Comparative effects of 1,25-dihydroxyvitamin D3 and EB 1089 on mouse renal and intestinal 25-hydroxyvitamin D3-24-hydroxylase

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Abstract
EB 1089 is a vitamin D analog that is less potent than 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) in its calcemic action but more potent in its antiproliferative action. We characterized the interaction of 1,25(OH)2D3 and EB 1089 with renal 25‐hydroxyvitamin D3‐24‐hydroxylase (24‐hydroxylase), the first enzyme in the C‐24 oxidation pathway, and compared the effects of 1,25(OH)2D3 and EB 1089 on induction of 24‐hydroxylase mRNA in mouse kidney and intestine. 1,25(OH)2D3 and EB 1089 were competitive inhibitors of 24‐hydroxylase activity. However, the K1 for 1,25(OH)2D3 (5.2 ± 2.5 nM) was significantly lower than that for EB 1089 (286 ± 59 nM). In the kidney, the time course and extent of 24‐hydroxylase mRNA induction, relative to 18S rRNA, was similar for 1,25(OH)2D3 and EB 1089 with a peak response at ≈ 6 h that was sustained for at least 16 h. In the intestine, however, induction of 24‐hydroxylase mRNA, relative to 18S rRNA, was ∼50% lower for EB 1089 than for 1,25(OH)2D3 at 3 h (p < 0.05) and 6 h (p < 0.05) while at 16 h 24‐hydroxylase mRNA was no longer detectable. Moreover, while both 1,25(OH)2D3 and EB 1089 elicited a similar dose‐dependent induction of 24‐hydroxylase mRNA in the kidney (EC50 = 0.4 ± 0.13 and 0.3 ± 0.08 ng/g for EB 1089 and 1,25(OH)2D3, respectively), the EC50 for EB 1089 (6.6 ± 1.7 ng/g) was significantly higher than that for 1,25(OH)2D3 (0.9 ± 0.32 ng/g) in the intestine (p < 0.01). EB 1089 was also less effective than 1,25(OH)2D3 in the induction of intestinal but not renal calbindin‐D9k mRNA. To determine the mechanism for tissue‐specific differences in potency, we determined the binding affinity of 1,25(OH)2D3 and EB 1089 for the vitamin D receptor. In the kidney, Kd values for 1,25(OH)2D3 (0.40 ± 0.05 nM) and EB 1089 (0.48 ± 0.04 nM) were not different. However, in the intestine, the Kd for EB 1089 (1.43 ± 0.19 nM) was significantly higher than that for 1,25(OH)2D3 (0.85 ± 0.06 nM; p < 0.05). Our results demonstrate that: (i) EB 1089 has a 50‐fold lower affinity than 1,25(OH)2D3 for renal 24‐hydroxylase, suggesting that it is more resistant to catabolism by the C‐24 oxidation pathway; and (ii) EB 1089 and 1,25(OH)2D3 exhibit tissue‐specific differences in vitamin D receptor‐mediated responses in vivo that may be ascribed, at least in part, to differences in binding affinities for the vitamin D receptor.
Funding Information
  • Kidney Foundation of Canada and Medical Research Council of Canada
  • Stéphane Roy is the recipient of a McGill University-Montreal Children's Hospital Research Institute Studentship Award