Intermittent cyclic mechanical tension promotes endplate cartilage degeneration via canonical Wnt signaling pathway and E-cadherin/β-catenin complex cross-talk

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Intermittent cyclic mechanical tension promotes endplate cartilage degeneration via canonical Wnt signaling pathway and E-cadherin/β-catenin complex cross-talk H.-g. Xu, Q. Zheng, J.-x. Song, J. Li, H. Wang, P. Liu, J. Wang, C.-d. Wang, X.-l. Zhang Osteoarthritis and Cartilage Volume 24, Issue 1, Pages 158-168 (January 2016) DOI: 10.1016/j.joca.2015.07.019 Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 ICMT induces disc and endplate chondrocyte matrix degeneration. (A) Human endplate cartilage was obtained from patients with cervical vertebrae fracture (normal disc group) or cervical disc degeneration (CDD group). MRIs of subjects and the vertebral surgery (green arrow) with the site of the obtained material (red arrow) are shown. (B) External loading device and animal model. (C) COL-2A, ACAN, and Sox9 expression in normal disc and CDD groups was measured by real-time RT-PCR (normal disc group: n = 8; CDD group n = 12). (D) Rabbit discs treated with (Loaded) or without (Sham) tensile loading for 0, 28, and 56 days, H&E and Safranin O-fast green staining showed changes in the disc structure and matrix distribution. Original magnification: ×40. Representative results from three samples are shown. (E) Real-time RT-PCR analysis of COL-2A, ACAN, and Sox9 expression in endplate cartilage after tensile strain application in vitro. (F) Relative expression of COL-2A, ACAN, and Sox9 in endplates was measured by real-time RT-PCR after ICMT stimulation in vitro. (G) Alcian Blue and Toluidine Blue staining showed decreases in the chondrocyte matrix after ICMT application for 7 days. Representative results from three donors are shown. GAPDH was used as an internal control for the Real-time RT-PCR. Real-time RT-PCR data are presented as the means and 95% CI from three independent experiments, each performed in triplicate. Each dot representing the average of one donor. *Statistically significant difference. Osteoarthritis and Cartilage 2016 24, 158-168DOI: (10.1016/j.joca.2015.07.019) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 ICMT does not induce endplate chondrocyte death but changes the cytoskeleton. (A and B) After mechanical loading in vivo, NBT/DAPI staining showed no obvious differences in the viable cell ratio of endplate chondrocytes between 0 d, sham operation, and tensile loading groups. NBT staining indicates live cells, while DAPI staining shows the total cells. Original magnification: ×100. Representative results from three samples are shown. (C and D) Endplate chondrocytes were subjected to ICMT in vitro for 3 days, and then cell apoptosis was examined by propidium iodide/annexin V staining (C) and cell proliferation was examined by an Alamar Blue assay (D). Data are presented as the means and 95% CI from three independent experiments. (E) F-actin staining showed changes in the chondrocyte cytoskeleton after 3 days of ICMT treatment. Representative results from three donors are shown, red indicates F-actin and blue indicates DAPI staining. Each experiment performed in triplicate. *Statistically significant difference. Osteoarthritis and Cartilage 2016 24, 158-168DOI: (10.1016/j.joca.2015.07.019) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 ICMT promotes activation of Wnt/β-catenin signaling in endplate chondrocytes. (A) Real-time RT-PCR and western blot analyses of β-catenin mRNA and protein expression in samples obtained from patients with cervical vertebrae fracture or cervical disc degeneration (normal disc group: n = 8; CDD group n = 12). For western blotting, Representative results from three patients are shown. (B) Immunohistochemical analysis showed up-regulation of β-catenin protein in samples from patients with disc degeneration. Original magnification: ×200. Representative results from three patients are shown. (C and D) β-Catenin mRNA and protein expression in endplate chondrocytes was assessed by real-time RT-PCR and western blotting after ICMT application in vivo (C) and in vitro (D). For western blotting, representative results from three donors are shown. (E) β-Catenin protein expression was examined by immunohistochemical analysis in rabbit endplate cartilage tissue after 28 days of tensile loading in vivo. Original magnification: ×200. Representative results from three samples are shown. (F) Endplate chondrocytes were subjected to ICMT for 3 days, and then the expression and distribution of β-catenin protein were detected by immunofluorescence. Representative results from three donors are shown. (G) Nuclear-cytoplasmic protein fractionation was performed to measure nuclear and cytoplasmic β-catenin protein expression after ICMT application. Representative results from three donors are shown. (H) Real-time RT-PCR showed changes in cyclinD1 and TCF-1 expression after endplate chondrocytes were subjected to ICMT for 3 days. GAPDH was used as an internal control for the Real-time RT-PCR and Western blots. Real-time RT-PCR data are presented as the means and 95% CI from three independent experiments, each performed in triplicate. Each dot representing the average of one donor. *Statistically significant difference. Osteoarthritis and Cartilage 2016 24, 158-168DOI: (10.1016/j.joca.2015.07.019) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Inhibition of Wnt/β-catenin signaling rescues ICMT-induced endplate chondrocyte anabolic genes decreasing. (A and B) Endplate chondrocytes were treated with or without XAV-939 during ICMT application. Expression of β-catenin mRNA (A) and protein (B) indicated that XAV-939 suppressed the Wnt/β-catenin signaling activated by ICMT. For western blotting, representative results from three donors are shown. (C) Immunofluorescence showed inhibition of β-catenin protein expression. Representative results from three patients are shown. Representative results from three donors are shown. (D) Real-time RT-PCR showed alterations in the expression levels of COL-2A, ACAN, and Sox9 after ICMT with or without XAV-939 treatment. GAPDH was used as an internal control for the Real-time RT-PCR and Western blots. Real-time RT-PCR data are presented as the means and 95% CI from three independent experiments, each performed in triplicate. Each dot representing the average of one donor. *Statistically significant difference. Osteoarthritis and Cartilage 2016 24, 158-168DOI: (10.1016/j.joca.2015.07.019) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 ICMT suppresses the interaction between E-cadherin and β-catenin by down-regulation of E-cadherin expression. (A – D) Expression of E-cadherin mRNA and protein was measured by real-time RT-PCR and western blotting after ICMT loaded in vivo (A and B) and in vitro (C and D). For western blotting, representative results from three donors are shown. (E) Endplate chondrocytes were treated with ICMT for 3 days. Double immunofluorescence staining revealed colocalization of E-cadherin and β-catenin proteins (yellow) in endplate chondrocytes. Representative results from three donors are shown. (F) Co-immunoprecipitation showed an interaction between E-cadherin and β-catenin proteins after treatment with or without ICMT for 3 days. Representative results from three donors are shown. GAPDH was used as an internal control for the Real-time RT-PCR and Western blots. Real-time RT-PCR data are presented as the means and 95% CI from three independent experiments, each performed in triplicate. Each dot representing the average of one donor. *Statistically significant difference. Osteoarthritis and Cartilage 2016 24, 158-168DOI: (10.1016/j.joca.2015.07.019) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 E-cadherin relieves ICMT-induced down-regulation of endplate chondrocyte anabolic genes via inhibition of β-catenin signaling. (A and B) Overexpression of GFP-labeled E-cadherin in rat endplate chondrocytes was confirmed by real-time RT-PCR (A) and western blotting (B). For western blotting, representative results from three donors are shown. (C) GFP-labeled E-cadherin was observed by fluorescence microscopy at 3 days post-transfection. Original magnification: ×100. Representative results from three donors are shown. (D) Endplate chondrocytes were transfected with an E-cadherin expression plasmid (E-cad) or pEGFPN1 vector (Veh). At 1 day after transfection, ICMT was applied for 3 days, and then the interaction between E-cadherin and β-catenin proteins was detected by co-immunoprecipitation. Representative results from three donors are shown. (E) Nuclear and cytoplasmic β-catenin protein expression was examined in nuclear and cytoplasmic protein fractions. Representative results from three donors are shown. (F) CyclinD1 and TCF-1 mRNA expression was detected by real-time RT-PCR. (G) Relative expression levels of COL-2A, ACAN, and Sox9 were measured by real-time RT-PCR. GAPDH was used as an internal control for the Real-time RT-PCR and Western blots. Real-time RT-PCR data are presented as the means and 95% CI from three independent experiments, each performed in triplicate. Each dot representing the average of one donor. *Statistically significant difference. (H) Schematic diagram of ICMT-induced endplate cartilage degeneration through E-cadherin/β-catenin complex and Wnt/β-catenin signaling cross-talk. Osteoarthritis and Cartilage 2016 24, 158-168DOI: (10.1016/j.joca.2015.07.019) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions