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Despite the recent development of improved methods of treating melanoma such as targeted therapy, immunotherapy or combined treatment, the number of new cases worldwide is increasing. It is well known that active metabolites of vitamin D 3 and lumisterol (L 3 ) exert photoprotective and antiproliferative effects on the skin, while UV radiation is a major environmental risk factor for melanoma. Thus, many natural metabolites and synthetic analogs of steroidal and secosteroidal molecules have been tested on various cancer cells and in animal models. In this study, we tested the anti-melanoma properties of several natural derivatives of vitamin D 3 and L 3 in comparison to 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ). A significant decrease in melanoma cell proliferation and cell mobility was observed for selected derivatives, with (25R)-27-hydroxyL 3 showing the highest potency (lowest IC50) in A375 cells but lower potency in SK-MEL-28 cells, whereas the parent L 3 failed to inhibit proliferation. The efficacy (% inhibition) by 1,24,25(OH) 3 D 3 and 1,25(OH) 2 D 3 were similar in both cell types. 1,25(OH) 2 D 3 showed higher potency than 1,24,25(OH) 3 D 3 in SK-MEL-28 cells, but lower potency in A375 cells for the inhibition of proliferation. As for 1,25(OH) 2 D 3 , but not the other derivatives tested, treatment of melanoma cells with 1,24,25(OH) 3 D 3 markedly increased the expression of CYP24A1, enhanced translocation of the vitamin D receptor (VDR) from the cytoplasm to the nucleus and also decreased the expression of the proliferation marker Ki67. The effects of the other compounds tested were weaker and occurred only under certain conditions. Our data indicate that 1,24,25(OH) 3 D 3 , which has undergone the first step in 1,25(OH) 2 D 3 inactivation by being hydroxylated at C24, still shows anti-melanoma properties, displaying higher potency than 1,25(OH) 2 D 3 in SK-MEL-28 cells. Furthermore, hydroxylation increases the potency of some of the lumisterol hydroxy-derivatives, as in contrast to L 3 , (25 R )-27(OH)L 3 effectively inhibits proliferation and migration of the human malignant melanoma cell line A375.

Melatonin, a product of tryptophan metabolism via serotonin, is a molecule with an indole backbone that is widely produced by bacteria, unicellular eukaryotic organisms, plants, fungi and all animal taxa. Aside from its role in the regulation of circadian rhythms, it has diverse biological actions including regulation of cytoprotective responses and other functions crucial for survival across different species. The latter properties are also shared by its metabolites including kynuric products generated by reactive oxygen species or phototransfomation induced by ultraviolet radiation. Vitamins D and related photoproducts originate from phototransformation of ∆5,7 sterols, of which 7-dehydrocholesterol and ergosterol are examples. Their ∆5,7 bonds in the B ring absorb solar ultraviolet radiation [290-315 nm, ultraviolet B (UVB) radiation] resulting in B ring opening to produce previtamin D, also referred to as a secosteroid. Once formed, previtamin D can either undergo thermal-induced isomerization to vitamin D or absorb UVB radiation to be transformed into photoproducts including lumisterol and tachysterol. Vitamin D, as well as the previtamin D photoproducts lumisterol and tachysterol, are hydroxylated by cyochrome P450 (CYP) enzymes to produce biologically active hydroxyderivatives. The best known of these is 1,25-dihydroxyvitamin D (1,25(OH) 2 D) for which the major function in vertebrates is regulation of calcium and phosphorus metabolism. Herein we review data on melatonin production and metabolism and discuss their functions in insects. We discuss production of previtamin D and vitamin D, and their photoproducts in fungi, plants and insects, as well as mechanisms for their enzymatic activation and suggest possible biological functions for them in these groups of organisms. For the detection of these secosteroids and their precursors and photoderivatives, as well as melatonin metabolites, we focus on honey produced by bees and on body extracts of Drosophila melanogaster. Common biological functions for melatonin derivatives and secosteroids such as cytoprotective and photoprotective actions in insects are discussed. We provide hypotheses for the photoproduction of other secosteroids and of kynuric metabolites of melatonin, based on the known photobiology of ∆5,7 sterols and of the indole ring, respectively. We also offer possible mechanisms of actions for these unique molecules and summarise differences and similarities of melatoninergic and secosteroidogenic pathways in diverse organisms including insects., (© 2024 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.)

Novel pathways of vitamin D3, lumisterol 3 (L3), and tachysterol 3 (T3) activation have been discovered, initiated by CYP11A1 and/or CYP27A1 in the case of L3 and T3. The resulting hydroxymetabolites enhance protection of skin against DNA damage and oxidative stress; stimulate keratinocyte differentiation; exert anti-inflammatory, antifibrogenic, and anticancer activities; and inhibit cell proliferation in a structure-dependent manner. They act on nuclear receptors, including vitamin D receptor, aryl hydrocarbon receptor, LXRα/β, RAR-related orphan receptor α/γ, and peroxisome proliferator-activated receptor-γ, with selectivity defined by their core structure and distribution of hydroxyl groups. They can activate NRF2 and p53 and inhibit NF-κB, IL-17, Shh, and Wnt/β-catenin signaling. Thus, they protect skin integrity and physiology., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Melanoma, originating through malignant transformation of melanin-producing melanocytes, is a formidable malignancy, characterized by local invasiveness, recurrence, early metastasis, resistance to therapy, and a high mortality rate. This review discusses etiologic and risk factors for melanoma, diagnostic and prognostic tools, including recent advances in molecular biology, omics, and bioinformatics, and provides an overview of its therapy. Since the incidence of melanoma is rising and mortality remains unacceptably high, we discuss its inherent properties, including melanogenesis, that make this disease resilient to treatment and propose to use AI to solve the above complex and multidimensional problems. We provide an overview on vitamin D and its anticancerogenic properties, and report recent advances in this field that can provide solutions for the prevention and/or therapy of melanoma. Experimental papers and clinicopathological studies on the role of vitamin D status and signaling pathways initiated by its active metabolites in melanoma prognosis and therapy are reviewed. We conclude that vitamin D signaling, defined by specific nuclear receptors and selective activation by specific vitamin D hydroxyderivatives, can provide a benefit for new or existing therapeutic approaches. We propose to target vitamin D signaling with the use of computational biology and AI tools to provide a solution to the melanoma problem.

We investigated multiple signaling pathways activated by CYP11A1-derived vitamin D3 hydroxymetabolites in human skin fibroblasts by assessing the actions of these molecules on their cognate receptors and by investigating the role of CYP27B1 in their biological activities. The actions of 20(OH)D3, 20,23(OH) 2 D3, 1,20(OH) 2 D3 and 1,20,23(OH) 3 D3 were compared to those of classical 1,25(OH) 2 D3. This was undertaken using wild type (WT) fibroblasts, as well as cells with VDR , RORs , or CYP27B1 genes knocked down with siRNA. Vitamin D3 hydroxymetabolites had an inhibitory effect on the proliferation of WT cells, but this effect was abrogated in cells with silenced VDR or RORs. The collagen expression by WT cells was reduced upon secosteroid treatment. This effect was reversed in cells where VDR or RORs were knocked down where the inhibition of collagen production and the expression of anti-fibrotic genes in response to the hydroxymetabolites was abrogated, along with ablation of their anti-inflammatory action. The knockdown of CYP27B 1 did not change the effect of either 20(OH)D3 or 20,23(OH) 2 D3, indicating that their actions are independent of 1α-hydroxylation. In conclusion, the expression of the VDR and/or RORα/γ receptors in fibroblasts is necessary for the inhibition of both the proliferation and fibrogenic activity of hydroxymetabolites of vitamin D3, while CYP27B1 is not required.

Lumisterol 2 (L2) is a photoproduct of UVB action on the fungal membrane sterol, ergosterol. Like vitamin D 2 , it is present in edible mushrooms, especially after UV irradiation. Lumisterol 3 is similarly produced in human skin from 7-dehydrocholesterol by UVB and can be converted to hydroxy-metabolites by CYP27A1 and CYP11A1. These products are biologically active on human cells with actions that include photoprotection and inhibition of proliferation. The aim of this study was to test the ability of CYP11A1 and CYP27A1 to metabolise L2. Purified CYP27A1 was found to efficiently metabolise L2 to three major products and several minor products, whilst CYP11A1 did not act appreciably on L2. The three major products of CYP27A1 action on L2 were identified by mass spectrometry and NMR as 24-hydroxyL2, 27-hydroxyL2 and 28-hydroxyL2. Minor products included two dihydroxy L2 species, one which was identified as 24,27(OH) 2 L2, and another metabolite with one oxo and one hydroxyl group added. A comparison on the kinetics of the metabolism of L2 by CYP27A1 with that of the structurally similar compounds, L3 and ergosterol, was carried out with substrates incorporated into phospholipid vesicles. CYP27A1 displayed a 12-fold lower K m with L2 as substrate compared to L3 and a 5-fold lower turnover number (k cat ), resulting in a 2.2 fold higher catalytic efficiency (k cat /K m ) for L2 metabolism. L2 was a much better substrate for CYP27A1 than its precursor, ergosterol, with a catalytic efficiency 18-fold higher. The major CYP27A1-derived hydroxy-L2 products, 24-hydroxyL2, 27-hydroxyL2 and 28-hydroxyL2, inhibited the proliferation of melanoma and epidermoid cancer cell lines. In conclusion, this study shows that L2 is not metabolized appreciably by CYP11A1, but it is a good substrate for CYP27A1 which hydroxylates its side chain to produce 3 major products that display anti-proliferative activity on skin-cancer cell lines., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Vitamin D is found in two forms in humans, D3 produced in the skin and D2 solely from the diet. Both 25-hydroxyvitamin D (25(OH)D) and 1,25-dihydroxyvitamin D (1,25(OH) 2 D) are oxidised and inactivated by CYP24A1, a tightly regulated mitochondrial enzyme that controls serum levels of these secosteroids. The pathways of oxidation of 25(OH)D2 and 1,25(OH) 2 D2, particularly 25(OH)D2, by human CYP24A1 are not well characterized. The aim of this study was to further elucidate these pathways, and to compare the kinetics of metabolism of 25(OH)D2 and 1,25(OH) 2 D2 with their vitamin D3 counterparts. We used expressed and partially purified human CYP24A1 with substrates dissolved in the membrane of phospholipid vesicles, to mimic the inner mitochondrial membrane. We found that the major pathways for side chain oxidation of 25(OH)D2 and 1,25(OH) 2 D2 were identical and that predominant intermediates of 25(OH)D2 metabolism could be converted to the corresponding intermediates in the pathway of 1,25(OH) 2 D2 oxidation by 1α-hydroxylation by CYP27B1. The initial steps in the CYP24A1-mediated oxidation involved hydroxylation at the C24R position, and another unknown position where the alcohol was oxidised to an aldehyde. The 24R-hydroxylation was followed by hydroxylation at C26 or C28, or cleavage between C24 and C25 to produce the 24-oxo-25,26,27-trinor derivative. All of these products were further oxidised, with 24-oxo-25,26,27-trinor-1(OH)D2 giving a product tentatively identified as 24-oxo-25,26,27-trinor-1,28(OH) 2 D2. The catalytic efficiency (k cat /K m ) of CYP24A1 for initial 25(OH)D2 hydroxylation was similar to that for 25(OH)D3, indicating that they have similar rates of inactivation at low substrate concentrations, supporting that vitamins D2 and D3 are equally effective in maintaining serum 25(OH)D concentrations. In contrast, the k cat /K m value for 1,25(OH) 2 D3 was almost double that for 1,25(OH) 2 D2 indicating a lower rate of inactivation of 1,25(OH) 2 D2 at a low substrate concentration, suggesting that it has increased metabolic stability in vivo., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The hormonal form of vitamin D 3 , 1,25(OH) 2 D 3 , reduces UV-induced DNA damage. UV exposure initiates pre-vitamin D 3 production in the skin, and continued UV exposure photoisomerizes pre-vitamin D 3 to produce "over-irradiation products" such as lumisterol 3 (L 3 ). Cytochrome P450 side-chain cleavage enzyme (CYP11A1) in skin catalyzes the conversion of L 3 to produce three main derivatives: 24-hydroxy-L 3 [24(OH)L 3 ], 22-hydroxy-L 3 [22(OH)L 3 ], and 20,22-dihydroxy-L 3 [20,22(OH)L 3 ]. The current study investigated the photoprotective properties of the major over-irradiation metabolite, 24(OH)L 3 , in human primary keratinocytes and human skin explants. The results indicated that treatment immediately after UV with either 24(OH)L 3 or 1,25(OH) 2 D 3 reduced UV-induced cyclobutane pyrimidine dimers and oxidative DNA damage, with similar concentration response curves in keratinocytes, although in skin explants, 1,25(OH) 2 D 3 was more potent. The reductions in DNA damage by both compounds were, at least in part, the result of increased DNA repair through increased energy availability via increased glycolysis, as well as increased DNA damage recognition proteins in the nucleotide excision repair pathway. Reductions in UV-induced DNA photolesions by either compound occurred in the presence of lower reactive oxygen species. The results indicated that under in vitro and ex vivo conditions, 24(OH)L 3 provided photoprotection against UV damage similar to that of 1,25(OH) 2 D 3 .

25-Hydroxyvitamin D3 (25(OH)D3) is present in the human circulation esterified to sulfate with some studies showing that 25(OH)D3 3-sulfate levels are almost as high as unconjugated 25(OH)D3. Vitamin D3 is also present in human serum in the sulfated form as are other metabolites. Our aim was to determine whether sulfated forms of vitamin D3 and vitamin D3 metabolites can be acted on by vitamin D-metabolizing cytochromes P450 (CYPs), one of which (CYP11A1) is known to act on cholesterol sulfate. We used purified, bacterially expressed CYPs to test if they could act on the sulfated forms of their natural substrates. Purified CYP27A1 converted vitamin D3 sulfate to 25(OH)D3 3-sulfate with a catalytic efficiency (k cat /K m ) approximately half that for the conversion of vitamin D3 to 25(OH)D3. Similarly, the rate of metabolism of vitamin D3 sulfate was half that of vitamin D3 for CYP27A1 in rat liver mitochondria. CYP2R1 which is also a vitamin D 25-hydroxylase did not act on vitamin D3 sulfate. CYP11A1 was able to convert vitamin D3 sulfate to 20(OH)D3 3-sulfate but at a considerably lower rate than for conversion of vitamin D3 to 20(OH)D3. 25(OH)D3 3-sulfate was not metabolized by the activating enzyme, CYP27B1, nor by the inactivating enzyme, CYP24A1. Thus, we conclude that 25(OH)D3 3-sulfate in the circulation may act as a pool of metabolically inactive vitamin D3 to be released by hydrolysis at times of need whereas vitamin D3 sulfate can be metabolized in a similar manner to free vitamin D3 by CYP27A1 and to a lesser degree by CYP11A1., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

CYP11A1 and CYP27A1 hydroxylate tachysterol 3 , a photoproduct of previtamin D 3 , producing 20S-hydroxytachysterol 3 [20S(OH)T 3 ] and 25(OH)T 3 , respectively. Both metabolites were detected in the human epidermis and serum. Tachysterol 3 was also detected in human serum at a concentration of 7.3 ± 2.5 ng/ml. 20S(OH)T 3 and 25(OH)T 3 inhibited the proliferation of epidermal keratinocytes and dermal fibroblasts and stimulated the expression of differentiation and anti-oxidative genes in keratinocytes in a similar manner to 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ]. They acted on the vitamin D receptor (VDR) as demonstrated by image flow cytometry and the translocation of VDR coupled GFP from the cytoplasm to the nucleus of melanoma cells, as well as by the stimulation of CYP24A1 expression. Functional studies using a human aryl hydrocarbon receptor (AhR) reporter assay system revealed marked activation of AhR by 20S(OH)T 3 , a smaller effect by 25(OH)T 3 , and a minimal effect for their precursor, tachysterol 3 . Tachysterol 3 hydroxyderivatives showed high-affinity binding to the ligan-binding domain (LBD) of the liver X receptor (LXR) α and β, and the peroxisome proliferator-activated receptor γ (PPARγ) in LanthaScreen TR-FRET coactivator assays. Molecular docking using crystal structures of the LBDs of VDR, AhR, LXRs, and PPARγ revealed high docking scores for 20S(OH)T 3 and 25(OH)T 3 , comparable to their natural ligands. The scores for the non-genomic-binding site of the VDR were very low indicating a lack of interaction with tachysterol 3 ligands. Our identification of endogenous production of 20S(OH)T 3 and 25(OH)T 3 that are biologically active and interact with VDR, AhR, LXRs, and PPARγ, provides a new understanding of the biological function of tachysterol 3 ., (© 2022 Federation of American Societies for Experimental Biology.)

Hydroxyderivatives of vitamin D3, including classical 1,25(OH) 2 D3 and novel CYP11A1‑derived hydroxyderivatives, exert their biological activity by acting as agonists on the vitamin D receptor (VDR) and inverse agonists on retinoid‑related orphan receptors (ROR)α and γ. The anticancer activities of CYP11A1‑derived hydroxyderivatives were tested using cell biology, tumor biology and molecular biology methods in human A431 and SCC13 squamous (SCC)‑ and murine ASZ001 basal (BCC)‑cell carcinomas, in comparison with classical 1,25(OH) 2 D3. Vitamin D3‑hydroxyderivatives with or without a C1α(OH) inhibited cell proliferation in a dose‑dependent manner. While all the compounds tested had similar effects on spheroid formation by A431 and SCC13 cells, those with a C1α(OH) group were more potent in inhibiting colony and spheroid formation in the BCC line. Potent anti‑tumorigenic activity against the BCC line was exerted by 1,25(OH) 2 D3, 1,20(OH) 2 D3, 1,20,23(OH) 3 D3, 1,20,24(OH) 3 D3, 1,20,25(OH) 3 D3 and 1,20,26(OH) 3 D3, with smaller effects seen for 25(OH)D3, 20(OH)D3 and 20,23(OH) 2 D3. 1,25(OH) 2 D3, 1,20(OH) 2 D3 and 20(OH)D3 inhibited the expression of GLI1 and β‑catenin in ASZ001 cells. In A431 cells, these compounds also decreased the expression of GLI1 and stimulated involucrin expression. VDR, RORγ, RORα and CYP27B1 were detected in A431, SCC13 and ASZ001 lines, however, with different expression patterns. Immunohistochemistry performed on human skin with SCC and BCC showed nuclear expression of all three of these receptors, as well as megalin (transmembrane receptor for vitamin D‑binding protein), the level of which was dependent on the type of cancer and antigen tested in comparison with normal epidermis. Classical and CYP11A1‑derived vitamin D3‑derivatives exhibited anticancer‑activities on skin cancer cell lines and inhibited GLI1 and β‑catenin signaling in a manner that was dependent on the position of hydroxyl groups. The observed expression of VDR, RORγ, RORα and megalin in human SCC and BCC suggested that they might provide targets for endogenously produced or exogenously applied vitamin D hydroxyderivatives and provide excellent candidates for anti‑cancer therapy.

To better understand the molecular and structural basis underlying the interaction of vitamin D3 hydroxyderivatives with AhR, molecular simulation was used to probe the binding of 1,20(OH) 2 D3, 1,25(OH) 2 D3, 20,23(OH) 2 D3 and 20(OH)D3 to AhR. qPCR showed that vitamin D3 derivatives stimulate expression of cyp1A1 and cyp1B1 genes that are downstream targets of AhR signaling. These secosteroids stimulated the translocation of the AhR to the nucleus, as measured by flow cytometry and western blotting. Molecular dynamics simulations were used to model the binding of vitamin D3 derivatives to AhR to examine their influence on the structure, conformation and dynamics of the AhR ligand binding domain (LBD). Binding thermodynamics, conformation, secondary structure, dynamical motion and electrostatic potential of AhR were analyzed. The molecular docking scores and binding free energy were all favorable for the binding of D3 derivatives to the AhR. These established ligands and the D3 derivatives are predicted to have different patterns of hydrogen bond formation with the AhR, and varied residue conformational fluctuations and dynamical motion for the LBD. These changes could alter the shape, size and electrostatic potential distribution of the ligand binding pocket, contributing to the different binding affinities of AhR for the natural ligands and D3 derivatives., (Copyright © 2022 Elsevier B.V. All rights reserved.)

New and more efficient routes of chemical synthesis of vitamin D 3 (D 3 ) hydroxy (OH) metabolites, including 20S(OH)D 3 , 20S,23S(OH) 2 D 3 and 20S,25(OH) 2 D 3 , that are endogenously produced in the human body by CYP11A1, and of 20S,23R(OH) 2 D 3 were established. The biological evaluation showed that these compounds exhibited similar properties to each other regarding inhibition of cell proliferation and induction of cell differentiation but with subtle and quantitative differences. They showed both overlapping and differential effects on T-cell immune activity. They also showed similar interactions with nuclear receptors with all secosteroids activating vitamin D, liver X, retinoic acid orphan and aryl hydrocarbon receptors in functional assays and also as indicated by molecular modeling. They functioned as substrates for CYP27B1 with enzymatic activity being the highest towards 20S,25(OH) 2 D 3 and the lowest towards 20S(OH)D 3 . In conclusion, defining new routes for large scale synthesis of endogenously produced D 3 -hydroxy derivatives by pathways initiated by CYP11A1 opens an exciting era to analyze their common and differential activities in vivo, particularly on the immune system and inflammatory diseases., (Copyright © 2022. Published by Elsevier Inc.)

We previously demonstrated that the non-calcemic pregnacalciferol (pD) analog 17,20S (OH) 2 pD suppressed TGF-β1-induced type I collagen production in cultured normal human dermal fibroblasts. In the present studies, we examined fibroblasts cultured from the lesional skin of patients with systemic sclerosis (scleroderma (SSc)) and assessed the effects of 17,20S(OH) 2 pD on fibrosis-related mediators. Dermal fibroblast lines were established from skin biopsies from patients with SSc and healthy controls. Fibroblasts were cultured with either 17,20S(OH) 2 pD or 1,25(OH) 2 D 3 (positive control) with/without TGF-β1 stimulation and extracted for protein and/or mRNA for collagen synthesis and mediators of fibrosis (MMP-1, TIMP-1, PAI-1, BMP-7, PGES, GLI1, and GLI2). 1 7,20S(OH) 2 pD (similar to 1,25(OH) 2 D 3 ) significantly suppressed net total collagen production in TGF-β1-stimulated normal donor fibroblast cultures and in cultures of SSc dermal fibroblasts. 17,20S(OH) 2 pD (similar to 1,25(OH) 2 D 3 ) also increased MMP-1, BMP-7, and PGES and decreased TIMP-1 and PAI1 expression in SSc fibroblasts. Although 17,20S(OH) 2 pD had no effect on Gli1 or Gli2 in SSc fibroblasts, it increased Gli2 expression when cultured with TGF-β1 in normal fibroblasts. These studies demonstrated that 17,20S(OH) 2 pD modulates mediators of fibrosis to favor the reduction of fibrosis and may offer new noncalcemic secosteroidal therapeutic approaches for treating SSc and fibrosis.

Vitamin D plays a crucial role in regulation of the immune response. However, treatment of autoimmune diseases with 1,25-dihydroxyvitamin D3 [1,25(OH) 2 D3] doses sufficient to be effective is prohibitive due to its calcemic and toxic effects. We use the collagen-induced arthritis (CIA) model to analyze the efficacy of the noncalcemic analog of vitamin D, 20 S -hydroxyvitamin D3 [20 S (OH)D3], as well as 1,25(OH) 2 D3, to attenuate arthritis and explore a potential mechanism of action. Mice fed a diet deficient in vitamin D developed a more severe arthritis characterized by enhanced secretion of T cell inflammatory cytokines, compared to mice fed a normal diet. The T cell inflammatory cytokines were effectively suppressed, however, by culture of the cells with 20 S (OH)D3. Interestingly, one of the consequences of culture with 1,25(OH) 2 D3 or 20 S (OH)D3, was upregulation of the natural inhibitory receptor leukocyte associated immunoglobulin-like receptor-1 (LAIR-1 or CD305). Polyclonal antibodies which activate LAIR-1 were also capable of attenuating arthritis. Moreover, oral therapy with active forms of vitamin D suppressed arthritis in LAIR-1 sufficient DR1 mice, but were ineffective in LAIR-1 -/- deficient mice. Taken together, these data show that the effect of vitamin D on inflammation is at least, in part, mediated by LAIR-1 and that non-calcemic 20 S (OH)D3 may be a promising therapeutic agent for the treatment of autoimmune diseases such as Rheumatoid Arthritis.

The vitamin D hormone, 1,25dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), and related compounds derived from vitamin D 3 or lumisterol as a result of metabolism via the enzyme CYP11A1, have been shown, when applied 24 hours before or immediately after UV irradiation, to protect human skin cells and skin from DNA damage due to UV exposure, by reducing both cyclobutane pyrimidine dimers (CPD) and oxidative damage in the form of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OHdG). We now report that knockdown of either the vitamin D receptor or the endoplasmic reticulum protein ERp57 by small, interfering RNA (siRNA) abolished the reductions in UV-induced DNA damage with 20-hydroxyvitamin D 3 or 24-hydroxylumisterol 3, as previously shown for 1,25(OH) 2 D 3 . Treatment with 1,25(OH) 2 D 3 reduced oxygen consumption rates in UV-exposed and sham-exposed human keratinocytes and reduced phosphorylation of cyclic AMP response binding element protein (CREB). Both these actions have been shown to inhibit skin carcinogenesis after chronic UV exposure, consistent with the anticarcinogenic activity of 1,25(OH) 2 D 3 . The requirement for a vitamin D receptor for the photoprotective actions of 1,25(OH) 2 D 3 and of naturally occurring CYP11A1-derived vitamin D-related compounds may explain why mice lacking the vitamin D receptor in skin are more susceptible to UV-induced skin cancers, whereas mice lacking the 1α-hydroxylase and thus unable to make 1,25(OH) 2 D 3 are not more susceptible. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research., Competing Interests: None., (© 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.)

7-Dehydrocholesterol reductase (DHCR7) catalyses the final step of cholesterol biosynthesis in the Kandutsch-Russel pathway, the reduction of 7-dehydrocholesterol (7DHC) to cholesterol. 7DHC can be acted on by a range of other enzymes including CYP27A1 and CYP11A1, as well as by UVB radiation, producing a number of derivatives including hydroxy-metabolites, some of which retain the C7-C8 double bond and are biologically active. These metabolites include lumisterol (L3) which is a stereoisomer of 7DHC produced in the skin by UVB radiation of 7DHC, as well as vitamin D3. The aim of this study was to test whether these metabolites could act as substrates or inhibitors of DHCR7 in rat liver microsomes. To initially screen the ability of these metabolites to interact with the active site of DHCR7, their ability to inhibit the conversion of ergosterol to brassicasterol was measured. Sterols that significantly inhibited this reaction included 7DHC (as expected), 20S(OH)7DHC, 27(OH)DHC, 8DHC, 20S(OH)L3 and 22(OH)L3 but not 7-dehydropregnenolone (7DHP), 25(OH)7DHC, L3 or vitamin D3 and its hydroxyderivatives. Sterols that inhibited ergosterol reduction were directly tested as substrates for DHCR7. 20S(OH)7DHC, 27(OH)DHC and 7-dehydrodesmosterol were confirmed to be substrates, giving the expected product with the C7-C8 double bond removed. No products were observed from 8DHC or 20S(OH)L3 indicating that these sterols are inhibitors and not substrates of DHCR7. The resistance of lumisterol and 7DHP to reduction by DHCR7 in cells will permit other enzymes to metabolise these sterols to their active forms retaining the C7-C8 double bond, conferring specificity to their biological actions., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Melatonin and serotonin, products of tryptophan metabolism, are endogenous neurotransmitters and hormones. We have identified and quantified these metabolites in natural honey from Australia, USA, and Poland using a Xevo G2 XS qTof LC-MS. To help ensure correct product identification, some samples were prepurified by RP-HPLC based on the retention times of standards, prior to LC-MS. The concentrations of the metabolites of interest depended on the source of the honey. For Australian honey, levels for melatonin and 2-hydroxymelatonin were 0.91 and 0.68 ng/g, respectively. Melatonin was detected in one brand of US commercial honey at 0.48 ng/g, while a second brand contained serotonin at 88.2 ng/g. In Polish natural honey, 20.6 ng/g of serotonin and 40.8 ng/g of N -acetylserotonin (NAS) were detected, while in Polish commercial honey 25.9 ng/g of serotonin and 7.30 ng/g of NAS were present. We suggest that addictive and health-related properties of honey may be in part dependent on the presence of serotonin, melatonin, and their metabolites, and that these compounds may play a role in the colony activities of bees.

Vitamin D deficiency significantly correlates with the severity of SARS-CoV-2 infection. Molecular docking-based virtual screening studies predict that novel vitamin D and related lumisterol hydroxymetabolites are able to bind to the active sites of two SARS-CoV-2 transcription machinery enzymes with high affinity. These enzymes are the main protease (M pro ) and RNA-dependent RNA polymerase (RdRP), which play important roles in viral replication and establishing infection. Based on predicted binding affinities and specific interactions, we identified 10 vitamin D3 (D3) and lumisterol (L3) analogs as likely binding partners of SARS-CoV-2 M pro and RdRP and, therefore, tested their ability to inhibit these enzymes. Activity measurements demonstrated that 25(OH)L3, 24(OH)L3, and 20(OH)7DHC are the most effective of the hydroxymetabolites tested at inhibiting the activity of SARS-CoV-2 M pro causing 10%-19% inhibition. These same derivatives as well as other hydroxylumisterols and hydroxyvitamin D3 metabolites inhibited RdRP by 50%-60%. Thus, inhibition of these enzymes by vitamin D and lumisterol metabolites may provide a novel approach to hindering the SARS-CoV-2 infection. NEW & NOTEWORTHY Active forms of vitamin D and lumisterol can inhibit SARS-CoV-2 replication machinery enzymes, which indicates that novel vitamin D and lumisterol metabolites are candidates for antiviral drug research.

The ability to use large doses of vitamin D3 (D3) to chronically treat autoimmune diseases such as rheumatoid arthritis (RA) is prohibitive due to its calcemic effect which can damage vital organs. Cytochrome P450scc (CYP11A1) is able to convert D3 into the noncalcemic analog 20 S -hydroxyvitamin D3 [20 S (OH)D3]. We demonstrate that 20 S (OH)D3 markedly suppresses clinical signs of arthritis and joint damage in a mouse model of RA. Furthermore, treatment with 20 S (OH)D3 reduces lymphocyte subsets such as CD4 + T cells and CD19 + B cells leading to a significant reduction in inflammatory cytokines. The ratio of T reg cells (CD4+CD25+Foxp3+ T cells) to CD3+CD4+ T cells is increased while there is a decrease in critical complement-fixing anti-CII antibodies. Since pro-inflammatory cytokines and antibodies against type II collagen ordinarily lead to destruction of cartilage and bone, their decline explains why arthritis is attenuated by 20(OH) D3. These results provide a basis for further consideration of 20 S (OH)D3 as a potential treatment for RA and other autoimmune disorders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Postlethwaite, Tuckey, Kim, Li, Bhattacharya, Myers, Brand and Slominski.)

Vitamin D3 is not only involved in calcium and phosphate metabolism in humans, but it can also affect proliferation and differentiation of normal and cancer cells, including melanoma. The mechanism of the anti-cancer action of vitamin D3 is not fully understood. The nuclear vitamin D receptor (VDR) is crucial for the phenotypic effects of vitamin D hydroxyderivatives. VDR expression shows an inverse correlation with melanoma progression and poor outcome of the disease. In this study we knocked out the VDR in a human melanoma cell line using CRISPR methodology. This enhanced the proliferation of melanoma cells grown in monolayer culture, spheroids or colonies and their migration. Activated forms of vitamin D, including classical 1,25(OH) 2 D3, 20(OH)D3 and 1,20(OH) 2 D3, inhibited cell proliferation, migration rate and the ability to form colonies and spheroids in the wild-type melanoma cell line, while VDR KO cells showed a degree of resistance to their action. These results indicate that expression of VDR is important for the inhibition of melanoma growth induced by activated forms of vitamin D. In conclusion, based on our previous clinicopathological analyses and the current study, we suggest that the VDR can function as a melanoma tumor suppressor gene.

CYP11A1, a member of the cytochrome P450 family, plays several key roles in the human body. It catalyzes the first and rate-limiting step in steroidogenesis, converting cholesterol to pregnenolone. Aside from the classical steroidogenic tissues such as the adrenals, gonads and placenta, CYP11A1 has also been found in the brain, gastrointestinal tract, immune systems, and finally the skin. CYP11A1 activity in the skin is regulated predominately by StAR protein and hence cholesterol levels in the mitochondria. However, UVB, UVC, CRH, ACTH, cAMP, and cytokines IL-1, IL-6 and TNFα can also regulate its expression and activity. Indeed, CYP11A1 plays several critical roles in the skin through its initiation of local steroidogenesis and specific metabolism of vitamin D, lumisterol, and 7-dehydrocholesterol. Products of these pathways regulate the protective barrier and skin immune functions in a context-dependent fashion through interactions with a number of receptors. Disturbances in CYP11A1 activity can lead to skin pathology., (Published by Elsevier B.V.)

Objectives: 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., Methods: 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., Results: 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) 2 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) 2 D3, 1,25(OH) 2 D3 and 1,20 S (OH) 2 D3) was consistently achieved in human serum samples., Conclusions: 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., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

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) 2 D3. 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) 3 D3, 1,25(OH) 2 D3, 1,20(OH) 2 D3 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) 2 D3, 1,20(OH) 2 D3, 25(OH)D3, 20(OH)D3, 20(OH)L3 and 20,22(OH) 2 L3, 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.

Previous studies showed that noncalcemic 20(OH)D3, a product of CYP11A1 action on vitamin D3, has antifibrotic activity in human dermal fibroblasts and in a bleomycin mouse model of scleroderma. In this study, we tested the role of retinoic acid-related orphan receptor γ (RORγ), which is expressed in skin, in the action of CYP11A1-derived secosteroids using murine fibroblasts isolated from the skin of wild-type (RORγ +/+), knockout (RORγ -/-), and heterozygote (RORγ +/-) mice. CYP11A1-derived 20(OH)D3, 20,23(OH)2D3, 1,20(OH)2D3, and 1,20,23(OH)3D3 inhibited proliferation of RORγ +/+ fibroblasts in a dose-dependent manner with a similar potency to 1,25(OH)2D3. Surprisingly, this effect was reversed in RORγ +/- and RORγ -/- fibroblasts, with the most pronounced stimulatory effect seen in RORγ -/- fibroblasts. All analogs tested inhibited TGF-β1-induced collagen synthesis in RORγ +/+ fibroblasts and the expression of other fibrosis-related genes. This effect was curtailed or reversed in RORγ -/- fibroblasts. These results show that the antiproliferative and antifibrotic activities of the vitamin D hydroxy derivatives are dependent on a functional RORγ. The dramatic changes in the transcriptomes of fibroblasts of RORγ -/- versus wild-type mice following treatment with 20(OH)D3 or 1,20(OH)2D3 provide a molecular basis to explain, at least in part, the observed phenotypic differences., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

Vitamin D and its derivatives, acting via the vitamin D receptor (VDR) and retinoic acid-related orphan receptors γ and α (RORγ and RORα), show anticancer properties. Since pathological conditions are characterized by disturbances in the expression of these receptors, in this study, we investigated their expression in ovarian cancers (OCs), as well as explored the phenotypic effects of vitamin D hydroxyderivatives and RORγ/α agonists on OC cells. The VDR and RORγ showed both a nuclear and a cytoplasmic location, and their expression levels were found to be reduced in the primary and metastatic OCs in comparison to normal ovarian epithelium, as well as correlated to the tumor grade. This reduction in VDR and RORγ expression correlated with a shorter overall disease-free survival. VDR, RORγ, and RORα were also detected in SKOV-3 and OVCAR-3 cell lines with increased expression in the latter line. 20-Hydroxy-lumisterol3 (20(OH)L 3 ) and synthetic RORα/RORγ agonist SR1078 inhibited proliferation only in the OVCAR-3 line, while 20-hydroxyvitamin-D 3 (20(OH)D 3 ) only inhibited SKOV-3 cell proliferation. 1,25(OH) 2 D 3 , 20(OH)L 3 , and SR1078, but not 20(OH)D 3 , inhibited spheroid formation in SKOV-3 cells. In summary, decreases in VDR, RORγ, and RORα expression correlated with an unfavorable outcome for OC, and compounds targeting these receptors had a context-dependent anti-tumor activity in vitro. We conclude that VDR and RORγ expression can be used in the diagnosis and prognosis of OC and suggest their ligands as potential candidates for OC therapy.

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., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Published by Elsevier Inc.)

20( S )-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) 2 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 m / z = 423.324 [M + Na] + for 20(OH)D3 corresponding to a concentration in honey of 256 ng/g. We also detected peaks of m / z = 383.331 [M + H - H 2 O] + for 20(OH)-7DHC and 25(OH)D3 with retention times corresponding to the standards. We further detected species with m/z = 407.329 [M + Na] + corresponding to the RT of 7DHC, D3 and lumisterol3 (L3). Similarly, peaks with m/z = 399.326 [M + H - H 2 O] + were detected at the RT of 1,25(OH) 2 D3 and 1,20-dihydroxyvitamin D3 (1,20(OH) 2 D3). Species corresponding to 20-monohydroxylumisterol3 (20(OH)L3), 22-monohydroxyvitamin D3 (22(OH)D3), 20,23-dihydroxyvitamin D3 (20,23(OH) 2 D3), 20,24/25/26-dihydroxyvitamin D3 (20,24/25/26(OH) 2 D3) and 1,20,23/24/25/26-trihydroxyvitamin D3 (1,20,23/24/25/26(OH) 3 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) 2 D3, 25(OH)D3 and 1,25(OH) 2 D3 in honey implies their production in bees, although the precise biochemistry and photochemistry of these processes remain to be defined.

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.

Background/aim: The hormonally-active form of vitamin D, 1,25(OH) 2 D 3 , demonstrated activity against oral squamous cell carcinoma (OSCC). Cytochrome P450scc (CYP11A1)-derived vitamin D hydroxyderivatives, such as 20(OH)D 3 and 1,20(OH) 2 D 3 , have overlapping beneficial effects with 1,25(OH) 2 D 3 without causing hypercalcemia. This study sought to determine (i) whether 20(OH)D 3 and 1,20(OH) 2 D 3 exhibit antitumor effects against OSCC comparable to those of 1,25(OH) 2 D 3 and (ii) whether these effects may stem from down-regulation of sonic hedgehog (SHH) or WNT/β-catenin signaling pathways., Materials and Methods: Effects on CAL-27 cells were assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt and spheroid assays. Signaling pathways were assessed by immunofluorescence and western blotting., Results: 20(OH)D 3 and 1,20(OH) 2 D 3 inhibited the growth of CAL-27 and demonstrated inhibition of WNT/β-catenin and the SHH signaling as evidenced by down-regulation of nuclear translocation of glioma-associated oncogene 1(GLI1) and β-catenin., Conclusion: Noncalcemic vitamin D hydroxyderivatives demonstrated antitumor activities against OSCC comparable to those of 1,25(OH) 2 D 3 Their activities against SHH and the WNT/β-catenin pathways provide insight for a possible target for OSCC treatment., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Glucocorticoid synthesis is a complex, multistep process that starts with cholesterol being delivered to the inner membrane of mitochondria by StAR and StAR-related proteins. Here its side chain is cleaved by CYP11A1 producing pregnenolone. Pregnenolone is converted to cortisol by the enzymes 3-βHSD, CYP17A1, CYP21A2, and CYP11B1. Glucocorticoids play a critical role in the regulation of the immune system and exert their action through the glucocorticoid receptor (GR). Although corticosteroids are primarily produced in the adrenal gland, they can also be produced in a number of extra-adrenal tissue including the immune system, skin, brain, and intestine. Glucocorticoid production is regulated by ACTH, CRH, and cytokines such as IL-1, IL-6, and TNFα. The bioavailability of cortisol is also dependent on its interconversion to cortisone, which is inactive, by 11βHSD1/2. Local and systemic glucocorticoid biosynthesis can be stimulated by ultraviolet B, explaining its immunosuppressive activity. In this review, we want to emphasize that dysregulation of extra-adrenal glucocorticoid production can play a key role in a variety of autoimmune diseases including multiple sclerosis (MS), lupus erythematosus (LE), rheumatoid arthritis (RA), and skin inflammatory disorders such as psoriasis and atopic dermatitis (AD). Further research on local glucocorticoid production and its bioavailability may open doors into new therapies for autoimmune diseases.

Nonmelanoma skin cancers including basal and squamous cell carcinomas (SCC and BCC) represent a significant clinical problem due to their relatively high incidence, imposing an economic burden to healthcare systems around the world. It is accepted that ultraviolet radiation (UVR: λ = 290-400 nm) plays a crucial role in the initiation and promotion of BCC and SCC with UVB (λ = 290-320 nm) having a central role in this process. On the other hand, UVB is required for vitamin D3 (D3) production in the skin, which supplies >90% of the body's requirement for this prohormone. Prolonged exposure to UVB can also generate tachysterol and lumisterol. Vitamin D3 itself and its canonical (1,25(OH) 2 D3) and noncanonical (CYP11A1-intitated) D3 hydroxyderivatives show photoprotective functions in the skin. These include regulation of keratinocyte proliferation and differentiation, induction of anti-oxidative responses, inhibition of DNA damage and induction of DNA repair mechanisms, and anti-inflammatory activities. Studies in animals have demonstrated that D3 hydroxyderivatives can attenuate UVB or chemically induced epidermal cancerogenesis and inhibit growth of SCC and BCC. Genomic and non-genomic mechanisms of action have been suggested. In addition, vitamin D3 itself inhibits hedgehog signaling pathways which have been implicated in many cancers. Silencing of the vitamin D receptor leads to increased propensity to develop UVB or chemically induced epidermal cancers. Other targets for vitamin D compounds include 1,25D3-MARRS, retinoic orphan receptors α and γ, aryl hydrocarbon receptor, and Wnt signaling. Most recently, photoprotective effects of lumisterol hydroxyderivatives have been identified. Clinical trials demonstrated a beneficial role of vitamin D compounds in the treatment of actinic keratosis. In summary, recent advances in vitamin D biology and pharmacology open new exciting opportunities in chemoprevention and treatment of skin cancers.

We tested whether novel CYP11A1-derived vitamin D 3 - and lumisterol-hydroxyderivatives, including 1,25(OH) 2 D 3 , 20(OH)D 3 , 1,20(OH) 2 D 3 , 20,23(OH) 2 D 3 , 1,20,23(OH) 3 D 3 , lumisterol, 20(OH)L 3 , 22(OH)L 3 , 20,22(OH) 2 L 3 , and 24(OH)L 3 , 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/cm 2 , 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/cm 2 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) 2 D 3 or CYP11A1-derived vitamin D 3 - 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 D 3 and lumisterol hydroxy-derivatives represent promising anti-photodamaging agents., (Copyright © 2019. Published by Elsevier B.V.)

25-Hydroxyvitamin D3 3-epimerase catalyzes the 3β → 3α epimerization of 25-hydroxyvitamin D3 (25(OH)D3) producing 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3). 3-Epi-25(OH)D3 is one of the most abundant forms of vitamin D present in the serum. It can be converted to 3-epi-1α,25-dihydroxyvitamin D3 by CYP27B1 which generally displays lower biological activity than 1α,25-dihydroxyvitamin D3 (1,25(OH) 2 D3). The 25(OH)D3 3-epimerase has been poorly characterized to date and the gene encoding it has not been identified. The 3-epimerase has been reported to be present in the microsomal fraction of cells, including liver cells, and to use NADPH as cofactor. It can also act on 1,25(OH) 2 D3 and 24,25(OH) 2 D3 forming the 3α-epimers. In this study we have characterized the activity of the 25(OH)D3 3-epimerase in rat and human liver microsomes, using 25(OH)D3 as substrate and HPLC to analyze product formation. For both rat and human liver microsomes the preferred cofactor was NADH, with the rat enzyme displaying a 6-fold greater catalytic efficiency (V max /K m ) for NADH over that for NADPH. No activity was observed with oxidized cofactor, either NAD + or NADP + . This was unexpected since the initial step in the epimerization, predicted to be the oxidation of the 3β-OH to a ketone, would require oxidized cofactor. The rat 3-epimerase in microsomes gave a K m for 25(OH)D3 of 14 μM. The reverse reaction, conversion of 3-epi-25(OH)D3 to 25(OH)D3, was catalyzed by both rat and human liver microsomes but at lower rates than the forward reaction. In conclusion, both rat and human 25-hydroxyvitamin D3 3-epimerase catalyze the reversible interconversion of 25(OH)D3 and 3-epi-25(OH)D3, and use NADH as the preferred cofactor. The lack of requirement for exogenous NAD + suggests that the enzyme has a tightly bound NAD + in its active site that is released only upon its reduction., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Melanoma represents a significant challenge in cancer treatment due to the high drug resistance of melanomas and the patient mortality rate. This study presents data indicating that nanomolar concentrations of the hormonally active form of vitamin D, 1α,25‑dihydroxyvitamin D3 [1α,25(OH)2D3], its non‑calcemic analogues 20S‑hydroxyvitamin D3 and 21‑hydroxypregnacalciferol, as well as the low‑calcemic synthetic analog calcipotriol, modulate the efficacy of the anticancer drugs cisplatin and dacarbazine. It was observed that vitamin D analogs sensitized melanoma A375 cells to hydrogen peroxide used as an inducer of oxidative stress. On the other hand, only 1α,25(OH)2D3 resulted in a minor, but significant effect on the proliferation of melanoma cells treated simultaneously with dacarbazine, but not cisplatin. Notably, cisplatin (300 µM) exhibited a higher overall antiproliferative activity than dacarbazine. Cisplatin treatment of melanoma cells resulted in an induction of apoptosis as demonstrated by flow cytometry (accumulation of cells at the subG1 phase of the cell cycle), whereas dacarbazine caused G1/G0 cell cycle arrest, with the effects being improved by pre‑treatment with vitamin D analogs. Treatment with cisplatin resulted in an initial increase in the level of reactive oxygen species (ROS). Dacarbazine caused transient stimulation of ROS levels and the mitochondrial membrane potential (Δψm) (after 1 or 3 h of treatment, respectively), but the effect was not detectable following prolonged (24 h) incubation with the drug. Vitamin D exhibited modulatory effects on the cells treated with dacarbazine, decreasing the half maximal inhibitory concentration (IC50) for the drug, stimulating G1/G0 arrest and causing a marked decrease in Δψm. Finally, cisplatin, dacarbazine and 1α,25(OH)2D3 displayed modulatory effects on the expression of ROS and vitamin D‑associated genes in the melanoma A375 cells. In conclusion, nanomolar concentrations of 1,25(OH)2D3 only had minor effects on the proliferation of melanoma cells treated with dacarbazine, decreasing the relative IC50 value. However, co‑treatment with vitamin D analogs resulted in the modulation of cell cycle and ROS responses, and affected gene expression, suggesting possible crosstalk between the signaling pathways of vitamin D and the anticancer drugs used in this study.

Vitamin D, referring to the two forms, D2 from the diet and D3 primarily derived from phototransformation in the skin, is a prohormone important in human health. The most hormonally active form, 1α,25-dihydroxyvitamin D (1α,25(OH) 2 D), formed from vitamin D via 25-hydroxyvitamin D (25(OH)D), is not only important for regulating calcium metabolism, but has many pleiotropic effects including regulation of the immune system and has anti-cancer properties. The major circulating form of vitamin D is 25(OH)D and both D2 and D3 forms are routinely measured by LC/MS/MS to assess vitamin D status, due to their relatively long half-lives and much higher concentrations compared to 1α,25(OH) 2 D. Inactivation of both 25(OH)D and 1α,25(OH) 2 D is catalyzed by CYP24A1 and 25-hydroxyvitamin D3 3-epimerase. Initial products from these enzymes acting on 25(OH)D3 are 24R,25(OH) 2 D3 and 3-epi-25(OH)D3, respectively, and both of these can also be measured routinely in some clinical laboratories to further document vitamin D status. With advances in LC/MS/MS and its increased availability, and with the help of studies with recombinant vitamin D-metabolizing enzymes, many other vitamin D metabolites have now been detected and in some cases quantitated, in human serum. CYP11A1 which catalyzes the first step in steroidogenesis, has been found to also act on vitamins D3 and D2 hydroxylating both at C20, but with some secondary metabolites produced by subsequent hydroxylations at other positions on the side chain. The major vitamin D3 metabolite, 20S-hydroxyvitamin D3 (20S(OH)D3), shows biological activity, often similar to 1α,25(OH) 2 D3 but without calcemic effects. Using standards produced enzymatically by purified CYP11A1 and characterized by NMR, many of these new metabolites have been detected in human serum, with semi-quantitative measurement of 20S(OH)D3 indicating it is present at comparable concentrations to 24R,25(OH) 2 D3 and 3-epi-25(OH)D3. Recently, vitamin D-related hydroxylumisterols derived from lumisterol3, a previtamin D3 photoproduct, have also been measured in human serum and displayed biological activity in initial in vitro studies. With the current extensive knowledge on the reactions and pathways of metabolism of vitamin D, especially those catalyzed by CYP24A1, CYP27A1, CYP27B1, CYP3A4 and CYP11A1, it is likely that many other of the resulting hydroxyvitamin D metabolites will be measured in human serum in the future, some contributing to a more detailed understanding of vitamin D status in health and disease., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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'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.

Prolonged exposure of the skin to UV radiation causes previtamin D3, the initial photoproduct formed by opening of the B ring of 7-dehydrocholesterol, to undergo a second photochemical reaction where the B-ring is reformed giving lumisterol3 (L3), a stereoisomer of 7-dehydrocholesterol. L3 was believed to be an inactive photoproduct of excessive UV radiation whose formation prevents excessive vitamin D production. Recently, we reported that L3 is present in serum and that CYP11A1 can act on L3 producing monohydroxy- and dihydroxy-metabolites which inhibit skin cell proliferation similarly to 1α,25-dihydroxyvitamin D3. In this study we tested the ability of human CYP27A1 to hydroxylate L3. L3 was metabolized by purified CYP27A1 to 3 major products identified as 25-hydroxyL3, (25R)-27-hydroxyL3 and (25S)-27-hydroxyL3, by NMR. These three products were also seen when mouse liver mitochondria containing CYP27A1 were incubated with L3. The requirement for CYP27A1 for their formation by mitochondria was confirmed by the inhibition of their synthesis by 5β-cholestane-3α,7α,12α-triol, an intermediate in bile acid synthesis which serves as an efficient competitive substrate for CYP27A1. CYP27A1 displayed a high k cat for the metabolism of L3 (76 mol product/min/mol CYP27A1) and a catalytic efficiency (k cat /K m ) that was 260-fold higher than that for vitamin D3. The CYP27A1-derived hydroxy-derivatives inhibited the proliferation of cultured human melanoma cells and colony formation with IC 50 values in the nM range. Thus, L3 is metabolized efficiently by CYP27A1 with hydroxylation at C25 or C27 producing metabolites potent in their ability to inhibit melanoma cell proliferation, supporting that L3 is a prohormone which can be activated by CYP-dependent hydroxylations., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Melanoma represents a significant clinical problem affecting a large segment of the population with a relatively high incidence and mortality rate. Ultraviolet radiation (UVR) is an important etiological factor in malignant transformation of melanocytes and melanoma development. UVB, while being a full carcinogen in melanomagenesis, is also necessary for the cutaneous production of vitamin D3 (D3). Calcitriol (1,25(OH) 2 D3) and novel CYP11A1-derived hydroxyderivatives of D3 show anti-melanoma activities and protective properties against damage induced by UVB. The former activities include inhibitory effects on proliferation, plating efficiency and anchorage-independent growth of cultured human and rodent melanomas in vitro, as well as the in vivo inhibition of tumor growth by 20(OH)D3 after injection of human melanoma cells into immunodeficient mice. The literature indicates that low levels of 25(OH)D3 are associated with more advanced melanomas and reduced patient survivals, while single nucleotide polymorphisms of the vitamin D receptor or the D3 binding protein gene affect development or progression of melanoma, or disease outcome. An inverse correlation of VDR and CYP27B1 expression with melanoma progression has been found, with low or undetectable levels of these proteins being associated with poor disease outcomes. Unexpectedly, increased expression of CYP24A1 was associated with better melanoma prognosis. In addition, decreased expression of retinoic acid orphan receptors α and γ, which can also bind vitamin D3 hydroxyderivatives, showed positive association with melanoma progression and shorter disease-free and overall survival. Thus, inadequate levels of biologically active forms of D3 and disturbances in expression of the target receptors, or D3 activating or inactivating enzymes, can affect melanomagenesis and disease progression. We therefore propose that inclusion of vitamin D into melanoma management should be beneficial for patients, at least as an adjuvant approach. The presence of multiple hydroxyderivatives of D3 in skin that show anti-melanoma activity in experimental models and which may act on alternative receptors, will be a future consideration when planning which forms of vitamin D to use for melanoma therapy., (Published by Elsevier Ltd.)

Recent studies indicate that CYP2R1 is the major 25-hydroxylase catalyzing the first step in vitamin D activation. Since the catalytic properties of CYP2R1 have been poorly studied to date and it is a membrane protein, we examined the purified enzyme in a membrane environment. CYP2R1 was expressed in E. coli and purified by nickel affinity- and hydrophobic interaction-chromatography and assayed in a reconstituted membrane system comprising phospholipid vesicles plus purified human NADPH-P450 oxidoreductase. CYP2R1 converted vitamin D3 in the vesicle membrane to 25-hydroxyvitamin D3 [25(OH)D3] with good adherence to Michaelis-Menten kinetics. The kinetic parameters for 25-hydroxylation of vitamin D3 by the two major vitamin D 25-hydroxylases, CYP2R1 and CYP27A1, were examined in vesicles under identical conditions. CYP2R1 displayed a slightly lower k cat than CYP27A1 but a much lower K m for vitamin D3, and thus an overall 17-fold higher catalytic efficiency (k cat /K m ), consistent with CYP2R1 being the major vitamin D 25-hydroxylase. 20-Hydroxyvitamin D3 [20(OH)D3], the main product of vitamin D3 activation by an alternative pathway catalyzed by CYP11A1, was metabolized by CYP2R1 to 20,25-dihydroxyvitamin D3 [20,25(OH) 2 D3], with catalytic efficiency similar to that for the 25-hydroxylation of vitamin D3. 20,25(OH) 2 D3 retained full, or somewhat enhanced activity compared to the parent 20(OH)D3 for the inhibition of the proliferation of melanocytes and dermal fibroblasts, with a potency comparable to 1,25-dihydroxyvitamin D3 [1,25(OH) 2 D3]. The 20,25(OH) 2 D3 was also able to act as an inverse agonist on retinoic acid-related orphan receptor α, like its parent 20(OH)D3. Thus, the major findings of this study are that CYP2R1 can metabolize substrates in a membrane environment, the enzyme displays higher catalytic efficiency than CYP27A1 for the 25-hydroxylation of vitamin D, it efficiently hydroxylates 20(OH)D3 at C25 and this product retains the biological activity of the parent compound., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

20S-hydroxyvitamin D 3 [20S(OH)D 3 ] is anti-inflammatory and not hypercalcemic, suggesting its potential as a lead compound. In this study, side chain modified 20S(OH)D 3 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)D 3 , 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)D 3 scaffold for the development of novel anti-inflammatory agents.

The rate-limiting step in the steroid synthesis pathway is catalyzed by CYP11A1 through three sequential reactions. The first two steps involve hydroxylations at positions 22 and 20, generating 20(R),22(R)-dihydroxycholesterol (20R,22R-DiOHCH), with the third stage leading to a C20-C22 bond cleavage, forming pregnenolone. This work provides detailed information about the active site structure of CYP11A1 in the resting state and substrate-bound ferric forms as well as the CO-ligated adducts. In addition, high-quality resonance Raman spectra are reported for the dioxygen complexes, providing new insight into the status of Fe-O-O fragments encountered during the enzymatic cycle. Results show that the three natural substrates of CYP11A1 have quite different effects on the active site structure, including variations of spin state populations, reorientations of heme peripheral groups, and, most importantly, substrate-mediated distortions of Fe-CO and Fe-O 2 fragments, as revealed by telltale shifts of the observed vibrational modes. Specifically, the vibrational mode patterns observed for the Fe-O-O fragments with the first and third substrates are consistent with H-bonding interactions with the terminal oxygen, a structural feature that tends to promote O-O bond cleavage to form the Compound I intermediate. Furthermore, such spectral data are acquired for complexes with the natural redox partner, adrenodoxin (Adx), revealing protein-protein-induced active site structural perturbations. While this work shows that Adx has an only weak effect on ferric and ferrous CO states, it has a relatively stronger impact on the Fe-O-O fragments of the functionally relevant oxy complexes.

The classical pathway of vitamin D activation follows the sequence D3→25(OH)D3→1,25(OH) 2 D3 with the final product acting on the receptor for vitamin D (VDR). An alternative pathway can be started by the action of CYP11A1 on the side chain of D3, primarily producing 20(OH)D3, 22(OH)D3, 20,23(OH) 2 D3, 20,22(OH) 2 D3 and 17,20,23(OH) 3 D3. Some of these metabolites are hydroxylated by CYP27B1 at C1α, by CYP24A1 at C24 and C25, and by CYP27A1 at C25 and C26. The products of these pathways are biologically active. In the epidermis and/or serum or adrenals we detected 20(OH)D3, 22(OH)D3, 20,22(OH) 2 D3, 20,23(OH) 2 D3, 17,20,23(OH) 3 D3, 1,20(OH) 2 D3, 1,20,23(OH) 3 D3, 1,20,22(OH) 3 D3, 20,24(OH) 2 D3, 1,20,24(OH) 3 D3, 20,25(OH) 2 D3, 1,20,25(OH) 3 D3, 20,26(OH) 2 D3 and 1,20,26(OH) 3 D3. 20(OH)D3 and 20,23(OH) 2 D3 are non-calcemic, while the addition of an OH at C1α confers some calcemic activity. Molecular modeling and functional assays show that the major products of the pathway can act as "biased" agonists for the VDR with high docking scores to the ligand binding domain (LBD), but lower than that of 1,25(OH) 2 D3. Importantly, cell based functional receptor studies and molecular modeling have identified the novel secosteroids as inverse agonists of both RORα and RORγ receptors. Specifically, they have high docking scores using crystal structures of RORα and RORγ LBDs. Furthermore, 20(OH)D3 and 20,23(OH) 2 D3 have been tested in a cell model that expresses a Tet-on RORα or RORγ vector and a RORE-LUC reporter (ROR-responsive element), and in a mammalian 2-hybrid model that test interactions between an LBD-interacting LXXLL-peptide and the LBD of RORα/γ. These assays demonstrated that the novel secosteroids have ROR-antagonist activities that were further confirmed by the inhibition of IL17 promoter activity in cells overexpressing RORα/γ. In conclusion, endogenously produced novel D3 hydroxy-derivatives can act both as "biased" agonists of the VDR and/or inverse agonists of RORα/γ. We suggest that the identification of large number of endogenously produced alternative hydroxy-metabolites of D3 that are biologically active, and of possible alternative receptors, may offer an explanation for the pleiotropic and diverse activities of vitamin D, previously assigned solely to 1,25(OH) 2 D3 and VDR., (Published by Elsevier Ltd.)

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.

1α,20S-Dihydroxyvitamin D3 [1,20S(OH) 2 D 3 ], a natural and bioactive vitamin D3 metabolite, was chemically synthesized for the first time. X-ray crystallography analysis of intermediate 15 confirmed its 1α-OH configuration. 1,20S(OH) 2 D 3 interacts with the vitamin D receptor (VDR), with similar potency to its native ligand, 1α,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] as illustrated by its ability to stimulate translocation of the VDR to the nucleus, stimulate VDRE-reporter activity, regulate VDR downstream genes (VDR, CYP24A1, TRPV6 and CYP27B1), and inhibit the production of inflammatory markers (IFNγ and IL1β). However, their co-crystal structures revealed differential molecular interactions of the 20S-OH moiety and the 25-OH moiety to the VDR, which may explain some differences in their biological activities. Furthermore, this study provides a synthetic route for the synthesis of 1,20S(OH) 2 D 3 using the intermediate 1α,3β-diacetoxypregn-5-en-20-one (3), and provides a molecular and biological basis for the development of 1,20S(OH) 2 D 3 and its analogs as potential therapeutic agents.