RESUMEN
Intestinal stromal cells (SCs), which synthesize the extracellular matrix that gives the mucosa its structure, are newly appreciated to play a role in mucosal inflammation. Here, we show that human intestinal vimentin+CD90+smooth muscle actin- SCs synthesize retinoic acid (RA) at levels equivalent to intestinal epithelial cells, a function in the human intestine previously attributed exclusively to epithelial cells. Crohn's disease SCs (Crohn's SCs), however, synthesized markedly less RA than SCs from healthy intestine (normal SCs). We also show that microbe-stimulated Crohn's SCs, which are more inflammatory than stimulated normal SCs, induced less RA-regulated differentiation of mucosal dendritic cells (DCs) (circulating pre-DCs and monocyte-derived DCs), leading to the generation of more potent inflammatory interferon-γhi/interleukin-17hi T cells than normal SCs. Explaining these results, Crohn's SCs expressed more DHRS3, a retinaldehyde reductase that inhibits retinol conversion to retinal and, thus, synthesized less RA than normal SCs. These findings uncover a microbe-SC-DC crosstalk in which luminal microbes induce Crohn's disease SCs to initiate and perpetuate inflammation through impaired synthesis of RA.
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The hair follicle (HF) is a self-renewing adult miniorgan that undergoes drastic metabolic and morphological changes during precisely timed cyclic organogenesis. The HF cycle is known to be regulated by steroid hormones, growth factors and circadian clock genes. Recent data also suggest a role for a vitamin A derivative, all-trans-retinoic acid (ATRA), the activating ligand of transcription factors, retinoic acid receptors, in the regulation of the HF cycle. Here we demonstrate that ATRA signaling cycles during HF regeneration and this pattern is disrupted by genetic deletion of epidermal retinol dehydrogenases 2 (RDHE2, SDR16C5) and RDHE2-similar (RDHE2S, SDR16C6) that catalyze the rate-limiting step in ATRA biosynthesis. Deletion of RDHEs results in accelerated anagen to catagen and telogen to anagen transitions, altered HF composition, reduced levels of HF stem cell markers, and dysregulated circadian clock gene expression, suggesting a broad role of RDHEs in coordinating multiple signaling pathways.
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Epidermis , Vitamina A , Adulto , Humanos , Vitamina A/farmacología , Cabello , Catálisis , Tretinoina , Células MadreRESUMEN
Rexinoids are agonists of nuclear rexinoid X receptors (RXR) that heterodimerize with other nuclear receptors to regulate gene transcription. A number of selective RXR agonists have been developed for clinical use but their application has been hampered by the unwanted side effects associated with the use of rexinoids and a limited understanding of their mechanisms of action across different cell types. Our previous studies showed that treatment of organotypic human epidermis with the low toxicity UAB30 and UAB110 rexinoids resulted in increased steady-state levels of all-trans-retinoic acid (ATRA), the obligatory ligand of the RXR-RAR heterodimers. Here, we investigated the molecular mechanism underlying the increase in ATRA levels using a dominant negative RXRα that lacks the activation function 2 (AF-2) domain. The results demonstrated that overexpression of dnRXRα in human organotypic epidermis markedly reduced signaling by resident ATRA, suggesting the existence of endogenous RXR ligand, diminished the biological effects of UAB30 and UAB110 on epidermis morphology and gene expression, and nearly abolished the rexinoid-induced increase in ATRA levels. Global transcriptome analysis of dnRXRα-rafts in comparison to empty vector-transduced rafts showed that over 95% of the differentially expressed genes in rexinoid-treated rafts constitute direct or indirect ATRA-regulated genes. Thus, the biological effects of UAB30 and UAB110 are mediated through the AF-2 domain of RXRα with minimal side effects in human epidermis. As ATRA levels are known to be reduced in certain epithelial pathologies, treatment with UAB30 and UAB110 may represent a promising therapy for normalizing the endogenous ATRA concentration and signaling in epithelial tissues.
Asunto(s)
Furilfuramida , Tretinoina , Humanos , Receptores X Retinoide/genética , Receptores X Retinoide/agonistas , Receptores X Retinoide/metabolismo , Ligandos , Tretinoina/farmacología , Tretinoina/metabolismo , Epidermis/metabolismo , Receptores Citoplasmáticos y NuclearesRESUMEN
Genes Sdr16c5 and Sdr16c6 encode proteins that belong to a superfamily of short-chain dehydrogenases/reductases (SDR16C5 and SDR16C6). Simultaneous inactivation of these genes in double-KO (DKO) mice was previously shown to result in a marked enlargement of the mouse Meibomian glands (MGs) and sebaceous glands, respectively. However, the exact roles of SDRs in physiology and biochemistry of MGs and sebaceous glands have not been established yet. Therefore, we characterized, for the first time, meibum and sebum of Sdr16c5/Sdr16c6-null (DKO) mice using high-resolution MS and LC. In this study, we demonstrated that the mutation upregulated the overall production of MG secretions (also known as meibogenesis) and noticeably altered their lipidomic profile, but had a more subtle effect on sebogenesis. The major changes in meibum of DKO mice included abnormal accumulation of shorter chain, sebaceous-type cholesteryl esters and wax esters (WEs), and a marked increase in the biosynthesis of monounsaturated and diunsaturated Meibomian-type WEs. Importantly, the MGs of DKO mice maintained their ability to produce typical extremely long chain Meibomian-type lipids at seemingly normal levels. These observations indicated preferential activation of a previously dormant biosynthetic pathway that produce shorter chain, and more unsaturated, sebaceous-type WEs in the MGs of DKO mice, without altering the elongation patterns of their extremely long chain Meibomian-type counterparts. We conclude that the Sdr16c5/Sdr16c6 pair may control a point of bifurcation in one of the meibogenesis subpathways at which biosynthesis of lipids can be redirected toward either abnormal sebaceous-type lipidome or normal Meibomian-type lipidome in WT mice.
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Glándulas Tarsales , Lágrimas , Animales , Ratones , Ésteres del Colesterol/metabolismo , Metabolismo de los Lípidos/fisiología , Espectrometría de Masas , Lágrimas/metabolismoRESUMEN
Retinoid X receptors (RXRs) are nuclear transcription factors that partner with other nuclear receptors to regulate numerous physiological processes. Although RXR represents a valid therapeutic target, only a few RXR-specific ligands (rexinoids) have been identified, in part due to the lack of clarity on how rexinoids selectively modulate RXR response. Previously, we showed that rexinoid UAB30 potentiates all-trans-retinoic acid (ATRA) signaling in human keratinocytes, in part by stimulating ATRA biosynthesis. Here, we examined the mechanism of action of next-generation rexinoids UAB110 and UAB111 that are more potent in vitro than UAB30 and the FDA-approved Targretin. Both UAB110 and UAB111 enhanced ATRA signaling in human organotypic epithelium at a 50-fold lower concentration than UAB30. This was consistent with the 2- to 5- fold greater increase in ATRA in organotypic epidermis treated with UAB110/111 versus UAB30. Furthermore, at 0.2 µM, UAB110/111 increased the expression of ATRA genes up to 16-fold stronger than Targretin. The less toxic and more potent UAB110 also induced more changes in differential gene expression than Targretin. Additionally, our hydrogen deuterium exchange mass spectrometry analysis showed that both ligands reduced the dynamics of the ligand-binding pocket but also induced unique dynamic responses that were indicative of higher affinity binding relative to UAB30, especially for Helix 3. UAB110 binding also showed increased dynamics towards the dimer interface through the Helix 8 and Helix 9 regions. These data suggest that UAB110 and UAB111 are potent activators of RXR-RAR signaling pathways but accomplish activation through different molecular responses to ligand binding.
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Tetrahidronaftalenos , Tretinoina , Humanos , Receptores X Retinoide/metabolismo , Bexaroteno , Ligandos , Tetrahidronaftalenos/farmacología , Tretinoina/farmacología , Tretinoina/metabolismo , Epidermis/metabolismoRESUMEN
Compound 1 is a potent rexinoid that is highly effective in cancer chemoprevention but elevates serum triglycerides. In an effort to separate the lipid toxicity from the anticancer activity of 1, we synthesized four new analogs of rexinoid 1, of which three rexinoids did not elevate serum triglycerides. Rexinoids 3 and 4 are twice as potent as rexinoid 1 in binding to Retinoid X receptor (RXR). All-trans retinoic acid (ATRA) plays a key role in maintaining skin homeostasis, and rexinoids 3-6 are highly effective in upregulating the genes responsible for the biosynthesis of ATRA. Inflammation plays a key role in skin cancer, and rexinoids 3 and 4 are highly effective in diminishing LPS-induced inflammation. Rexinoids 3 and 4 are highly effective in preventing UVB-induced nonmelanoma skin cancer (NMSC) without displaying any overt toxicities. Biophysical studies of rexinoids 3 and 5 bound to hRXRα-ligand binding domain (LBD) reveal important conformational and dynamical differences in the ligand binding domain.
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Neoplasias Cutáneas , Tetrahidronaftalenos , Humanos , Tetrahidronaftalenos/química , Ligandos , Receptores X Retinoide/metabolismo , Tretinoina/química , Tretinoina/metabolismo , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/prevención & control , Inflamación/tratamiento farmacológico , Inflamación/prevención & control , TriglicéridosRESUMEN
Bioactive oxylipins play multiple roles during inflammation and in the immune response, with termination of their actions partly dependent on the activity of yet-to-be characterized dehydrogenases. Here, we report that human microsomal dehydrogenase reductase 9 (DHRS9, also known as SDR9C4 of the short-chain dehydrogenase/reductase (SDR) superfamily) exhibits a robust oxidative activity toward oxylipins with hydroxyl groups located at carbons C9 and C13 of octadecanoids, C12 and C15 carbons of eicosanoids, and C14 carbon of docosanoids. DHRS9/SDR9C4 is also active toward lipid inflammatory mediator dihydroxylated Leukotriene B4 and proresolving mediators such as tri-hydroxylated Resolvin D1 and Lipoxin A4, although notably, with lack of activity on the 15-hydroxyl of prostaglandins. We also found that the SDR enzymes phylogenetically related to DHRS9, i.e., human SDR9C8 (or retinol dehydrogenase 16), the rat SDR9C family member known as retinol dehydrogenase 7, and the mouse ortholog of human DHRS9 display similar activity toward oxylipin substrates. Mice deficient in DHRS9 protein are viable, fertile, and display no apparent phenotype under normal conditions. However, the oxidative activity of microsomal membranes from the skin, lung, and trachea of Dhrs9-/- mice toward 1 µM Leukotriene B4 is 1.7- to 6-fold lower than that of microsomes from wild-type littermates. In addition, the oxidative activity toward 1 µM Resolvin D1 is reduced by about 2.5-fold with DHRS9-null microsomes from the skin and trachea. These results strongly suggest that DHRS9 might play an important role in the metabolism of a wide range of bioactive oxylipins in vivo.
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Oxilipinas , Deshidrogenasas-Reductasas de Cadena Corta , Animales , Leucotrieno B4/metabolismo , Ratones , Microsomas/metabolismo , Oxilipinas/metabolismo , Prostaglandinas , Ratas , Deshidrogenasas-Reductasas de Cadena Corta/genética , Deshidrogenasas-Reductasas de Cadena Corta/metabolismoRESUMEN
Elevated aldehyde dehydrogenase (ALDH) activity correlates with poor outcome for many solid tumors as ALDHs may regulate cell proliferation and chemoresistance of cancer stem cells (CSCs). Accordingly, potent, and selective inhibitors of key ALDH enzymes may represent a novel CSC-directed treatment paradigm for ALDH+ cancer types. Of the many ALDH isoforms, we and others have implicated the elevated expression of ALDH1A3 in mesenchymal glioma stem cells (MES GSCs) as a target for the development of novel therapeutics. To this end, our structure of human ALDH1A3 combined with in silico modeling identifies a selective, active-site inhibitor of ALDH1A3. The lead compound, MCI-INI-3, is a selective competitive inhibitor of human ALDH1A3 and shows poor inhibitory effect on the structurally related isoform ALDH1A1. Mass spectrometry-based cellular thermal shift analysis reveals that ALDH1A3 is the primary binding protein for MCI-INI-3 in MES GSC lysates. The inhibitory effect of MCI-INI-3 on retinoic acid biosynthesis is comparable with that of ALDH1A3 knockout, suggesting that effective inhibition of ALDH1A3 is achieved with MCI-INI-3. Further development is warranted to characterize the role of ALDH1A3 and retinoic acid biosynthesis in glioma stem cell growth and differentiation.
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Aldehído Oxidorreductasas/antagonistas & inhibidores , Glioma/metabolismo , Células Madre Neoplásicas/metabolismo , Tretinoina/metabolismo , HumanosRESUMEN
The hetero-oligomeric retinoid oxidoreductase complex (ROC) catalyzes the interconversion of all-trans-retinol and all-trans-retinaldehyde to maintain the steady-state output of retinaldehyde, the precursor of all-trans-retinoic acid that regulates the transcription of numerous genes. The interconversion is catalyzed by two distinct components of the ROC: the NAD(H)-dependent retinol dehydrogenase 10 (RDH10) and the NADP(H)-dependent dehydrogenase reductase 3 (DHRS3). The binding between RDH10 and DHRS3 subunits in the ROC results in mutual activation of the subunits. The molecular basis for their activation is currently unknown. Here, we applied site-directed mutagenesis to investigate the roles of amino acid residues previously implied in subunit interactions in other SDRs to obtain the first insight into the subunit interactions in the ROC. The results of these studies suggest that the cofactor binding to RDH10 subunit is critical for the activation of DHRS3 subunit and vice versa. The C-terminal residues 317-331 of RDH10 are critical for the activity of RDH10 homo-oligomers but not for the binding to DHRS3. The C-terminal residues 291-295 are required for DHRS3 subunit activity of the ROC. The highly conserved C-terminal cysteines appear to be involved in inter-subunit communications, affecting the affinity of the cofactor binding site in RDH10 homo-oligomers as well as in the ROC. Modeling of the ROC quaternary structure based on other known structures of SDRs suggests that its integral membrane-associated subunits may be inserted in adjacent membranes of the endoplasmic reticulum (ER), making the formation and function of the ROC dependent on the dynamic nature of the tubular ER network.
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Oxidorreductasas de Alcohol/metabolismo , Carbonil Reductasa (NADPH)/metabolismo , Proteínas de la Membrana/metabolismo , Retinaldehído/metabolismo , Tretinoina/metabolismo , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/genética , Secuencia de Aminoácidos , Animales , Biocatálisis , Carbonil Reductasa (NADPH)/química , Carbonil Reductasa (NADPH)/genética , Dominio Catalítico , Retículo Endoplásmico/metabolismo , Células HEK293 , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Mutagénesis Sitio-Dirigida/métodos , Estructura Cuaternaria de Proteína , Spodoptera/citología , Relación Estructura-ActividadRESUMEN
Hair follicles cycle through periods of growth (anagen), regression (catagen), rest (telogen), and release (exogen). Telogen is further divided into refractory and competent telogen based on expression of bone morphogenetic protein 4 (BMP4) and wingless-related MMTV integration site 7A (WNT7A). During refractory telogen hair follicle stem cells (HFSC) are inhibited. Retinoic acid synthesis proteins localized to the hair follicle and this localization pattern changed throughout the hair cycle. In addition, excess retinyl esters arrested hair follicles in telogen. The purpose of this study was to further define these hair cycle changes. BMP4 and WNT7A expression was also used to distinguish refractory from competent telogen in C57BL/6J mice fed different levels of retinyl esters from two previous studies. These two studies produced opposite results; and differed in the amount of retinyl esters the dams consumed and the age of the mice when the different diet began. There were a greater percentage of hair follicles in refractory telogen both when mice were bred on an unpurified diet containing copious levels of retinyl esters (study 1) and consumed excess levels of retinyl esters starting at 12 weeks of age, as well as when mice were bred on a purified diet containing adequate levels of retinyl esters (study 2) and remained on this diet at 6 weeks of age. WNT7A expression was consistent with these results. Next, the localization of vitamin A metabolism proteins in the two stages of telogen was examined. Keratin 6 (KRT6) and cellular retinoic acid binding protein 2 (CRABP2) localized almost exclusively to refractory telogen hair follicles in study 1. However, KRT6 and CRABP2 localized to both competent and refractory telogen hair follicles in mice fed adequate and high levels of retinyl esters in study 2. In mice bred and fed an unpurified diet retinol dehydrogenase SDR16C5, retinal dehydrogenase 2 (ALDH1A2), and cytochrome p450 26B1 (CYP26B1), enzymes and proteins involved in RA metabolism, localized to BMP4 positive refractory telogen hair follicles. This suggests that vitamin A may contribute to the inhibition of HFSC during refractory telogen in a dose dependent manner.
RESUMEN
Liver is the central metabolic hub that coordinates carbohydrate and lipid metabolism. The bioactive derivative of vitamin A, retinoic acid (RA), was shown to regulate major metabolic genes including phosphoenolpyruvate carboxykinase, fatty acid synthase, carnitine palmitoyltransferase 1, and glucokinase among others. Expression levels of these genes undergo profound changes during adaptation to fasting or in metabolic diseases such as type 1 diabetes (T1D). However, it is unknown whether the levels of hepatic RA change during metabolic remodeling. This study investigated the dynamics of hepatic retinoid metabolism and signaling in the fed state, in fasting, and in T1D. Our results show that fed-to-fasted transition is associated with significant decrease in hepatic retinol dehydrogenase (RDH) activity, the rate-limiting step in RA biosynthesis, and downregulation of RA signaling. The decrease in RDH activity correlates with the decreased abundance and altered subcellular distribution of RDH10 while Rdh10 transcript levels remain unchanged. In contrast to fasting, untreated T1D is associated with upregulation of RA signaling and an increase in hepatic RDH activity, which correlates with the increased abundance of RDH10 in microsomal membranes. The dynamic changes in RDH10 protein levels in the absence of changes in its transcript levels imply the existence of posttranscriptional regulation of RDH10 protein. Together, these data suggest that the downregulation of hepatic RA biosynthesis, in part via the decrease in RDH10, is an integral component of adaptation to fasting. In contrast, the upregulation of hepatic RA biosynthesis and signaling in T1D might contribute to metabolic inflexibility associated with this disease.
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Oxidorreductasas de Alcohol/genética , Diabetes Mellitus Tipo 1/metabolismo , Retinoides/metabolismo , Tretinoina/metabolismo , Animales , Carnitina O-Palmitoiltransferasa/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patología , Modelos Animales de Enfermedad , Ayuno/metabolismo , Regulación Enzimológica de la Expresión Génica/genética , Glucoquinasa/genética , Humanos , Hígado/enzimología , Hígado/metabolismo , Metabolismo/genética , Ratones , Microsomas Hepáticos/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/genética , Retinoides/genética , Transducción de Señal/genéticaRESUMEN
BACKGROUND AND AIMS: 17-Beta hydroxysteroid dehydrogenase 13 (HSD17B13) is genetically associated with human nonalcoholic fatty liver disease (NAFLD). Inactivating mutations in HSD17B13 protect humans from NAFLD-associated and alcohol-associated liver injury, fibrosis, cirrhosis, and hepatocellular carcinoma, leading to clinical trials of anti-HSD17B13 therapeutic agents in humans. We aimed to study the in vivo function of HSD17B13 using a mouse model. APPROACH AND RESULTS: Single-cell RNA-sequencing and quantitative RT-PCR data revealed that hepatocytes are the main HSD17B13-expressing cells in mice and humans. We compared Hsd17b13 whole-body knockout (KO) mice and wild-type (WT) littermate controls fed regular chow (RC), a high-fat diet (HFD), a Western diet (WD), or the National Institute on Alcohol Abuse and Alcoholism model of alcohol exposure. HFD and WD induced significant weight gain, hepatic steatosis, and inflammation. However, there was no difference between genotypes with regard to body weight, liver weight, hepatic triglycerides (TG), histological inflammatory scores, expression of inflammation-related and fibrosis-related genes, and hepatic retinoid levels. Compared to WT, KO mice on the HFD had hepatic enrichment of most cholesterol esters, monoglycerides, and certain sphingolipid species. Extended feeding with the WD for 10 months led to extensive liver injury, fibrosis, and hepatocellular carcinoma, with no difference between genotypes. Under alcohol exposure, KO and WT mice showed similar hepatic TG and liver enzyme levels. Interestingly, chow-fed KO mice showed significantly higher body and liver weights compared to WT mice, while KO mice on obesogenic diets had a shift toward larger lipid droplets. CONCLUSIONS: Extensive evaluation of Hsd17b13 deficiency in mice under several fatty liver-inducing dietary conditions did not reproduce the protective role of HSD17B13 loss-of-function mutants in human NAFLD. Moreover, mouse Hsd17b13 deficiency induces weight gain under RC. It is crucial to understand interspecies differences prior to leveraging HSD17B13 therapies.
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17-Hidroxiesteroide Deshidrogenasas/deficiencia , Dieta Alta en Grasa/efectos adversos , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , Animales , Dieta Occidental/efectos adversos , Etanol/efectos adversos , Hígado Graso/etiología , Lípidos/análisis , Hígado/química , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Aumento de PesoRESUMEN
Human genetic studies recently identified an association of SNPs in the 17-ß hydroxysteroid dehydrogenase 13 (HSD17B13) gene with alcoholic and nonalcoholic fatty liver disease development. Mutant HSD17B13 variants devoid of enzymatic function have been demonstrated to be protective from cirrhosis and liver cancer, supporting the development of HSD17B13 as a promising therapeutic target. Previous studies have demonstrated that HSD17B13 is a lipid droplet (LD)-associated protein. However, the critical domains that drive LD targeting or determine the enzymatic activity have yet to be defined. Here we used mutagenesis to generate multiple truncated and point-mutated proteins and were able to demonstrate in vitro that the N-terminal hydrophobic domain, PAT-like domain, and a putative α-helix/ß-sheet/α-helix domain in HSD17B13 are all critical for LD targeting. Similarly, we characterized the predicted catalytic, substrate-binding, and homodimer interaction sites and found them to be essential for the enzymatic activity of HSD17B13, in addition to our previous identification of amino acid P260 and cofactor binding site. In conclusion, we identified critical domains and amino acid sites that are essential for the LD localization and protein function of HSD17B13, which may facilitate understanding of its function and targeting of this protein to treat chronic liver diseases.
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17-Hidroxiesteroide Deshidrogenasas/metabolismo , Hepatopatías/tratamiento farmacológico , 17-Hidroxiesteroide Deshidrogenasas/análisis , 17-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Células Cultivadas , Enfermedad Crónica , Humanos , Hepatopatías/metabolismo , Hepatopatías/patología , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Several human enzymes of the short-chain dehydrogenase/reductase (SDR) superfamily of proteins exhibit catalytic oxidoreductive activity toward retinoid substrates in vitro. For some retinoid-active enzymes, their physiological significance for retinoid metabolism is supported by phenotypes linked to naturally occurring mutations in human genes or by targeted gene knockout studies of their murine homologs. However, for those enzymes that are not well conserved or display properties different from their murine counterparts, evaluation of their physiological roles can be challenging. Here, we describe the adaptation of stratified organotypic culture of human epidermis for evaluating the contribution of human putative SDR retinol dehydrogenases to biosynthesis of all-trans-retinoic acid in a three-dimensional cellular model highly sensitive to the levels of all-trans-retinol and all-trans-retinoic acid. In addition to providing a valuable readout of metabolic changes occurring in the presence or absence of the enzyme of interest, this model allows for evaluation of the effects of various retinoid and rexinoid therapeutic compounds on retinoic acid signaling, cell growth and differentiation.
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Oxidorreductasas de Alcohol , Tretinoina , Animales , Epidermis , Humanos , Ratones , Retinoides , Vitamina ARESUMEN
All-trans-retinoic acid (RA) is a bioactive lipid that influences many processes in embryonic and adult tissues. Given its bioactive nature, cellular concentrations of this molecule are highly regulated. The oxidation of all-trans-retinol to all-trans-retinaldehyde represents the first and rate-limiting step of the RA synthesis pathway. As such, it is the target of mechanisms that fine-tune RA levels within the cell. RDH10 is one enzyme responsible for the oxidation of all-trans-retinol to all-trans-retinaldehyde, and together with the all-trans-retinaldehyde reductase DHRS3 forms an oligomeric protein complex. The resulting retinoid oxidoreductase complex (ROC) is bifunctional and has the capacity to regulate steady-state levels of the direct precursor of RA, all-trans-retinaldehyde. As ROC represents a major regulatory element within the RA synthesis pathway, it is essential that methods are in place that allow for the study of this complex. Here we describe the production and isolation of recombinant ROC using a baculovirus expression system. Recombinant proteins retain enzymatic activities in intact microsomes and can be affinity purified for analysis. These methods can be used to assist in the assessment of ROC properties and the regulation of this protein complex's functional attributes.
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Oxidorreductasas de Alcohol , Retinoides , Oxidorreductasas de Alcohol/genética , Oxidorreductasas , Retinaldehído , TretinoinaRESUMEN
The concentration of all-trans-retinoic acid, the bioactive derivative of vitamin A, is critically important for the optimal performance of numerous physiological processes. Either too little or too much of retinoic acid in developing or adult tissues is equally harmful. All-trans-retinoic acid is produced by the irreversible oxidation of all-trans-retinaldehyde. Thus, the concentration of retinaldehyde as the immediate precursor of retinoic acid has to be tightly controlled. However, the enzymes that produce all-trans-retinaldehyde for retinoic acid biosynthesis and the mechanisms responsible for the control of retinaldehyde levels have not yet been fully defined. The goal of this review is to summarize the current state of knowledge regarding the identities of physiologically relevant retinol dehydrogenases, their enzymatic properties, and tissue distribution, and to discuss potential mechanisms for the regulation of the flux from retinol to retinaldehyde.
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Retinaldehído/metabolismo , Tretinoina/metabolismo , Animales , Vías Biosintéticas , Humanos , Retinaldehído/química , Tretinoina/químicaRESUMEN
Retinol dehydrogenases catalyze the rate-limiting step in the biosynthesis of retinoic acid, a bioactive lipid molecule that regulates the expression of hundreds of genes by binding to nuclear transcription factors, the retinoic acid receptors. Several enzymes exhibit retinol dehydrogenase activities in vitro; however, their physiological relevance for retinoic acid biosynthesis in vivo remains unclear. Here, we present evidence that two murine epidermal retinol dehydrogenases, short-chain dehydrogenase/reductase family 16C member 5 (SDR16C5) and SDR16C6, contribute to retinoic acid biosynthesis in living cells and are also essential for the oxidation of retinol to retinaldehyde in vivo Mice with targeted knockout of the more catalytically active SDR16C6 enzyme have no obvious phenotype, possibly due to functional redundancy, because Sdr16c5 and Sdr16c6 exhibit an overlapping expression pattern during later developmental stages and in adulthood. Mice that lack both enzymes are viable and fertile but display accelerated hair growth after shaving and also enlarged meibomian glands, consistent with a nearly 80% reduction in the retinol dehydrogenase activities of skin membrane fractions from the Sdr16c5/Sdr16c6 double-knockout mice. The up-regulation of hair-follicle stem cell genes is consistent with reduced retinoic acid signaling in the skin of the double-knockout mice. These results indicate that the retinol dehydrogenase activities of murine SDR16C5 and SDR16C6 enzymes are not critical for survival but are responsible for most of the retinol dehydrogenase activity in skin, essential for the regulation of the hair-follicle cycle, and required for the maintenance of both sebaceous and meibomian glands.
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Epidermis/enzimología , Epidermis/crecimiento & desarrollo , Glándulas Tarsales/anatomía & histología , Deshidrogenasas-Reductasas de Cadena Corta/deficiencia , Animales , Técnicas de Inactivación de Genes , Cinética , Ratones , Fenotipo , Deshidrogenasas-Reductasas de Cadena Corta/genética , Tretinoina/metabolismoRESUMEN
Retinol dehydrogenase 11 (RDH11) is an NADPH-dependent retinaldehyde reductase that was previously reported to function in the visual cycle. Recently, we have shown that RDH11 contributes to the maintenance of retinol levels in extraocular tissues under conditions of vitamin A deficiency or reduced vitamin A availability. RDH11 is also expressed in the embryo. Rdh11 knockout animals do not display embryonic defects and appear to develop normally to the adult stage, but the exact function of RDH11 during development is not yet known. In contrast to RDH11-null mice, animals that lack dehydrogenase/reductase 3 (DHRS3), the enzyme that functions as a retinaldehyde reductase and is essential for the maintenance of retinoid homeostasis during embryogenesis, rarely survive until birth. Here, we investigated whether inactivation of RDH11 together with DHRS3 exacerbates the severity of retinoid homeostasis disruption in embryos that lack both enzymes compared to DHRS3-null mice. The results of this study indicate that in vitamin A sufficient animals, the loss of RDH11 in addition to DHRS3 does not appear to significantly impact the total levels of retinoic acid, free retinol, or retinyl esters in Rdh11-/-/Dhrs3-/-embryos in comparison to Dhrs3-/- embryos. Surprisingly, Rdh11-/- single gene knockout embryos obtained from breeding of Rdh11-/- dams display elevated levels of embryonic retinyl esters compared to wild type embryos. The mechanism of the maternal effect of Rdh11 status on fetal retinoid stores remains to be elucidated.
Asunto(s)
Oxidorreductasas/genética , Retinoides/metabolismo , Oxidorreductasas de Alcohol/deficiencia , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/metabolismo , Ésteres/química , Ratones , Ratones Noqueados , Oxidorreductasas/deficiencia , Retinaldehído/análisis , Tretinoina/análisis , Vitamina A/farmacologíaRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease. A single-nucleotide polymorphism (SNP), rs6834314, was associated with serum liver enzymes in the general population, presumably reflecting liver fat or injury. We studied rs6834314 and its nearest gene, 17-beta hydroxysteroid dehydrogenase 13 (HSD17B13), to identify associations with histological features of NAFLD and to characterize the functional role of HSD17B13 in NAFLD pathogenesis. The minor allele of rs6834314 was significantly associated with increased steatosis but decreased inflammation, ballooning, Mallory-Denk bodies, and liver enzyme levels in 768 adult Caucasians with biopsy-proven NAFLD and with cirrhosis in the general population. We found two plausible causative variants in the HSD17B13 gene. rs72613567, a splice-site SNP in high linkage with rs6834314 (r2 = 0.94) generates splice variants and shows a similar pattern of association with NAFLD histology. Its minor allele generates simultaneous expression of exon 6-skipping and G-nucleotide insertion variants. Another SNP, rs62305723 (encoding a P260S mutation), is significantly associated with decreased ballooning and inflammation. Hepatic expression of HSD17B13 is 5.9-fold higher (P = 0.003) in patients with NAFLD. HSD17B13 is targeted to lipid droplets, requiring the conserved amino acid 22-28 sequence and amino acid 71-106 region. The protein has retinol dehydrogenase (RDH) activity, with enzymatic activity dependent on lipid droplet targeting and cofactor binding site. The exon 6 deletion, G insertion, and naturally occurring P260S mutation all confer loss of enzymatic activity. Conclusion: We demonstrate the association of variants in HSD17B13 with specific features of NAFLD histology and identify the enzyme as a lipid droplet-associated RDH; our data suggest that HSD17B13 plays a role in NAFLD through its enzymatic activity.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/genética , Enfermedad del Hígado Graso no Alcohólico/genética , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , Adulto , Secuencia de Aminoácidos , Estudios de Cohortes , Femenino , Células HEK293 , Células Hep G2 , Humanos , Hígado/metabolismo , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Polimorfismo de Nucleótido Simple , Retinoides/metabolismoRESUMEN
Retinol dehydrogenase 11 (RDH11) is a microsomal short-chain dehydrogenase/reductase that recognizes all-trans- and cis-retinoids as substrates and prefers NADPH as a cofactor. Previous work has suggested that RDH11 contributes to the oxidation of 11-cis-retinol to 11-cis-retinaldehyde during the visual cycle in the eye's retinal pigment epithelium. However, the role of RDH11 in metabolism of all-trans-retinoids remains obscure. Here, we report that microsomes isolated from the testes and livers of Rdh11-/- mice fed a regular diet exhibited a 3- and 1.7-fold lower rate of all-trans-retinaldehyde conversion to all-trans-retinol, respectively, than the microsomes of WT littermates. Testes and livers of Rdh11-/- mice fed a vitamin A-deficient diet had â¼35% lower levels of all-trans-retinol than those of WT mice. Furthermore, the conversion of ß-carotene to retinol via retinaldehyde as an intermediate appeared to be impaired in the testes of Rdh11-/-/retinol-binding protein 4-/-(Rbp4-/-) mice, which lack circulating holo RBP4 and rely on dietary supplementation with ß-carotene for maintenance of their retinoid stores. Together, these results indicate that in mouse testis and liver, RDH11 functions as an all-trans-retinaldehyde reductase essential for the maintenance of physiological levels of all-trans-retinol under reduced vitamin A availability.