RESUMEN
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.
Asunto(s)
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.
Asunto(s)
Tetrahidronaftalenos , Tretinoina , Humanos , Receptores X Retinoide/metabolismo , Bexaroteno , Ligandos , Tetrahidronaftalenos/farmacología , Tretinoina/farmacología , Tretinoina/metabolismo , Epidermis/metabolismoRESUMEN
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.
Asunto(s)
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
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.
Asunto(s)
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.
Asunto(s)
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
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.
Asunto(s)
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
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.
Asunto(s)
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
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.
Asunto(s)
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
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.
Asunto(s)
Microsomas Hepáticos/metabolismo , Microsomas/metabolismo , Oxidorreductasas/metabolismo , Testículo/metabolismo , Vitamina A/metabolismo , Animales , Dieta , Femenino , Expresión Génica , Homeostasis , Masculino , Ratones , Ratones Noqueados , Oxidorreductasas/genética , Retinaldehído/metabolismo , Proteínas Plasmáticas de Unión al Retinol/metabolismo , Transducción de Señal , Deficiencia de Vitamina A/metabolismo , beta Caroteno/administración & dosificación , beta Caroteno/metabolismoRESUMEN
All-trans-retinoic acid (RA), a bioactive derivative of vitamin A, exhibits diverse effects on gene transcription and non-genomic regulatory pathways. The steady-state levels of RA are therefore tightly controlled, but the mechanisms responsible for RA homeostasis are not fully understood. We report a molecular mechanism that allows cells to maintain a stable rate of RA biosynthesis by utilizing a biological circuit generated by a bifunctional retinoid oxidoreductive complex (ROC). We show that ROC is composed of at least two subunits of NAD+-dependent retinol dehydrogenase 10 (RDH10), which catalyzes the oxidation of retinol to retinaldehyde, and two subunits of NADPH-dependent dehydrogenase reductase 3 (DHRS3), which catalyzes the reduction of retinaldehyde back to retinol. RDH10 and DHRS3 also exist as homo-oligomers. When complexed, RDH10 and DHRS3 mutually activate and stabilize each other. These features of ROC ensure that the rate of RA biosynthesis in whole cells is largely independent of the concentration of the individual ROC components. ROC operates in various subcellular fractions including microsomes, mitochondria, and lipid droplets; however, lipid droplets display weaker mutual activation between RDH10 and DHRS3, suggesting reduced formation of ROC. Importantly, disruption of the ROC-generated circuit by a knockdown of DHRS3 results in an increased flux through the RA biosynthesis pathway and elevated RA levels despite the decrease in RDH10 protein destabilized by the absence of DHRS3, hence demonstrating a loss of control. Thus, the bifunctional nature of ROC provides the RA-based signaling system with robustness by safeguarding appropriate RA concentration despite naturally occurring fluctuations in RDH10 and DHRS3.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Complejos Multienzimáticos/metabolismo , Transducción de Señal/fisiología , Tretinoina/metabolismo , Oxidorreductasas de Alcohol/genética , Humanos , Complejos Multienzimáticos/genéticaRESUMEN
The bloom of genomics is revealing gene loss as a pervasive evolutionary force generating genetic diversity that shapes the evolution of species. Outside bacteria and yeast, however, the understanding of the process of gene loss remains elusive, especially in the evolution of animal species. Here, using the dismantling of the retinoic acid metabolic gene network (RA-MGN) in the chordate Oikopleura dioica as a case study, we combine approaches of comparative genomics, phylogenetics, biochemistry, and developmental biology to investigate the mutational robustness associated to biased patterns of gene loss. We demonstrate the absence of alternative pathways for RA-synthesis in O. dioica, which suggests that gene losses of RA-MGN were not compensated by mutational robustness, but occurred in a scenario of regressive evolution. In addition, the lack of drastic phenotypic changes associated to the loss of RA-signaling provides an example of the inverse paradox of Evo-Devo. This work illustrates how the identification of patterns of gene coelimination-in our case five losses (Rdh10, Rdh16, Bco1, Aldh1a, and Cyp26)-is a useful strategy to recognize gene network modules associated to distinct functions. Our work also illustrates how the identification of survival genes helps to recognize neofunctionalization events and ancestral functions. Thus, the survival and extensive duplication of Cco and RdhE2 in O. dioica correlated with the acquisition of complex compartmentalization of expression domains in the digestive system and a process of enzymatic neofunctionalization of the Cco, while the surviving Aldh8 could be related to its ancestral housekeeping role against toxic aldehydes.
Asunto(s)
Evolución Molecular , Eliminación de Gen , Redes Reguladoras de Genes , Tretinoina/metabolismo , Urocordados/genética , Animales , Evolución Biológica , Biología Computacional/métodos , Femenino , Variación Genética , Genómica , Masculino , Filogenia , Transducción de Señal , Relación Estructura-Actividad , Urocordados/metabolismoRESUMEN
Retinoic acid (RA) was identified as the biologically active form of vitamin A almost 70 years ago and work on its function and mechanism of action is still of major interest both from a scientific and a clinical perspective. The currently accepted model postulates that RA is produced in two sequential oxidative steps: first, retinol is oxidized reversibly to retinaldehyde, and then retinaldehyde is oxidized irreversibly to RA. Excess RA is inactivated by conversion to hydroxylated derivatives. Much is left to learn, especially about retinoid binding proteins and the trafficking of the hydrophobic retinoid substrates between membrane bound and cytosolic enzymes. Here, background on development of the field and an update on recent advances in our understanding of the enzymatic pathways and mechanisms that control the rate of RA production and degradation are presented with a focus on the many questions that remain unanswered.
Asunto(s)
Tretinoina/metabolismo , Aldehído Deshidrogenasa/metabolismo , Animales , Transporte Biológico , Membrana Celular/enzimología , Familia 26 del Citocromo P450/metabolismo , Citosol/enzimología , Retroalimentación Fisiológica , Predicción , Humanos , Isoenzimas/metabolismo , Ratones , Microsomas Hepáticos/enzimología , Oxidación-Reducción , Oxidorreductasas/metabolismo , Ratas , Proteínas Recombinantes/metabolismo , Retinaldehído/metabolismo , Vitamina A/metabolismoRESUMEN
The retinoic acid-inducible dehydrogenase reductase 3 (DHRS3) is thought to function as a retinaldehyde reductase that controls the levels of all-trans-retinaldehyde, the immediate precursor for bioactive all-trans-retinoic acid. However, the weak catalytic activity of DHRS3 and the lack of changes in retinaldehyde conversion to retinol and retinoic acid in the cells overexpressing DHRS3 undermine its role as a physiologically important all-trans-retinaldehyde reductase. This study demonstrates that DHRS3 requires the presence of retinol dehydrogenase 10 (RDH10) to display its full catalytic activity. The RDH10-activated DHRS3 acts as a robust high affinity all-trans-retinaldehyde-specific reductase that effectively converts retinaldehyde back to retinol, decreasing the rate of retinoic acid biosynthesis. In turn, the retinol dehydrogenase activity of RDH10 is reciprocally activated by DHRS3. At E13.5, DHRS3-null embryos have â¼4-fold lower levels of retinol and retinyl esters, but only slightly elevated levels of retinoic acid. The membrane-associated retinaldehyde reductase and retinol dehydrogenase activities are decreased by â¼4- and â¼2-fold, respectively, in Dhrs3(-/-) embryos, and Dhrs3(-/-) mouse embryonic fibroblasts exhibit reduced metabolism of both retinaldehyde and retinol. Neither RDH10 nor DHRS3 has to be itself catalytically active to activate each other. The transcripts encoding DHRS3 and RDH10 are co-localized at least in some tissues during development. The mutually activating interaction between the two related proteins may represent a highly sensitive and conserved mechanism for precise control over the rate of retinoic acid biosynthesis.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Homeostasis , Retinaldehído/metabolismo , Oxidorreductasas de Alcohol/genética , Animales , Biocatálisis , Western Blotting , Células Cultivadas , Activación Enzimática , Femenino , Células HEK293 , Células Hep G2 , Humanos , Hibridación in Situ , Esbozos de los Miembros/embriología , Esbozos de los Miembros/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Unión Proteica , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Sf9 , Vitamina A/metabolismoRESUMEN
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.
Asunto(s)
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
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.
RESUMEN
All-trans-retinoic acid is a biologically active derivative of vitamin A that regulates numerous physiological processes. The concentration of retinoic acid in the cells is tightly regulated, but the exact mechanisms responsible for this regulation are not completely understood, largely because the enzymes involved in the biosynthesis of retinoic acid have not been fully defined. Recent studies using in vitro and in vivo models suggest that several members of the short-chain dehydrogenase/reductase superfamily of proteins are essential for retinoic acid biosynthesis and the maintenance of retinoic acid homeostasis. However, the exact roles of some of these recently identified enzymes are yet to be characterized. The properties of the known contributors to retinoid metabolism have now been better defined and allow for more detailed understanding of their interactions with retinoid-binding proteins and other retinoid enzymes. At the same time, further studies are needed to clarify the interactions between the cytoplasmic and membrane-bound proteins involved in the processing of hydrophobic retinoid metabolites. This review summarizes current knowledge about the roles of various biosynthetic and catabolic enzymes in the regulation of retinoic acid homeostasis and outlines the remaining questions in the field.
Asunto(s)
Enzimas/metabolismo , Tretinoina/metabolismoRESUMEN
The enzymes responsible for the rate-limiting step in retinoic acid biosynthesis, the oxidation of retinol to retinaldehyde, during embryogenesis and in adulthood have not been fully defined. Here, we report that a novel member of the short chain dehydrogenase/reductase superfamily, frog sdr16c5, acts as a highly active retinol dehydrogenase (rdhe2) that promotes retinoic acid biosynthesis when expressed in mammalian cells. In vivo assays of rdhe2 function show that overexpression of rdhe2 in frog embryos leads to posteriorization and induction of defects resembling those caused by retinoic acid toxicity. Conversely, antisense morpholino-mediated knockdown of endogenous rdhe2 results in phenotypes consistent with retinoic acid deficiency, such as defects in anterior neural tube closure, microcephaly with small eye formation, disruption of somitogenesis, and curved body axis with bent tail. Higher doses of morpholino induce embryonic lethality. Analyses of retinoic acid levels using either endogenous retinoic acid-sensitive gene hoxd4 or retinoic acid reporter cell line both show that the levels of retinoic acid are significantly decreased in rdhe2 morphants. Taken together, these results provide strong evidence that Xenopus rdhe2 functions as a retinol dehydrogenase essential for frog embryonic development in vivo. Importantly, the retinol oxidizing activity of frog rdhe2 is conserved in its mouse homologs, suggesting that rdhe2-related enzymes may represent the previously unrecognized physiologically relevant retinol dehydrogenases that contribute to retinoic acid biosynthesis in higher vertebrates.
Asunto(s)
Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/metabolismo , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Xenopus laevis/genética , Oxidorreductasas de Alcohol/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Alineación de Secuencia , Tretinoina/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/metabolismoRESUMEN
Retinoic acid is essential for skin growth and differentiation, and its concentration in skin is controlled tightly. In humans, four different members of the short-chain dehydrogenase/reductase (SDR) superfamily of proteins were proposed to catalyze the rate-limiting step in the biosynthesis of retinoic acid (the oxidation of retinol to retinaldehyde). Epidermis contains at least three of these enzymes, but their relative importance for retinoic acid biosynthesis and regulation of gene expression during growth and differentiation of epidermis is not known. Here, we investigated the effect of the four human SDRs on retinoic acid biosynthesis, and their impact on growth and differentiation of keratinocytes using organotypic skin raft culture model of human epidermis. The results of this study demonstrate that ectopic expression of retinol dehydrogenase 10 (RDH10, SDR16C4) in skin rafts dramatically increases proliferation and inhibits differentiation of keratinocytes, consistent with the increased steady-state levels of retinoic acid and activation of retinoic acid-inducible genes in RDH10 rafts. In contrast, SDRs with dual retinol/sterol substrate specificity, namely retinol dehydrogenase 4 (RoDH4, SDR9C8), RoDH-like 3α-hydroxysteroid dehydrogenase (RL-HSD, SDR9C6), and RDH-like SDR (RDHL, SDR9C4) do not affect the expression of retinoic acid-inducible genes but alter the expression levels of several components of extracellular matrix. These results reveal essential differences in the metabolic contribution of RDH10 versus retinol/sterol dehydrogenases to retinoic acid biosynthesis and provide the first evidence that non-retinoid metabolic products of retinol/sterol dehydrogenases affect gene expression in human epidermis.