C-25 hydroxylation of 1alpha,24(R)-dihydroxyvitamin D3 is catalyzed by 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1): metabolism studies with human keratinocytes and rat recombinant CYP24A1.
Recently, 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) has been shown to catalyze not only hydroxylation at C-24 but also hydroxylations at C-23 and C-26 of the secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3). It remains to be determined whether CYP24A1 has the ability to hydroxylate vitamin D3 compounds at C-25. 1alpha,24(R)-dihydroxyvitamin D3 (1alpha,24(R)(OH)2D3) is a non-25-hydroxylated synthetic vitamin D3 analog that is presently being used as an antipsoriatic drug. In the present study, we investigated the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes in order to examine the ability of CYP24A1 to hydroxylate 1alpha,24(R)(OH)2D3 at C-25. The results indicated that keratinocytes metabolize 1alpha,24(R)(OH)2D3 into several previously known both 25-hydroxylated and non-25-hydroxylated metabolites along with two new metabolites, namely 1alpha,23,24(OH)3D3 and 1alpha,24(OH)2-23-oxo-D3. Production of the metabolites including the 25-hydroxylated ones was detectable only when CYP24A1 activity was induced in keratinocytes 1alpha,25(OH)2D3. This finding provided indirect evidence to indicate that CYP24A1 catalyzes C-25 hydroxylation of 1alpha,24(R)(OH)2D3. The final proof for this finding was obtained through our metabolism studies using highly purified recombinant rat CYP24A1 in a reconstituted system. Incubation of this system with 1alpha,24(R)(OH)2D3 resulted in the production of both 25-hydroxylated and non-25-hydroxylated metabolites. Thus, in our present study, we identified CYP24A1 as the main enzyme responsible for the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes, and provided unequivocal evidence to indicate that the multicatalytic enzyme CYP24A1 has the ability to hydroxylate 1alpha,24(R)(OH)2D3 at C-25.
Department of Chemistry, Brown University, Box H, Providence, RI 02912, USA.
Norbert
N
Astecker
Ekaterina A
EA
Bobrovnikova
John L
JL
Omdahl
Lynn
L
Gennaro
Paul
P
Vouros
Inge
I
Schuster
Milan R
MR
Uskokovic
Seiichi
S
Ishizuka
Guochun
G
Wang
G Satyanarayana
GS
Reddy
D000818:Animals; D002112:Calcifediol / Q000031:analogs & derivatives* / Q000378:metabolism*; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000378:metabolism*; D002384:Catalysis; D002460:Cell Line; D002849:Chromatography, Gas; D002851:Chromatography, High Pressure Liquid; D016895:Culture Media, Serum-Free; D006801:Humans; D006900:Hydroxylation; D015603:Keratinocytes / Q000378:metabolism*; D013058:Mass Spectrometry; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins; D002117:Calcitriol; D002112:Calcifediol