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Tumor necrosis factor alpha decreases 1,25-dihydroxyvitamin D3 receptors in osteoblastic ROS 17/2.8 cells
N Mayur1, S Lewis, B D Catherwood
1Division of Endocrinology, Emory University School of Medicine, Atlanta, Georgia.
Abstract:
Bone remodeling is a complex process regulated by systemic hormones, local cytokines, and growth factors. One cytokine, tumor necrosis factor alpha (TNF-alpha), is known to have potent inhibitory effects on osteoblast matrix protein production and to stimulate osteoclast recruitment. We have previously shown that TNF-alpha inhibits 1,25-(OH)2D3-stimulated synthesis of bone gla protein (BGP), an abundant and osteoblast-specific matrix constituent. We hypothesized that the mechanism of TNF-alpha action included inhibition of intracellular 1,25-(OH)2D3 receptor (VDR) number or function. To test this, the osteoblastic cell line ROS 17/2.8 was cultured in the presence or absence of TNF-alpha (100 ng/ml), and binding of [3H]1,25-(OH)2D3 to 0.3 M KCl extracts of cytosol was measured by equilibrium assay. Specific [3H]1,25-(OH)2D3 binding decreased 70%, 25 h after addition of TNF-alpha. The decrease in [3H]1,25-(OH)2D3 binding was seen by 18 h, was sustained throughout the 72 h culture period, and was greater in low-density cultures. Scatchard analysis confirmed that TNF-alpha (100 ng/ml for 24 h) caused a decrease in the number of binding sites without change in VDR affinity. Northern analysis with a VDR riboprobe revealed that the decrease in VDR occurred without a change in the 4.4 kb steady-state VDR mRNA [VDR/cyclophilin mRNA signal ratio: control, 2.25; TNF-alpha, 2.24 (24 h), 2.17 (40 h), n = 2 flasks/time point]. These results suggest that TNF-alpha action on osteoblastic cells includes an inhibitory effect on VDR number at a point distal to the synthesis of VDR mRNA.
Insights
Tumor necrosis factor alpha (TNF-alpha) reduces vitamin D receptor (VDR) number in osteoblasts, impacting bone remodeling. This suggests TNF-alpha interferes with vitamin D signaling at a post-transcriptional level.
Area of Science:
- Molecular Endocrinology
- Bone Biology
- Cell Signaling
Background:
- Bone remodeling is a dynamic process influenced by hormones and cytokines.
- Tumor necrosis factor alpha (TNF-alpha) negatively impacts osteoblast function and promotes osteoclast activity.
- TNF-alpha inhibits 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-stimulated synthesis of bone gla protein (BGP).
Purpose of the Study:
- To investigate the hypothesis that TNF-alpha affects the number or function of the intracellular 1,25-(OH)2D3 receptor (VDR).
- To elucidate the mechanism by which TNF-alpha influences vitamin D signaling in osteoblasts.
Main Methods:
- Osteoblastic ROS 17/2.8 cells were cultured with or without TNF-alpha.
- Specific binding of [3H]1,25-(OH)2D3 to VDR in cytosol extracts was measured.
- VDR mRNA levels were analyzed using Northern blot.
Main Results:
- TNF-alpha treatment significantly decreased specific [3H]1,25-(OH)2D3 binding by 70% within 25 hours.
- Scatchard analysis indicated TNF-alpha reduced the number of VDR binding sites without altering VDR affinity.
- VDR mRNA levels remained unchanged, suggesting TNF-alpha acts post-transcriptionally.
Conclusions:
- TNF-alpha exerts an inhibitory effect on VDR number in osteoblastic cells.
- This reduction in VDR occurs at a level distal to VDR mRNA synthesis.
- The findings reveal a novel mechanism for TNF-alpha's interference with vitamin D action in bone cells.