Selective inhibitors of CYP24: mechanistic tools to explore vitamin D metabolism in human keratinocytes

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.     

Characterization and regulation of the vitamin D hydroxylases

The metabolism of vitamin D is regulated by three major cytochrome P450-containing h hydroxylases—the hepatic 25-hydroxylase, the renal 1-hydroxylase, and the renal and intestinal 24-hydroxylase. In the liver, the 25-hydroxylation reaction is catalyzed by microsomal and mitochondrial cytochrome P450cc25. The microsomal P450 accepts electrons from the NADPH-cytochrome P450 reductase, and the mitochondrial P450 accepts electrons from NADPH-ferredoxin reductase and ferredoxin. In the kidney, the 1- and 24-hydroxylation reactions are catalyzed by mitochondrial cytochromes P450cc1 and P450cc24, respectively. The 24-hydroxylase is also found in vitamin D target tissues such as the intestine. The rat hepatic mitochondrial P450cc25 and the rat renal mitochondrial P450cc24 have been purified, and their cDNAs have been cloned and sequenced. 1,25-Dihydroxyvitamin D, the active metabolite of vitamin D, markedly stimulates renal P450cc24 mRNA and 24-hydroxylase activity in the intact animal and in renal cell lines. This stimulation occurs via a receptor-mediated mechanism requiring new protein synthesis. Despite the availability of a clone, no studies have yet been reported of the regulation of hepatic P450cc25 at the mRNA level. The study of one of the most important enzymes in vitamin D metabolism, the renal 1-hydroxylase which produces the active metabolite, awaits the definitive cloning of the cDNA for the P450cc1.     

Inhibitors of vitamin D hydroxylases: structure-activity relationships

Aiming at new drugs to efficiently treat diseases, in which either increased or decreased levels of active vitamin D are desirable, we have designed some 400 structurally different azole-type inhibitors and examined their capacity to selectively block vitamin D metabolism by CYP24 or synthesis by CYP27B, in human keratinocytes. Based on resulting data, we built pharmacophore models of the active sites using commercial software. The overlay of potent selective compounds indicated similar docking modes in the two-substrate pockets and allowed for identification of bioactive conformations. Superimposing these bioactive conformations with low energy conformers of 25(OH)D(3) suggested that the substrate-mimicked by strong inhibitors in size, shape and lipophilic character-binds to both enzymes in 6s-trans configuration. Pharmacophoric models implied a similar geometry of the substrate sites, nevertheless specific features of CYP24 and CYP27B could be defined. Bulky substituents in alpha-position to the azole caused selectivity for CYP24, whereas bulky substituents in beta-position could result in selectivity for CYP27B. Moreover, studies with small sterically restricted inhibitors revealed a probable location of the 3-OH-group of 25(OH)D(3) in CYP27B. In the absence of crystal structures, our inhibitors are valuable tools to model and understand the active sites of vitamin D hydroxylases, resulting in the design of powerful, selective therapeutics.     

The flavonoid apigenin suppresses vitamin D receptor expression and vitamin D responsiveness in normal human keratinocytes

Apigenin, a flavonoid with chemopreventive properties, induces cellular growth arrest, with concomitant inhibition of intracellular signaling cascades and decreased proto-oncogene expression. We report that apigenin potently inhibited vitamin D receptor (VDR) mRNA and protein expression in human keratinocytes without changes in VDR mRNA half-life. Concurrently, downregulation of retinoid X receptor alpha, a dramatic loss of c-myc mRNA, and upregulation of p21(WAF1) took place. Furthermore, a nearly complete suppression of vitamin D responsiveness was observed as estimated by induction of 24-hydroxylase mRNA. The apigenin effect on VDR expression was shared by some other (quercetine and fisetine) but not all tested flavonoids. Interestingly, the apigenin-mediated VDR suppression was counteracted by the NFkappaB inhibitors sodium salicylate and caffeic acid phenethyl ester. The presented results propose suppression of nuclear receptor levels as a novel mechanism whereby flavonoids exert their pleiotropic effects. This study may also contribute to the understanding of the regulation of VDR expression in epidermal keratinocytes.     

Identification of vitamin D target genes in human keratinocytes by subtractive screening

1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2) D(3)) imposes cell cycle block in late G1 phase in cultured human keratinocytes. We wanted to identify early vitamin D target genes using a subtractive screening approach. Human foreskin keratinocytes were grown to about 70% confluence, treated with 2 x 10(-7) M 1alpha,25(OH)(2) D(3) or left untreated and RNA from both populations were isolated after 22h of incubation. cDNA was synthesised and cloned into plasmid vectors. For screening of the libraries, cDNA was amplified in vitro using T7 RNA polymerase and then the amplified RNA (driver, control population) and single stranded cDNA (tester) were used for subtractive hybridisation. Heterohybrids were then separated from single stranded nucleotides using a hydroxyapatite column. The radiolabeled single stranded cDNA was used for screening a colony blot. Positive clones were rescreened, plasmid DNA was isolated and used for verifying the results by Northern blot analysis, using RNA isolated from untreated keratinocytes, as well as RNA isolated after 6h, 12h and 24h of vitamin D treatment.     

Mechanism of vitamin D3-induced transcription of phospholipase D1 in HaCat human keratinocytes

1α,25-Dihydroxyvitamin D₃ (VitD₃) increases protein and gene expression of phospholipase D1 (PLD1), but not PLD2, in HaCaT human keratinocytes. We show that VitD₃ increases PLD1 gene expression through a vitamin D responsive element (VDRE) on the 5′ PLD1 promoter (−260 bp to −246 bp from exon 1). Similar results were obtained by transfecting VitD₃ receptor (VDR) into HEK293 cells, which are originally VitD₃-unresponsive. Electrophoresis mobility shift assays (EMSA) and chromatin immunoprecipitation (CHIP) assays showed that the complex of VitD₃, VDR and retinoid X receptor α (RXRα) binds to the VDRE and increases PLD1 gene expression in HaCaT cells.     

Cultured human keratinocytes cannot metabolize vitamin D3 to 25-hydroxyvitamin D3.

The metabolism of [3H]vitamin D3 was studied in cultured human keratinocytes (CHK). Intact CHK were incubated for 1, 6, 12, 24 and 48 h with [3H]vitamin D3 and the lipid soluble fractions from the media and cells were extracted by high-performance liquid chromatography (HPLC). Vitamin D3 and its metabolites, 25-OH-D3, 24,25(OH)2D3 and 1,25(OH)2D3 were added to the extracts, as markers, prior to HPLC. HPLC analysis of the lipid extracts did not reveal any monohydroxylated metabolites. CHK incubated for one hour with [3H]25-OH-D3 showed a 10 +/- 4% conversion to [3H]1,25(OH)2D3 whereas no conversion to [3H]1,25(OH)2D3 was observed in control CHKs that were boiled prior to incubation with [3H]25-OH-D3. These findings suggest that cultured neonatal keratinocytes are incapable of metabolizing vitamin D3 to 25-OH-D3.     

An in vitro study of vitamin D2 hydroxylases in the chick.

An in vitro study of the liver 25-hydroxylation of vitamin D2 and the kidney 1- and 24-hydroxylations of 25-hydroxyvitamin D2 was undertaken in order to determine whether the discrimination against vitamin D2 seen in chicks in vivo is the result of a block of one or more of the steps in the activation of the vitamin D2 molecule. Vitamin D2 hydroxylation reactions in the chick are virtually identical with those observed with the vitamin D3 series. It is, therefore, concluded that the chick possesses the required enzymatic machinery to synthesize 1,25-dihydroxyvitamin D2 and that the discrimination must be because of some unknown metabolic reaction of the vitamin D2 compounds, to a defect in the transport of vitamin D2 metabolites, or to target organ discrimination.     

Vitamin D hydroxylases.

There are three mixed function oxidases which catalyze hydroxylations of vitamin D and its derivatives. These include the hepatic mitochondrial or microsomal vitamin D3-25-hydroxylase and the two renal mitochondrial enzymes which further hydroxylate 25-hydroxyvitamin-D3 (25-OH-D3) to form 24R,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the primary steroid hormonal derivative of vitamin D3. All three enzymes are cytochrome P450 dependent. The two renal mitochondrial enzymes are regulated, usually in a reciprocal fashion. The intracellular signalling systems involved in this regulation include 1,25(OH)2D3 itself and both protein kinases A and C. Recent progress has been made in the purification and cloning of the vitamin D3-25-hydroxylase and the 25-OH-D3-24-hydroxylase. When the 25-OH-D3-1-hydroxylase is purified and cloned, efforts which have thus far been frustrated by its low abundance, fertile new ground for the study of the regulation of vitamin D metabolism at the molecular level will be opened up.     

UVB-induced production of 1,25-dihydroxyvitamin D3 and vitamin D activity in human keratinocytes pretreated with a sterol delta7-reductase inhibitor

The skin fulfills an important role in the vitamin D photo-endocrine system. Epidermis is not only the site of vitamin D3 photoproduction. In addition, epidermal keratinocytes contain the vitamin D receptor (VDR) and possess 25-hydroxylase and 1alpha-hydroxylase activity indicating that all components of the vitamin D system are present. We investigated whether these components cooperate in inducing vitamin D activity upon treatment with physiological UVB doses. Upon irradiation, 24-hydroxylase mRNA was induced in keratinocytes pretreated with a sterol Delta7-reductase inhibitor (BM15766) whereby the 7-dehydrocholesterol content increased by 300-fold. Transfection experiments with a vitamin D response element containing construct confirmed VDR-dependent gene activation. Furthermore, the UVB-dependent induction of 24-hydroxylase was blocked by the cytochrome-P450 inhibitor ketoconazole. The 24-hydroxylase inducing photoproduct was transferable to unirradiated keratinocytes by medium and cellular homogenates of UVB-irradiated, BM15766-pretreated cells and was identified as 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] by high-performance liquid chromatography with tandem mass spectrometric detection. Addition of vitamin D binding protein blunted UVB-induced 24-hydroxylase suggesting the possibility of a paracrine or autocrine role for 1,25(OH)2D3. In conclusion, epidermal keratinocytes can produce vitamin D3, convert it to 1,25(OH)2D3 and respond to it upon UVB irradiation in the absence of exogenous 7-dehydrocholesterol and therefore contain a unique and complete photo-endocrine vitamin D system.     

Selective expression of 15-lipoxygenase activity by cultured human keratinocytes

Human keratinocytes isolated from neonatal skin express 15-lipoxygenase activity at a level far greater than that of any of the other pathways for lipoxygenation of arachidonic acid. The 10,000 x g supernatant of sonicates of 10(6) keratinocytes generates 15-hydroxy-eicosatetraenoic acid from 5 micrograms/ml of arachidonic acid at a mean maximum rate of 38 ng/30 min at 37 degrees C, that is similar to the activity of the 15-lipoxygenase of human airway epithelial cells and greater than that of endothelial cells and leukocytes. The unique mediators derived from the 15-lipoxygenation of arachidonic acid, that stimulate secretion and exert hyperalgesic effects, may achieve a concentration in skin sufficient to regulate local cellular and neural functions.     

Coordinate control by vitamin A of keratin gene expression in human keratinocytes

In the present study, we examine the effects of vitamin A on keratin protein and mRNA levels in human keratinocytes. In epidermal keratinocytes, the levels of keratins 5, 6, 14, and 17 decrease and keratins 13 and 19 increase in response to increasing concentrations of a potent synthetic trans-retinoic acid analog, arotinoid Ro 13-6298. In tracheal keratinocytes, a similar suppression is observed for keratins 5, 6, 14, 17, and 18 and an increase in keratin 19. Both induction and suppression responses show identical kinetics and both processes are half-maximal at 5 nM arotinoid and maximal at 10 nM. Utilizing cDNAs specific for keratins 5, 6, 13, and 19, we demonstrate that the mRNA levels for these keratins change coordinately with the corresponding amount of keratin protein, indicating that the control of keratin protein expression most likely resides at the level of mRNA synthesis and/or degradation. The identical kinetics for all of the responses, both inductive and suppressive, suggests that a common mechanism controls the expression of these genes. These results indicate that vitamin A produces more sweeping changes in keratinocyte function than previously appreciated in that many and perhaps all keratins are modulated by vitamin A. Moreover, these responses are 10- to 100-fold less sensitive to retinoid than the process of envelope formation, suggesting that at least two sets of processes with different sensitivities to vitamin A are present in keratinocytes.     

Regulation of terminal differentiation of cultured human keratinocytes by vitamin A

Vitamin A is known to exert an important influence on epithelial differentiation. The fetal calf serum supplement of cell-culture medium contains enough of the vitamin to affect the differentiation of cultured keratinocytes derived from epidermis and from other stratified squamous epithelia. The cellular and molecular properties of the cultures are altered when the medium is supplemented with serum from which the vitamin A has been removed by solvent extraction (delipidized serum). Cell motility is reduced, the adhesiveness of cells increases and pattern formation is prevented. In both epidermal and conjunctival keratinocytes, removal of vitamin A leads to the synthesis of a 67 kd keratin characteristic of terminally differentiating epidermis and to much reduced synthesis of the 52 kd and 40 kd keratins typical of conjunctiva. These changes, both cellular and molecular, are reversed by the addition of retinyl acetate to the medium containing delipidized serum. Cell motility and pattern formation are restored, and detachment of the most mature cells from the surface of the stratified epithelium is promoted. Synthesis of the 67 kd keratin is prevented and the synthesis of the 40 and 52 kd keratins is stimulated. The nature of the keratins synthesized is regulated by the concentration of vitamin A, and each cell type adjusts its synthesis differently at a given vitamin concentration.     

Environment-dependent growth inhibition of human epidermal keratinocytes by recombinant human transforming growth factor-beta

Transforming growth factor-beta (TGF-beta) purified from platelets is a potent growth inhibitor of several normal epithelial cell types in culture. In contrast, some carcinoma cell lines derived from tumors of these same tissues are resistant to this factor. Using recombinant human TGF-beta, the authors have confirmed these results with six normal human epidermal keratinocyte strains and four human epidermal squamous carcinoma cell lines. However, the sensitivity of normal cells to TGF-beta was found to depend on the culture conditions. When grown in a specialized nutrient medium supplemented with pituitary extract, keratinocytes were completely inhibited by the addition of 0.3 ng/ml TGF-beta. In contrast, when their growth was supported by cocultivation with 3T3 fibroblast feeder cells, 30- to 100-fold higher concentrations of TGF-beta were required to achieve comparable growth inhibition. This differential sensitivity occurred despite the fact that in both culture systems TGF-beta in the culture medium had a half-life of about 50 minutes, becoming tightly bound to the surface of the culture dish. Bound TGF-beta proved to be biologically active and stable for about a week in the absence of 3T3 feeder cells. Incubating 3T3 cells on TGF-beta-coated dishes, however, resulted in nearly quantitative removal and degradation of the TGF-beta within 2 days, permitting normal rates of keratinocyte growth. The binding of TGF-beta to surfaces and the ability of fibroblasts to attenuate its inhibitory activity for epithelial cells must be considered when evaluating in vitro models and in planning strategies for the use of this factor in vivo.