Skin is an autonomous organ in synthesis, two-step activation and degradation of vitamin D(3): CYP27 in epidermis completes the set of essential vitamin D(3)-hydroxylases.
The current understanding of the vitamin D(3) system shows skin as the unique site of vitamin D(3) production and liver is thought to be the main site of conversion to 25(OH)D(3). Skin is capable of activating 25(OH)D(3) via 1alpha-hydroxylation and the resulting 1alpha,25(OH)(2)D(3) plays a role in epidermal homeostasis in normal and diseased skin. It also rapidly up-regulates the major vitamin D(3) metabolizing enzyme 24-hydroxylase at the mRNA level, which is an established indicator for 1alpha,25(OH)(2)D(3)-presence. We investigated the capability of primary human keratinocytes to produce 25(OH)D(3) and subsequent metabolites from vitamin D(3). Thus, by orchestrating the entire system of production, activation and inactivation, skin could be independent of other organs in supply of hormonally active vitamin D(3). First, we demonstrated substantial conversion of (3)H-D(3) to (3)H-25(OH)D(3) in primary human keratinocytes. 25-Hydroxylation was slow, followed first order rate kinetics and was not saturable under our experimental conditions. Then we showed expression of 25-hydroxylase mRNA and compared it to levels of 1alpha-hydroxylase and 24-hydroxylase. Pre-incubation with vitamin D(3) resulted in dose and time dependent up-regulation of 24-hydroxylase mRNA, whereas neither 1alpha-hydroxylase nor 25-hydroxylase expression was affected. Since both, D(3) and 25(OH)D(3) are lacking intrinsic 24-hydroxylase-inducing capacity, up-regulation had to be the consequence of a two-step activation process via 25-hydroxylation and subsequent 1alpha-hydroxylation. 24-Hydroxylase-activities closely followed the corresponding mRNA levels. When 1alpha,25(OH)(2)D(3) itself or its precursor 25(OH)D(3) were used as inducing agents, 24-hydroxylase mRNA and enzyme activity followed a transient time course. In contrast, induction observed with physiological doses of D(3) remained high, even after a 20 h-time period. These differing characteristics may be explained by the slow but constant formation of 1alpha,25(OH)(2)D(3) from a large reservoir of D(3) in the target cell, providing constant supplies for induction.
Schuessler
M
M
Novartis Research Institute Vienna, Brunnerstrasse 59, A-1230, Vienna, Austria. ms@oejab.at
Astecker
N
N
Herzig
G
G
Vorisek
G
G
Schuster
I
I
D002117:Calcitriol / Q000096:biosynthesis; D002478:Cells, Cultured; D002762:Cholecalciferol / Q000378:metabolism* / Q000494:pharmacology; D053493:Cholestanetriol 26-Monooxygenase; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012867:Skin / Q000166:cytology / Q000378:metabolism*; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism; D065668:Vitamin D3 24-Hydroxylase
D012333:RNA, Messenger; D002762:Cholecalciferol; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase; D065668:Vitamin D3 24-Hydroxylase; D002117:Calcitriol