Evidence for the activation of 1α-hydroxyvitamin D 2 by 25-hydroxyvitamin D-24-hydroxylase: Delineation of pathways involving 1α,24-dihydroxyvitamin D 2 and 1α,25-dihydroxyvitamin D 2 Sonoko Masuda a , Stephen A. Strugnell b , Joyce C. Knutson b , René St-Arnaud c,d , Glenville Jones a, ⁎ a Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6 b Bone Care International Inc., Madison, WI 53713, USA c Genetics Unit, Shriner’s Hospital for Children, Montreal, Quebec, Canada H3G 1A6 d Departments of Medicine, Surgery, and Human Genetics, McGill University, Montreal, Quebec, Canada H3A 2T5 Received 1 November 2005; accepted 12 January 2006 Available online 2 February 2006 Abstract 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, 1α-hydroxyvitamin D 2 (1α(OH)D 2 ) in vitro. Mammalian cells stably transfected with CYP24A1 (V79-CYP24A1) converted 1α(OH)D 2 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 1α(OH)D 2 included the active metabolites 1α,24-dihydroxyvitamin D 2 (1α,24(OH) 2 D 2 ) and 1α,25-dihydroxyvitamin D 2 (1α,25 (OH) 2 D 2 ); the formation of both indicating the existence of distinct activation pathways. A novel water-soluble metabolite, identified as 26-carboxy-1α,24(OH) 2 D 2 , was the presumed terminal degradation product of 1α(OH)D 2 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 1α(OH)D 2 and 1α,24(OH) 2 D 2 relative to 1α,25(OH) 2 D 2 and 1α,25(OH) 2 D 3 were noted, arguing for a role of 24- hydroxylated metabolites in the altered biological activity profile of 1α(OH)D 2 . 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. © 2006 Elsevier B.V. All rights reserved. Keywords: CYP24A1; 1α-hydroxyvitamin D 2 ;1α,24-dihydroxyvitamin D 2 ;1α,25-dihydroxyvitamin D 2 ;1α,25-dihydroxyvitamin D 3 ; Vitamin D catabolism 1. Introduction The active form of vitamin D 3 – 1α,25-dihydroxyvitamin D 3 (1α,25(OH) 2 D 3 ) – plays a dual role as both a major regulator of calcium homeostasis and as a powerful antiproliferative agent and differentiation factor [1,2]. Consequently, much effort has been expended in the design and synthesis of vitamin D analogs that might be used in clinical conditions involving calcium imbalance and/or uncontrolled hyperproliferation [3]. One of the initial and most successful of these strategies has been the development of analogs based upon vitamin D 2 , a sterol which differs from the skin-derived natural vitamin D 3 in that it possesses two additional features in the cholesterol/vitamin D 3 Biochimica et Biophysica Acta 1761 (2006) 221 – 234 http://www.elsevier.com/locate/bba Abbreviations: CYP, cytochrome P450; 1α,25(OH) 2 D 3 ,1α,25-dihydroxy- vitamin D 3 ; 25(OH)D 3 , 25-hydroxyvitamin D 3 ;1α(OH)D 3 ,1α-hydroxyvitamin D 3 ;1α(OH)D 2 ,1α-hydroxyvitamin D 2 ;1α,24(OH) 2 D 2 ,1α,24-dihydroxyvita- min D 2 ; 1α,25(OH) 2 D 2 , 1α,25-dihydroxyvitamin D 2 ; 1α,24,25(OH) 3 D 2 , 1 α,24,25-trihydroxyvitamin D 2 ; 1 α ,24,26(OH) 3 D 2 , 1 α ,24,26-tri- hydroxyvitamin D 2 ; PTH, parathyroid hormone; VDR, vitamin D receptor; HPLC, High Performance Liquid Chromatography; LC-MS, Liquid Chromato- graphic-Mass Spectrometry; NMR, Nuclear magnetic resonance; DMEM, Dulbecco's modified Eagle's medium; DPPD, N′N-diphenyl-p-phenylenedia- mine; GAA, Glacial acetic acid ⁎ Corresponding author. Tel.: +1 613 533 2494; fax: +1 613 533 2022. E-mail address: gj1@post.queensu.ca (G. Jones). 1388-1981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2006.01.004