Comparative effects of 1,25-dihydroxyvitamin D3 and EB 1089 on mouse renal and intestinal 25-hydroxyvitamin D3-24-hydroxylase.
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 Ki 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 approximately equal to 6 h that was sustained for at least 16 h. In the intestine, however, induction of 24-hydroxylase mRNA, relative to 18S rRNA, was approximately 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 10898 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 +/- 0.95 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.
Roy
S
S
McGill University-Montreal Children's Hospital Research Institute, Department of Pediatrics, McGill University, Quebec, Canada.
Martel
J
J
Tenenhouse
H S
HS
D000704:Analysis of Variance; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001665:Binding Sites; D015152:Blotting, Northern; D064026:Calbindins; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004790:Enzyme Induction / Q000187:drug effects; D007422:Intestines / Q000187:drug effects* / Q000201:enzymology; D007668:Kidney / Q000187:drug effects* / Q000201:enzymology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D012333:RNA, Messenger / Q000378:metabolism; D012337:RNA, Ribosomal, 18S / Q000378:metabolism; D018167:Receptors, Calcitriol / Q000187:drug effects / Q000378:metabolism; D064030:S100 Calcium Binding Protein G / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism*; D065668:Vitamin D3 24-Hydroxylase
D000970:Antineoplastic Agents; D064026:Calbindins; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S; D018167:Receptors, Calcitriol; D064030:S100 Calcium Binding Protein G; C576507:S100g protein, mouse; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D002117:Calcitriol; C078903:seocalcitol