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 1alpha,25(OH)(2)D(3) and subsequently, to metabolize the hormone by self-induced CYP24. These reactions generate a cascade of highly transient products and, eventually terminate biologic activity. To elucidate regulatory principles in the VD cascade in more detail, we made use of novel selective CYP24 inhibitors, recently synthesized by our group. Here, we describe the effects of VID400 and SDZ 89-443 on the metabolism of 20 nM (3)H-25(OH)D(3) in human keratinocytes, analyzed by sensitive HPLC methods. First, we present evidence that freshly generated 1alpha,25(OH)(2)D(3) does not down-regulate 1alpha-hydroxylation, as commonly assumed. The transient time course of 1alpha,25(OH)(2)D(3), could be explained by its fast 24-hydroxylation to polar products, undetectable by usual HPLC-analysis of organic extracts. On inhibition of CYP24, 1alpha-hydroxylation continued throughout extended periods, indicating its constitutive nature. Asking whether 1alpha,25(OH)(2)D(3) derived metabolites [1alpha,25(OH)(2)-3epi-D(3), 1alpha,24(R),25(OH)(3)D(3), 1alpha,25(OH)(2)-24oxo-D(3), 1alpha,23(S),25(OH)(3)-24-oxo-D(3) and calcitroic acid] would regulate 1alpha-hydroxylase, we pre-treated cells with 20 nM of these metabolites for 5 h and 24 h. Subsequent incubation with (3)H-25(OH)D(3) demonstrated that neither metabolite substantially impaired 1alpha-hydroxylase, while all of them transiently induced CYP24 activity. Analyzing the effects of VID400 on the kinetics of (3)H-25(OH)D(3), we showed that 1alpha-hydroxylation rather than 24-hydroxylation was rate-limiting in the C-24 oxidation pathway - again suggesting constitutive expression of 1alpha-hydroxylase. CYP24 inhibitors effectively increased the levels and lifetime of all transient 1alpha-hydroxylated metabolites, especially of 1alpha,25(OH)(2)-3epi-D(3) that became the predominant lipid soluble metabolite. Highly increased levels of 1alpha,23(S),25(OH)(3)-24-oxo-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 appear to be valuable to discover the intrinsic biologic functions of distinct metabolites.
Schuster
I
I
Novartis Research Institute, Vienna, Austria. inge@tbi.univie.ac.at
Egger
H
H
Astecker
N
N
Herzig
G
G
Schüssler
M
M
Vorisek
G
G
D002117:Calcitriol / Q000378:metabolism* / Q000494:pharmacology*; D053493:Cholestanetriol 26-Monooxygenase; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors* / Q000187:drug effects; D014316:Tritium; D014807:Vitamin D / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D065668:Vitamin D3 24-Hydroxylase
D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D007093:Imidazoles; C429905:SDZ 89-443; C429906:VID 400; C118756:dihydroxy-vitamin D3; D014316:Tritium; D014807:Vitamin D; D013250:Steroid Hydroxylases; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase; D065668:Vitamin D3 24-Hydroxylase; D002117:Calcitriol