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We have previously described new pathways of vitamin D3 activation by CYP11A1 to produce a variety of metabolites including 20(OH)D3 and 20,23(OH)(2)D3. These can be further hydroxylated by CYP27B1 to produce their C1α-hydroxyderivatives. CYP11A1 similarly initiates the metabolism of lumisterol (L3) through sequential hydroxylation of the side chain to produce 20(OH)L3, 22(OH)L3, 20,22(OH)(2)L3 and 24(OH)L3. CYP11A1 also acts on 7-dehydrocholesterol (7DHC) producing 22(OH)7DHC, 20,22(OH)(2)7DHC and 7-dehydropregnenolone (7DHP) which can be converted to the D3 and L3 configurations following exposure to UVB. These CYP11A1-derived compounds are produced in vivo and are biologically active displaying anti-proliferative, anti-inflammatory, anti-cancer and pro-differentiation properties. Since the protective role of the classical form of vitamin D3 (1,25(OH)(2)D3) against UVB induced damage is recognized, we recently tested whether novel CYP11A1-derived D3- and L3-hydroxyderivatives protect against UVB-induced damage in epidermal human keratinocytes and melanocytes. We found that along with 1,25(OH)(2)D3, CYP11A1-derived D3-hydroxyderivatives and L3 and its hydroxyderivatives exert photoprotective effects. These included induction of intracellular free radical scavenging and attenuation and repair of DNA damage. The protection of human keratinocytes against DNA damage included the activation of the NRF2-regulated antioxidant response, p53-phosphorylation and its translocation to the nucleus, and DNA repair induction. These data indicate that novel derivatives of vitamin D3 and lumisterol are promising photoprotective agents. However, detailed mechanisms of action, and the involvement of specific nuclear receptors, other vitamin D binding proteins or mitochondria, remain to be established.

Lumisterol (L3) is a stereoisomer of 7-dehydrocholesterol and is produced through the photochemical transformation of 7-dehydrocholesteol induced by high doses of UVB. L3 is enzymatically hydroxylated by CYP11A1, producing 20(OH)L3, 22(OH)L3, 20,22(OH)2L3, and 24(OH)L3. Hydroxylumisterols function as reverse agonists of the retinoic acid-related orphan receptors &alpha, and &gamma, (ROR&alpha, /&gamma, ) and can interact with the non-genomic binding site of the vitamin D receptor (VDR). These intracellular receptors are mediators of photoprotection and anti-inflammatory activity. In this study, we show that L3-hydroxyderivatives significantly increase the expression of VDR at the mRNA and protein levels in keratinocytes, both non-irradiated and after UVB irradiation. L3-hydroxyderivatives also altered mRNA and protein levels for ROR&alpha, in non-irradiated cells, while the expression was significantly decreased in UVB-irradiated cells. In UVB-irradiated keratinocytes, L3-hydroxyderivatives inhibited nuclear translocation of NF&kappa, B p65 by enhancing levels of I&kappa, B&alpha, in the cytosol. This anti-inflammatory activity mediated by L3-hydroxyderivatives through suppression of NF&kappa, B signaling resulted in the inhibition of the expression of UVB-induced inflammatory cytokines, including IL-17, IFN-&gamma, and TNF-&alpha, The L3-hydroxyderivatives promoted differentiation of UVB-irradiated keratinocytes as determined from upregulation of the expression at the mRNA of involucrin (IVL), filaggrine (FLG), and keratin 14 (KRT14), downregulation of transglutaminase 1 (TGM1), keratins including KRT1, and KRT10, and stimulation of ILV expression at the protein level. We conclude that CYP11A1-derived hydroxylumisterols are promising photoprotective agents capable of suppressing UVB-induced inflammatory responses and restoring epidermal function through targeting the VDR and RORs.

The interactions of derivatives of lumisterol (L3) and vitamin D3 (D3) with liver X receptors (LXRs) were investigated. Molecular docking using crystal structures of the ligand binding domains (LBDs) of LXRα and β revealed high docking scores for L3 and D3 hydroxymetabolites, similar to those of the natural ligands, predicting good binding to the receptor. RNA sequencing of murine dermal fibroblasts stimulated with D3-hydroxyderivatives revealed LXR as the second nuclear receptor pathway for several D3-hydroxyderivatives, including 1,25(OH)2D3. This was validated by their induction of genes downstream of LXR. L3 and D3-derivatives activated an LXR-response element (LXRE)-driven reporter in CHO cells and human keratinocytes, and by enhanced expression of LXR target genes. L3 and D3 derivatives showed high affinity binding to the LBD of the LXRα and β in LanthaScreen TR-FRET LXRα and β coactivator assays. The majority of metabolites functioned as LXRα/β agonists; however, 1,20,25(OH)3D3, 1,25(OH)2D3, 1,20(OH)2D3 and 25(OH)D3 acted as inverse agonists of LXRα, but as agonists of LXRβ. Molecular dynamics simulations for the selected compounds, including 1,25(OH)2D3, 1,20(OH)2D3, 25(OH)D3, 20(OH)D3, 20(OH)L3 and 20,22(OH)2L3, showed different but overlapping interactions with LXRs. Identification of D3 and L3 derivatives as ligands for LXRs suggests a new mechanism of action for these compounds.

UVB radiation mediates inflammatory responses causing skin damage and defects in epidermal differentiation. 1α,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) interacts with the vitamin D(3) receptor (VDR) to regulate inflammatory responses. Additionally, 1,25(OH)(2)D(3)/VDR signaling represents a potential therapeutic target in the treatment of skin disorders associated with inflammation and poor differentiation of keratinocytes. Since the protective effect of 1,25(OH)(2)D(3) against UVB-induced skin damage and inflammation is recognized, CYP11A1-derived vitamin D(3)-hydroxyderivatives including 20(OH)D(3), 1,20(OH)(2)D(3), 20,23(OH)(2)D(3) and 1,20,23(OH)(3)D(3) were tested for their anti-inflammatory and skin protection properties in UVB-irradiated human epidermal keratinocytes (HEKn). HEKn were treated with secosteroids for 24 h pre- and post-UVB (50 mJ/cm(2)) irradiation. Secosteroids modulated the expression of the inflammatory response genes (IL-17, NF-κB p65, and IκB-α), reducing nuclear-NF-κB-p65 activity and increasing cytosolic-IκB-α expression as well as that of pro-inflammatory mediators, IL-17, TNF-α, and IFN-γ. They stimulated the expression of involucrin (IVL) and cytokeratin 10 (CK10), the major markers of epidermal differentiation, in UVB-irradiated cells. We conclude that CYP11A1-derived hydroxyderivatives inhibit UVB-induced epidermal inflammatory responses through activation of IκB-α expression and suppression of NF-kB-p65 activity and its downstream signaling cytokines, TNF-α, and IFN-γ, as well as by inhibiting IL-17 production and activating epidermal differentiation.

Melatonin is produced in almost all living taxa and is probably 2-3 billion years old. Its pleiotropic activities are related to its local concentration that is secondary to its local synthesis, delivery from distant sites and metabolic or non-enzymatic consumption. This consumption generates metabolites through indolic, kynuric and cytochrome P450 (CYP) mediated hydroxylations and O-demethylation or non-enzymatic processes, with potentially diverse phenotypic effects. While melatonin acts through receptor-dependent and receptor-independent mechanisms, receptors for melatonin metabolites remain to be identified, while their receptor-independent activities are well documented. The human skin with its main cellular components including malignant cells can both produce and rapidly metabolize melatonin in cell-type and context-dependent fashion. The predominant metabolism in human skin occurs through indolic, CYP-mediated and kynuric pathways with main metabolites represented by 6-hydroxymelatonin, N1 -acetyl-N2 -formyl-5-methoxykynuramine (AFMK), N1 -acetyl-5-methoxykynuramine (AMK), 5-methoxytryptamine, 5-methoxytryptophol and 2-hydroxymelatonin. AFMK, 6-hydroxymelatonin, 2-hydroxymelatonin and probably 4-hydroxymelatonin can potentially be produced in epidermis through UVB-induced non-enzymatic melatonin transformation. The skin metabolites are also the same as those produced in lower organisms and plants indicating phylogenetic conservation across diverse species and adaptation by skin of the primordial defense mechanism. As melatonin and its metabolites counteract or buffer environmental stresses to maintain its homeostasis through broad-spectrum activities, both melatoninergic and degradative pathways must be precisely regulated, because the nature of phenotypic regulations will depend on local concentration of melatonin and its metabolites. These can be receptor-mediated or represent non-receptor regulatory mechanisms.

We tested whether novel CYP11A1-derived vitamin D3- and lumisterol-hydroxyderivatives, including 1,25(OH)2D3, 20(OH)D3, 1,20(OH)2D3, 20,23(OH)2D3, 1,20,23(OH)3D3, lumisterol, 20(OH)L3, 22(OH)L3, 20,22(OH)2L3, and 24(OH)L3, can protect against UVB-induced damage in human epidermal keratinocytes. Cells were treated with above compounds for 24 h, then subjected to UVB irradiation at UVB doses of 25, 50, 75, or 200 mJ/cm2, and then examined for oxidant formation, proliferation, DNA damage, and the expression of genes at the mRNA and protein levels. Oxidant formation and proliferation were determined by the DCFA-DA and MTS assays, respectively. DNA damage was assessed using the comet assay. Expression of antioxidative genes was evaluated by real-time RT-PCR analysis. Nuclear expression of CPD, phospho-p53, and Nrf2 as well as its target proteins including HO-1, CAT, and MnSOD, were assayed by immunofluorescence and western blotting. Treatment of cells with the above compounds at concentrations of 1 or 100 nM showed a dose-dependent reduction in oxidant formation. At 100 nM they inhibited the proliferation of cultured keratinocytes. When keratinocytes were irradiated with 50–200 mJ/cm2 of UVB they also protected against DNA damage, and/or induced DNA repair by enhancing the repair of 6-4PP and attenuating CPD levels and the tail moment of comets. Treatment with test compounds increased expression of Nrf2-target genes involved in the antioxidant response including GR, HO-1, CAT, SOD1, and SOD2, with increased protein expression for HO-1, CAT, and MnSOD. The treatment also stimulated the phosphorylation of p53 at Ser-15, increased its concentration in the nucleus and enhanced Nrf2 translocation into the nucleus. In conclusion, pretreatment of keratinocytes with 1,25(OH)2D3 or CYP11A1-derived vitamin D3- or lumisterol hydroxy-derivatives, protected them against UVB-induced damage via activation of the Nrf2-dependent antioxidant response and p53-phosphorylation, as well as by the induction of the DNA repair system. Thus, the new vitamin D3 and lumisterol hydroxy-derivatives represent promising anti-photodamaging agents. Keywords: Epidermal keratinocytes, Lumisterol (L3), Nuclear factor E2-related factor 2 (Nrf2), Photoprotective effects, Ultraviolet B (UVB), Vitamin D3 hydroxy-derivatives

Tryptophan hydroxylase (TPH) activity was detected in cultured epidermal melanocytes and dermal fibroblasts with respective Km of 5.08 and 2.83 mM and Vmax of 80.5 and 108.0 µmol/min. Low but detectable TPH activity was also seen in cultured epidermal keratinocytes. Serotonin and/or its metabolite and precursor to melatonin, N-acetylserotonin (NAS), were identified by LC/MS in human epidermis and serum. Endogenous epidermal levels were 113.18 ± 13.34 and 43.41 ± 12.45 ng/mg protein for serotonin (n = 8/8) and NAS (n = 10/13), respectively. Their production was independent of race, gender, and age. NAS was also detected in human serum (n = 13/13) at a concentration 2.44 ± 0.45 ng/mL, while corresponding serotonin levels were 295.33 ± 17.17 ng/mL (n = 13/13). While there were no differences in serum serotonin levels, serum NAS levels were slightly higher in females. Immunocytochemistry studies showed localization of serotonin to epidermal and follicular keratinocytes, eccrine glands, mast cells, and dermal fibrocytes. Endogenous production of serotonin in cultured melanocytes, keratinocytes, and dermal fibroblasts was modulated by UVB. In conclusion, serotonin and NAS are produced endogenously in the epidermal, dermal, and adnexal compartments of human skin and in cultured skin cells. NAS is also detectable in human serum. Both serotonin and NAS inhibited melanogenesis in human melanotic melanoma at concentrations of 10-4 -10-3 M. They also inhibited growth of melanocytes. Melanoma cells were resistant to NAS inhibition, while serotonin inhibited cell growth only at 10-3 M. In summary, we characterized a serotonin-NAS system in human skin that is a part of local neuroendocrine system regulating skin homeostasis.

A novel pathway of vitamin D activation by CYP11A has previously been elucidated. To define the mechanism of action of its major dihydroxy-products, we tested the divergence and overlap between the gene expression profiles of human epidermal keratinocytes treated with either CYP11A1-derived 20,23(OH)2D3 or classical 1,25(OH)2D3. Both secosteroids have significant chemical similarity with the only differences being the positions of the hydroxyl groups. mRNA was isolated and examined by microarray analysis using Illumina&rsquo, s HumanWG-6 chip/arrays and subsequent bioinformatics analyses. Marked differences in the up- and downregulated genes were observed between 1,25(OH)2D3- and 20,23(OH)2D3-treated cells. Hierarchical clustering identified both distinct, opposite and common (overlapping) gene expression patterns. CYP24A1 was a common gene strongly activated by both compounds, a finding confirmed by qPCR. Ingenuity pathway analysis identified VDR/RXR signaling as the top canonical pathway induced by 1,25(OH)2D3. In contrast, the top canonical pathway induced by 20,23(OH)2D3 was AhR, with VDR/RXR being the second nuclear receptor signaling pathway identified. QPCR analyses validated the former finding by revealing that 20,23(OH)2D3 stimulated CYP1A1 and CYP1B1 gene expression, effects located downstream of AhR. Similar stimulation was observed with 20(OH)D3, the precursor to 20,23(OH)2D3, as well as with its downstream metabolite, 17,20,23(OH)3D3. Using a Human AhR Reporter Assay System we showed marked activation of AhR activity by 20,23(OH)2D3, with weaker stimulation by 20(OH)D3. Finally, molecular modeling using an AhR LBD model predicted vitamin D3 hydroxyderivatives to be good ligands for this receptor. Thus, our microarray, qPCR, functional studies and molecular modeling indicate that AhR is the major receptor target for 20,23(OH)2D3, opening an exciting area of investigation on the interaction of different vitamin D3-hydroxyderivatives with AhR and the subsequent downstream activation of signal transduction pathways in a cell-type-dependent manner.

CYP11A1 hydroxylates the side chain of vitamin D3 (D3) in a sequential fashion [D3→20S(OH)D3→20,23(OH)2D3→17,20,23(OH)3D3], in an alternative to the classical pathway of activation [D3→25(OH)D3→1,25(OH)2D3]. The products/intermediates of the pathway can be further modified by the action of CYP27B1. The CYP11A1-derived products are biologically active with functions determined by the lineage of the target cells. This pathway can operate in epidermal keratinocytes. To further define the role of these novel secosteroids we tested them for protective effects against UVB-induced damage in human epidermal keratinocytes, melanocytes and HaCaT keratinocytes, cultured in vitro. The secosteroids attenuated ROS, H2O2 and NO production by UVB-irradiated keratinocytes and melanocytes, with an efficacy similar to 1,25(OH)2D3, while 25(OH)D3 had lower efficacy. These attenuations were also seen to some extent for the 20(OH)D3 precursor, 20S-hydroxy-7-dehydrocholesterol. These effects were accompanied by upregulation of genes encoding enzymes responsible for defense against oxidative stress. Using immunofluorescent staining we observed that the secosteroids reduced the generation cyclobutane pyrimidine dimers in response to UVB and enhanced expression of p53 phosphorylated at Ser-15, but not at Ser-46. Additional evidence for protection against DNA damage in cells exposed to UVB and treated with secosteroids was provided by the Comet assay where DNA fragmentation was markedly reduced by 20(OH)D3 and 20,23(OH)2D3. In conclusion, novel secosteroids that can be produced by the action of CYP11A1 in epidermal keratinocytes have protective effects against UVB radiation. This article is part of a special issue entitled '17th Vitamin D Workshop'.

20S-hydroxyvitamin D3 [20S(OH)D3] is anti-inflammatory and not hypercalcemic, suggesting its potential as a lead compound. In this study, side chain modified 20S(OH)D3 analogs (4, 13, 23 and 33) together with their 1α-OH derivatives were synthesized and their metabolism and biological activities tested. 4, 13 and 23 are good substrates for CYP27B1, enabling enzymatic synthesis of their 1α-OH derivatives 5, 14 and 24. However, 33 could not be hydroxylated by CYP27B1 and acts as an inhibitor. All analogs were poorer substrates for CYP24A1 than calcitriol, indicating improved catabolic stability. While the parent analogs showed minimal VDR stimulating activity, their 1α-OH derivatives were potent VDR agonists. 4, 5, 14 and 24 significantly upregulated the expression of CYP24A1 at the mRNA level, consistent with their VDR activation abilities and indicating that 1α-hydroxylation is required to produce analogs with strong activity. These analogs have anti-inflammatory activities that are influenced by side chain composition and by 1α-hydroxylation. To understand their molecular interactions with the VDR, 20S(OH)D3, 4 and 33 were co-crystalized with the VDR ligand binding domain, which revealed subtle differences to the calcitriol-bound receptor. This study demonstrates the potential of the 20S(OH)D3 scaffold for the development of novel anti-inflammatory agents.

Using LC/qTOF-MS we detected lumisterol, 20-hydroxylumisterol, 22-hydroxylumisterol, 24-hydroxylumisterol, 20,22-dihydroxylumisterol, pregnalumisterol, 17-hydroxypregnalumisterol and 17,20-dihydroxypregnalumisterol in human serum and epidermis, and the porcine adrenal gland. The hydroxylumisterols inhibited proliferation of human skin cells in a cell type-dependent fashion with predominant effects on epidermal keratinocytes. They also inhibited melanoma proliferation in both monolayer and soft agar. 20-Hydroxylumisterol stimulated the expression of several genes, including those associated with keratinocyte differentiation and antioxidative responses, while inhibiting the expression of others including RORA and RORC. Molecular modeling and studies on VDRE-transcriptional activity excludes action through the genomic site of the VDR. However, their favorable interactions with the A-pocket in conjunction with VDR translocation studies suggest they may act on this non-genomic VDR site. Inhibition of RORα and RORγ transactivation activities in a Tet-on CHO cell reporter system, RORα co-activator assays and inhibition of (RORE)-LUC reporter activity in skin cells, in conjunction with molecular modeling, identified RORα and RORγ as excellent receptor candidates for the hydroxylumisterols. Thus, we have discovered a new biologically relevant, lumisterogenic pathway, the metabolites of which display biological activity. This opens a new area of endocrine research on the effects of the hydroxylumisterols on different pathways in different cells and the mechanisms involved.

RORα and RORγ are expressed in human skin cells that produce the noncalcemic 20-hydroxyvitamin D3 [20(OH)D3] and 20,23-dihydroxyvitamin D3 [20,23(OH)2D3]. Chinese hamster ovary (CHO) cells stably expressing a Tet-on RORα or RORγ expression vector and a ROR-responsive element (RORE)-LUC reporter, and a mammalian 2-hybrid model examining the interaction between the ligand binding domain (LBD) of RORα or RORγ with an LBD-interacting LXXLL-peptide, were used to study ROR-antagonist activities. These assays revealed that 20(OH)D3 and 20,23(OH)2D3 function as antagonists of RORα and RORγ. Moreover, 20(OH)D3 inhibited the activation of the promoter of the Bmal1 and G6pase genes, targets of RORα, and 20(OH)D3 and 20,23(OH)2D3 inhibited Il17 promoter activity in Jurkat cells overexpressing RORα or RORγ. Molecular modeling using crystal structures of the LBDs of RORα and RORγ revealed docking scores for 20(OH)D3, 20,23(OH)2D3 and 1,25(OH)2D3 similar to those of the natural ligands, predicting good binding to the receptor. Notably, 20(OH)D3, 20,23(OH)2D3, and 1,25(OH)2D3 inhibited RORE-mediated activation of a reporter in keratinocytes and melanoma cells and inhibited IL-17 production by immune cells. Our study identifies a novel signaling pathway, in which 20(OH)D3 and 20,23(OH)2D3 act as antagonists or inverse agonists of RORα and RORγ, that opens new possibilities for local (skin) or systemic regulation.—Slominski, A. T., Kim, T.-K., Takeda, Y., Janjetovic, Z., Broz˙yna, A. A., Skobowiat, C., Wang, J., Postlethwaite, A., Li, W., Tuckey, R. C., Jetten, A. M. RORα and ROR γ are expressed in human skin and serve as receptors for endogenously produced noncalcemic 20-hydroxy- and 20,23-dihydroxyvitamin D.

Indolic and kynuric pathways of skin melatonin metabolism were monitored by liquid chromatography mass spectrometry in human keratinocytes, melanocytes, dermal fibroblasts, and melanoma cells. Production of 6-hydroxymelatonin [6(OH)M], N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and 5-methoxytryptamine (5-MT) was detected in a cell type-dependent fashion. The major metabolites, 6(OH)M and AFMK, were produced in all cells. Thus, in immortalized epidermal (HaCaT) keratinocytes, 6(OH)M was the major product with Vmax = 63.7 ng/106 cells and Km = 10.2 μM, with lower production of AFMK and 5-MT. Melanocytes, keratinocytes, and fibroblasts transformed melatonin primarily into 6(OH)M and AFMK. In melanoma cells, 6(OH)M and AFMK were produced endogenously, a process accelerated by exogenous melatonin in the case of AFMK. In addition, N-acetylserotonin was endogenously produced by normal and malignant melanocytes. Metabolites showed selective antiproliferative effects on human primary epidermal keratinocytes in vitro. In ex vivo human skin, both melatonin and AFMK-stimulated expression of involucrin and keratins-10 and keratins-14 in the epidermis, indicating their stimulatory role in building and maintaining the epidermal barrier. In summary, the metabolism of melatonin and its endogenous production is cell type-dependent and expressed in all three main cell populations of human skin. Furthermore, melatonin and its metabolite AFMK stimulate differentiation in human epidermis, indicating their key role in building the skin barrier.—Kim, T.-K., Kleszczyński, K., Janjetovic, Z., Sweatman, T., Lin, Z., Li, W., Reiter, R. J., Fischer, T. W., Slominski, A. T. Metabolism of melatonin and biological activity of intermediates of melatoninergic pathway in human skin cells.

Vitamin D3 (D3) can be metabolized by cytochrome P450scc (CYP11A1) into 20S-hydroxyvitamin D3 (20D3) as a major metabolite. This bioactive metabolite has shown strong antiproliferative, antifibrotic, pro-differentiation and anti-inflammatory effects while being non-toxic (non-calcemic) at high concentrations. Since D3 analogs with two symmetric side chains (Gemini analogs) result in potent activation of the vitamin D receptor (VDR), we hypothesized that the chain length and composition of these types of analogs also containing a 20-hydroxyl group would affect their biological activities. In this study, we designed and synthesized a series of Gemini 20D3 analogs. Biological tests showed that some of these analogs are partial VDR activators and can significantly stimulate the expression of mRNA for VDR and VDR-regulated genes including CYP24A1 and transient receptor potential cation channel V6 (TRPV6). These analogs inhibited the proliferation of melanoma cells with potency comparable to that of 1α,25-dihydroxyvitamin D3. Moreover, these analogs reduced the level of interferon γ and up-regulated the expression of leukocyte associated immunoglobulin-like receptor 1 in splenocytes, indicating that they have potent anti-inflammatory activities. There are no clear correlations between the Gemini chain length and their VDR activation or biological activities, consistent with the high flexibility of the ligand-binding pocket of the VDR.

We define previously unrecognized in vivo pathways of vitamin D3 (D3) metabolism generating novel D3-hydroxyderivatives different from 25-hydroxyvitamin D3 [25(OH)D3] and 1,25(OH)2D3. Their novel products include 20-hydroxyvitamin D3 [20(OH)D3], 22(OH)D3, 20,23(OH)2D3, 20,22(OH)2D3, 1,20(OH)2D3, 1,20,23(OH)3D3, and 17,20,23(OH)3D3 and were produced by placenta, adrenal glands, and epidermal keratinocytes. We detected the predominant metabolite [20(OH)D3] in human serum with a relative concentration ∼20 times lower than 25(OH)D3. Use of inhibitors and studies performed with isolated mitochondria and purified enzymes demonstrated involvement of the steroidogenic enzyme cytochrome P450scc (CYP11A1) as well as CYP27B1 (1α-hydroxylase). In placenta and adrenal glands with high CYP11A1 expression, the predominant pathway was D3 → 20(OH)D3 → 20,23(OH)2D3 → 17,20,23(OH)3D3 with further 1α-hydroxylation, and minor pathways were D3 → 25(OH)D3 → 1,25(OH)2D3 and D3 → 22(OH)D3 → 20,22(OH)2D3. In epidermal keratinocytes, we observed higher proportions of 22(OH)D3 and 20,22(OH)2D3. We also detected endogenous production of 20(OH)D3, 22(OH) D3, 20,23(OH)2D3, 20,22(OH)2D3, and 17,20,23(OH)3D3 by immortalized human keratinocytes. Thus, we provide in vivo evidence for novel pathways of D3 metabolism initiated by CYP11A1, with the product profile showing organ/cell type specificity and being modified by CYP27B1 activity. These findings define the pathway intermediates as natural products/endogenous bioregulators and break the current dogma that vitamin D is solely activated through the sequence D3 → 25(OH)D3 → 1,25(OH)2D3.—Slominski, A. T., Kim, T.-K., Shehabi, H. Z., Semak, I., Tang, E. K. Y., Nguyen, M. N., Benson, H. A. E., Korik, E., Janjetovic, Z., Chen, J., Yates, C. R., Postlethwaite, A., Li, W., Tuckey, R. C. In vivo evidence for a novel pathway of vitamin D3 metabolism initiated by P450scc and modified by CYP27B1.

The new steroidal 5,7-diene, 3beta-hydroxyandrosta-5,7-diene-17beta-carboxylic acid (17-COOH-7DA), was synthesized from 21-acetoxypregnenolone, with the oxidative cleavage of the side chain being dependent on the presence of oxygen. In human epidermal (HaCaT) keratinocytes, 17-COOH-7DA inhibited proliferation in a dose-dependent manner, starting at a dose as low as 10(-11) M. This inhibition was accompanied by decreased expression of epidermal growth factor receptor, bcl2 and cyclin E2 mRNAs and by increased expression of involucrin mRNA. Inhibition of proliferation was associated with slowing of the cell cycle in G1/G0 phases but not with cell death. 17-COOH-7DA was significantly more potent than pregnenolone, 17-COOH-pregnenolone, 17-COOCH(3)-7DA and calcitriol. 17-COOH-7DA also inhibited proliferation of normal human epidermal melanocytes and human and hamster melanoma lines, however, with lower potency than for keratinocytes. In normal human dermal fibroblasts 17-COOH-7DA stimulated proliferation in serum-free media but inhibited it in the presence of 5% serum. 17-COOH-7DA inhibited cell colony formation of human and hamster melanoma cells, and induced monocyte-like differentiation of human HL60 leukemia cells. Thus, the new steroidal 5,7-diene, 17-COOH-7DA, can serve as an inhibitor of proliferation of normal keratinocytes and normal and malignant melanocytes, as a condition-dependent regulator of fibroblast proliferation and a stimulator of leukemia cell differentiation.

Previously, we demonstrated that skin cells metabolize melatonin to 6-hydroxymelatonin, N1-acetyl-N2-formyl-5-methoxykynuramine and 5-methoxytryptamine. In this study, we determined that N1-acetyl-5-methoxykynuramine (AMK) is endogenously produced in the human epidermis from melatonin through the kynuric pathway. The epidermal content of AMK (average from 13 subjects) is 0.99 ± 0.21 ng/mg protein, being significantly higher in African Americans (1.50 ± 0.36 ng/mg protein) than in Caucasians (0.56 ± 0.09 ng/mg protein). It is especially high in young African Americans. The levels do not differ significantly between males and females. In vitro testing using HaCaT keratinocytes has shown that exogenously added melatonin is metabolized to AMK in a dose dependent manner with a Vmax = 388 pg/million cells and Km = 185 μM. AMK production is higher in melanized than in amelanotic melanoma cells. Testing of DNA incorporation shows that AMK has antiproliferative effects in HaCaT and SKMEL-188 cells (nonpigmented and pigmented). AMK also inhibits growth of normal melanocytes but has no significant effect on melanogenesis or cell morphology. These findings indicate that antiproliferative effects of AMK are not related to melanin pigmentation. In summary, we show for the first time that AMK is produced endogenously in the human epidermis, that its production is affected by melanin skin pigmentation, and that AMK exhibits antiproliferative effects in cultured keratinocytes and melanoma cells.

The human skin is not only a target for the protective actions of melatonin, but also a site of melatonin synthesis and metabolism, suggesting an important role for a local melatoninergic system in protection against ultraviolet radiation (UVR) induced damages. While melatonin exerts many effects on cell physiology and tissue homeostasis via membrane bound melatonin receptors, the strong protective effects of melatonin against the UVR-induced skin damage including DNA repair/protection seen at its high (pharmocological) concentrations indicate that these are mainly mediated through receptor-independent mechanisms or perhaps through activation of putative melatonin nuclear receptors. The destructive effects of the UVR are significantly counteracted or modulated by melatonin in the context of a complex intracutaneous melatoninergic anti-oxidative system with UVR-enhanced or UVR-independent melatonin metabolites. Therefore, endogenous intracutaneous melatonin production, together with topically-applied exogenous melatonin or metabolites would be expected to represent one of the most potent anti-oxidative defense systems against the UV-induced damage to the skin. In summary, we propose that melatonin can be exploited therapeutically as a protective agent or as a survival factor with anti-genotoxic properties or as a “guardian” of the genome and cellular integrity with clinical applications in UVR-induced pathology that includes carcinogenesis and skin aging.

We investigated the protective effects of melatonin and its metabolites: 6-hydroxymelatonin (6-OHM), N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), N-acetylserotonin (NAS), and 5-methoxytryptamine (5-MT) in human keratinocytes against a range of doses (25, 50, and 75 mJ/cm2) of ultraviolet B (UVB) radiation. There was significant reduction in the generation of reactive oxygen species (50-60%) when UVB-exposed keratinocytes were treated with melatonin or its derivatives. Similarly, melatonin and its metabolites reduced the nitrite and hydrogen peroxide levels that were induced by UVB as early as 30 min after the exposure. Moreover, melatonin and its metabolites enhanced levels of reduced glutathione in keratinocytes within 1 hr after UVB exposure in comparison with control cells. Using proliferation assay, we observed a dose-dependent increase in viability of UVB-irradiated keratinocytes that were treated with melatonin or its derivatives after 48 hr. Using the dot-blot technique and immunofluorescent staining we also observed that melatonin and its metabolites enhanced the DNA repair capacity of UVB-induced pyrimidine photoproducts (6-4)or cyclobutane pyrimidine dimers generation in human keratinocytes. Additional evidence for induction of DNA repair in cells exposed to UVB and treated with the indole compounds was shown using the Comet assay. Finally, melatonin and its metabolites further enhanced expression of p53 phosphorylated at Ser-15 but not at Ser-46 or its nonphosphorylated form. In conclusion, melatonin, its precursor NAS, and its metabolites 6-OHM, AFMK, 5-MT, which are endogenously produced in keratinocytes, protect these cells against UVB-induced oxidative stress and DNA damage.

We investigated the metabolism of vitamin D2 to hydroxyvitamin D2 metabolites ((OH)D2) by human placentas ex-utero, adrenal glands ex-vivo and cultured human epidermal keratinocytes and colonic Caco-2 cells, and identified 20(OH)D2, 17,20(OH)2D2, 1,20(OH)2D2, 25(OH)D2 and 1,25(OH)2D2 as products. Inhibition of product formation by 22R-hydroxycholesterol indicated involvement of CYP11A1 in 20- and 17-hydroxylation of vitamin D2, while use of ketoconazole indicated involvement of CYP27B1 in 1α-hydroxylation of products. Studies with purified human CYP11A1 confirmed the ability of this enzyme to convert vitamin D2 to 20(OH)D2 and 17,20(OH)2D2. In placentas and Caco-2 cells, production of 20(OH)D2 was higher than 25(OH)D2 while in human keratinocytes the production of 20(OH)D2 and 25(OH)D2 were comparable. HaCaT keratinocytes showed high accumulation of 1,20(OH)2D2 relative to 20(OH)D2 indicating substantial CYP27B1 activity. This is the first in vivo evidence for a novel pathway of vitamin D2 metabolism initiated by CYP11A1 and modified by CYP27B1, with the product profile showing tissue- and cell-type specificity.

Novel metabolic pathways initiated by the enzymatic action of CYP11A1 on 7DHC (7-dehydrocholesterol), ergosterol, vitamins D3 and D2 were characterized with help of chemical synthesis, UV and mass spectrometry and NMR analyses. The first pathway follows the sequence 7DHC→22(OH)7DHC → 20,22(OH)27DHC → 7DHP (7-dehydropregnenolone), which can further be metabolized by steroidogenic enzymes. The resulting 5,7-dienes can be transformed by UVB to corresponding, biologically active, secosteroids. Action of CYP11A1 on vitamin D3 and D2 produces novel hydroxyderivatives with OH added at positions C17, C20, C22, C23 and C24, some of which can be hydroxylated by CYP27B1 and/or by CYP27A1 and/ or by CYP24A1.The main products of these pathways are biologically active with a potency related to their chemical structure and the target cell type. Main products of CYP11A1-mediated metabolism on vitamin D are non-calcemic and non-toxic at relatively high doses and serve as partial agonists on the vitamin D receptor. New secosteroids are excellent candidates for therapy of fibrosing, inflammatory or hyperproliferative disorders including cancers and psoriasis.

To define the interaction of novel secosteroids produced by the action of cytochrome P450scc with vitamin D receptor (VDR), we used a human melanoma line overexpressing VDR fused with enhanced green fluorescent protein (EGFP) and tested the ligand induced translocation of VDR from the cytoplasm to the nucleus. Hydroxyderivatives of vitamin D(3) with a full length (D(3)) side chain and hydroxy-secosteroids with a shortened side chain (pD) stimulated VDR translocation and inhibited proliferation, however, with different potencies. In general the D(3) were more potent than pD analogues. Molecular modeling of the binding of the secosteroids to the VDR genomic binding pocket (G-pocket) correlated well with the experimental data for VDR translocation. In contrast, docking scores for the non-genomic binding site of the VDR were poor. In conclusion, both the length of the side chain and the number and position of hydroxyl groups affect the activation of VDR by novel secosteroids.

The discovery that 7-dehydrocholesterol (7DHC) is an excellent substrate for cytochrome P450scc (CYP11A1) opens up new possibilities in biochemistry. To elucidate its biological significance we tested ex vivo P450scc-dependent metabolism of 7DHC by tissues expressing high and low levels of P450scc activity, placenta and epidermal keratinocytes, respectively. Incubation of human placenta fragments with 7DHC led to its conversion to 7-dehydropregnenolone (7DHP), which was inhibited by dl-aminoglutethimide, and stimulated by forskolin. Final proof for P450scc involvement was provided in isolated placental mitochondria where production of 7DHP was almost completely inhibited by 22R-hydroxycholesterol. 7DHC was metabolized by placental mitochondria at a faster rate than exogenous cholesterol, under both limiting and saturating conditions of substrate transport, consistent with higher catalytic efficiency (k(cat)/K(m)) with 7DHC as substrate than with cholesterol. Ex vivo experiments showed five 5,7-dienal intermediates with MS spectra of dihydroxy and mono-hydroxy-7DHC and retention time corresponding to 20,22(OH)(2)7DHC and 22(OH)7DHC. The chemical structure of 20,22(OH)(2)7DHC was defined by NMR. 7DHP was further metabolized by either placental fragments or placental microsomes to 7-dehydroprogesterone as defined by UV, MS and NMR, and to an additional product with a 5,7-dienal structure and MS corresponding to hydroxy-7DHP. Furthermore, epidermal keratinocytes transformed either exogenous or endogenous 7DHC to 7DHP. 7DHP inhibited keratinocytes proliferation, while the product of its pholytic transformation, pregcalciferol, lost this capability. In conclusion, tissues expressing P450scc can metabolize 7DHC to biologically active 7DHP with 22(OH)7DHC and 20,22(OH)(2)7DHC serving as intermediates, and with further metabolism to 7-dehydroprogesterone and (OH)7DHP.

Cytochrome P450scc (CYP11A1) can hydroxylate vitamin D(3), producing 20S-hydroxyvitamin D(3) [20(OH)D(3)] and 20S,23-dihydroxyvitamin D(3) [20,23(OH)(2)D(3)] as the major metabolites. These are biologically active, acting as partial vitamin D receptor (VDR) agonists. Minor products include 17-hydroxyvitamin D(3), 17,20-dihydroxyvitamin D(3), and 17,20,23-trihydroxyvitamin D(3). In the current study, we have further analyzed the reaction products from cytochrome P450scc (P450scc) action on vitamin D(3) and have identified two 22-hydroxy derivatives as products, 22-hydroxyvitamin D(3) [22(OH)D(3)] and 20S,22-dihydroxyvitamin D(3) [20,22(OH)(2)D(3)]. The structures of both of these derivatives were determined by NMR. P450scc could convert purified 22(OH)D(3) to 20,22(OH)(2)D(3). The 20,22(OH)(2)D(3) could also be produced from 20(OH)D(3) and was metabolized to a trihydroxyvitamin D(3) product. We compared the biological activities of these new derivatives with those of 20(OH)D(3), 20,23(OH)(2)D(3), and 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. 1,25(OH)(2)D(3), 20(OH)D(3), 22(OH)D(3), 20,23(OH)(2)D(3), and 20,22(OH)(2)D(3) significantly inhibited keratinocyte proliferation in a dose-dependent manner. The strongest inducers of involucrin expression (a marker of keratinocyte differentiation) were 20,23(OH)(2)D(3), 20,22(OH)(2)D(3), 20(OH)D(3), and 1,25(OH)(2)D(3), with 22(OH)D(3) having a heterogeneous effect. Little or no stimulation of CYP24 mRNA expression was observed for all the analogs tested except for 1,25(OH)(2)D(3). All the compounds stimulated VDR translocation from the cytoplasm to the nucleus with 22(OH)D(3) and 20,22(OH)(2)D(3) having less effect than 1,25(OH)(2)D(3) and 20(OH)D(3). Thus, we have identified 22(OH)D(3) and 20,22(OH)(2)D(3) as products of CYP11A1 action on vitamin D(3) and shown that, like 20(OH)D(3) and 20,23(OH)(2)D(3), they are active on keratinocytes via the VDR, however, showing a degree of phenotypic heterogeneity in comparison with other P450scc-derived hydroxy metabolites of vitamin D(3).

20-hydroxyvitamin D2[20(OH)D2] inhibits DNA synthesis in epidermal keratinocytes, melanocytes, and melanoma cells in a dose- and time-dependent manner. This inhibition is dependent on cell type, with keratinocytes and melanoma cells being more sensitive than normal melanocytes. The antiproliferative activity of 20(OH)D2is similar to that of 1,25(OH)2D3and of newly synthesized 1,20(OH)2D2but significantly higher than that of 25(OH)D3. 20(OH)D2also displays tumorostatic effects. In keratinocytes 20(OH)D2inhibits expression of cyclins and stimulates involucrin expression. It also stimulates CYP24 expression, however, to a significantly lower degree than that by 1,25(OH)2D3or 25(OH)D3. 20(OH)D2is a poor substrate for CYP27B1 with overall catalytic efficiency being 24- and 41-fold lower than for 25(OH)D3with the mouse and human enzymes, respectively. No conversion of 20(OH)D2to 1,20(OH)2D2was detected in intact HaCaT keratinocytes. 20(OH)D2also demonstrates anti-leukemic activity but with lower potency than 1,25(OH)2D3. The phenotypic effects of 20(OH)D2are mediated through interaction with the vitamin D receptor (VDR) as documented by attenuation of cell proliferation after silencing of VDR, by enhancement of the inhibitory effect through stable overexpression of VDR and by the demonstration that 20(OH)D2induces time-dependent translocation of VDR from the cytoplasm to the nucleus at a comparable rate to that for 1,25(OH)2D3. In vivo tests show that while 1,25(OH)2D3at doses as low as 0.8 μg/kg induces calcium deposits in the kidney and heart, 20(OH)D2is devoid of such activity even at doses as high as 4 μg/kg. Silencing of CY27B1 in human keratinocytes showed that 20(OH)D2does not require its transformation to 1,20(OH)2D2for its biological activity. Thus 20(OH)D2shows cell-type dependent antiproliferative and prodifferentiation activities through activation of VDR, while having no detectable toxic calcemic activity, and is a poor substrate for CYP27B1.

20S-hydroxyvitamin D3 (20S-(OH)D3), an in vitro product of vitamin D3 metabolism by the cytochrome P450scc, was recently isolated, identified and shown to possess antiproliferative activity without inducing hypercalcemia. The enzymatic production of 20S-(OH)D3 is tedious, expensive, and cannot meet the requirements for extensive chemical and biological studies. Here we report for the first time the chemical synthesis of 20S-(OH)D3 which exhibited biological properties characteristic of the P450scc-generated compound. Specifically, it was hydroxylated to 20,23-dihydroxyvitamin D3 and 17,20-dihydroxyvitamin D3 by P450scc and was converted to 1alpha,20-dihydroxyvitamin D3 by CYP27B1. It inhibited proliferation of human epidermal keratinocytes with lower potency than 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) in normal epidermal human keratinocytes, but with equal potency in immortalized HaCaT keratinocytes. It also stimulated VDR gene expression with similar potency to 1,25(OH)2D3, and stimulated involucrin (a marker of differentiation) and CYP24 gene expression, showing a lower potency for the latter gene than 1,25(OH)2D3. Testing performed with hamster melanoma cells demonstrated a dose-dependent inhibition of cell proliferation and colony forming capabilities similar or more pronounced than those of 1,25(OH)2D3. Thus, we have developed a chemical method for the synthesis of 20S-(OH)D3, which will allow the preparation of a series of 20S-(OH)D3 analogs to study structure-activity relationships to further optimize this class of compound for therapeutic use.