d, e Representative immunofluorescence images are shown to visualize the expression of p-Smad2 and p-Smad3 in NP cells (magnification: ×400). The data are expressed as the mean ± SD (n = 3); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. p-Smad2 phosphorylated Smad-2. p-Smad3 phosphorylated Smad-3.

Fig. 6. Grem1 regulates IVDD by suppressing the TGF-β/Smad signaling pathway.

a, e The protein expression of cleaved Caspase-3, Bax, and Bcl2 and aggrecan, collagen II, MMP3, and Adamts5 in groups treated with rh-Grem1 (50 ng/mL, 24 h) or TGF-β1. b–d, f–i ImageJ or Image Lab software was used to determine the band intensities, which were normalized to β-actin. j, k Representative images of flow cytometry and TUNEL staining (magnification: ×400) are shown in groups after the different treatments. l, m Quantitative analysis was performed. The data are expressed as the mean ± SD (n = 3); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 7. Grem1 siRNA alleviated surgery-induced IVDD in a rat model.

a, b The four MRI images shown are representative of the different groups (CTR, AFP, AFP + si-CTR, AFP + si-Grem1) 8 weeks after surgery, and quantitative analysis based on the Pfirrmann grade was performed. c, d Representative images of HE staining, safranin-O staining, and histological scores in the different groups are shown. e–g Immunohistochemical staining of aggrecan and collagen II and qualification analysis are presented (magnification: ×400). The data are expressed as the mean ± SD (n = 5); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. CTR control, AFP annulus fibrosus puncture, si-CTR control siRNA, si-Grem1 Grem1 siRNA.

Fig. 8. The mechanism by which Grem1 regulates the TGF-β1/Smad2/3 signaling pathway in IVDD.

Grem1 inhibited Grem1-induced apoptosis and ECM component alteration by mediating the TGF-β/Smad signaling pathway. ECM extracellular matrix.