Melanoma is one of the most lethal types of skin cancer, with a poor prognosis once the disease enters metastasis. The efficacy of currently available treatment schemes for advanced melanomas is low, ...expensive, and burdened by significant side-effects. Therefore, there is a need to develop new treatment options. Skin cells are able to activate vitamin D via classical and non-classical pathways. Vitamin D derivatives have anticancer properties which promote differentiation and inhibit proliferation. The role of systemic vitamin D in patients with melanoma is unclear as epidemiological studies are not definitive. In contrast, experimental data have clearly shown that vitamin D and its derivatives have anti-melanoma properties. Furthermore, molecular and clinicopathological studies have demonstrated a correlation between defects in vitamin D signaling and progression of melanoma and disease outcome. Therefore, adequate vitamin D signaling can play a role in the treatment of melanoma.
Human skin has the ability to synthesize glucocorticoids de novo from cholesterol or from steroid intermediates of systemic origin. By interacting with glucocorticoid receptors, they regulate skin ...immune functions as well as functions and phenotype of the epidermal, dermal and adnexal compartments. Most of the biochemical (enzyme and transporter activities) and regulatory (neuropeptides mediated activation of cAMP and protein kinase A dependent pathways) principles of steroidogenesis in the skin are similar to those operating in classical steroidogenic organs. However, there are also significant differences determined by the close proximity of synthesis and action (even within the same cells) allowing para‐, auto‐ or intracrine modes of regulation. We also propose that ultraviolet light B (UVB) can regulate the availability of 7‐dehydrocholesterol for transformation to cholesterol with its further metabolism to steroids, oxysterols or ∆7 steroids, because of its transformation to vitamin D3. In addition, UVB can rearrange locally produced ∆7 steroids to the corresponding secosteroids with a short‐ or no‐side chain. Thus, different mechanisms of regulation occur in the skin that can be either stochastic or structuralized. We propose that local glucocorticosteroidogenic systems and their regulators, in concert with cognate receptors operate to stabilize skin homeostasis and prevent or attenuate skin pathology.
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 α and γ (RORα/γ) 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 RORα/γ in non-irradiated cells, while the expression was significantly decreased in UVB-irradiated cells. In UVB-irradiated keratinocytes, L3-hydroxyderivatives inhibited nuclear translocation of NFκB p65 by enhancing levels of IκBα in the cytosol. This anti-inflammatory activity mediated by L3-hydroxyderivatives through suppression of NFκB signaling resulted in the inhibition of the expression of UVB-induced inflammatory cytokines, including IL-17, IFN-γ, and TNF-α. 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.
ABSTRACT
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., Brożyna, 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. FASEB J. 28, 2775–2789 (2014). www.fasebj.org
Clinical evaluation of vitamin D status is conventionally performed by measuring serum levels of a single vitamin D metabolite, 25-hydroxyvitamin D predominantly by immunoassay methodology. However, ...this neglects the complex metabolic pathways involved in vitamin D bioactivity, including two canonical forms D3 and D2, bioactive 1,25-dihydroxy metabolites and inactive 24-hydroxy and other metabolites.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can measure multiple analytes in a sample during a single run with high sensitivity and reference level specificity. We therefore aimed to develop and validate a LC-MS/MS method to measure simultaneously 13 circulating vitamin D metabolites and apply it to 103 human serum samples.
The LC-MS/MS method using a Cookson-type derivatization reagent phenyl-1,2,4-triazoline-3,5-dione (PTAD) quantifies 13 vitamin D metabolites, including mono and dihydroxy-metabolites, as well as CYP11A1-derived D3 and D2 metabolites in a single run. The lower limit of quantitation was 12.5 pg/mL for 1,25(OH)
D3 with accuracy verified by analysis of National Institute of Standards and Technology (NIST) 972a standards. Quantification of seven metabolites (25(OH)D3, 25(OH)D2, 3-epi-25(OH)D3, 20(OH)D3, 24,25(OH)
D3, 1,25(OH)
D3 and 1,20
(OH)
D3) was consistently achieved in human serum samples.
This profiling method can provide new insight into circulating vitamin D metabolite pathways forming the basis for improved understanding of the role of vitamin D in health and disease.
Melatonin (Mel) is the major biologically active molecule secreted by the pineal gland. Mel and its metabolites, 6-hydroxymelatonin (6(OH)Mel) and 5-methoxytryptamine (5-MT), possess a variety of ...functions, including the scavenging of free radicals and the induction of protective or reparative mechanisms in the cell. Their amphiphilic character allows them to cross cellular membranes and reach subcellular organelles, including the mitochondria. Herein, the action of Mel, 6(OH)Mel, and 5-MT in human MNT-1 melanoma cells against ultraviolet B (UVB) radiation was investigated. The dose of 50 mJ/cm² caused a significant reduction of cell viability up to 48%, while investigated compounds counteracted this deleterious effect. UVB exposure increased catalase activity and led to a simultaneous Ca
influx (16%), while tested compounds prevented these disturbances. Additional analysis focused on mitochondrial respiration performed in isolated mitochondria from the liver of BALB/cJ mice where Mel, 6(OH)Mel, and 5-MT significantly enhanced the oxidative phosphorylation at the dose of 10
M with lower effects seen at 10
or 10
M. In conclusion, Mel, 6(OH)Mel and 5-MT protect MNT-1 cells, which express melatonin receptors (MT1 and MT2) against UVB-induced oxidative stress and mitochondrial dysfunction, including the uncoupling of oxidative phosphorylation.
20(
)-Hydroxyvitamin D3 (20(OH)D3) is an endogenous metabolite produced by the action of CYP11A1 on the side chain of vitamin D3 (D3). 20(OH)D3 can be further hydroxylated by CYP11A1, CYP27A1, ...CYP24A1 and/or CYP27B1 to several hydroxyderivatives. CYP11A1 also hydroxylates D3 to 22-monohydroxyvitamin D3 (22(OH)D3), which is detectable in the epidermis. 20-Hydroxy-7-dehydrocholesterol (20(OH)-7DHC) has been detected in the human epidermis and can be phototransformed into 20(OH)D3 following the absorption of ultraviolet B (UVB) energy by the B-ring. 20(OH)D3 and its hydroxyderivatives have anti-inflammatory, pro-differentiation and anti-proliferative effects, comparable to 1,25-dihydroxyvitamin D3 (1,25(OH)
D3). Since cytochromes P450 with 20- or 25-hydroxylase activity are found in insects participating in ecdysone synthesis from 7-dehydrocholesterol (7DHC), we tested whether D3-hydroxyderivatives are present in honey, implying their production in bees. Honey was collected during summer in the Birmingham area of Alabama or purchased commercially and extracted and analyzed using LC-MS. We detected a clear peak of
/
= 423.324 M + Na
for 20(OH)D3 corresponding to a concentration in honey of 256 ng/g. We also detected peaks of
/
= 383.331 M + H - H
O
for 20(OH)-7DHC and 25(OH)D3 with retention times corresponding to the standards. We further detected species with
= 407.329 M + Na
corresponding to the RT of 7DHC, D3 and lumisterol3 (L3). Similarly, peaks with
= 399.326 M + H - H
O
were detected at the RT of 1,25(OH)
D3 and 1,20-dihydroxyvitamin D3 (1,20(OH)
D3). Species corresponding to 20-monohydroxylumisterol3 (20(OH)L3), 22-monohydroxyvitamin D3 (22(OH)D3), 20,23-dihydroxyvitamin D3 (20,23(OH)
D3), 20,24/25/26-dihydroxyvitamin D3 (20,24/25/26(OH)
D3) and 1,20,23/24/25/26-trihydroxyvitamin D3 (1,20,23/24/25/26(OH)
D3) were not detectable above the background. In conclusion, the presence of 7DHC and D3 and of species corresponding to 20(OH)-7DHC, 20(OH)D3, 1,20(OH)
D3, 25(OH)D3 and 1,25(OH)
D3 in honey implies their production in bees, although the precise biochemistry and photochemistry of these processes remain to be defined.
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