Selective inhibitors of CYP24: mechanistic tools to explore vitamin D metabolism in human keratinocytes

Human keratinocytes are fully competent cells of the vitamin D (VD) hormone system. They have the capacity to generate VD, to convert it to hormonally active 1 alpha ,25(OH)(2)D-3 and subsequently, to metabolize the hormone b y self-induced CYP24. These reactions generate a cascade of highly transien t products and, eventually terminate biologic activity. To elucidate regula tory principles in the VD cascade in more detail, we made use of novel sele ctive CYP24 inhibitors, recently synthesized by our group. Here, we describ e the effects of VID400 and SDZ 89-443 on the metabolism of 20 nM H-3-25(OH )D-3 in human keratinocytes, analyzed by sensitive HPLC methods. First, we present evidence that freshly generated 1 alpha ,25(OH)(2)D-3 does not down -regulate 1 alpha -hydroxylation, as commonly assumed. The transient time c ourse of 1 alpha ,25(OH)(2)D-3, could be explained by its fast 24-hydroxyla tion to polar products, undetectable by usual HPLC-analysis of organic extr acts. On inhibition of CYP24, 1 alpha -hydroxylation continued throughout e xtended periods, indicating its constitutive nature. Asking whether 1 alpha ,25(OH)(2)D-3 derived metabolites [1 alpha ,25(OH)(2)-3epi-D-3. 1 alpha ,2 4(R),25(OH)(3)D-3, 1 alpha ,25(OH)(2)-24oxo-D-3, 1 alpha ,23(S),25(OH)(3)-2 4-oxo-D-3 and calcitroic acid] would regulate 1 alpha -hydroxylase, we pre- treated cells with 20 nM of these metabolites for 5 h and 24 h. Subsequent incubation with H-3-25(OH)D-3 demonstrated that neither metabolite substant ially impaired 1 alpha -hydroxylase, while all of them transiently induced CYP24 activity. Analyzing the effects of VID400 on the kinetics of H-3-25(O H)D-3, we showed that 1 alpha -hydroxylation rather than 24-hydroxylation w as rate-limiting in the C-24 oxidation pathway - again suggesting constitut ive expression of 1 alpha -hydroxylase. CYP24 inhibitors effectively increa sed the levels and lifetime of all transient la-hydroxylated metabolites, e specially of 1 alpha ,25(OH), 3epi-D-3 that became the predominant lipid so luble metabolite. Highly increased levels of 1 alpha ,23(S),25(OH)(3)-24-ox o-D-3, the metabolite preceding side chain cleavage, indicated involvement of CYP24 also in the terminal step of the cascade. Besides using inhibitors of CYP24 as tools to explore mechanisms in the VD cascade, they also appea r to be valuable to discover the intrinsic biologic functions of distinct m etabolites. (C) 2001 Elsevier Science Inc. All rights reserved.