| BackgroundChronic low back pain(LBP) caused by degenerative disc disease(DDD) is the most common musculoskeletal complaints for middle aged and old people. The total health care and socioeconomic cost associated to LBP is enormous. The pathophysiology of DDD has been extensively studied in recent years. Various factors have been suggested as influencing its aetiology, including ageing, genetic, nutrition, metabolic, infection and mechanical factors. However, the etiological basis of this degeneration is poorly understood yet.DDD is the result of the development and severity of intervertebral disc degeneration(IVD). In this degenerative process, cellular loss of nucleus pulposus(NP) from apoptosis has been demonstrated. And it has also been reported that apoptosis of NP cells is one of the initial triggers of IVD degeneration. Additionally, inflammatory cytokines including IL-1β and TNF-α increased significantly in IVD degeneration. Through a series of signaling networks, inflammatory cytokines induce NP cells apoptosis, which results in progressive IVD degeneration. While the exact mechanism of NP cells apoptosis remains to be determined.There are a number of genes found to play important roles in IVD degeneration, including Caveolin-1. As a marker of Caveolea, Caveolin-1 can be expressed in multiple tissues and regulates lipid transport, membrane trafficking and intracellular signalling pathways. Heathfield et al demonstrated the expression of Caveolin-1 in NP cells of human IVD for the first time. In recent years, Caveolin-1 has been found to be a crucial factor in the initiation of IVD degeneration. NP cells of degenerate discs express high levels of Caveolin-1 and show evidence of cellular senescence. In addition, the role of Caveolin-1 in the regulation of apoptosis in bronchial epithelial cells, vascular smooth muscle cells and human leukemia cell lines HL-60 has been reported previously. While, there is little information concerning the relationship between Caveolin-1 and apoptosis in NP cells.Caveolin-1 can activate Wnt/β-catenin signaling pathway and regulate downstream gene expression in osteocyte and HepG2 cells. Increased Wnt/β-catenin signaling activity was found in early IVD degeneration. Researches have suggested that Wnt/β-catenin signaling plays an important role in IVD. Here we hypothesize that cross-talk between Caveolin-1 and Wnt/β-catenin may play a role in the regulation of IVD degeneration. And the goal of the present study is to examine the effect of Caveolin-1 in the process of IVD degeneration and the interaction between Caveolin-1 and Wnt/β-catenin signaling during NP cells apoptosis.Objectives1. The expression of Caveolin-1 in the nucleus pulposus cells, and its role in the process of nucleus pulposus cells apoptosis.2. Role of Wnt/β-catenin signaling pathway in the apoptosis of nucleus pulposus cells.3. the interaction between Caveolin-1 and Wnt/β-catenin signaling during NP cells apoptosis.Methods1. To investigate the effect of inflammatory cytokines on Caveolin-1 expression in NP cells, cells were treated by IL-1β(10ng/ml) or TNF-α(50ng/ml). Then the protein and mRNA level of Caveolin-1 were measured in designated time(0, 1, 6, 12, 24, 48h) by Western blotting and Real time RT-PCR respectively.2. To investigate the effect of Caveolin-1 on NP cells apoptosis, NP cells were treated by inflammatory cytokines and/or Caveolin-1 expression was silenced by siRNA technique. FITC-Annexin V/PI FCM assay was performed to assess the NP cells apoptosis rate.3. To investigate the effect of inflammatory cytokines on Wnt/β-catenin signaling pathway, NP cells were treated by IL-1β(10ng/ml) or TNF-α(50ng/ml) as previously. Expression of β-catenin in NP cells was measured in designated time(0, 6, 12, 24h) by Western blotting and Real time RT-PCR respectively.4. To investigate the role of Wnt/β-catenin signaling in NP cells apoptosis, β-catenin expression was silenced by siRNA technique. FITC-Annexin V/PI FCM assay was performed to assess the NP cells apoptosis rate.5. To investigate the interaction between Caveolin-1 and WNT/β-catenin signaling in NP cells apoptosis, Caveolin-1 silence and over expression were performed in NP cells respectively. WNT/β-catenin signaling was inhibited by β-Catenin si RNA technique. Activation of Wnt/β-Catenin signaling was tested as above. Apoptosis of NP cells was evaluated by both FITC-Annexin V/PI FCM assay and Caspase-3, 8, 9 assay.Results1. Inflammatory cytokines increased expression of Caveolin-1After 6h inflammatory cytokines treatment, Caveolin-1 expression in NP cells increased significantly in both protein and mRNA level compared with control. The mRNA expression of Caveolin-1 peaked at 4.0 or 3.3 folds of the baseline value respectively after 12 h treatment.2. Caveolin-1 regulated apoptosis of NP cellsAfter inflammatory cytokines treatment, the NP cells apoptosis rate was increased from 5.2%(control group) to 29.3%(IL-1β group) and 26.2%(TNF-α group) respectively. These increases can be reversed by Caveolin-1 siRNA as apoptosis rate was decreased to 16.0%(IL-1β + Caveolin-1 siRNA group) and 14.9%(TNF-α + Caveolin-1 siRNA group) respectively after Caveolin-1 siRNA treatment.3. Inflammatory cytokines induced activation of Wnt/β-catenin signalingNuclear translocation of β-catenin was increased after inflammatory cytokines treatment. The mRNA expression of β-catenin increased in a time-dependent manner after treatment, which was 3.47(IL-1β group) and 2.81(TNF-α group) folds of the baseline value at 24 h. After inflammatory cytokines treatment, transactivation of TOP Flash was significantly high. Meanwhile, FOP Flash was not activated.4. Activation of Wnt/β-catenin signaling promoted NP cells apoptosisIn β-catenin siRNA group, nuclear translocation decreased significantly compared with IL-1β and TNF-α group. After treated by inflammatory cytokines, apoptosis rate of NP cells was increased from 5.0%(control group) to 26.9%(IL-1β group) and 22.0%(TNF-α group) respectively. This effect was reversed by β-catenin siRNA as apoptosis rate was decreased to 13.8%(IL-1β + β-catenin siRNA group) and 11.2%(TNF-α +β-catenin siRNA group) respectively.5. Caveolin-1 regulated NP cells apoptosis through Wnt/β-catenin signalingIncreased nuclear translocation of β-catenin was detected after inflammatory cytokines treatment, which can be reversed by Caveolin-1 si RNA. Activity of Caspase-3, 9 increased significantly after inflammatory cytokines treatment, and this increase can be inhibited by both Caveolin-1 siRNA and β-catenin siRNA. Nuclear translocation of β-catenin, activity of Caspase-3, 9 and apoptosis rate of NP cells were all increased after Caveolin-1 overexpression. While, these increases were abolished in β-catenin siRNA group.ConclusionOur results identified Caveolin-1 as a novel factor regulating NP cells apoptosis in IVD degeneration through Wnt/β-Catenin Signaling. Caveolin-1 and Wnt/β-catenin Signaling can be expected to provide a potential therapeutic target for IVD degeneration. |
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