Enhancement of intervertebral disc cell senescence by WNT/β-catenin signaling-induced matrix metalloproteinase expression

Abstract

Objective: To determine whether intervertebral disc (IVD) cells express β-catenin and to assess the role of the WNT/β-catenin signaling pathway in cellular senescence and aggrecan synthesis.

Methods: The expression of β-catenin messenger RNA (mRNA) and protein in rat IVD cells was assessed by using several real-time reverse transcription-polymerase chain reaction, Western blot, immunohistochemical, and immunofluorescence analyses. The effect of WNT/β-catenin on nucleus pulposus (NP) cells was examined by transfection experiments, an MTT assay, senescence-associated β-galactosidase staining, a cell cycle analysis, and a transforming growth factor (TGFβ)/bone morphogenetic protein (BMP) pathway-focused microarray analysis.

Results: We found that β-catenin mRNA and protein were expressed in discs in vivo and that rat NP cells exhibited increased β-catenin mRNA and protein upon stimulation with lithium chloride, a known activator of WNT signaling. LiCl treatment inhibited the proliferation of NP cells in a time- and dose-dependent manner. In addition, there was an increased level of cellular senescence in LiCl-treated cells. Long-term treatment with LiCl induced cell cycle arrest and promoted subsequent apoptosis in NP cells. Activation of WNT/β-catenin signaling also regulated the expression of aggrecan. We also demonstrated that WNT/β-catenin signaling induced the expression of matrix metalloproteinases (MMPs) and TGFβ in NP cells.

Conclusion: The activation of WNT/β-catenin signaling promotes cellular senescence and may modulate MMP and TGFβ signaling in NP cells. We hypothesize that the activation of WNT/β-catenin signaling may lead to an increased breakdown of the matrix, thereby promoting IVD degeneration.

Figure 1

A and B, Sagittal sections of an intervertebral disc from a neonatal rat (A) and an embryonic mouse (obtained on day 15.0 of embryogenesis) (B). Sections were treated with an anti–β-catenin antibody, and counterstained with hematoxylin. Nucleus pulposus (NP) cells expressed β-catenin protein (arrows). In A, the boxed area in the left panel is shown at higher magnification in the right panel. C, Real-time reverse transcription–polymerase chain reaction analysis of β-catenin mRNA levels in NP cells and in anulus fibrosus (AF) cells cultured for 24 hours in the presence or absence of 20 mM LiCl. Values are the mean and SD. * = P < 0.05 versus controls. D, Detection of β-catenin expression by immunofluorescence microscopy. After 24 hours of culture in the presence or absence of 20 mM LiCl, NP cells were fixed and stained with an antibody raised against β-catenin as well as with DAPI (to identify healthy nuclei). There was nuclear localization of β-catenin in cells treated with LiCl as compared with the positive control. The merge image represents cells stained with β-catenin and DAPI. Bars = 200 μm (original magnification × 10). E, Representative Western blot showing a detectable increase in β-catenin protein levels at 6 hours after treatment with 20 mM LiCl. F, Basal activities of TOPflash in both NP cells and AF cells were determined by dual luciferase assay. G, NP cells were cotransfected with the TOPflash reporter plasmid (left) or with the FOPflash reporter plasmid and pGL4.74 plasmid (right). Values in F and G are the mean and SD. * = P < 0.05. NS = not significant.

Figure 2

A and B, Determination of cell viability in nucleus pulposus (NP) cells. NP cells were pretreated for 48 hours (A) or for up to 72 hours (B) with various concentrations (1–20 mM) of LiCl, and cell viability was determined by MTT assay. Values are the mean and SD. * = P < 0.05 versus controls (Cr). C, Photomicrographs of NP cells harvested from 12-week-old Sprague-Dawley rats and cultured for 24 hours in the presence or absence of 20 mM LiCl. Bars = 500 μm (original magnification × 4). D, Photomicrographs showing staining of NP cells and anulus fibrosus (AF) cells for senescence-associated β-galactosidase (SA-β-gal), which was determined as the percentage of positive cells (top). Positive staining for SA-β-gal was detectable for 24 hours after LiCl treatment. Bars = 500 μm (original magnification × 4). SA-β-gal staining in NP (left) and AF (right) cells was also quantified (bottom). A minimum of 100 cells spanning 5 different microscopy fields were scored for staining. Values are the mean and SD. * = P < 0.05.

Figure 3

A, Cell cycle analysis of nucleus pulposus (NP) and anulus fibrosus (AF) cells. Analysis of the cell cycle was performed using combined 5-bromo-2′-deoxyuridine (BrdU) and 7-aminoactinomycin D (7-AAD) double staining (top row) and allophycocyanin (APC)–labeled Ki-67 staining (bottom row) in NP cells and AF cells treated for 24 hours with 20 mM LiCl. The percentages of cells in phases G₁, S, and G₂/M are indicated in each of the flow cytometric dot plots (top). The mean ± SD percentages of Ki-67–negative (G₀) and Ki-67–positive (G₁) cells in the total population were also calculated (bottom). FITC = fluorescein isothiocyanate; PE = phycoerythrin. B, Determination of NP cell proliferation and caspase 3/7, 8, and 9 activities. The activity of caspases 3/7, 8, and 9 and NP cell proliferation were quantified in 2 separate microtiter plates using the Caspase-Glo 3/7, 8, and 9 Assay and the CellTiter-Glo Luminescent Cell Viability Assay, respectively. The luminescence values in the caspase assays were normalized to the luminescence in the cell viability assay. Values are the mean and SD of 8 replicates per treatment. * = P < 0.05 versus controls.

Figure 4

A, Photomicrographs showing matrix metalloproteinase 9 (MMP-9) and MMP-10 expression in nucleus pulposus (NP) cells, as determined by immunofluorescence. NP cells were grown in 96-well plates and exposed to LiCl (20 mM) for 24 hours. Cells were stained with the appropriate primary and fluorescence-labeled secondary antibodies. Results are representative of 3 independent experiments per protein. Bars = 200 μm (original magnification × 20). B, Effects of the canonical WNT/β-catenin signal in NP cells on the expression of MMP-2, MMP-3, MMP-7, MMP-9, and MMP-13 (left), as well as MMP-10 (right). Relative expression of the MMPs and GAPDH was determined by real-time polymerase chain reaction analysis, quantified, and normalized to the expression in untreated cells, which was arbitrarily set at 1.0. Values are the mean and SD. * = P < 0.05 versus controls. C, Expression of MMP-9 (left) and MMP-10 (right) in NP cells cotransfected with WT-β-catenin expression plasmid. Levels of MMPs 9 and 10 expression were determined in NP cells cotransfected with MMP-9-Luc (400 ng) or MMP-10-Luc (400 ng) and an increasing concentration of the WT-β-catenin expression plasmid (100–500 ng). Values are the mean and SD. * = P < 0.05.

Figure 5

Expression of aggrecan and type II collagen in rat nucleus pulposus (NP) cells. A–D, Aggrecan reporter plasmid (A and B) or type II collagen reporter plasmid (C and D) was transfected into rat NP cells with the pGL4.74 vector. Cells were then stimulated for 24 hours with increasing concentrations of LiCl (A and C) or were cotransfected with 400 ng of aggrecan or type II collagen reporter plasmid and an increasing concentration of a WT-β-catenin expression plasmid (B and D), and reporter activity was measured. E, Relative expression of the aggrecan mRNA (top) and Col2a1 mRNA (bottom) was determined by real-time polymerase chain reaction analysis. NP cells were left untreated or were treated with LiCl, and the levels of aggrecan, Col2a1, and GAPDH expression were quantified and normalized to the expression in untreated cells, which was arbitrarily set at 1.0. Values in A–E are the mean and SD of 3 independent transfections. * = P < 0.05. NS = not significant.

Figure 6

A, Profiling of genes regulated by transforming growth factor β (TGFβ)/bone morphogenetic protein (BMP) signaling in nucleus pulposus (NP) cells. Representative Oligo GEArray profiling results are shown. The intensity of several spots was up-regulated or down-regulated after 24 hours of treatment of NP cells with LiCl (20 mM) (right) as compared with controls (left). Regions containing Col1a1, Col3a1, and TGFβ3 are shown at higher magnification in the middle and bottom panels, and demonstrate that the intensity of these spots was markedly increased in the LiCl-treated cells. Band intensities were quantified by densitometry and normalized to control (without treatment) gene levels using CS Analyzer software (version 2.01; Atto). Spots of interest are numbered in the control array and are enclosed in boxes in both the control and the LiCl treatment arrays; the numbers are defined at bottom right. B, Relative expression of TGFβ3, Col1a1, and Col3a1 (left) and TGFβ3 following LiCl treatment (right) in NP cells, as determined by real-time polymerase chain reaction analysis. Levels of TGFβ3, Col1a1, Col3a1, and GAPDH expression were quantified and normalized to the expression in untreated cells, which was arbitrarily set at 1.0. TGFβ3 gene expression was significantly increased after 24 hours of treatment of NP cells with LiCl (20 mM), with 6.8-fold higher levels than in untreated control cells. Values are the mean and SD. * = P < 0.05 versus control.