Evidence for the activation of 1alpha-hydroxyvitamin D2 by 25-hydroxyvitamin D-24-hydroxylase: delineation of pathways involving 1alpha,24-dihydroxyvitamin D2 and 1alpha,25-dihydroxyvitamin D2.
While current dogma argues that vitamin D prodrugs require side-chain activation by liver enzymes, recent data suggest that hydroxylation may also occur extrahepatically. We used keratinocytes and recombinant human enzyme to test if the 25-hydroxyvitamin D-24-hydroxylase (CYP24A1) is capable of target cell activation and inactivation of a model prodrug, 1alpha-hydroxyvitamin D2 (1alpha(OH)D2) in vitro. Mammalian cells stably transfected with CYP24A1 (V79-CYP24A1) converted 1alpha(OH)D2 to a series of metabolites similar to those observed in murine keratinocytes and the human cell line HPK1A-ras, confirming the central role of CYP24A1 in metabolism. Products of 1alpha(OH)D2 included the active metabolites 1alpha,24-dihydroxyvitamin D2 (1alpha,24(OH)2D2) and 1alpha,25-dihydroxyvitamin D2 (1alpha,25(OH)2D2); the formation of both indicating the existence of distinct activation pathways. A novel water-soluble metabolite, identified as 26-carboxy-1alpha,24(OH)2D2, was the presumed terminal degradation product of 1alpha(OH)D2 synthesized by CYP24A1 via successive 24-hydroxylation, 26-hydroxylation and further oxidation at C-26. This acid was absent in keratinocytes from Cyp24a1 null mice. Slower clearance rates of 1alpha(OH)D2 and 1alpha,24(OH)2D2 relative to 1alpha,25(OH)2D2 and 1alpha,25(OH)2D3 were noted, arguing for a role of 24-hydroxylated metabolites in the altered biological activity profile of 1alpha(OH)D2. Our findings suggest that CYP24A1 can activate and inactivate vitamin D prodrugs in skin and other target cells in vitro, offering the potential for treatment of hyperproliferative disorders such as psoriasis by topical administration of these prodrugs.
Masuda
Sonoko
S
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6.
Strugnell
Stephen A
SA
Knutson
Joyce C
JC
St-Arnaud
René
R
Jones
Glenville
G
D000818:Animals; D002460:Cell Line; D004872:Ergocalciferols / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D006801:Humans; D006900:Hydroxylation; D066298:In Vitro Techniques; D015603:Keratinocytes / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008954:Models, Biological; D011355:Prodrugs / Q000378:metabolism / Q000493:pharmacokinetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013250:Steroid Hydroxylases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D065668:Vitamin D3 24-Hydroxylase; D014867:Water
D004872:Ergocalciferols; D011355:Prodrugs; D011994:Recombinant Proteins; D014867:Water; C042533:1 alpha-hydroxyergocalciferol; C003564:1,25-dihydroxyergocalciferol; D013250:Steroid Hydroxylases; C585549:CYP24A1 protein, human; C585550:Cyp24a1 protein, mouse; D065668:Vitamin D3 24-Hydroxylase