Roy S, Martel J, Tenenhouse H S
McGill University-Montreal Children's Hospital Research Institute, Department of Pediatrics, McGill University, Quebec, Canada.
J Bone Miner Res. 1995 Dec;10(12):1951-9. doi: 10.1002/jbmr.5650101215.
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.
EB 1089是一种维生素D类似物，其在血钙作用方面比1,25 - 二羟基维生素D3（1,25(OH)2D3）的效力低，但在抗增殖作用方面效力更强。我们对1,25(OH)2D3和EB 1089与肾脏25 - 羟基维生素D3 - 24 - 羟化酶（24 - 羟化酶）的相互作用进行了表征，该酶是C - 24氧化途径中的首个酶，并比较了1,25(OH)2D3和EB 1089对小鼠肾脏和肠道中24 - 羟化酶mRNA诱导的影响。1,25(OH)2D3和EB 1089是24 - 羟化酶活性的竞争性抑制剂。然而，1,25(OH)2D3的Ki（5.2±2.5 nM）显著低于EB 1089的Ki（286±59 nM）。在肾脏中，相对于18S rRNA，1,25(OH)2D3和EB 1089诱导24 - 羟化酶mRNA的时间进程和程度相似，在约6小时达到峰值反应，并持续至少16小时。然而，在肠道中，相对于18S rRNA，EB 1089在3小时（p < 0.05）和6小时（p < 0.05）时诱导24 - 羟化酶mRNA的水平比1,25(OH)2D3低约50%，而在16小时时不再能检测到24 - 羟化酶mRNA。此外，虽然1,25(OH)2D3和EB 1089在肾脏中均引发了类似的24 - 羟化酶mRNA剂量依赖性诱导（EB 1089和1,25(OH)2D3的EC50分别为0.4±0.13和0.3±0.08 ng/g），但EB 1089在肠道中的EC50（6.6±1.7 ng/g）显著高于1,25(OH)2D3的EC50（0.9±0.32 ng/g，p < 0.01）。在诱导肠道而非肾脏钙结合蛋白 - D9k mRNA方面，EB 1089也比1,25(OH)2D3效果差。为了确定效力存在组织特异性差异的机制，我们测定了1,25(OH)2D3和EB 1089对维生素D受体的结合亲和力。在肾脏中，1,25(OH)2D3的Kd值（0.40±0.95 nM）和EB 1089的Kd值（0.48±0.04 nM）没有差异。然而，在肠道中，EB 1089的Kd值（1.43±0.19 nM）显著高于1,25(OH)2D3的Kd值（0.85±0.06 nM；p < 0.05）。我们的结果表明：（i）EB 1089对肾脏24 - 羟化酶的亲和力比1,25(OH)2D3低50倍，这表明它对C - 24氧化途径的分解代谢更具抗性；（ii）EB 1089和1,25(OH)2D3在体内维生素D受体介导的反应中表现出组织特异性差异，这至少部分可归因于对维生素D受体结合亲和力的差异。
