Search

Summary Objective This study aimed to investigate the role of the Wnt/β-catenin signaling pathway and E-cadherin/β-catenin complex in intermittent cyclic mechanical tension (ICMT)-induced endplate cartilage degeneration. Design β-Catenin expression was measured in disc samples obtained from patients with disc degeneration and those with cervical vertebrae fracture or dislocation. Histological staining was performed to examine the disc tissue morphology and extracellular matrix after application of ICMT in vitro and in vivo. Multiple strategies were employed to examine activation of Wnt/β-catenin signaling after ICMT application in vivo and in vitro . Co-immunoprecipitation was performed to examine the interaction between E-cadherin and β-catenin. Pathway-specific inhibitors and an E-cadherin expression plasmid were used to regulate Wnt/β-catenin signaling and E-cadherin expression. Results β-Catenin protein expression was elevated significantly, whereas cartilaginous genes were down-regulated in endplate cartilage samples obtained from patients with disc degeneration. ICMT loading led to Wnt/β-catenin signaling activation and the loss of the chondrogenic phenotype of endplate chondrocytes in both an in vivo rabbit model and in vitro endplate chondrocyte culture system. Inhibition of Wnt/β-catenin signaling suppressed the decrease in ICMT-induced cartilaginous gene expression. Furthermore, E-cadherin expression was inhibited by ICMT stimulation, resulting in a decrease in the interaction between E-cadherin and β-catenin proteins. Over-expression of E-cadherin rescued the cartilaginous gene expression by enhancing the interaction between E-cadherin and β-catenin proteins. Conclusions ICMT promotes endplate cartilage degeneration via activation of Wnt/β-catenin signaling and suppression of physical protein–protein interactions between E-cadherin and β-catenin.

Objective To investigate the relationship between ectonucleotide pyrophosphatase phosphodiesterase-1(ENPP-1) expression and transforming growth factor beta 1 (TGF-β1) of end-plate chondrocytes after stimulation with intermittent cyclic mechanical tension (ICMT) by using an FX-4000T Flexercell Tension Plus unit. Methods Rat end-plate chondrocytes were cultured and ICMT (strain at 0.5 Hz sinusoidal curve at 10% elongation) applied for 7 days for 4 h/day and cultured for a further 2 days. End-plate chondrocytes were also exposed to 10 ng/mL of TGF-β1. Then, using small interfering RNA technology, small interfering TGF-β1 (siTGF-β1) was transfected. Expression of ENPP-1 and TGF-β1 was measured by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and western blotting. Results Expression of both ENPP-1 and TGF-β1 was up-regulated after ICMT. Both RT-PCR and western blot showed that ENPP-1 expression decreases with siRNA TGF-β1 after 3% elongation 40 min, and cultured for an additional 2 days. Conclusion It was found that down-regulation of ENPP-1 gene expression induced by ICMT is likely dependent on TGF-β1 in end-plate chondrocytes.

Calcification of end plate chondrocytes is a major cause of intervertebral disc (IVD) degeneration. However, the underlying molecular mechanism of end plate chondrocyte calcification is still unclear. The aim of this study was to clarify whether autophagy in end plate chondrocytes could protect the calcification of end plate chondrocytes. Previous studies showed that intermittent cyclic mechanical tension (ICMT) contributes to the calcification of end plate chondrocytes in vitro. While autophagy serves as a cell survival mechanism, the relationship of autophagy and induced end plate chondrocyte calcification by mechanical tension in vitro is unknown. Thus, we investigated autophagy, the expression of the autophagy genes, Beclin-1 and LC3, and rat end plate chondrocyte calcification by ICMT. The viability of end plate chondrocytes was examined using the LIVE/DEAD viability/cytotoxicity kit. The reverse transcription-polymerase chain reaction and western blotting were used to detect the expression of Beclin-1; LC3; type I, II and X collagen; aggrecan; and Sox-9 genes. Immunofluorescent and fluorescent microscopy showed decreased autophagy in the 10- and 20-day groups loaded with ICMT. Additionally, Alizarin red and alkaline phosphatase staining detected the palpable calcification of end plate chondrocytes after ICMT treatment. We found that increased autophagy induced by short-term ICMT treatment was accompanied by an insignificant calcification of end plate chondrocytes. To the contrary, the suppressive autophagy inhibited by long-term ICMT was accompanied by a more significant calcification. The process of calcification induced by ICMT was partially resisted by increased autophagy activity induced by rapamycin, implicating that autophagy may prevent end plate chondrocyte calcification.

Ectonucleotide pyrophosphatase/phosphodiesterase (ENPP)-1 is a membrane-bound protein that catalyzes the hydrolysis of extracellular nucleoside triphosphates to monophosphate and extracellular inorganic pyrophosphate (ePPi). Mechanical stimulation regulates ENPP-1 expression. This study sought to investigate the changes in ENPP-1 expression after stimulation using cyclic mechanical tension (CMT).Rat end-plate chondrocytes were cultured and subjected to CMT (at 3%, 6%, and 9% elongation) for 20, 40, and 60 minutes to observe changes in the expression of ENPP-1. To investigate the pathway, end-plate chondrocytes were exposed to 10 ng/ml of transforming growth factor beta 1 (TGF-β1), TGF-β1 siRNA, or a specific extracellular signalregulated kinase (ERK)1/2 inhibitor, U0126, in addition to CMT. Changes in ENPP-1 expression were measured by reverse transcription PCR (RT-PCR) and Western blotting.We observed the largest increase in ENPP-1 expression following 3% elongation CMT stimulation. ENPP-1 expression was also increased when end-plate chondrocytes were exposed to 10 ng/ml of TGF-β1, but decreased after TGF-β knockdown with siRNA. ERK1/2 phosphorylation was activated after 3% elongation for 40 minutes, and the stimulatory effect of TGF-β1 on ENPP-1 mRNA and protein expression was inhibited by the suppression of the ERK1/2 pathway using U0126.CMT increases the expression of ENPP-1 in end-plate chondrocytes in a manner likely dependent on TGF-β induction by the ERK1/2 signaling pathway.

OBJECTIVE: To investigate the relationship between ectonucleotide pyrophosphatase phosphodiesterase‐1(ENPP‐1) expression and transforming growth factor beta 1 (TGF‐β1) of end‐plate chondrocytes after stimulation with intermittent cyclic mechanical tension (ICMT) by using an FX‐4000T Flexercell Tension Plus unit. METHODS: Rat end‐plate chondrocytes were cultured and ICMT (strain at 0.5 Hz sinusoidal curve at 10% elongation) applied for 7 days for 4 h/day and cultured for a further 2 days. End‐plate chondrocytes were also exposed to 10 ng/mL of TGF‐β1. Then, using small interfering RNA technology, small interfering TGF‐β1 (siTGF‐β1) was transfected. Expression of ENPP‐1 and TGF‐β1 was measured by real‐time reverse‐transcriptase polymerase chain reaction (RT‐PCR) and western blotting. RESULTS: Expression of both ENPP‐1 and TGF‐β1 was up‐regulated after ICMT. Both RT‐PCR and western blot showed that ENPP‐1 expression decreases with siRNA TGF‐β1 after 3% elongation 40 min, and cultured for an additional 2 days. CONCLUSION: It was found that down‐regulation of ENPP‐1 gene expression induced by ICMT is likely dependent on TGF‐β1 in end‐plate chondrocytes.