Exp Dermatol 2000: 9: 97–103 Copyright C Munksgaard 2000 Printed in Denmark ¡ All rights reserved EXPERIMENTAL DERMATOLOGY ISSN 0906-6705 Conversion of vitamin D 3 to 1 a ,25-dihydroxyvitamin D 3 in human skin equivalents Lehmann B, Rudolph T, Pietzsch J, Meurer M. Conversion of vitamin D 3 B. Lehmann 1 , T. Rudolph 1 , to 1a,25-dihydroxyvitamin D 3 in human skin equivalents. J. Pietzsch 2 and M. Meurer 1 Exp Dermatol 2000: 9: 97–103. C Munksgaard, 2000 1 Department of Dermatology and 2 Institute and Policlinic of Clinical Metabolic Research, Abstract: These results demonstrate for the first time that human kera- Carl Gustav Carus Medical School, tinocytes under in vivo-like conditions have the capacity of the enzymatic Dresden University of Technology, hydroxylation of vitamin D 3 to hormonally active calcitriol D-01307, Germany (1a,25(OH) 2 D 3 ). Supplementation of the culture medium with bovine serum albumin (BSA) up to 1.5% (w/v) amplifies the conversion of vit- amin D 3 to 1a,25(OH) 2 D 3 . The maximum turnover rate of this reaction at 780 nM vitamin D 3 in presence of 1.0% (w/v) BSA amounts to approxi- Key words: keratinocytes – vitamin D 3 – calcitriol – metabolism mately 3 pmol 1a,25(OH) 2 D 3 per 10 6 cells after 6 h of incubation. The hydroxylation of vitamin D 3 to 1a,25(OH) 2 D 3 is inhibited by the P-450 Dr B. Lehmann, Dept of Dermatology, Carl oxidase inhibitor ketoconazole. The generation of 1a,25(OH) 2 D 3 from vit- Gustav Carus Medical School, Dresden University of Technology, Fetscherstr. 74, amin D 3 has an apparent Michaelis constant (K m ) of 2.3¿10 ª6 M. The D-0351 Dresden, Germany intrinsic conversion of vitamin D 3 to biologically active 1a,25(OH) 2 D 3 may Tel.: 49 351 458 2692; Fax: 49 351 458 4338 be of importance for the regulation of proliferation and differentiation of Accepted for publication 30 June 1999 keratinocytes. Introduction Calcitriol (1a,25-dihydroxyvitamin D 3 , 1a,25(OH) 2 D 3 ) 1 , the biologically active form of vitamin D 3 , is produced by a cascade of reactions which includes photochemical vitamin D 3 syn- thesis in the skin and subsequent hydroxylation of vitamin D 3 at the C-25 atom in the liver and at the C-1a position in the kidney (1). Calcitriol exerts antiproliferative and prodifferentiative actions on epidermal keratinocytes (2–4). However, the calci- triol concentrations required for these effects in vi- tro are approximately three orders of magnitude higher than present in human serum (5). The con- centration of 25-hydroxyvitamin D 3 (calcidiol, 25OHD 3 ) in serum is about 1000 times higher and 1 The abbreviations used are: 1a25(OH) 2 D 3 ,1a,25-dihydroxy- vitamin D 3 ; 25OHD 3 , 25-hydroxyvitamin D 3 ;1a-OHD 3 ,1a- hydroxyvitamin D 3 ; KGM, keratinocyte growth medium; KBM, keratinocyte basal medium; DMEM, Dulbecco’s modi- fied Eagle’s medium; FCS, fetal calf serum, BSA, bovine serum albumin; EDTA, ethylenediaminetetraacetic acid; PBS, phos- phate buffered saline; HPLC, high-performance liquid chrom- atography; NP, normal phase; RP, reversed phase; GC-MS, gas chromatography-mass spectrometry. 97 cultured keratinocytes possess a high capacity to convert exogenous 25OHD 3 to calcitriol (6–8). But, the physiological importance of this pathway is probably insignificant: ex vivo perfusion experi- ments in pig skin have shown that only trace amounts of calcitriol can be synthesized in the presence of calcidiol (9). Our previous results showed that the transform- ed HaCaT cell line converts vitamin D 3 as well as the synthetic vitamin D 3 analogue alphacalcidol (1a-hydroxyvitamin D 3 ,1a-OHD 3 ) to calcitriol (10). In preliminary experiments we had found that normal human keratinocytes in organotypic culture (human skin equivalent, HSE) also can convert 1a-OHD 3 to calcitriol 2 . This conversion is inhibited by the P450 oxidase inhibitor ketocona- zole. In addition, we were able to detect the vit- amin D 3 -25-hydroxylase (CYP27) mRNA in kera- tinocytes. However, the CYP27 mRNA is not constitutively expressed but inducible by vitamin D 3 or UVB radiation (11). Thus, normal human keratinocytes are supposed to express both 1a-hy- 2 Unpublished results.