Regulation of catabolic gene expression in normal and degenerate human intervertebral disc cells: implications for the pathogenesis of intervertebral disc degeneration.

INTRODUCTION: The aim of this study was to compare the effects of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1beta) on protease and catabolic cytokine and receptor gene expression in normal and degenerate human nucleus pulposus cells in alginate culture. METHODS: Cells isolated from normal and degenerate nucleus pulposus regions of human intervertebral discs were cultured in alginate pellets and stimulated by the addition of 10 ng/mL TNF-alpha or IL-1beta for 48 hours prior to RNA extraction. Quantitative real-time polymerase chain reaction was used to assess the effect of TNF-alpha or IL-beta stimulation on the expression of matrix metalloproteinase (MMP)-3, -9 and -13, TNF-alpha, TNF receptor 1 (TNF-R1), TNF receptor 2 (TNF-R2), IL-1alpha, IL-1beta, IL-1 receptor 1 (IL-1R1) and IL-1 receptor antagonist (IL-1Ra). RESULTS: MMP-3 and MMP-9 gene expressions were upregulated to a greater level by IL-1beta than TNF-alpha. MMP-13 was upregulated by each cytokine to a similar extent. TNF-alpha and TNF-R2 expressions were upregulated by both TNF-alpha and IL-beta, whereas TNF-R1 expression was not significantly affected by either cytokine. IL-1beta and IL-1Ra expressions were significantly upregulated by TNF-alpha, whereas IL-1alpha and IL-1R1 were unchanged. CONCLUSIONS: TNF-alpha does not induce MMP expression to the same degree as stimulation by IL-1beta, but it does act to upregulate IL-1beta expression as well as TNF-alpha and TNF-R2. The net result of this would be an increased inflammatory environment and accelerated degradation of the matrix. These results support the hypothesis that, while TNF-alpha may be an important initiating factor in matrix degeneration, IL-1beta plays a greater role in established pathological degradation.

Human meniscus cells express hypoxia inducible factor-1alpha and increased SOX9 in response to low oxygen tension in cell aggregate culture.

In previous work we demonstrated that the matrix-forming phenotype of cultured human cells from whole meniscus was enhanced by hypoxia (5% oxygen). Because the meniscus contains an inner region that is devoid of vasculature and an outer vascular region, here we investigate, by gene expression analysis, the separate responses of cells isolated from the inner and outer meniscus to lowered oxygen, and compared it with the response of articular chondrocytes. In aggregate culture of outer meniscus cells, hypoxia (5% oxygen) increased the expression of type II collagen and SOX9 (Sry-related HMG box-9), and decreased the expression of type I collagen. In contrast, with inner meniscus cells, there was no increase in SOX9, but type II collagen and type I collagen increased. The articular chondrocytes exhibited little response to 5% oxygen in aggregate culture, with no significant differences in the expression of these matrix genes and SOX9. In both aggregate cultures of outer and inner meniscus cells, but not in chondrocytes, there was increased expression of collagen prolyl 4-hydroxylase (P4H)alpha(I) in response to 5% oxygen, and this hypoxia-induced expression of P4H alpha(I) was blocked in monolayer cultures of meniscus cells by the hypoxia-inducible factor (HIF)-1alpha inhibitor (YC-1). In fresh tissue from the outer and inner meniscus, the levels of expression of the HIF-1alpha gene and downstream target genes (namely, those encoding P4H alpha(I) and HIF prolyl 4-hydroxylase) were significantly higher in the inner meniscus than in the outer meniscus. Thus, this study revealed that inner meniscus cells were less responsive to 5% oxygen tension than were outer meniscus cells, and they were both more sensitive than articular chondrocytes from a similar joint. These results suggest that the vasculature and greater oxygen tension in the outer meniscus may help to suppress cartilage-like matrix formation.