RESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Deshidrocolesteroles/farmacología , Liposomas/farmacología , Fármacos Fotosensibilizantes/farmacología , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Deshidrocolesteroles/química , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Liposomas/química , Imagen Óptica , Tamaño de la Partícula , Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Especies Reactivas de Oxígeno/análisis , Especies Reactivas de Oxígeno/metabolismoRESUMEN
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.
Asunto(s)
Deshidrocolesteroles/farmacología , Síndrome de Smith-Lemli-Opitz/tratamiento farmacológico , Canal Catiónico TRPA1/efectos de los fármacos , Canales Catiónicos TRPV/efectos de los fármacos , Canales de Potencial de Receptor Transitorio/efectos de los fármacos , Animales , Células Cultivadas , Ganglios Espinales/efectos de los fármacos , Masculino , Ratones , Neuronas/efectos de los fármacosRESUMEN
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.
Asunto(s)
Calcitriol/farmacología , Colesterol/análogos & derivados , Deshidrocolesteroles/farmacología , Hidrocarburos Fluorados/farmacología , Receptores X del Hígado/genética , Sulfonamidas/farmacología , Células CACO-2 , Calcitriol/química , Línea Celular Tumoral , Colesterol/química , Colesterol/farmacología , Deshidrocolesteroles/química , Regulación de la Expresión Génica , Genes Reporteros , Células HEK293 , Células Hep G2 , Humanos , Hidrocarburos Fluorados/química , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Receptores X del Hígado/agonistas , Receptores X del Hígado/química , Receptores X del Hígado/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Células MCF-7 , Simulación del Acoplamiento Molecular , Especificidad de Órganos , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Transducción de Señal , Relación Estructura-Actividad , Sulfonamidas/químicaRESUMEN
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.
Asunto(s)
Colecalciferol/metabolismo , Materiales Biocompatibles Revestidos , Deshidrocolesteroles/farmacología , Implantes Dentales , Rayos Ultravioleta , Vitamina E/farmacología , Animales , Relación Dosis-Respuesta a Droga , Femenino , Expresión Génica/efectos de los fármacos , Osteocalcina/genética , ConejosRESUMEN
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.
Asunto(s)
Aterosclerosis/metabolismo , Colesterol/farmacología , Deshidrocolesteroles/farmacología , Factor de Crecimiento Transformador beta/metabolismo , Caveolas/metabolismo , Línea Celular , Humanos , Microdominios de Membrana/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína Smad2/metabolismoRESUMEN
Smith-Lemli-Opitz syndrome (SLOS) is a rare disorder of cholesterol synthesis. Affected individuals exhibit growth failure, intellectual disability and a broad spectrum of developmental malformations. Among them, renal agenesis or hypoplasia, decreased innervation of the gut, and ptosis are consistent with impaired Ret signaling. Ret is a receptor tyrosine kinase that achieves full activity when recruited to lipid rafts. Mice mutant for Ret are born with no kidneys and enteric neurons, and display sympathetic nervous system defects causing ptosis. Since cholesterol is a critical component of lipid rafts, here we tested the hypothesis of whether the cause of the above malformations found in SLOS is defective Ret signaling owing to improper lipid raft composition or function. No defects consistent with decreased Ret signaling were found in newborn Dhcr7(-/-) mice, or in Dhcr7(-/-) mice lacking one copy of Ret. Although kidneys from Dhcr7(-/-) mice showed a mild branching defect in vitro, GDNF was able to support survival and downstream signaling of sympathetic neurons. Consistently, GFRα1 correctly partitioned to lipid rafts in brain tissue. Finally, replacement experiments demonstrated that 7-DHC efficiently supports Ret signaling in vitro. Taken together, our findings do not support a role of Ret signaling in the pathogenesis of SLOS.
Asunto(s)
Deshidrocolesteroles/farmacología , Proteínas Proto-Oncogénicas c-ret/metabolismo , Transducción de Señal/efectos de los fármacos , Síndrome de Smith-Lemli-Opitz/tratamiento farmacológico , Síndrome de Smith-Lemli-Opitz/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Células Cultivadas , Colesterol/metabolismo , Modelos Animales de Enfermedad , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Microdominios de Membrana/metabolismo , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismoRESUMEN
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.
Asunto(s)
Deshidrocolesteroles/metabolismo , Proteínas Hedgehog/metabolismo , Transducción de Señal , Receptor Smoothened/metabolismo , Animales , Chlamydomonas reinhardtii/química , Deshidrocolesteroles/química , Deshidrocolesteroles/farmacología , Flagelos/química , Células HEK293 , Proteínas Hedgehog/genética , Humanos , Ratones , Células 3T3 NIH , Síndrome de Smith-Lemli-Opitz/genética , Síndrome de Smith-Lemli-Opitz/metabolismo , Receptor Smoothened/genética , Alcaloides de Veratrum/farmacología , beta-Ciclodextrinas/farmacologíaRESUMEN
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.
Asunto(s)
Antozoos/química , Antineoplásicos/aislamiento & purificación , Colesterol/análogos & derivados , Deshidrocolesteroles/aislamiento & purificación , Esteroides/administración & dosificación , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Colesterol/química , Colesterol/aislamiento & purificación , Colesterol/farmacología , Deshidrocolesteroles/química , Deshidrocolesteroles/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Células HL-60 , Humanos , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Esteroides/química , VietnamRESUMEN
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.
Asunto(s)
Deshidrocolesteroles/farmacología , Células Ependimogliales/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Oxiesteroles/farmacología , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Retina/citología , Animales , Recuento de Células , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Deshidrocolesteroles/química , Deshidrocolesteroles/metabolismo , Macaca mulatta , Modelos Animales , Ratas , Retina/metabolismo , Epitelio Pigmentado de la Retina/citologíaRESUMEN
Vitamin D metabolites are essential for bone regeneration and mineral homeostasis. The vitamin D precursor 7-dehydrocholesterol can be used after UV irradiation to locally produce active vitamin D by osteoblastic cells. Furthermore, UV-irradiated 7-dehydrocholesterol is a biocompatible coating for titanium implants with positive effects on osteoblast differentiation. In this study, we examined the impact of titanium implants surfaces coated with UV-irradiated 7-dehydrocholesterol on the osteogenic differentiation of human umbilical cord mesenchymal stem cells. First, the synthesis of cholecalciferol (D3) was achieved through the incubation of the UV-activated 7-dehydrocholesterol coating for 48 h at 23â. Further, we investigated in vitro the biocompatibility of this coating in human umbilical cord mesenchymal stem cells and its potential to enhance their differentiation towards the osteogenic lineage. Human umbilical cord mesenchymal stem cells cultured onto UV-irradiated 7-dehydrocholesterol-coated titanium implants surfaces, combined with osteogenic supplements, upregulated the gene expression of several osteogenic markers and showed higher alkaline phosphatase activity and calcein blue staining, suggesting increased mineralization. Thus, our results show that the use of UV irradiation on 7-dehydrocholesterol -treated titanium implants surfaces generates a bioactive coating that promotes the osteogenic differentiation of human umbilical cord mesenchymal stem cells, with regenerative potential for improving osseointegration in titanium-based bone anchored implants.
Asunto(s)
Deshidrocolesteroles/química , Deshidrocolesteroles/farmacología , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Osteogénesis/fisiología , Titanio/química , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/efectos de la radiación , Deshidrocolesteroles/efectos de la radiación , Femenino , Sangre Fetal/citología , Humanos , Ensayo de Materiales , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/fisiología , Osteoblastos/fisiología , Osteogénesis/efectos de los fármacos , Prótesis e Implantes , Propiedades de Superficie , Rayos UltravioletaRESUMEN
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.
Asunto(s)
Materiales Biocompatibles Revestidos/farmacología , Implantes Dentales , Fibroblastos/efectos de los fármacos , Encía/efectos de los fármacos , Titanio/química , Rayos Ultravioleta , Vitamina D/farmacología , Vitamina E/farmacología , Adulto , Pérdida de Hueso Alveolar/terapia , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Colágeno Tipo III/análisis , Deshidrocolesteroles/farmacología , Deshidrocolesteroles/efectos de la radiación , Femenino , Fibroblastos/química , Fibronectinas/análisis , Expresión Génica , Humanos , Interleucina-8/análisis , Masculino , Metaloproteinasa 1 de la Matriz/análisis , Persona de Mediana Edad , Ligando RANK/análisis , ARN Mensajero/análisis , Propiedades de Superficie , Inhibidor Tisular de Metaloproteinasa-1/análisis , Vitamina D/efectos de la radiación , Vitamina E/efectos de la radiación , Cicatrización de HeridasRESUMEN
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.
Asunto(s)
Colecalciferol/metabolismo , Deshidrocolesteroles/farmacología , Riñón/metabolismo , Hígado/metabolismo , Provitaminas/farmacología , Administración Oral , Alimentación Animal/análisis , Animales , Calcifediol/análisis , Calcifediol/sangre , Calcifediol/metabolismo , Colecalciferol/análisis , Colecalciferol/sangre , Colesterol/análisis , Colesterol/sangre , Colesterol/metabolismo , Deshidrocolesteroles/administración & dosificación , Suplementos Dietéticos/análisis , Masculino , Ratones Endogámicos C57BL , Provitaminas/administración & dosificación , Triglicéridos/análisis , Triglicéridos/sangre , Triglicéridos/metabolismoRESUMEN
Primary cicatricial alopecia (PCA) is a group of inflammatory hair disorders that cause scarring and permanent hair loss. Previous studies have implicated PPARγ, a transcription factor that integrates lipogenic and inflammatory signals, in the pathogenesis of PCA. However, it is unknown what triggers the inflammatory response in these disorders, whether the inflammation is a primary or secondary event in disease pathogenesis, and whether the inflammatory reaction reflects an autoimmune process. In this paper, we show that the cholesterol biosynthetic pathway is impaired in the skin and hair follicles of PCA patients. Treatment of hair follicle cells with BM15766, a cholesterol biosynthesis inhibitor, or 7-dehydrocholesterol (7-DHC), a sterol precursor, stimulates the expression of pro-inflammatory chemokine genes. Painting of mouse skin with 7-DHC or BM15766 inhibits hair growth, causes follicular plugging and induces the infiltration of inflammatory cells into the interfollicular dermis. Our results demonstrate that cholesterologenic changes within hair follicle cells trigger an innate immune response that is associated with the induction of toll-like receptor (TLR) and interferon (IFN) gene expression, and the recruitment of macrophages that surround the hair follicles and initiate their destruction. These findings reveal a previously unsuspected role for cholesterol precursors in PCA pathogenesis and identify a novel link between sterols and inflammation that may prove transformative in the diagnosis and treatment of these disorders.
Asunto(s)
Alopecia/genética , Colesterol/biosíntesis , Cicatriz/genética , Cabello/metabolismo , Inmunidad Innata/genética , Esteroles/metabolismo , Adolescente , Adulto , Alopecia/inmunología , Alopecia/patología , Animales , Vías Biosintéticas/genética , Células Cultivadas , Quimiocinas/genética , Colesterol/química , Cicatriz/inmunología , Cicatriz/patología , Deshidrocolesteroles/farmacología , Femenino , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Cabello/efectos de los fármacos , Cabello/crecimiento & desarrollo , Folículo Piloso/efectos de los fármacos , Folículo Piloso/metabolismo , Folículo Piloso/patología , Humanos , Inmunidad Innata/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Piperazinas/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esteroles/químicaRESUMEN
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.
Asunto(s)
Encéfalo/metabolismo , Colestenonas/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Síndrome de Smith-Lemli-Opitz/patología , Síndrome de Smith-Lemli-Opitz/fisiopatología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Encéfalo/patología , Supervivencia Celular , Células Cultivadas , Colestenonas/farmacología , Cromatografía Líquida de Alta Presión/métodos , Deshidrocolesteroles/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/deficiencia , Síndrome de Smith-Lemli-Opitz/genética , Espectrometría de Masas en TándemRESUMEN
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.
Asunto(s)
Proteínas Portadoras/fisiología , Colesterol/metabolismo , Proteínas de Drosophila/fisiología , Drosophila/fisiología , Espermatogénesis , Animales , Deshidrocolesteroles/farmacología , Ecdisona/fisiología , Femenino , Infertilidad Masculina/etiología , Masculino , Proteínas de la Membrana , Microscopía Electrónica de Transmisión , Proteína Niemann-Pick C1 , Receptores de Esteroides/fisiología , Temperatura , Testículo/ultraestructuraRESUMEN
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.
Asunto(s)
Antozoos/química , Antibacterianos/química , Deshidrocolesteroles/química , Esfingolípidos/química , Esteroides/química , Animales , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Deshidrocolesteroles/aislamiento & purificación , Deshidrocolesteroles/farmacología , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Conformación Molecular , Esfingolípidos/aislamiento & purificación , Esfingolípidos/farmacología , Esteroides/aislamiento & purificación , Esteroides/farmacologíaRESUMEN
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.
Asunto(s)
Deshidrocolesteroles/metabolismo , Síndrome de Smith-Lemli-Opitz/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Deshidrocolesteroles/farmacología , Relación Dosis-Respuesta a Droga , Perfilación de la Expresión Génica , Ratones , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/deficiencia , Peróxidos/química , Peróxidos/toxicidad , Síndrome de Smith-Lemli-Opitz/patología , Factores de TiempoRESUMEN
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.
Asunto(s)
Caveolas/efectos de los fármacos , Membrana Celular/metabolismo , Colesterol/biosíntesis , Microdominios de Membrana/efectos de los fármacos , Células 3T3-L1 , Animales , Caveolas/metabolismo , Caveolina 1/metabolismo , Deshidrocolesteroles/farmacología , Inhibidores Enzimáticos/farmacología , Gangliósido G(M1)/metabolismo , Lanosterol/análogos & derivados , Lanosterol/farmacología , Ratones , Receptor de Insulina/efectos de los fármacos , Receptor de Insulina/metabolismo , Triparanol/farmacología , Diclorhidrato de trans-1,4-Bis(2-clorobenzaminometil)ciclohexano/farmacologíaRESUMEN
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.
Asunto(s)
Colesterol/metabolismo , Esteroles/farmacología , Animales , Células CACO-2 , Deshidrocolesteroles/farmacología , Humanos , Absorción Intestinal/efectos de los fármacos , Poríferos , ARN Mensajero/análisis , Sitoesteroles/farmacología , Esterol O-Aciltransferasa/genética , Esterol O-Aciltransferasa 2RESUMEN
The serotonin1A receptor is an important member of the G-protein coupled receptor family, and is involved in the generation and modulation of a variety of cognitive, behavioral, and developmental functions. Solubilization of the hippocampal serotonin1A receptor by 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) is accompanied by loss of membrane cholesterol which results in a reduction in specific agonist binding activity. Replenishment of cholesterol to solubilized membranes restores the cholesterol content of the membrane and significantly enhances specific agonist binding activity. In order to test the stringency of the requirement of cholesterol in this process, we solubilized native hippocampal membranes followed by replenishment with 7-dehydrocholesterol (7-DHC). 7-DHC is an immediate biosynthetic precursor of cholesterol differing only in a double bond at the 7th position in its sterol ring. Our results show, for the first time, that replenishment of solubilized hippocampal membranes with 7-DHC does not restore ligand binding activity of the serotonin1A receptor, in spite of recovery of the overall membrane order. This observation shows that the requirement for restoration of ligand binding activity is more stringent than the requirement for the recovery of overall membrane order. These novel results have potential implications in understanding the interaction of membrane sterols with this important neuronal receptor under pathogenic conditions such as the Smith-Lemli-Opitz syndrome.