Mechanism of 24,25‐dihydroxyvitamin D₃‐mediated inhibition of rapid, 1,25‐dihydroxyvitamin D₃‐induced responses: Role of reactive oxygen species

In intestine, 24,25(OH)₂D₃, which is made under conditions of calcium‐, phosphate‐, and 1,25(OH)₂D₃ sufficiency, inhibits the stimulatory actions of 1,25(OH)₂D₃ on phosphate and calcium absorption. In the current work, we provide evidence that 24,25(OH)₂D₃‐mediated signal transduction occurs mechanistically through increased H₂O₂ production which involves binding of 24,25(OH)₂D₃ to catalase and resultant decreases in enzyme activity. Physiological levels of H₂O₂ mimicked the action of 24,25(OH)₂D₃ on inhibiting 1,25(OH)₂D₃‐stimulated phosphate uptake in isolated enterocytes. Moreover, the molecular basis of such inhibition was suggested by the presence of two thioredoxin domains in the 1,25D₃‐MARRS protein/ERp57: Exposure of cells to either 24,25(OH)₂D₃ or H₂O₂ gradually reduced 1,25(OH)₂D₃ binding to 1,25D₃‐MARRS protein, between 10 and 20 min of incubation, but not to VDR. Feeding studies with diets enriched in the antioxidants vitamins C and E showed that net phosphate absorption in vivo nearly doubled relative to chicks on control diet. Antioxidant diets also resulted in increased [³H]1,25(OH)₂D₃ binding to both 1,25D₃‐MARRS and VDR, suggesting benefits to both transcription‐ and membrane‐initiated signaling pathways. Intriguingly, phosphorous content of bones from birds on antioxidant diets was reduced, suggesting increased osteoclast activity. Because mature osteoclasts lack VDR, we analyzed a clonal osteoclast cell line by RT‐PCR and found it contained the 1,25D₃‐MARRS mRNA. The combined data provide mechanistic details for the 1,25(OH)₂D₃/24,25(OH)₂D₃ endocrine system, and point to a role for the 1,25D₃‐MARRS protein as a redox‐sensitive mediator of osteoclast activity and potential therapeutic target. J. Cell. Biochem. 99: 1572–1581, 2006. © 2006 Wiley‐Liss, Inc.