RESUMO
Recent studies of metabolism using pharmacological substrate concentrations of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)(2)D3] in several tissues including primary cultures of human keratinocytes, bovine parathyroid cells and bone cells led to the identification of 1alpha,25-dihydroxy-3-epi-vitamin D3 [1alpha,25(OH)(2)-3-epi-D3] as a major natural metabolite of 1alpha,25(OH)(2)D3. In the present study, we demonstrate that human keratinocytes incubated with 25-hydroxy[26,27-(3)H] vitamin D3 produce 1alpha,25(OH)(2)-3-epi-D3 along with 1alpha,25(OH)(2)D3. The production of 1alpha,25(OH)(2)-3-epi-D3 is also identified in human keratinocytes incubated with physiological substrate concentrations of 1alpha,25(OH)(2)D3. Unlike 24-hydroxylase, the major enzyme involved in the further metabolism of 1alpha,25(OH)(2)D3 in human keratinocytes, the enzyme(s) responsible for the production of 1alpha,25(OH)(2)-3-epi-D3 is constitutive and is not inhibited by ketoconazole. It is also noted that 1alpha,25(OH)(2)-3-epi-D3 is further metabolised in human keratinocytes into several as yet unidentified metabolites, the production of which is inhibited to a great extent by SDZ 89-443, an inhibitor of 24-hydroxylase. This finding indicates that the 24-hydroxylase like in the case of 1alpha,25(OH)(2)D3, also plays a major role in the metabolism of 1alpha,25(OH)(2)-3-epi-D3. The results obtained from the metabolism studies performed in parallel among 25OHD3, 1alpha,25(OH)(2)D3 and 1alpha,25(OH)(2)-3-epi-D3 indicate that 1alpha,25(OH)(2)-3-epi-D3 and its metabolites exhibit higher metabolic stability. In summary, we demonstrate for the first time that 1alpha,25(OH)(2)-3-epi-D3 is a physiological metabolite of 1alpha,25(OH)(2)D3 in human keratinocytes. Also, 1alpha,25(OH)(2)-3-epi-D(3) is further metabolised in human keratinocytes mainly through the activity of 24-hydroxylase. Furthermore, our finding of the relative metabolic stability of 1alpha,25(OH)(2)-3-epi-D3 and especially its metabolites when compared to 1alpha,25(OH)(2)D3 and its metabolites provides an important explanation for its previously observed potent inhibitory effect on keratinocyte growth in spite of its low affinity to vitamin D receptor.
Assuntos
Calcitriol/metabolismo , Sistema Enzimático do Citocromo P-450 , Vitamina D/metabolismo , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Deutério , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Imidazóis/farmacologia , Queratinócitos/metabolismo , Bicamadas Lipídicas/metabolismo , Esteroide Hidroxilases/antagonistas & inibidores , Vitamina D/análogos & derivados , Vitamina D/biossíntese , Vitamina D3 24-HidroxilaseRESUMO
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.
Assuntos
Colecalciferol/metabolismo , Pele/metabolismo , Calcitriol/biossíntese , Células Cultivadas , Colecalciferol/farmacologia , Colestanotriol 26-Mono-Oxigenase , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Queratinócitos/citologia , Queratinócitos/enzimologia , Queratinócitos/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/citologia , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo , Vitamina D3 24-HidroxilaseRESUMO
Human keratinocytes are fully competent cells of the vitamin D (VD) hormone system. They have the capacity to generate VD, to convert it to hormonally active 1alpha,25(OH)(2)D(3) and subsequently, to metabolize the hormone by self-induced CYP24. These reactions generate a cascade of highly transient products and, eventually terminate biologic activity. To elucidate regulatory principles in the VD cascade in more detail, we made use of novel selective CYP24 inhibitors, recently synthesized by our group. Here, we describe the effects of VID400 and SDZ 89-443 on the metabolism of 20 nM (3)H-25(OH)D(3) in human keratinocytes, analyzed by sensitive HPLC methods. First, we present evidence that freshly generated 1alpha,25(OH)(2)D(3) does not down-regulate 1alpha-hydroxylation, as commonly assumed. The transient time course of 1alpha,25(OH)(2)D(3), could be explained by its fast 24-hydroxylation to polar products, undetectable by usual HPLC-analysis of organic extracts. On inhibition of CYP24, 1alpha-hydroxylation continued throughout extended periods, indicating its constitutive nature. Asking whether 1alpha,25(OH)(2)D(3) derived metabolites [1alpha,25(OH)(2)-3epi-D(3), 1alpha,24(R),25(OH)(3)D(3), 1alpha,25(OH)(2)-24oxo-D(3), 1alpha,23(S),25(OH)(3)-24-oxo-D(3) and calcitroic acid] would regulate 1alpha-hydroxylase, we pre-treated cells with 20 nM of these metabolites for 5 h and 24 h. Subsequent incubation with (3)H-25(OH)D(3) demonstrated that neither metabolite substantially impaired 1alpha-hydroxylase, while all of them transiently induced CYP24 activity. Analyzing the effects of VID400 on the kinetics of (3)H-25(OH)D(3), we showed that 1alpha-hydroxylation rather than 24-hydroxylation was rate-limiting in the C-24 oxidation pathway - again suggesting constitutive expression of 1alpha-hydroxylase. CYP24 inhibitors effectively increased the levels and lifetime of all transient 1alpha-hydroxylated metabolites, especially of 1alpha,25(OH)(2)-3epi-D(3) that became the predominant lipid soluble metabolite. Highly increased levels of 1alpha,23(S),25(OH)(3)-24-oxo-D(3), the metabolite preceding side chain cleavage, indicated involvement of CYP24 also in the terminal step of the cascade. Besides using inhibitors of CYP24 as tools to explore mechanisms in the VD cascade, they also appear to be valuable to discover the intrinsic biologic functions of distinct metabolites.
Assuntos
Calcitriol/metabolismo , Calcitriol/farmacologia , Inibidores das Enzimas do Citocromo P-450 , Esteroide Hidroxilases/antagonistas & inibidores , Colestanotriol 26-Mono-Oxigenase , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Imidazóis/farmacologia , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Cinética , Oxirredução , Esteroide Hidroxilases/efeitos dos fármacos , Trítio , Vitamina D/análogos & derivados , Vitamina D/metabolismo , Vitamina D/farmacologia , Vitamina D3 24-HidroxilaseRESUMO
Human keratinocytes convert 25(OH)D(3) to hormonally active 1alpha,25(OH)(2)D(3) and respond to its antiproliferative/prodifferentiating action in vitro and in vivo. Levels and activity of 1alpha,25(OH)(2)D(3) are short-lived. 1alpha,25(OH)(2)D(3) induces 24-hydroxylase (CYP24) that rapidly metabolizes the hormone, yielding a cascade of side-chain oxidized products and this eventually results in the loss of activity. Aiming at stabilizing the levels of active hormone, we have searched for potent, selective inhibitors of CYP24. Selective inhibition was crucial in order to avoid impairment of 1alpha,25(OH)(2)D(3) synthesis, catalyzed by 1alpha-hydroxylase - a related member of cytochrome P-450 (CYP) superfamily. We describe here the testing protocol, using primary human keratinocyte cultures as an appropriate source of CYP24 and 1alpha-hydroxylase, (3)H-25(OH)D(3) (at physiological concentrations) as substrate and sensitive HPLC techniques to analyze the complex metabolite profiles. Four hundred potential inhibitors were screened by this method; most of them were synthesized in our laboratory. These compounds (entitled azoles) were capable of direct binding to the heme iron and of additional interactions with other parts of the enzyme. In this paper, we present VID400 and SDZ 89-443, as first examples of powerful selective CYP24 inhibitors. As anticipated, these compounds increased the levels of 1alpha-hydroxylated products generated from (3)H-25(OH)D(3) and extended their lifetime. Importantly, blocking of 24-hydroxylation led to a switch in metabolism, namely to preferential conversion of 1alpha,25(OH)(2)D(3) to 1alpha,25(OH)(2)-3epi-D(3). As spin-off from our program, selective inhibitors of 1alpha-hydroxylase were also found (e.g. SDZ 88-357). Using (3)H-25(OH)D(3) as substrate in the absence of SDZ 88-357, CYP24 showed high preference for freshly generated 1alpha-hydroxylated metabolites over abundant 25(OH)D(3). In the presence of SDZ 88-357, we noticed a great increase in 24-hydroxylation of (3)H-25(OH)D(3). Besides their use as valuable tools in elucidating regulatory mechanisms, inhibitors of VD hydroxylases may give rise to novel therapeutic strategies, especially in defects of cell growth and differentiation.