Abstract
BackgroundThe degenerative disc disease (DDD) is a major cause of low back pain. The physiological low-glucose microenvironment of the cartilage endplate (CEP) is disrupted in DDD. Glucose influences protein O-GlcNAcylation via the hexosamine biosynthetic pathway (HBP), which is the key to stem cell fate. Thiamet-G is an inhibitor of O-GlcNAcase for accumulating O-GlcNAcylated proteins while 6-diazo-5-oxo-l-norleucine (DON) inhibits HBP. Mechanisms of DDD are incompletely understood but include CEP degeneration and calcification. We aimed to identify the molecular mechanisms of glucose in CEP calcification in DDD.MethodsWe assessed normal and degenerated CEP tissues from patients, and the effects of chondrogenesis and osteogenesis of the CEP were determined by western blot and immunohistochemical staining. Cartilage endplate stem cells (CESCs) were induced with low-, normal-, and high-glucose medium for 21 days, and chondrogenic and osteogenic differentiations were measured by Q-PCR, western blot, and immunohistochemical staining. CESCs were induced with low-glucose and high-glucose medium with or without Thiamet-G or DON for 21 days, and chondrogenic and osteogenic differentiations were measured by Q-PCR, western blot, and immunohistochemical staining. Sox9 and Runx2 O-GlcNAcylation were measured by immunofluorescence. The effects of O-GlcNAcylation on the downstream genes of Sox9 and Runx2 were determined by Q-PCR and western blot.ResultsDegenerated CEPs from DDD patients lost chondrogenesis, acquired osteogenesis, and had higher protein O-GlcNAcylation level compared to normal CEPs from LVF patients. CESC chondrogenic differentiation gradually decreased while osteogenic differentiation gradually increased from low- to high-glucose differentiation medium. Furthermore, Thiamet-G promoted CESC osteogenic differentiation and inhibited chondrogenic differentiation in low-glucose differentiation medium; however, DON acted opposite role in high-glucose differentiation medium. Interestingly, we found that Sox9 and Runx2 were O-GlcNAcylated in differentiated CESCs. Finally, O-GlcNAcylation of Sox9 and Runx2 decreased chondrogenesis and increased osteogenesis in CESCs.ConclusionsOur findings demonstrate the effect of glucose concentration on regulating the chondrogenic and osteogenic differentiation potential of CESCs and provide insight into the mechanism of how glucose concentration regulates Sox9 and Runx2 O-GlcNAcylation to affect the differentiation of CESCs, which may represent a target for CEP degeneration therapy.
Highlights
The degenerative disc disease (DDD) is a major cause of low back pain
Our findings demonstrate the effect of glucose concentration on regulating the chondrogenic and osteogenic differentiation potential of Cartilage endplate stem cells (CESCs) and provide insight into the mechanism of how glucose concentration regulates Sox9 and Runt-related transcription factor 2 (Runx2) O-GlcNAcylation to affect the differentiation of CESCs, which may represent a target for cartilage endplate (CEP) degeneration therapy
Degenerated CEP in a normal glucose microenvironment lost chondrogenesis and acquired osteogenesis To explore the crosstalk in the chondrogenesis, osteogenesis, glucose, and O-GlcNAcylation conditions during CEP degeneration, we selected relatively normal CEP tissues from lumbar vertebral fracture (LVF) patients as the normal group
Summary
The degenerative disc disease (DDD) is a major cause of low back pain. The physiological low-glucose microenvironment of the cartilage endplate (CEP) is disrupted in DDD. Mechanisms of DDD are incompletely understood but include CEP degeneration and calcification. We aimed to identify the molecular mechanisms of glucose in CEP calcification in DDD. Degenerative disc disease (DDD) plays an important role in back pain, which can cause activity limitation [1, 2]. The main mechanism of DDD is decreased nutrition of the disc, leading to cell waste product accumulation, microenvironment disturbance, and matrix molecule degradation that further disrupts cell activities [6]. Poor nutritional supply of the IVD is an important factor in the pathophysiology of DDD. CEP degeneration and insufficient nutrition supply may initiate DDD [10]
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