Endogenous B-ring oxysterols inhibit the Hedgehog component Smoothened in a manner distinct from cyclopamine or side-chain oxysterols.

Cellular lipids are speculated to act as key intermediates in Hedgehog signal transduction, but their precise identity and function remain enigmatic. In an effort to identify such lipids, we pursued a Hedgehog pathway inhibitory activity that is particularly abundant in flagellar lipids of Chlamydomonas reinhardtii, resulting in the purification and identification of ergosterol endoperoxide, a B-ring oxysterol. A mammalian analog of ergosterol, 7-dehydrocholesterol (7-DHC), accumulates in Smith-Lemli-Opitz syndrome, a human genetic disease that phenocopies deficient Hedgehog signaling and is caused by genetic loss of 7-DHC reductase. We found that depleting endogenous 7-DHC with methyl-ß-cyclodextrin treatment enhances Hedgehog activation by a pathway agonist. Conversely, exogenous addition of 3ß,5α-dihydroxycholest-7-en-6-one, a naturally occurring B-ring oxysterol derived from 7-DHC that also accumulates in Smith-Lemli-Opitz syndrome, blocked Hedgehog signaling by inhibiting activation of the essential transduction component Smoothened, through a mechanism distinct from Smoothened modulation by other lipids.

7-Dehydrocholesterol (7-DHC), But Not Cholesterol, Causes Suppression of Canonical TGF-ß Signaling and Is Likely Involved in the Development of Atherosclerotic Cardiovascular Disease (ASCVD).

For several decades, cholesterol has been thought to cause ASCVD. Limiting dietary cholesterol intake has been recommended to reduce the risk of the disease. However, several recent epidemiological studies do not support a relationship between dietary cholesterol and/or blood cholesterol and ASCVD. Consequently, the role of cholesterol in atherogenesis is now uncertain. Much evidence indicates that TGF-ß, an anti-inflammatory cytokine, protects against ASCVD and that suppression of canonical TGF-ß signaling (Smad2-dependent) is involved in atherogenesis. We had hypothesized that cholesterol causes ASCVD by suppressing canonical TGF-ß signaling in vascular endothelium. To test this hypothesis, we determine the effects of cholesterol, 7-dehydrocholesterol (7-DHC; the biosynthetic precursor of cholesterol), and other sterols on canonical TGF-ß signaling. We use Mv1Lu cells (a model cell system for studying TGF-ß activity) stably expressing the Smad2-dependent luciferase reporter gene. We demonstrate that 7-DHC (but not cholesterol or other sterols) effectively suppresses the TGF-ß-stimulated luciferase activity. We also demonstrate that 7-DHC suppresses TGF-ß-stimulated luciferase activity by promoting lipid raft/caveolae formation and subsequently recruiting cell-surface TGF-ß receptors from non-lipid raft microdomains to lipid rafts/caveolae where TGF-ß receptors become inactive in transducing canonical signaling and undergo rapid degradation upon TGF-ß binding. We determine this by cell-surface 125 I-TGF-ß-cross-linking and sucrose density gradient ultracentrifugation. We further demonstrate that methyl-ß-cyclodextrin (MßCD), a sterol-chelating agent, reverses 7-DHC-induced suppression of TGF-ß-stimulated luciferase activity by extrusion of 7-DHC from resident lipid rafts/caveolae. These results suggest that 7-DHC, but not cholesterol, promotes lipid raft/caveolae formation, leading to suppression of canonical TGF-ß signaling and atherogenesis. J. Cell. Biochem. 118: 1387-1400, 2017. © 2016 Wiley Periodicals, Inc.

Titanium implants coated with UV-irradiated vitamin D precursor and vitamin E: in vivo performance and coating stability.

OBJECTIVES: This study aimed at evaluating the biological response of titanium implants coated with UV-irradiated 7-dehydrocholesterol (7-DHC) and vitamin E (VitE) in vivo and analyzing the effects of aging on their stability and bioactivity in vitro. MATERIAL AND METHODS: Titanium surfaces were coated with 7-DHC and VitE, UV-irradiated and incubated for 48 h at 23°C to allow cholecalciferol synthesis. The in vivo biological response was tested using a rabbit tibia model after 8 weeks of healing by analyzing the wound fluid and the mRNA levels of several markers at the bone-implant interface (N = 8). The stability of the coating after storage up to 12 weeks was determined using HPLC analysis, and the bioactivity of the stored modified implants was studied by an in vitro study with MC3T3-E1 cells (N = 6). RESULTS: A significant increase in gene expression levels of osteocalcin was found in the bone tissue attached to implants coated with the low dose of 7-DHC and VitE, together with a higher ALP activity in the wound fluid. Implants treated with the high dose of 7-DHC and VitE showed increased tissue necrosis and inflammation. Regarding the aging effects, coated implants were stable and bioactive up to 12 weeks when stored at 4°C and avoiding oxygen, light and moisture. CONCLUSION: This study demonstrates that Ti implants coated with UV-irradiated 7-DHC and VitE promote in vivo gene expression of bone formation markers and ALP activity, while they keep their osteopromotive potential in vitro and composition when stored up to 12 weeks at 4°C.

Differential cytotoxic effects of 7-dehydrocholesterol-derived oxysterols on cultured retina-derived cells: Dependence on sterol structure, cell type, and density.

Tissue accumulation of 7-dehydrocholesterol (7DHC) is a hallmark of Smith-Lemli-Opitz Syndrome (SLOS), a human inborn error of the cholesterol (CHOL) synthesis pathway. Retinal 7DHC-derived oxysterol formation occurs in the AY9944-induced rat model of SLOS, which exhibits a retinal degeneration characterized by selective loss of photoreceptors and associated functional deficits, Müller cell hypertrophy, and engorgement of the retinal pigment epithelium (RPE) with phagocytic inclusions. We evaluated the relative effects of four 7DHC-derived oxysterols on three retina-derived cell types in culture, with respect to changes in cellular morphology and viability. 661W (photoreceptor-derived) cells, rMC-1 (Müller glia-derived) cells, and normal diploid monkey RPE (mRPE) cells were incubated for 24 h with dose ranges of either 7-ketocholesterol (7kCHOL), 5,9-endoperoxy-cholest-7-en-3ß,6α-diol (EPCD), 3ß,5α-dihydroxycholest-7-en-6-one (DHCEO), or 4ß-hydroxy-7-dehydrocholesterol (4HDHC); CHOL served as a negative control (same dose range), along with appropriate vehicle controls, while staurosporine (Stsp) was used as a positive cytotoxic control. For 661W cells, the rank order of oxysterol potency was: EPCD > 7kCHOL >> DHCEO > 4HDHC ≈ CHOL. EC50 values were higher for confluent vs. subconfluent cultures. 661W cells exhibited much higher sensitivity to EPCD and 7kCHOL than either rMC-1 or mRPE cells, with the latter being the most robust when challenged, either at confluence or in sub-confluent cultures. When tested on rMC-1 and mRPE cells, EPCD was again an order of magnitude more potent than 7kCHOL in compromising cellular viability. Hence, 7DHC-derived oxysterols elicit differential cytotoxicity that is dose-, cell type-, and cell density-dependent. These results are consistent with the observed progressive, photoreceptor-specific retinal degeneration in the rat SLOS model, and support the hypothesis that 7DHC-derived oxysterols are causally linked to that retinal degeneration as well as to SLOS.

Improved human gingival fibroblast response to titanium implants coated with ultraviolet-irradiated vitamin D precursor and vitamin E.

BACKGROUND AND OBJECTIVE: Ultraviolet (UV)-irradiated 7-dehydrocholesterol (7-DHC) and vitamin E (VitE)-coated titanium (Ti) implants have a beneficial effect on bone cells. Human gingival fibroblasts (HGFs) are the most abundant cells in periodontal tissues and are involved in the wound healing and repair. The objective of this study was to evaluate the response of HGFs to Ti implants coated with UV-irradiated 7-DHC and VitE, for improved soft-tissue integration of dental implants. MATERIAL AND METHODS: Ti surfaces were coated with 7-DHC and VitE, irradiated with UV light and incubated for 48 h at 23°C to allow cholecalciferol (D3 ) synthesis from 7-DHC onto the Ti surface. HGFs were cultured on the modified surfaces and the influence of the coating on these cells was evaluated through the analysis of: (i) biocompatibility; (ii) the mRNA levels of genes involved in the composition and turnover of the extracellular matrix, the inflammatory response, periodontal bone resorption and wound healing; and (iii) the levels of MMP-1 and TIMP-1 proteins. RESULTS: We found a beneficial effect of UV-irradiated 7-DHC:VitE-coated Ti implants on HGFs. Besides being biocompatible with HGFs, the UV-irradiated 7-DHC and VitE coating increased the levels of collagen III α1 and fibronectin mRNAs. and decreased the level of interleukin-8 mRNA. TIMP-1 was increased at both mRNA and protein levels in HGFs cultured on UV-irradiated 7-DHC:VitE-coated Ti implants. Finally, the UV-irradiated 7-DHC and VitE coating decreased the level of RANKL mRNA in HGFs. CONCLUSION: UV-irradiated 7-DHC:VitE-coated Ti implants have a positive effect on HGFs in vitro by reducing the inflammatory response and extracellular matrix breakdown.

Steroid constituents from the soft coral Sinularia microspiculata.

A methanol extract of the soft coral Sinularia microspiculata revealed five sterols, including two new compounds. Using combined chromatographic and spectroscopic experiments, the new compounds were found to be 7-oxogorgosterol (1) and 16α-hydroxysarcosterol (2). Their structures were determined on the basis of spectroscopic data ((1)H and (13)C NMR, HSQC, HMBC, (1)H-(1)H COSY, NOESY, and FT-ICR-MS) and by comparing obtained results to the values indicated in previous studies. Among the isolated compounds, 3 showed weak cytotoxic effects against HL-60 (IC50  =  89.02  ±  9.93 µM) cell line, whereas 5 was weakly active against HL-60 (IC50  =  82.80  ±  13.65 µM) and SK-Mel2 (IC50  =  72.32  ±  1.30 µM) cell lines.

The cholesterol trafficking protein NPC1 is required for Drosophila spermatogenesis.

Niemann-Pick C (NPC) disease is a lethal neurodegenerative disorder affecting cellular sterol trafficking. Besides neurodegeneration, NPC patients also exhibit other pleiotropic conditions, indicating that NPC protein is required for other physiological processes. Previous studies indicated that a sterol shortage that in turn leads to a shortage of steroid hormones (for example, ecdysone in Drosophila) is likely to be the cause of NPC disease pathology. We have shown that mutations in Drosophila npc1, one of the two NPC disease-related genes, leads to larval lethal and male infertility. Here, we reported that npc1 mutants are defective in spermatogenesis and in particular in the membrane-remodeling individualization process. Interestingly, we found that ecdysone, the steroid hormone responsible for the larval lethal phenotype in npc1 mutants, is not required for individualization. However, supplying 7-dehydrocholesterol can partially rescue the male infertility of npc1 mutants, suggesting that a sterol shortage is responsible for the spermatogenesis defects. In addition, the individualization defects of npc1 mutants were enhanced at high temperature, suggesting that the sterol shortage may lead to temperature-sensitive defects in the membrane-remodeling process. Together, our study reveals a sterol-dependent, ecdysone-independent mechanism of NPC1 function in Drosophila spermatogenesis.

DHCEO accumulation is a critical mediator of pathophysiology in a Smith-Lemli-Opitz syndrome model.

Smith-Lemli-Opitz syndrome (SLOS) is an inborn error of metabolism caused by defective cholesterol biosynthesis. Mutations within the gene encoding 7-dehydrocholesterol reductase (DHCR7), the last enzyme in the pathway, lead to the accumulation of 7-dehydrocholesterol (7-DHC) in the brain tissue and blood of the SLOS patients. The objective of this study was to determine the consequences of the accumulation of an immediate cholesterol precursor, 7-DHC and its oxysterol metabolite, 3ß,5α-dihydroxycholest-7-en-6-one (DHCEO), in the brain tissue of Dhcr7-KO mouse, a model for SLOS. We found that cholesterol, 7-DHC and DHCEO show region-specific distribution, suggesting that the midbrain and the cortex are the primary sites of vulnerability. We also report that neurons are ten fold more susceptible to a 7-DHC-derived oxysterol mixture than glial cells, and that DHCEO accelerates differentiation and arborization of cortical neurons. The overall results suggest that 7-DHC oxidative metabolites are critical contributors to altered neural development in SLOS. The future studies will test if antioxidant supplementation will ameliorate some of the clinical symptoms associated with this devastating disease.

Hormonal control of C. elegans dauer formation and life span by a Rieske-like oxygenase.

C. elegans diapause, gonadal outgrowth, and life span are regulated by a lipophilic hormone, which serves as a ligand to the nuclear hormone receptor DAF-12. A key step in hormone production is catalyzed by the CYP450 DAF-9, but the extent of the biosynthetic pathway is unknown. Here, we identify a conserved Rieske-like oxygenase, DAF-36, as a component in hormone metabolism. Mutants display larval developmental and adult aging phenotypes, as well as patterns of epistasis similar to that of daf-9. Larval phenotypes are potently reversed by crude lipid extracts, 7-dehydrocholesterol, and a recently identified DAF-12 sterol ligand, suggesting that DAF-36 works early in the hormone biosynthetic pathway. DAF-36 is expressed primarily within the intestine, a major organ of metabolic and endocrine control, distinct from DAF-9. These results imply that C. elegans hormone production has multiple steps and is distributed, and that it may provide one way that tissues register their current physiological state during organismal commitments.

Biological activities of 7-dehydrocholesterol-derived oxysterols: implications for Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is a metabolic and developmental disorder caused by mutations in the gene encoding the enzyme 7-dehydrocholesterol reductase (Dhcr7). This reductase catalyzes the last step in cholesterol biosynthesis, and levels of 7-dehydrocholesterol (7-DHC), the substrate for this enzyme, are elevated in SLOS patients as a result of this defect. Our group has previously shown that 7-DHC is extremely prone to free radical autoxidation, and we identified about a dozen different oxysterols formed from oxidation of 7-DHC. We report here that 7-DHC-derived oxysterols reduce cell viability in a dose- and time-dependent manner, some of the compounds showing activity at sub-micromolar concentrations. The reduction of cell survival is caused by a combination of reduced proliferation and induced differentiation of the Neuro2a cells. The complex 7-DHC oxysterol mixture added to control Neuro2a cells also triggers the gene expression changes that were previously identified in Dhcr7-deficient Neuro2a cells. Based on the identification of overlapping gene expression changes in Dhcr7-deficient and 7-DHC oxysterol-treated Neuro2a cells, we hypothesize that some of the pathophysiological findings in the mouse SLOS model and SLOS patients might be due to accumulated 7-DHC oxysterols.

Inhibition of cholesterol biosynthesis disrupts lipid raft/caveolae and affects insulin receptor activation in 3T3-L1 preadipocytes.

Lipid rafts are plasma membrane microdomains that are highly enriched with cholesterol and sphingolipids and in which various receptors and other proteins involved in signal transduction reside. In the present work, we analyzed the effect of cholesterol biosynthesis inhibition on lipid raft/caveolae composition and functionality and assessed whether sterol precursors of cholesterol could substitute for cholesterol in lipid rafts/caveolae. 3T3-L1 preadipocytes were treated with distal inhibitors of cholesterol biosynthesis or vehicle (control) and then membrane rafts were isolated by sucrose density gradient centrifugation. Inhibition of cholesterol biosynthesis with either SKF 104976, AY 9944, 5,22-cholestadien-3beta-ol or triparanol, which inhibit different enzymes on the pathway, led to a marked reduction in cholesterol content and accumulation of different sterol intermediates in both lipid rafts and non-raft domains. These changes in sterol composition were accompanied by disruption of lipid rafts, with redistribution of caveolin-1 and Fyn, impairment of insulin-Akt signaling and the inhibition of insulin-stimulated glucose transport. Cholesterol repletion abrogated the effects of cholesterol biosynthesis inhibitors, reflecting they were specific. Our results show that cholesterol is required for functional raft-dependent insulin signaling.

Antibacterial sphingolipid and steroids from the black coral Antipathes dichotoma.

From the black coral Antipathies dichotoma, a sphingolipid (2S*,3S*,4E,8E)-2N-[tetradecanoyl]-4(E),8(E)-icosadiene-1,3-diol (1) and a steroid (22E)-methylcholesta-5,22-diene-1α,3ß,7α-triol (2) were isolated. Other known compounds, 3ß,7α-dihydroxy-cholest-5-ene (3), (22E,24S),5α,8α-epidioxy-24-methylcholesta-6,22-dien-3ß-ol (4) and (22E,24S),5α,8α-epidioxy-24-methylcholesta-6,9(11),22-trien-3ß-ol (5). The structures were established on the basis of NMR spectroscopic analysis and comparison with literature. The antibacterial activity of five compounds was evaluated.

Unusual sterolic mixture, and 24-isopropylcholesterol, from the sponge Ciocalypta sp. reduce cholesterol uptake and basolateral secretion in Caco-2 cells.

An unusual sterolic mixture (82.3% of 24-isopropylated sterols) and its major component, 24-isopropylcholesterol, isolated from a marine sponge, Ciocalypta sp. (Halichondriidae), reduce cholesterol uptake, basolateral secretion and ACAT-2 mRNA expression and increase the expression of ABCA1 mRNA in Caco-2 cells. The decreases of cholesterol uptake and secretion induced by 24-isopropylcholesterol alone were more than that of both the sterolic mixture and beta-sitosterol. These data add a new sterol, 24-isopropylcholesterol, to sterols that may reduce intestinal cholesterol absorption.

Repression of smoothened by patched-dependent (pro-)vitamin D3 secretion.

The developmentally important hedgehog (Hh) pathway is activated by binding of Hh to patched (Ptch1), releasing smoothened (Smo) and the downstream transcription factor glioma associated (Gli) from inhibition. The mechanism behind Ptch1-dependent Smo inhibition remains unresolved. We now show that by mixing Ptch1-transfected and Ptch1 small interfering RNA-transfected cells with Gli reporter cells, Ptch1 is capable of non-cell autonomous repression of Smo. The magnitude of this non-cell autonomous repression of Smo activity was comparable to the fusion of Ptch1-transfected cell lines and Gli reporter cell lines, suggesting that it is the predominant mode of action. CHOD-PAP analysis of medium conditioned by Ptch1-transfected cells showed an elevated 3beta-hydroxysteroid content, which we hypothesized to mediate the Smo inhibition. Indeed, the inhibition of 3beta-hydroxysteroid synthesis impaired Ptch1 action on Smo, whereas adding the 3beta-hydroxysteroid (pro-)vitamin D3 to the medium effectively inhibited Gli activity. Vitamin D3 bound to Smo with high affinity in a cyclopamine-sensitive manner. Treating zebrafish embryos with vitamin D3 mimicked the smo(-/-) phenotype, confirming the inhibitory action in vivo. Hh activates its signalling cascade by inhibiting Ptch1-dependent secretion of the 3beta-hydroxysteroid (pro-)vitamin D3. This action not only explains the seemingly contradictory cause of Smith-Lemli-Opitz syndrome (SLOS), but also establishes Hh as a unique morphogen, because binding of Hh on one cell is capable of activating Hh-dependent signalling cascades on other cells.

Incorporation of 7-dehydrocholesterol into liposomes as a simple, universal and efficient way to enhance anticancer activity by combining PDT and photoactivated chemotherapy.

Cholesterol (CHOL) is an indispensable component of liposomes. Incorporation of 7-dehydrocholesterol (7-DHC) instead of CHOL can efficiently enhance the anticancer activity of photosensitizer-encapsulated liposomes upon irradiation, yielding an IC50 value about half of that of CHOL-based controls. The photo-oxidation of 7-DHC into its endoperoxide form by singlet oxygen may account for the enhanced therapeutic effect, realizing an efficient combination of photodynamic therapy (PDT) and photoactivated chemotherapy.

Photosensitization of TRPA1 and TRPV1 by 7-dehydrocholesterol: implications for the Smith-Lemli-Opitz syndrome.

Loss-of-function mutations in the enzyme 7-dehydrocholesterol reductase are responsible for the Smith-Lemli-Opitz syndrome, in which 7-dehydrocholesterol (7-DHC) levels are markedly increased in the plasma and tissues of patients. This increase in 7-DHC is probably associated with the painful and itchy photosensitivity reported by the majority of patients with Smith-Lemli-Opitz syndrome. To identify the molecular targets involved in the activation and photosensitization of primary afferents by 7-DHC, we focused on TRPA1 and TRPV1, two ion channels expressed in nociceptive nerve endings and previously shown to respond to ultraviolet and visible light under pathophysiological circumstances. Recombinant human TRPA1 is activated and photosensitized in the presence of 7-DHC. Prolonged preexposure to 7-DHC causes more pronounced photosensitization, and while TRPV1 contributes less to the acute effect, it too becomes highly photosensitive upon preincubation with 7-DHC for 1 to 15 hours. Dorsal root ganglion neurons in primary culture display acute sensitivity to 7-DHC in the dark and also light-evoked responses in the presence of 7-DHC, which are exclusively dependent on TRPA1 and TRPV1. Similarly, prolonged exposure of mouse dorsal root ganglion neurons to 7-DHC renders these cells photosensitive in a largely TRPA1- and TRPV1-dependent manner. Single-fiber recordings in mouse skin-nerve preparations demonstrate violet light-evoked activation and a sensitization to 7-DHC exposure. Vice versa, 7-DHC pretreatment of the isolated trachea leads to a TRPA1- and TRPV1-dependent increase of the light-induced calcitonin gene-related peptide release. Taken together, our results implicate TRPA1 and TRPV1 channels as potential pharmacological targets to address the 7-DHC-induced hypersensitivity to light in patients.

1α-Hydroxy derivatives of 7-dehydrocholesterol are selective liver X receptor modulators.

The nuclear receptors liver X receptor (LXR) α and LXRß are involved in the regulation of lipid metabolism, inflammation, immunity, cellular proliferation, and apoptosis. Oxysterols are endogenous LXR ligands, and also interact with other nuclear and membrane receptors. We previously reported that a phytosterol derivative with a 1α-hydroxy group acts as a potent LXR agonist with intestine-selective action and that 25-hydroxy and 26/27-hydroxy metabolites of 7-dehydrocholesterol (7-DHC) exhibit partial LXR agonism. In this study, we report that 1α-hydroxy derivatives of 7-DHC, 1α-OH-7-DHC and 1,25-(OH)2-7-DHC, act as LXR modulators. Luciferase reporter gene assays showed that 1α-OH-7-DHC activates LXRα and LXRß and that 1,25-(OH)2-7-DHC activates both LXRs and vitamin D receptor. Examination of cofactor peptide association showed that the 1α-hydroxy derivatives, specifically 1,25-(OH)2-7-DHC, induce association of coactivator/corepressor peptide in a different manner from the agonist T0901317. Docking modeling and alanine mutational analysis of LXRα demonstrated that 1,25-(OH)2-7-DHC interacts with LXRα residues in a manner distinct from potent agonists, such as T0901317 and 24(S),25-epoxycholesterol. 1α-OH-7-DHC and 1,25-(OH)2-7-DHC induced expression of LXR target genes in a cell type- and gene-selective manner. 1,25-(OH)2-7-DHC effectively suppressed lipopolysaccharide-stimulated proinflammatory gene expression in an LXR-dependent manner. Therefore, 1α-hydroxy derivatives, such as 1,25-(OH)2-7-DHC, are unique LXR modulators with selective agonistic activity and potent transrepression function. These oxysterols have potential as LXR-targeted therapeutics for inflammatory disease.

Ultraviolet A induces apoptosis via reactive oxygen species in a model for Smith-Lemli-Opitz syndrome.

Solar ultraviolet A (UVA) radiation induces many responses in skin including oxidative stress, DNA damage, inflammation, and skin cancer. Smith-Lemli-Opitz syndrome (SLO-S) patients show dramatically enhanced immediate (5 min) and extended (24-48 h) skin inflammation in response to low UVA doses compared to normal skin. Mutations in Delta7-dehydrocholesterol reductase, which converts 7-dehydrocholesterol to cholesterol, produces high levels of 7-dehydrocholesterol in SLO-S patient's serum. Since 7-dehydrocholesterol is more rapidly oxidized than cholesterol, we hypothesized that 7-dehydrocholesterol enhances UVA-induced oxidative stress leading to keratinocyte death and inflammation. When keratinocytes containing high 7-dehydrocholesterol and low cholesterol were exposed to UVA (10 J/cm2), eightfold greater reactive oxygen species (ROS) were produced than in normal keratinocytes after 15 min. UVA induced 7-dehydrocholesterol concentration-dependent cell death at 24 h. These responses were inhibited by antioxidants, reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor (diphenyleneiodonium) and a mitochondria-specific radical quencher. Cell death was characterized by activation of caspases-3, -8, and -9 and by phosphatidylserine translocation. Studies using antioxidants and specific caspase inhibitors indicated that activation of caspase-8, but not caspase-9, mediates ROS-dependent caspase-3 activation and suggested that ROS from NADPH oxidase activate caspase-8. These results support a ROS-mediated apoptotic mechanism for the enhanced UVA-induced inflammation in SLO-S patients.

7-Dehydrocholesterol enhances ultraviolet A-induced oxidative stress in keratinocytes: roles of NADPH oxidase, mitochondria, and lipid rafts.

Long wavelength solar UVA radiation stimulates formation of reactive oxygen species (ROS) and prostaglandin E(2) (PGE(2)), which are involved in skin photosensitivity and tumor promotion. High levels of 7-dehydrocholesterol (7-DHC), the precursor to cholesterol, cause exaggerated photosensitivity to UVA in patients with Smith-Lemli-Opitz syndrome (SLOS). Partially replacing cholesterol with 7-DHC in keratinocytes rapidly (<5 min) increased UVA-induced ROS, intracellular calcium, phospholipase A(2) activity, PGE(2), and NADPH oxidase activity. UVA-induced ROS and PGE(2) production were inhibited in these cells by depleting the Nox1 subunit of NADPH oxidase using siRNA or using a mitochondrial radical quencher, MitoQ. Partial replacement of cholesterol with 7-DHC also disrupted membrane lipid raft domains, although depletion of cholesterol, which also disrupts lipid rafts, did not affect UVA-induced increases in ROS and PGE(2). Phospholipid liposomes containing 7-DHC were more rapidly oxidized by a free radical mechanism than those containing cholesterol. These results indicate that 7-DHC enhances rapid UVA-induced ROS and PGE(2) formation by enhancing free radical-mediated membrane lipid oxidation and suggests that this mechanism might underlie the UVA photosensitivity in SLOS.

Oral intake of 7-dehydrocholesterol increases vitamin D3 concentrations in the liver and kidney.

INTRODUCTION: Due to the high prevalence of vitamin D deficiency, strategies are needed to improve vitamin D status. Food components can affect vitamin D metabolism and have to be considered when estimating the efficacy of vitamin D supplements. 7-dehydrocholesterol (7-DHC) occurs naturally in food, but its impact on vitamin D metabolism has not yet been examined. METHODS: Three groups of male C57BL/6 mice (n=12 per group) were placed on a diet that contained 0, 2.5 or 5mg 7-DHC per kg diet over a period of 6 weeks. Vitamin D and other sterols in the serum, skin, liver and kidney were quantified by LC-MS/MS. The relative mRNA abundance of hepatic genes encoding vitamin D hydroxylation enzymes and transporters was analyzed by real-time RT-PCR. RESULTS: We found a substantial dose-dependent increase of non-hydroxylated vitamin D3 in the liver and kidney of mice fed a diet containing 7-DHC. The vitamin D3 content in the liver was 2.80±0.61pmol/g, 7.34±4.28pmol/g and 12.9±3.58pmol/g in groups that received 0, 2.5 and 5mg/kg 7-DHC, respectively. In the kidney, the vitamin D3 content of these groups was 1.78±1.17pmol/g, 3.55±1.06 and 6.36±2.29pmol/g, respectively. The serum and tissue concentrations of 25-hydroxyvitamin D3 (25(OH)D3) remained unaffected by 7-DHC. The relative mRNA data provided no plausible mechanism for the observed effects of 7-DHC on vitamin D3. All groups of mice had similar concentrations of cholesterol, desmosterol and 7-DHC in their serum and tissues. CONCLUSION: The current findings provide the first evidence that dietary 7-DHC seems to affect vitamin D metabolism. The underlying mechanism remains elusive and needs further investigation.