lithium-chloride and Intervertebral-Disc-Degeneration

Intervertebral disc degeneration (IVDD) greatly affects the quality of life. The nucleus pulposus (NP) of chondrodystrophic dog breeds (CDBs) is similar to the human NP because the cells disappear with age and are replaced by fibrochondrocyte-like cells. Because IVDD develops as early as within the first year of life, we used canines as a model to investigate the in vitro mechanisms underlying IVDD. The mechanism underlying age-related IVDD, however, is poorly understood. Several research groups have suggested that Wnt/β-catenin signaling plays an important role in IVDD. However, the role of Wnt/β-catenin signals in IVD cells is not yet well understood. Here, we demonstrate that Wnt/β-catenin signaling could enhance Runx2 expression in IVDD and lead to IVD calcification. Nucleus pulposus (NP) tissue was obtained from Beagle dogs after evaluation of the degeneration based on magnetic resonance imaging (MRI). Histological analysis showed that lack of Safranin-O staining, calcified area, and matrix metalloproteinase (MMP) 13-positive cells increased with progression of the degeneration. Furthermore, the levels of β-catenin- and Runx2-positive cells also increased. Real-time reverse-transcription polymerase chain reaction analysis showed that the MRI signal intensity and mRNA expression levels of β-catenin and Runx2 are correlated in NP tissues. Moreover, supplementation of LiCl induced β-catenin accumulation and Runx2 expression. In contrast, FH535 inhibited LiCl-induced upregulation. These results suggest that Runx2 transcript and protein expression, potentially in combination with β-catenin accumulation, are enhanced in degenerated and calcified intervertebral discs of CDBs.

Topics: Animals; beta Catenin; Calcinosis; Cells, Cultured; Core Binding Factor Alpha 1 Subunit; Dogs; Extracellular Matrix Proteins; Gene Expression; Intervertebral Disc; Intervertebral Disc Degeneration; Lithium Chloride; Male; Matrix Metalloproteinase 13; RNA, Messenger; Sulfonamides; Wnt Proteins; Wnt Signaling Pathway

Degeneration of the lumbar intervertebral disc (IVD) is a cause of low back pain. In osteoarthritis patients, an increase in β-catenin accumulation has been reported. However, the molecular mechanisms involved in IVD remain unclear. In the present study, we examined the relationship of Wnt/β-catenin and transforming growth factor-β (TGF-β)/bone morphogenetic protein (BMP) signals in the IVDs. We found that treatment of nucleus pulposus (NP) cells with the Wnt/β-catenin activator lithium chloride (LiCl) results in the increased expression of β-catenin mRNA and protein, and cell proliferation is decreased due to the activation of the Wnt/β-catenin signals through the suppression of c-myc and cyclin-D1. In addition, T-cell-specific transcription factor (TCF) promoter activity was found to increase the following stimulation with LiCl alone, and was further increased when BMP2 was added, in comparison to the control group. We further observed the effects of treatment with PD98059, a specific inhibitor of the mitogen-activated protein kinase pathway, on TCF promoter activity in NP cells. These effects were largely attenuated by PD98059. Moreover, when transfected IVDs were co-transfected with R-Smad expression plasmids, there was a significant decrease in TCF reporter activity. We thereafter evaluated the effects of increased Wnt/β-catenin activity on the transcriptional activity of the Smad binding element (SBE). As a result, LiCl suppressed the activity of SBE reporter activity. The present study demonstrates for the first time that there are opposing effects between the Wnt/β-catenin and TGF-β/BMP signals in IVDs, which is consistent with the Wnt/β-catenin signals contributing to the pathogenesis of IVD degeneration.

Topics: Animals; beta Catenin; Bone Morphogenetic Protein 2; Cell Line; Cell Proliferation; Cyclin D1; Extracellular Signal-Regulated MAP Kinases; Female; Flavonoids; Intervertebral Disc; Intervertebral Disc Degeneration; Lithium Chloride; Mice; Promoter Regions, Genetic; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-myc; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Smad Proteins, Receptor-Regulated; TCF Transcription Factors; Time Factors; Transcriptional Activation; Transfection; Transforming Growth Factor beta; Wnt Proteins; Wnt3 Protein