Many researchers now recognize that the steroid 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] has biological activity. In a previous work from our lab, it has been reported that 24,25(OH)2D3 decreases catalase protein levels in cultured intestinal cells over a period of several hours. An effect on catalase gene expression combined with rapid signaling would conclusively demonstrate that 24,25(OH)2D3 is not a ‘scrap’ metabolite. In the current work, we report that 24R,25(OH)2D3 is the effective metabolite for decreasing 32P uptake and catalase gene expression, and protein regulation. We used male chick intestinal cells treated with either vehicle, 24,25(OH)2D3 or 1,25(OH)2D3 for each condition. 32P uptake assays showed a decrease in uptake after 24R,25(OH)2D3 treatment in both suspension cells and cultured cells. The inhibitory effect was eliminated after enterocytes were transfected with catalase siRNA. RT-PCR (reverse transcription polymerase chain reaction) further indicated a down-regulation of catalase after treatment with 24R,25(OH)2D3 compared to vehicle control and 1,25(OH)2D3. When transfected with catalase siRNA, mRNA down-regulation effect was eliminated. Protein expression was then checked using Western Blotting, and a decrease in catalase immunoreactivity was found. By understanding the molecular and cellular actions of 24,25(OH)2D3 in chick intestine, progress could be made in enhancing phosphate and calcium absorption in animals to supply the minerals for adequate bone growth, and phosphate in manure of production animals could be diminished.
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