Beneficial Effects of Ursodeoxycholic Acid on Metabolic Parameters and Oxidative Stress in Patients with Type 2 Diabetes Mellitus: A Randomized Double-Blind, Placebo-Controlled Clinical Study.

Background: Oxidative stress and inflammation are closely related pathophysiological processes, both occurring in type 2 diabetes mellitus (T2DM). In addition to the standard treatment of T2DM, a potential strategy has been focused on the use of bile acids (BAs) as an additional treatment. Ursodeoxycholic acid (UDCA), as the first BA used in humans, improves glucose and lipid metabolism and attenuates oxidative stress. The aim of this study was to evaluate the potential metabolic, anti-inflammatory, and antioxidative effects of UDCA in patients with T2DM. Methods: This prospective, double-blind, placebo-controlled clinical study included 60 patients with T2DM, randomly allocated to receive UDCA or placebo. Subjects were treated with 500 mg tablets of UDCA or placebo administered three times per day (total dose of 1500 mg/day) for eight weeks. Two study visits, at the beginning (F0) and at the end (F1) of the study, included the interview, anthropometric and clinical measurements, and biochemical analyses. Results: UDCA treatment showed a significant reduction in body mass index (p = 0.024) and in diastolic blood pressure (p = 0.033), compared to placebo. In addition, there was a statistically significant difference in waist circumference in the UDCA group before and after treatment (p < 0.05). Although no statistical significance was observed at the two-month follow-up assessment, an average decrease in glucose levels in the UDCA group was observed. After two months of the intervention period, a significant decrease in the activity of liver enzymes was noticed. Furthermore, a significant reduction in prooxidative parameters (TBARS, NO2-, H2O2) and significant elevation in antioxidative parameters such as SOD and GSH were found (p < 0.001). Conclusions: The eight-week UDCA administration showed beneficial effects on metabolic and oxidative stress parameters in patients with T2DM. Thus, UDCA could attenuate the progression and complications of diabetes and should be considered as an adjuvant to other diabetes treatment modalities. This trial is registered with NCT05416580.

Novel Vitamin D3 Hydroxymetabolites Require Involvement of the Vitamin D Receptor or Retinoic Acid-Related Orphan Receptors for Their Antifibrogenic Activities in Human Fibroblasts.

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)2D3, 1,20(OH)2D3 and 1,20,23(OH)3D3 were compared to those of classical 1,25(OH)2D3. 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 CYP27B1 did not change the effect of either 20(OH)D3 or 20,23(OH)2D3, 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.

Melatonin and Its Metabolites Can Serve as Agonists on the Aryl Hydrocarbon Receptor and Peroxisome Proliferator-Activated Receptor Gamma.

Melatonin is widely present in Nature. It has pleiotropic activities, in part mediated by interactions with high-affinity G-protein-coupled melatonin type 1 and 2 (MT1 and MT2) receptors or under extreme conditions, e.g., ischemia/reperfusion. In pharmacological concentrations, it is given to counteract the massive damage caused by MT1- and MT2-independent mechanisms. The aryl hydrocarbon receptor (AhR) is a perfect candidate for mediating the latter effects because melatonin has structural similarity to its natural ligands, including tryptophan metabolites and indolic compounds. Using a cell-based Human AhR Reporter Assay System, we demonstrated that melatonin and its indolic and kynuric metabolites act as agonists on the AhR with EC50's between 10-4 and 10-6 M. This was further validated via the stimulation of the transcriptional activation of the CYP1A1 promoter. Furthermore, melatonin and its metabolites stimulated AhR translocation from the cytoplasm to the nucleus in human keratinocytes, as demonstrated by ImageStream II cytometry and Western blot (WB) analyses of cytoplasmic and nuclear fractions of human keratinocytes. These functional analyses are supported by in silico analyses. We also investigated the peroxisome proliferator-activated receptor (PPAR)γ as a potential target for melatonin and metabolites bioregulation. The binding studies using a TR-TFRET kit to assay the interaction of the ligand with the ligand-binding domain (LBD) of the PPARγ showed agonistic activities of melatonin, 6-hydroxymelatonin and N-acetyl-N-formyl-5-methoxykynuramine with EC50's in the 10-4 M range showing significantly lower affinities that those of rosiglitazone, e.g., a 10-8 M range. These interactions were substantiated by stimulation of the luciferase activity of the construct containing PPARE by melatonin and its metabolites at 10-4 M. As confirmed by the functional assays, binding mode predictions using a homology model of the AhR and a crystal structure of the PPARγ suggest that melatonin and its metabolites, including 6-hydroxymelatonin, 5-methoxytryptamine and N-acetyl-N-formyl-5-methoxykynuramine, are excellent candidates to act on the AhR and PPARγ with docking scores comparable to their corresponding natural ligands. Melatonin and its metabolites were modeled into the same ligand-binding pockets (LBDs) as their natural ligands. Thus, functional assays supported by molecular modeling have shown that melatonin and its indolic and kynuric metabolites can act as agonists on the AhR and they can interact with the PPARγ at high concentrations. This provides a mechanistic explanation for previously reported cytoprotective actions of melatonin and its metabolites that require high local concentrations of the ligands to reduce cellular damage under elevated oxidative stress conditions. It also identifies these compounds as therapeutic agents to be used at pharmacological doses in the prevention or therapy of skin diseases.

Metabolic activation of tachysterol3 to biologically active hydroxyderivatives that act on VDR, AhR, LXRs, and PPARγ receptors.

CYP11A1 and CYP27A1 hydroxylate tachysterol3 , a photoproduct of previtamin D3 , producing 20S-hydroxytachysterol3 [20S(OH)T3 ] and 25(OH)T3 , respectively. Both metabolites were detected in the human epidermis and serum. Tachysterol3 was also detected in human serum at a concentration of 7.3 ± 2.5 ng/ml. 20S(OH)T3 and 25(OH)T3 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 D3 [1,25(OH)2 D3 ]. 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)T3 , a smaller effect by 25(OH)T3 , and a minimal effect for their precursor, tachysterol3 . Tachysterol3 hydroxyderivatives showed high-affinity binding to the ligan-binding domain (LBD) of the liver X receptor (LXR) α and ß, 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)T3 and 25(OH)T3 , 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 tachysterol3 ligands. Our identification of endogenous production of 20S(OH)T3 and 25(OH)T3 that are biologically active and interact with VDR, AhR, LXRs, and PPARγ, provides a new understanding of the biological function of tachysterol3 .

CYP11A1­derived vitamin D hydroxyderivatives as candidates for therapy of basal and squamous cell carcinomas.

Hydroxyderivatives of vitamin D3, including classical 1,25(OH)2D3 and novel CYP11A1­derived hydroxyderivatives, exert their biological activity by acting as agonists on the vitamin D receptor (VDR) and inverse agonists on retinoid­related orphan receptors (ROR)α and γ. The anticancer activities of CYP11A1­derived 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)2D3. Vitamin D3­hydroxyderivatives with or without a C1α(OH) inhibited cell proliferation in a dose­dependent 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 anti­tumorigenic activity against the BCC line was exerted by 1,25(OH)2D3, 1,20(OH)2D3, 1,20,23(OH)3D3, 1,20,24(OH)3D3, 1,20,25(OH)3D3 and 1,20,26(OH)3D3, with smaller effects seen for 25(OH)D3, 20(OH)D3 and 20,23(OH)2D3. 1,25(OH)2D3, 1,20(OH)2D3 and 20(OH)D3 inhibited the expression of GLI1 and ß­catenin 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 D­binding protein), the level of which was dependent on the type of cancer and antigen tested in comparison with normal epidermis. Classical and CYP11A1­derived vitamin D3­derivatives exhibited anticancer­activities on skin cancer cell lines and inhibited GLI1 and ß­catenin 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 anti­cancer therapy.

Molecular and structural basis of interactions of vitamin D3 hydroxyderivatives with aryl hydrocarbon receptor (AhR): An integrated experimental and computational study.

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)2D3, 1,25(OH)2D3, 20,23(OH)2D3 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.

Chemical synthesis, biological activities and action on nuclear receptors of 20S(OH)D3, 20S,25(OH)2D3, 20S,23S(OH)2D3 and 20S,23R(OH)2D3.

New and more efficient routes of chemical synthesis of vitamin D3 (D3) hydroxy (OH) metabolites, including 20S(OH)D3, 20S,23S(OH)2D3 and 20S,25(OH)2D3, that are endogenously produced in the human body by CYP11A1, and of 20S,23R(OH)2D3 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)2D3 and the lowest towards 20S(OH)D3. In conclusion, defining new routes for large scale synthesis of endogenously produced D3-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.

Protective Role of Melatonin and Its Metabolites in Skin Aging.

The skin, being the largest organ in the human body, is exposed to the environment and suffers from both intrinsic and extrinsic aging factors. The skin aging process is characterized by several clinical features such as wrinkling, loss of elasticity, and rough-textured appearance. This complex process is accompanied with phenotypic and functional changes in cutaneous and immune cells, as well as structural and functional disturbances in extracellular matrix components such as collagens and elastin. Because skin health is considered one of the principal factors representing overall "well-being" and the perception of "health" in humans, several anti-aging strategies have recently been developed. Thus, while the fundamental mechanisms regarding skin aging are known, new substances should be considered for introduction into dermatological treatments. Herein, we describe melatonin and its metabolites as potential "aging neutralizers". Melatonin, an evolutionarily ancient derivative of serotonin with hormonal properties, is the main neuroendocrine secretory product of the pineal gland. It regulates circadian rhythmicity and also exerts anti-oxidative, anti-inflammatory, immunomodulatory, and anti-tumor capacities. The intention of this review is to summarize changes within skin aging, research advances on the molecular mechanisms leading to these changes, and the impact of the melatoninergic anti-oxidative system controlled by melatonin and its metabolites, targeting the prevention or reversal of skin aging.

17,20S(OH)2pD Can Prevent the Development of Skin Fibrosis in the Bleomycin-Induced Scleroderma Mouse Model.

Systemic sclerosis (SSc; scleroderma) is a chronic fibrotic disease involving TGF-ß1. Low serum vitamin D (vit D) correlates with the degree of fibrosis and expression of TGF-ß1. This study was designed to determine whether the noncalcemic vit D analog, 17,20S(OH)2pD, suppresses fibrosis and mediators of the TGF-ß1 pathway in the bleomycin (BLM) model of fibrosis. Fibrosis was induced into the skin of female C57BL/6 mice by repeated injections of BLM (50 µg/100 µL) subcutaneously. Mice received daily oral gavage with either vehicle (propylene glycol) or 17,20S(OH)2pD using 5, 15, or 30 µg/kg for 21 days. The injected skin was biopsied; analyzed histologically; examined for total collagen by Sircol; and examined for mRNA expression of MMP-13, BMP-7, MCP-1, Gli1, and Gli2 by TR-PCR. Spleen was analyzed for lymphocytes using flow cytometry. Serum was analyzed for cytokines using a multiplexed ELISA. Results showed that all three doses of 17,20S(OH)2pD suppressed net total collagen production, dermal thickness, and total collagen content in the BLM fibrosis model. 17,20S(OH)2pD also increased MMP-13 expression, decreased MCP-1 and Gli-2 expression in vivo, and suppressed serum levels of IL-13, TNF-α, IL-6, IL-10, IL-17, and IL-12p70. In summary, 17,20S(OH)2pD modulates the mediators of fibrosis in vivo and suppresses total collagen production and dermal thickness. This antifibrotic property of 17,20S(OH)2pD offers new therapeutic approaches for fibrotic disorders.

Knocking out the Vitamin D Receptor Enhances Malignancy and Decreases Responsiveness to Vitamin D3 Hydroxyderivatives in Human Melanoma Cells.

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)2D3, 20(OH)D3 and 1,20(OH)2D3, 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.

Vitamin D and lumisterol derivatives can act on liver X receptors (LXRs).

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 ß 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 ß in LanthaScreen TR-FRET LXRα and ß coactivator assays. The majority of metabolites functioned as LXRα/ß 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 LXRß. 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.

Differential and Overlapping Effects of Melatonin and Its Metabolites on Keratinocyte Function: Bioinformatics and Metabolic Analyses.

We investigated the effects of melatonin and its selected metabolites, i.e., N1-Acetyl-N2-formyl-5-methoxykynurenamine (AFMK) and 6-hydroxymelatonin (6(OH)Mel), on cultured human epidermal keratinocytes (HEKs) to assess their homeostatic activities with potential therapeutic implications. RNAseq analysis revealed a significant number of genes with distinct and overlapping patterns, resulting in common regulation of top diseases and disorders. Gene Set Enrichment Analysis (GSEA), Reactome FIViZ, and Ingenuity Pathway Analysis (IPA) showed overrepresentation of the p53-dependent G1 DNA damage response gene set, activation of p53 signaling, and NRF2-mediated antioxidative pathways. Additionally, GSEA exhibited an overrepresentation of circadian clock and antiaging signaling gene sets by melatonin derivatives and upregulation of extension of telomere signaling in HEKs, which was subsequently confirmed by increased telomerase activity in keratinocytes, indicating possible antiaging properties of metabolites of melatonin. Furthermore, Gene Ontology (GO) showed the activation of a keratinocyte differentiation program by melatonin, and GSEA indicated antitumor and antilipidemic potential of melatonin and its metabolites. IPA also indicated the role of Protein Kinase R (PKR) in interferon induction and antiviral response. In addition, the test compounds decreased lactate dehydrogenase A (LDHA) and lactate dehydrogenase C (LDHC) gene expression. These results were validated by qPCR and by Seahorse metabolic assay with significantly decreased glycolysis and lactate production under influence of AFMK or 6(OH)Mel in cells with a low oxygen consumption rate. In summary, melatonin and its metabolites affect keratinocytes' functions via signaling pathways that overlap for each tested molecule with some distinctions.

Modulation by 17,20S(OH)2pD of Fibrosis-Related Mediators in Dermal Fibroblast Lines from Healthy Donors and from Patients with Systemic Sclerosis.

We previously demonstrated that the non-calcemic pregnacalciferol (pD) analog 17,20S (OH)2pD suppressed TGF-ß1-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)2pD 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)2pD or 1,25(OH)2D3 (positive control) with/without TGF-ß1 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)2pD (similar to 1,25(OH)2D3) significantly suppressed net total collagen production in TGF-ß1-stimulated normal donor fibroblast cultures and in cultures of SSc dermal fibroblasts. 17,20S(OH)2pD (similar to 1,25(OH)2D3) also increased MMP-1, BMP-7, and PGES and decreased TIMP-1 and PAI1 expression in SSc fibroblasts. Although 17,20S(OH)2pD had no effect on Gli1 or Gli2 in SSc fibroblasts, it increased Gli2 expression when cultured with TGF-ß1 in normal fibroblasts. These studies demonstrated that 17,20S(OH)2pD modulates mediators of fibrosis to favor the reduction of fibrosis and may offer new noncalcemic secosteroidal therapeutic approaches for treating SSc and fibrosis.

Antifibrogenic Activities of CYP11A1-derived Vitamin D3-hydroxyderivatives Are Dependent on RORγ.

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-ß1-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.

Hydroxylumisterols, Photoproducts of Pre-Vitamin D3, Protect Human Keratinocytes against UVB-Induced Damage.

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.

The Role of Classical and Novel Forms of Vitamin D in the Pathogenesis and Progression of Nonmelanoma Skin Cancers.

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)2D3) 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.

Photoprotective Properties of Vitamin D and Lumisterol Hydroxyderivatives.

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)2D3. 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)2L3 and 24(OH)L3. CYP11A1 also acts on 7-dehydrocholesterol (7DHC) producing 22(OH)7DHC, 20,22(OH)27DHC 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)2D3) 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)2D3, 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.

Noncalcemic Vitamin D Hydroxyderivatives Inhibit Human Oral Squamous Cell Carcinoma and Down-regulate Hedgehog and WNT/ß-Catenin Pathways.

BACKGROUND/AIM: The hormonally-active form of vitamin D, 1,25(OH)2D3, demonstrated activity against oral squamous cell carcinoma (OSCC). Cytochrome P450scc (CYP11A1)-derived vitamin D hydroxyderivatives, such as 20(OH)D3 and 1,20(OH)2D3, have overlapping beneficial effects with 1,25(OH)2D3 without causing hypercalcemia. This study sought to determine (i) whether 20(OH)D3 and 1,20(OH)2D3 exhibit antitumor effects against OSCC comparable to those of 1,25(OH)2D3 and (ii) whether these effects may stem from down-regulation of sonic hedgehog (SHH) or WNT/ß-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)D3 and 1,20(OH)2D3 inhibited the growth of CAL-27 and demonstrated inhibition of WNT/ß-catenin and the SHH signaling as evidenced by down-regulation of nuclear translocation of glioma-associated oncogene 1(GLI1) and ß-catenin. CONCLUSION: Noncalcemic vitamin D hydroxyderivatives demonstrated antitumor activities against OSCC comparable to those of 1,25(OH)2D3 Their activities against SHH and the WNT/ß-catenin pathways provide insight for a possible target for OSCC treatment.

CYP11A1-derived vitamin D3 products protect against UVB-induced inflammation and promote keratinocytes differentiation.

UVB radiation mediates inflammatory responses causing skin damage and defects in epidermal differentiation. 1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3) interacts with the vitamin D3 receptor (VDR) to regulate inflammatory responses. Additionally, 1,25(OH)2D3/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)2D3 against UVB-induced skin damage and inflammation is recognized, CYP11A1-derived vitamin D3-hydroxyderivatives including 20(OH)D3, 1,20(OH)2D3, 20,23(OH)2D3 and 1,20,23(OH)3D3 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/cm2) 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.

Characterization of serotonin and N-acetylserotonin systems in the human epidermis and skin cells.

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.