1,25(OH)2-Vitamin D3 Inhibits Proliferation and Decreases Production of Monocyte Chemoattractant Protein-1, Thrombopoietin, VEGF, and Angiogenin by Human Annulus Cells In Vitro

Abstract

Human lumbar anulus tissue and cultured human lumbar anulus cells were used in retrospective studies of the immunocytochemical localization of the vitamin D receptor (VDR) in disc tissue, and of the in vitro effects of the active metabolite of vitamin D, 1,25(OH)2D3, on anulus cell proliferation, cytokine, and proteoglycan (PG) production. 24,25-D3 was also analyzed. Studies were approved by the authors' Human Subjects Institutional Review Board. Discs were obtained from surgical specimens and from control donors. To determine if human anulus cells express the VDR in vivo, and to test the effect of in vitro exposure to 1,25(OH)2D3 and 24,25-D3 on anulus cell proteoglycan and cytokine production in 3-dimensional culture. Intragenic polymorphisms in the VDR gene have been associated with disc degeneration. 1,25(OH)2D3 has well-recognized effects on calcium homeostasis and bone mineralization, and is a negative growth regulator of a variety of normal and tumor cells. Its effects on human disc cells, however, are unexplored. Immunocytochemistry was performed on human lumbar disc anulus tissue from 19 subjects; human disc cells were cultured to test the effect of 1,25(OH)2D3 on proliferation of anulus cells from 5 subjects. A paired experimental design was used to determine proteoglycan production in control or 1,25(OH)2D3-treated cells, or in control or 24,25-D3-treated cells using the dimethylmethylene blue assay. A paired experimental design was also used to identify differences in cytokine production in conditioned media from control or 1,25(OH)2D3-treated cells, or in control or 24,25-D3-treated cells using ELISA assays. Immunocytochemistry documented expression of the VDR in anulus cells. Young donor discs (aged newborn, 15 years) showed positive localization in all cells of the outer anulus, and some inner anulus cells. In adults (mean age, 38.9 years), some, but not all anulus cells, showed positive localization. Exposure to 10M 1,25(OH)2D3 in monolayer significantly reduced cell proliferation in vitro (P = 0.03). PG production in 3-dimensional was unchanged from control in both 1,25(OH)2D3- and 24,25-D3-treated cells. Cytokine production differed, however. 1,25(OH)2D3-treated cells showed significantly decreased production of vascular endothelial growth factor (VEGF) (P = 0.01), monocyte chemoattractant protein-1 (MCP-1) (P = 0.0006), angiogenin (P = 0.002), and thrombopoietin (P = 0.03) compared with controls. 24,25-D3-treated cells showed significantly elevated vascular endothelial growth factor-D (P = 0.01), beta-fibroblast growth factor (0.03), and significantly decreased interleukin-8, interferon-gamma, leptin, MCP-1, and TIMP-2 (tissue inhibitor of metalloproteinases-2) compared with controls (P <or= 0.01). Data suggest that 1,25(OH)2D3 and 24,25-D3 may play roles as regulators of cell proliferation and production of specific cytokines in the lumbar anulus.