Evolutionary formation of melatonin and vitamin D in early life forms: insects take centre stage.

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)2D) 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.

Photo-neuro-immuno-endocrinology: How the ultraviolet radiation regulates the body, brain, and immune system.

Ultraviolet radiation (UVR) is primarily recognized for its detrimental effects such as cancerogenesis, skin aging, eye damage, and autoimmune disorders. With exception of ultraviolet B (UVB) requirement in the production of vitamin D3, the positive role of UVR in modulation of homeostasis is underappreciated. Skin exposure to UVR triggers local responses secondary to the induction of chemical, hormonal, immune, and neural signals that are defined by the chromophores and extent of UVR penetration into skin compartments. These responses are not random and are coordinated by the cutaneous neuro-immuno-endocrine system, which counteracts the action of external stressors and accommodates local homeostasis to the changing environment. The UVR induces electrical, chemical, and biological signals to be sent to the brain, endocrine and immune systems, as well as other central organs, which in concert regulate body homeostasis. To achieve its central homeostatic goal, the UVR-induced signals are precisely computed locally with transmission through nerves or humoral signals release into the circulation to activate and/or modulate coordinating central centers or organs. Such modulatory effects will be dependent on UVA and UVB wavelengths. This leads to immunosuppression, the activation of brain and endocrine coordinating centers, and the modification of different organ functions. Therefore, it is imperative to understand the underlying mechanisms of UVR electromagnetic energy penetration deep into the body, with its impact on the brain and internal organs. Photo-neuro-immuno-endocrinology can offer novel therapeutic approaches in addiction and mood disorders; autoimmune, neurodegenerative, and chronic pain-generating disorders; or pathologies involving endocrine, cardiovascular, gastrointestinal, or reproductive systems.

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.

Environmental Air Pollutants Affecting Skin Functions with Systemic Implications.

The increase in air pollution worldwide represents an environmental risk factor that has global implications for the health of humans worldwide. The skin of billions of people is exposed to a mixture of harmful air pollutants, which can affect its physiology and are responsible for cutaneous damage. Some polycyclic aromatic hydrocarbons are photoreactive and could be activated by ultraviolet radiation (UVR). Therefore, such UVR exposure would enhance their deleterious effects on the skin. Air pollution also affects vitamin D synthesis by reducing UVB radiation, which is essential for the production of vitamin D3, tachysterol, and lumisterol derivatives. Ambient air pollutants, photopollution, blue-light pollution, and cigarette smoke compromise cutaneous structural integrity, can interact with human skin microbiota, and trigger or exacerbate a range of skin diseases through various mechanisms. Generally, air pollution elicits an oxidative stress response on the skin that can activate the inflammatory responses. The aryl hydrocarbon receptor (AhR) can act as a sensor for small molecules such as air pollutants and plays a crucial role in responses to (photo)pollution. On the other hand, targeting AhR/Nrf2 is emerging as a novel treatment option for air pollutants that induce or exacerbate inflammatory skin diseases. Therefore, AhR with downstream regulatory pathways would represent a crucial signaling system regulating the skin phenotype in a Yin and Yang fashion defined by the chemical nature of the activating factor and the cellular and tissue context.

How cancer hijacks the body's homeostasis through the neuroendocrine system.

During oncogenesis, cancer not only escapes the body's regulatory mechanisms, but also gains the ability to affect local and systemic homeostasis. Specifically, tumors produce cytokines, immune mediators, classical neurotransmitters, hypothalamic and pituitary hormones, biogenic amines, melatonin, and glucocorticoids, as demonstrated in human and animal models of cancer. The tumor, through the release of these neurohormonal and immune mediators, can control the main neuroendocrine centers such as the hypothalamus, pituitary, adrenals, and thyroid to modulate body homeostasis through central regulatory axes. We hypothesize that the tumor-derived catecholamines, serotonin, melatonin, neuropeptides, and other neurotransmitters can affect body and brain functions. Bidirectional communication between local autonomic and sensory nerves and the tumor, with putative effects on the brain, is also envisioned. Overall, we propose that cancers can take control of the central neuroendocrine and immune systems to reset the body homeostasis in a mode favoring its expansion at the expense of the host.

Novel CYP11A1-Derived Vitamin D and Lumisterol Biometabolites for the Management of COVID-19.

Vitamin D deficiency is associated with a higher risk of SARS-CoV-2 infection and poor outcomes of the COVID-19 disease. However, a satisfactory mechanism explaining the vitamin D protective effects is missing. Based on the anti-inflammatory and anti-oxidative properties of classical and novel (CYP11A1-derived) vitamin D and lumisterol hydroxymetabolites, we have proposed that they would attenuate the self-amplifying damage in lungs and other organs through mechanisms initiated by interactions with corresponding nuclear receptors. These include the VDR mediated inhibition of NFκß, inverse agonism on RORγ and the inhibition of ROS through activation of NRF2-dependent pathways. In addition, the non-receptor mediated actions of vitamin D and related lumisterol hydroxymetabolites would include interactions with the active sites of SARS-CoV-2 transcription machinery enzymes (Mpro;main protease and RdRp;RNA dependent RNA polymerase). Furthermore, these metabolites could interfere with the binding of SARS-CoV-2 RBD with ACE2 by interacting with ACE2 and TMPRSS2. These interactions can cause the conformational and dynamical motion changes in TMPRSS2, which would affect TMPRSS2 to prime SARS-CoV-2 spike proteins. Therefore, novel, CYP11A1-derived, active forms of vitamin D and lumisterol can restrain COVID-19 through both nuclear receptor-dependent and independent mechanisms, which identify them as excellent candidates for antiviral drug research and for the educated use of their precursors as nutrients or supplements in the prevention and attenuation of the COVID-19 disease.

WINNER: A network biology tool for biomolecular characterization and prioritization.

Background and contribution: In network biology, molecular functions can be characterized by network-based inference, or "guilt-by-associations." PageRank-like tools have been applied in the study of biomolecular interaction networks to obtain further the relative significance of all molecules in the network. However, there is a great deal of inherent noise in widely accessible data sets for gene-to-gene associations or protein-protein interactions. How to develop robust tests to expand, filter, and rank molecular entities in disease-specific networks remains an ad hoc data analysis process. Results: We describe a new biomolecular characterization and prioritization tool called Weighted In-Network Node Expansion and Ranking (WINNER). It takes the input of any molecular interaction network data and generates an optionally expanded network with all the nodes ranked according to their relevance to one another in the network. To help users assess the robustness of results, WINNER provides two different types of statistics. The first type is a node-expansion p-value, which helps evaluate the statistical significance of adding "non-seed" molecules to the original biomolecular interaction network consisting of "seed" molecules and molecular interactions. The second type is a node-ranking p-value, which helps evaluate the relative statistical significance of the contribution of each node to the overall network architecture. We validated the robustness of WINNER in ranking top molecules by spiking noises in several network permutation experiments. We have found that node degree-preservation randomization of the gene network produced normally distributed ranking scores, which outperform those made with other gene network randomization techniques. Furthermore, we validated that a more significant proportion of the WINNER-ranked genes was associated with disease biology than existing methods such as PageRank. We demonstrated the performance of WINNER with a few case studies, including Alzheimer's disease, breast cancer, myocardial infarctions, and Triple negative breast cancer (TNBC). In all these case studies, the expanded and top-ranked genes identified by WINNER reveal disease biology more significantly than those identified by other gene prioritizing software tools, including Ingenuity Pathway Analysis (IPA) and DiAMOND. Conclusion: WINNER ranking strongly correlates to other ranking methods when the network covers sufficient node and edge information, indicating a high network quality. WINNER users can use this new tool to robustly evaluate a list of candidate genes, proteins, or metabolites produced from high-throughput biology experiments, as long as there is available gene/protein/metabolic network information.

Neuroendocrine signaling in the skin with a special focus on the epidermal neuropeptides.

The skin, which is comprised of the epidermis, dermis, and subcutaneous tissue, is the largest organ in the human body and it plays a crucial role in the regulation of the body's homeostasis. These functions are regulated by local neuroendocrine and immune systems with a plethora of signaling molecules produced by resident and immune cells. In addition, neurotransmitters, endocrine factors, neuropeptides, and cytokines released from nerve endings play a central role in the skin's responses to stress. These molecules act on the corresponding receptors in an intra-, juxta-, para-, or autocrine fashion. The epidermis as the outer most component of skin forms a barrier directly protecting against environmental stressors. This protection is assured by an intrinsic keratinocyte differentiation program, pigmentary system, and local nervous, immune, endocrine, and microbiome elements. These constituents communicate cross-functionally among themselves and with corresponding systems in the dermis and hypodermis to secure the basic epidermal functions to maintain local (skin) and global (systemic) homeostasis. The neurohormonal mediators and cytokines used in these communications regulate physiological skin functions separately or in concert. Disturbances in the functions in these systems lead to cutaneous pathology that includes inflammatory (i.e., psoriasis, allergic, or atopic dermatitis, etc.) and keratinocytic hyperproliferative disorders (i.e., seborrheic and solar keratoses), dysfunction of adnexal structure (i.e., hair follicles, eccrine, and sebaceous glands), hypersensitivity reactions, pigmentary disorders (vitiligo, melasma, and hypo- or hyperpigmentary responses), premature aging, and malignancies (melanoma and nonmelanoma skin cancers). These cellular, molecular, and neural components preserve skin integrity and protect against skin pathologies and can act as "messengers of the skin" to the central organs, all to preserve organismal survival.

Vitamin D Signaling in Psoriasis: Pathogenesis and Therapy.

Psoriasis is a systemic, chronic, immune-mediated disease that affects approximately 2-3% of the world's population. The etiology and pathophysiology of psoriasis are still unknown, but the activation of the adaptive immune system with the main role of T-cells is key in psoriasis pathogenesis. The modulation of the local neuroendocrine system with the downregulation of pro-inflammatory and the upregulation of anti-inflammatory messengers represent a promising adjuvant treatment in psoriasis therapies. Vitamin D receptors and vitamin D-mediated signaling pathways function in the skin and are essential in maintaining the skin homeostasis. The active forms of vitamin D act as powerful immunomodulators of clinical response in psoriatic patients and represent the effective and safe adjuvant treatments for psoriasis, even when high doses of vitamin D are administered. The phototherapy of psoriasis, especially UVB-based, changes the serum level of 25(OH)D, but the correlation of 25(OH)D changes and psoriasis improvement need more clinical trials, since contradictory data have been published. Vitamin D derivatives can improve the efficacy of psoriasis phototherapy without inducing adverse side effects. The anti-psoriatic treatment could include non-calcemic CYP11A1-derived vitamin D hydroxyderivatives that would act on the VDR or as inverse agonists on RORs or activate alternative nuclear receptors including AhR and LXRs. In conclusion, vitamin D signaling can play an important role in the natural history of psoriasis. Selective targeting of proper nuclear receptors could represent potential treatment options in psoriasis.

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.

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 .

PAGER Web APP: An Interactive, Online Gene Set and Network Interpretation Tool for Functional Genomics.

Functional genomics studies have helped researchers annotate differentially expressed gene lists, extract gene expression signatures, and identify biological pathways from omics profiling experiments conducted on biological samples. The current geneset, network, and pathway analysis (GNPA) web servers, e.g., DAVID, EnrichR, WebGestaltR, or PAGER, do not allow automated integrative functional genomic downstream analysis. In this study, we developed a new web-based interactive application, "PAGER Web APP", which supports online R scripting of integrative GNPA. In a case study of melanoma drug resistance, we showed that the new PAGER Web APP enabled us to discover highly relevant pathways and network modules, leading to novel biological insights. We also compared PAGER Web APP's pathway analysis results retrieved among PAGER, EnrichR, and WebGestaltR to show its advantages in integrative GNPA. The interactive online web APP is publicly accessible from the link, https://aimed-lab.shinyapps.io/PAGERwebapp/.

Melanoma, Melanin, and Melanogenesis: The Yin and Yang Relationship.

Melanin pigment plays a critical role in the protection against the harmful effects of ultraviolet radiation and other environmental stressors. It is produced by the enzymatic transformation of L-tyrosine to dopaquinone and subsequent chemical and biochemical reactions resulting in the formation of various 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindole (DHI) oligomers-main constituents of eumelanin, and benzothiazine and benzothiazole units of pheomelanin. The biosynthesis of melanin is regulated by sun exposure and by many hormonal factors at the tissue, cellular, and subcellular levels. While the presence of melanin protects against the development of skin cancers including cutaneous melanoma, its presence may be necessary for the malignant transformation of melanocytes. This shows a complex role of melanogenesis in melanoma development defined by chemical properties of melanin and the nature of generating pathways such as eu- and pheomelanogenesis. While eumelanin is believed to provide radioprotection and photoprotection by acting as an efficient antioxidant and sunscreen, pheomelanin, being less photostable, can generate mutagenic environment after exposure to the short-wavelength UVR. Melanogenesis by itself and its highly reactive intermediates show cytotoxic, genotoxic, and mutagenic activities, and it can stimulate glycolysis and hypoxia-inducible factor 1-alpha (HIF-1α) activation, which, combined with their immunosuppressive effects, can lead to melanoma progression and resistance to immunotherapy. On the other hand, melanogenesis-related proteins can be a target for immunotherapy. Interestingly, clinicopathological analyses on advanced melanomas have shown a negative correlation between tumor pigmentation and diseases outcome as defined by overall survival and disease-free time. This indicates a "Yin and Yang" role for melanin and active melanogenesis in melanoma development, progression, and therapy. Furthermore, based on the clinical, experimental data and diverse effects of melanogenesis, we propose that inhibition of melanogenesis in advanced melanotic melanoma represents a realistic adjuvant strategy to enhance immuno-, radio-, and chemotherapy.

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.

Revisiting the role of melatonin in human melanocyte physiology: A skin context perspective.

The evolutionarily ancient methoxyindoleamine, melatonin, has long perplexed investigators by its versatility of functions and mechanisms of action, which include the regulation of vertebrate pigmentation. Although first discovered through its potent skin-lightening effects in amphibians, melatonin's role in human skin and hair follicle pigmentation and its impact on melanocyte physiology remain unclear. Synthesizing our limited current understanding of this role, we specifically examine its impact on melanogenesis, oxidative biology, mitochondrial function, melanocyte senescence, and pigmentation-related clock gene activity, with emphasis on human skin, yet without ignoring instructive pointers from nonhuman species. Given the strict dependence of melanocyte functions on the epithelial microenvironment, we underscore that melanocyte responses to melatonin are best interrogated in a physiological tissue context. Current evidence suggests that melatonin and some of its metabolites inhibit both, melanogenesis (via reducing tyrosinase activity) and melanocyte proliferation by stimulating melatonin membrane receptors (MT1, MT2). We discuss whether putative melanogenesis-inhibitory effects of melatonin may occur via activation of Nrf2-mediated PI3K/AKT signaling, estrogen receptor-mediated and/or melanocortin-1 receptor- and cAMP-dependent signaling, and/or via melatonin-regulated changes in peripheral clock genes that regulate human melanogenesis, namely Bmal1 and Per1. Melatonin and its metabolites also accumulate in melanocytes where they exert net cyto- and senescence-protective as well as antioxidative effects by operating as free radical scavengers, stimulating the synthesis and activity of ROS scavenging enzymes and other antioxidants, promoting DNA repair, and enhancing mitochondrial function. We argue that it is clinically and biologically important to definitively clarify whether melanocyte cell culture-based observations translate into melatonin-induced pigmentary changes in a physiological tissue context, that is, in human epidermis and hair follicles ex vivo, and are confirmed by clinical trial results. After defining major open questions in this field, we close by suggesting how to begin answering them in clinically relevant, currently available preclinical in situ research models.

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.

Disturbed expression of vitamin D and retinoic acid-related orphan receptors α and γ and of megalin in inflammatory skin diseases.

The pathogenesis of inflammatory skin diseases is associated with the abnormal activity of keratinocytes and immune cells infiltrate. Vitamin D3 deficiency can correlate with the increased incidence, severity and duration of inflammatory skin disorders. The exact mechanism on how vitamin D3 influences inflammatory skin diseases still requires clarification. However, it can be associated with the disturbances in transmembrane glycoprotein-LRP2/megalin, which is implicated in vitamin D3 transport to the cell, and defects in vitamin D-signalling through the nuclear receptors. Therefore, by using immunohistochemistry, we analysed the expression of LRP2/megalin, VDR, RORα and RORγ in allergic contact dermatitis, lichen simplex chronicus, sarcoidosis and psoriasis in comparison with the normal skin. We observed decreased expression of LRP2/megalin in all inflammatory lesions in comparison with the normal skin. Significant differences were also noticed in VDR, RORα and RORγ levels between inflammatory lesions and normal skin. Our research indicates disturbed expression of LRP2/megalin, VDR, RORα and RORγ in inflammatory skin lesions in comparison with normal skin. Therefore, we suggest that changes in the activity of these proteins may play role in pathogenesis of inflammatory skin disorders. Furthermore, we suggest that LRP2/megalin, VDR, RORα and RORy may serve as targets in therapy of these diseases.

Vitamin D3 and its hydroxyderivatives as promising drugs against COVID-19: a computational study.

The epidemiologic correlation between the poor prognosis of SARS-CoV-2 infection and vitamin D deficiency has been observed worldwide, however, their molecular mechanisms are not fully understood. In this study, we used combined molecular docking, molecular dynamics simulations and binding free energy analyses to investigate the potentials of vitamin D3 and its hydroxyderivatives as TMPRSS2 inhibitor and to inhibit the SARS-CoV-2 receptor binding domain (RBD) binding to angiotensin-converting enzyme 2 (ACE2), as well as to unveil molecular and structural basis of 1,25(OH)2D3 capability to inhibit ACE2 and SARS-CoV-2 RBD interactions. The results show that vitamin D3 and its hydroxyderivatives are favorable to bind active site of TMPRSS2 and the binding site(s) between ACE2 and SARS-CoV2-RBD, which indicate that vitamin D3 and its biologically active hydroxyderivatives can serve as TMPRSS2 inhibitor and can inhibit ACE2 binding of SARS-CoV-2 RBD to prevent SARS-CoV-2 entry. Interaction of 1,25(OH)2D3 with SARS-CoV-2 RBD and ACE2 resulted in the conformation and dynamical motion changes of the binding surfaces between SARS-CoV-2 RBD and ACE2 to interrupt the binding of SARS-CoV-2 RBD with ACE2. The interaction of 1,25(OH)2D3 with TMPRSS2 also caused the conformational and dynamical motion changes of TMPRSS2, which could affect TMPRSS2 to prime SARS-CoV-2 spike proteins. Our results propose that vitamin D3 and its biologically active hydroxyderivatives are promising drugs or adjuvants in the treatment of COVID-19. Communicated by Ramaswamy H. Sarma.