PubMed · 7039263
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J G Haddad. 1982. Vitamin D binding proteins.. https://doi.org/10.1007/978-1-4613-9934-6_2
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The steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly stimulates the uptake of phosphate in isolated chick intestinal cells, while the steroid 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] inhibits the rapid stimulation by 1,25(OH)2D3. Earlier work in this laboratory has indicated that a cellular binding protein for 24,25(OH)2D3 is the enzyme catalase. Since binding resulted in decreased catalase activity and increased H2O2 production, studies were undertaken to determine if pro-oxidant conditions mimicked the inhibitory actions of 24,25(OH)2D3, and anti-oxidant conditions prevented the inhibitory actions of 24,25(OH)2D3. An antibody against the 24,25(OH)2D3 binding protein was found to neutralize the inhibitory effect of the steroid on 1,25(OH)2D3-mediated 32P uptake. Incubation of cells in the presence of 50 nM catalase was also found to alleviate inhibition. In another series of experiments, isolated intestinal epithelial cells were incubated as controls or with 1,25(OH)2D3, each in the presence of the catalase inhibitor 3-amino-1,2,4-triazole, or with 1,25(OH)2D3 alone. Cells exposed to hormone alone again showed an increased accumulation of 32P, while cells treated with catalase inhibitor and hormone had uptake levels that were indistinguishable from controls. We tested whether inactivation of protein kinase C (PKC), the signaling pathway for 32P uptake, occurred. Incubation of cells with phorbol-13-myristate (PMA) increased 32P uptake, while cells pretreated with 50 microM H2O2 prior to PMA did not exhibit increased uptake. Likewise, PMA significantly increased PKC activity while cells exposed to H2O2 prior to PMA did not. It is concluded that catalase has a central role in mediating rapid responses to steroid hormones.
In intestine, 24,25(OH)(2)D(3), which is made under conditions of calcium-, phosphate-, and 1,25(OH)(2)D(3) sufficiency, inhibits the stimulatory actions of 1,25(OH)(2)D(3) on phosphate and calcium absorption. In the current work, we provide evidence that 24,25(OH)(2)D(3)-mediated signal transduction occurs mechanistically through increased H(2)O(2) production which involves binding of 24,25(OH)(2)D(3) to catalase and resultant decreases in enzyme activity. Physiological levels of H(2)O(2) mimicked the action of 24,25(OH)(2)D(3) on inhibiting 1,25(OH)(2)D(3)-stimulated phosphate uptake in isolated enterocytes. Moreover, the molecular basis of such inhibition was suggested by the presence of two thioredoxin domains in the 1,25D(3)-MARRS protein/ERp57: Exposure of cells to either 24,25(OH)(2)D(3) or H(2)O(2) gradually reduced 1,25(OH)(2)D(3) binding to 1,25D(3)-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 [(3)H]1,25(OH)(2)D(3) binding to both 1,25D(3)-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 1,25D(3)-MARRS mRNA. The combined data provide mechanistic details for the 1,25(OH)(2)D(3)/24,25(OH)(2)D(3) endocrine system, and point to a role for the 1,25D(3)-MARRS protein as a redox-sensitive mediator of osteoclast activity and potential therapeutic target.
Phosphate homeostasis is controlled in part by absorption from the intestine, and reabsorption in the kidney. While the effect of Vitamin D metabolites on enterocytes is well documented, in the current study we assess selected responses in primary cultures of kidney cells. Time course studies revealed a rapid stimulation of phosphate uptake in cells treated with 1,25(OH)(2)D(3), relative to controls. Dose-response studies indicated a biphasic curve with optimal stimulation at 300 pM 1,25(OH)(2)D(3) and inhibition at 600 pM seco-steroid. Antibody 099--against the 1,25D(3)-MARRS receptor - abolished stimulation by the steroid hormone. Moreover, phosphate uptake was mediated by the protein kinase C pathway. The metabolite 24,25(OH)(2)D(3), which was found to inhibit the rapid stimulation of phosphate uptake in intestinal cells, had a parallel effect in cultured kidney cells. Finally, the 24,25(OH)(2)D(3) binding protein, catalase, was assessed for longer term down regulation. In both intestinal epithelial cells and kidney cells incubated with 24,25(OH)(2)D(3) for 5-24h, both the specific activity of the enzyme and protein levels were decreased relative to controls, while 1,25(OH)(2)D(3) increased both parameters over the same time periods. We conclude that the Vitamin D metabolites have similar effects in both kidney and intestine, and that 24,25(OH)(2)D(3) may have effects at the level of gene expression.