RESUMEN
In brief: Mammalian spermatozoa actively generate reactive oxygen species (ROS) during capacitation, a maturational process necessary for fertilization in vivo. This study shows that hypotaurine, a precursor of taurine present in the oviduct, is incorporated and concentrated in hamster sperm cells via the taurine transporter, TauT, for cytoprotection against self-produced ROS. Abstract: To achieve fertilization competence, mammalian spermatozoa undergo capacitation, during which they actively generate reactive oxygen species (ROS). Therefore, mammalian spermatozoa must protect themselves from these self-generated ROS. The mammalian oviductal fluid is rich in hypotaurine, a taurine precursor, which reportedly protects mammalian spermatozoa, including those of hamsters, from ROS; however, its precise mechanism remains unknown. This study aimed to elucidate the mechanism underlying hypotaurine-mediated protection of spermatozoa from ROS using hamsters, particularly focusing on the taurine/hypotaurine transporter TauT. The effect of hypotaurine on sperm motility and ROS levels was tested using sperm motility analysis and the CellROX dye and luminol assays. RNA sequencing analysis was performed to verify TauT expression. We found that hypotaurine was necessary for maintaining sperm motility and hyperactivated motility. Hypotaurine did not scavenge extracellular ROS but lowered intracellular ROS levels and was incorporated and concentrated in hamster spermatozoa. TauT was detected at both mRNA and protein levels. ß-Alanine blocked hypotaurine transport, increased intracellular ROS levels, and inhibited hyperactivation. Elimination of Na+ or Cl- ions inhibited hypotaurine transport and increased intracellular ROS levels. Thus, these results indicated that hamster spermatozoa incorporated and concentrated hypotaurine in sperm cells via TauT to protect themselves from self-generated ROS.
Asunto(s)
Capacitación Espermática , Motilidad Espermática , Espermatozoides , Taurina , Animales , Cricetinae , Masculino , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Mesocricetus , Especies Reactivas de Oxígeno/metabolismo , Capacitación Espermática/efectos de los fármacos , Motilidad Espermática/efectos de los fármacos , Espermatozoides/metabolismo , Espermatozoides/efectos de los fármacos , Taurina/análogos & derivados , Taurina/farmacologíaRESUMEN
Ether phospholipids are synthesized by a series of enzymes localized in peroxisomes, the endoplasmic reticulum (ER), and the Golgi apparatus. During this process, the lipid intermediate alkylacylglycerol (AAG) synthesized in the ER is transferred from the site of its synthesis to the Golgi apparatus. In this study, we determined whether ceramide transport protein (CERT) is a candidate for AAG transfer. A lipid transfer assay revealed that CERT can mediate AAG transfer between phospholipid liposomes. AAG transport activity was markedly inhibited by the CERT inhibitor HPA-12 and reduced when the lipid transport domain of CERT was deleted. Suppression of CERT in HEK293 cells resulted in increased levels of plasmanyl-PC, which is synthesized by the ER-residing choline/ethanolamine phosphotransferase 1 (CEPT1). The mRNA levels and enzymatic activity of plasmanyl-PC synthesizing enzymes were not increased in CERT-deficient cells, indicating that the increase in plasmanyl-PC results from AAG accumulation in the ER. Re-introduction of CERT into CERT-deficient cells caused a decrease in plasmanyl-PC. Taken together, our findings suggest for the first time that CERT is involved in the transfer of AAG from the ER to the Golgi apparatus and plays a role in the biosynthesis of ether phospholipids.
Asunto(s)
Proteínas Portadoras , Ceramidas , Humanos , Transporte Biológico , Proteínas Portadoras/metabolismo , Ceramidas/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Células HEK293 , Éteres Fosfolípidos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Metabolic alteration is increasingly recognized as an important pathogenic process that underlies fibrosis across many organ types, and metabolically targeted therapies could become important strategies for reducing fibrosis. In present study, target enzymes that are involved in changes in phospholipid metabolism during fibroblast-to-myofibroblast transition induced by transforming growth factor beta 1 (TGF-ß1) were examined. Different amounts of phospholipids were found in the 2 groups. In response to TGF-ß1 stimulation, 17 lipids decreased and 17 increased. The latter included the phospholipids phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE). Furthermore, among the rate-limiting enzymes that regulate these phospholipids, phosphatidylserine decarboxylase (PISD), which controls conversion of PS to PE and is localized in mitochondria, decreased in response to TGF-ß1. Knockdown of PISD alone without TGF-ß1 stimulation increased expression of α-smooth muscle actin mRNA and production of total collagen. Taken together, these results indicate that PISD is involved in the mechanism of fibrogenesis by regulating phospholipid metabolism.
RESUMEN
Choline phospholipids (PLs) such as phosphatidylcholine (PC) and 1-alkyl-2-acyl-sn-glycerophosphocholine are important components for cell membranes and also serve as a source of several lipid mediators. These lipids are biosynthesized in mammals in the final step of the CDP-choline pathway by the choline phosphotransferases choline phosphotransferase 1 (CPT1) and choline/ethanolamine phosphotransferase 1 (CEPT1). However, the contributions of these enzymes to the de novo biosynthesis of lipids remain unknown. Here, we established and characterized CPT1- and CEPT1-deficient human embryonic kidney 293 cells. Immunohistochemical analyses revealed that CPT1 localizes to the trans-Golgi network and CEPT1 to the endoplasmic reticulum. Enzyme assays and metabolic labeling with radiolabeled choline demonstrated that loss of CEPT1 dramatically decreases choline PL biosynthesis. Quantitative PCR and reintroduction of CPT1 and CEPT1 revealed that the specific activity of CEPT1 was much higher than that of CPT1. LC-MS/MS analysis of newly synthesized lipid molecular species from deuterium-labeled choline also showed that these enzymes have similar preference for the synthesis of PC molecular species, but that CPT1 had higher preference for 1-alkyl-2-acyl-sn-glycerophosphocholine with PUFA than did CEPT1. The endogenous level of PC was not reduced by the loss of these enzymes. However, several 1-alkyl-2-acyl-sn-glycerophosphocholine molecular species were reduced in CPT1-deficient cells and increased in CEPT1-deficient cells when cultured in 0.1% FBS medium. These results suggest that CEPT1 accounts for most choline PL biosynthesis activity, and that both enzymes are responsible for the production of different lipid molecular species in distinct organelles.
Asunto(s)
Colina/biosíntesis , Diacilglicerol Colinafosfotransferasa/metabolismo , Fosfolípidos/biosíntesis , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Células Cultivadas , Células HEK293 , HumanosRESUMEN
The final step of the CDP-ethanolamine pathway is catalyzed by ethanolamine phosphotransferase 1 (EPT1) and choline/EPT1 (CEPT1). These enzymes are likely involved in the transfer of ethanolamine phosphate from CDP-ethanolamine to lipid acceptors such as 1,2-diacylglycerol (DAG) for PE production and 1-alkyl-2-acyl-glycerol (AAG) for the generation of 1-alkyl-2-acyl-glycerophosphoethanolamine. Here, we investigated the intracellular location and contribution to ethanolamine phospholipid (EP) biosynthesis of EPT1 and CEPT1 in HEK293 cells. Immunohistochemical analyses revealed that EPT1 localizes to the Golgi apparatus and CEPT1 to the ER. We created EPT1-, CEPT1-, and EPTI-CEPT1-deficient cells, and labeling of these cells with radio- or deuterium-labeled ethanolamine disclosed that EPT1 is more important for the de novo biosynthesis of 1-alkenyl-2-acyl-glycerophosphoethanolamine than is CEPT1. EPT1 also contributed to the synthesis of PE species containing the fatty acids 36:1, 36:4, 38:5, 38:4, 38:3, 40:6, 40:5, and 40:4. In contrast, CEPT1 was important for PE formation from shorter fatty acids such as 32:2, 32:1, 34:2, and 34:1. Brefeldin A treatment did not significantly affect the levels of the different PE species, indicating that the subcellular localization of the two enzymes is not responsible for their substrate preferences. In vitro enzymatic analysis revealed that EPT1 prefers AAG 16-20:4 > DAG 18:0-20:4 > DAG 16:0-18:1 = AAG 16-18:1 as lipid acceptors and that CEPT1 greatly prefers DAG 16:0-18:1 to other acceptors. These results suggest that EPT1 and CEPT1 differ in organelle location and are responsible for the biosynthesis of distinct EP species.
Asunto(s)
Etanolamina/química , Etanolamina/metabolismo , Etanolaminofosfotransferasa/metabolismo , Fosfolípidos/química , Células HEK293 , Humanos , Espacio Intracelular/metabolismo , Transporte de ProteínasRESUMEN
We previously purified lysophospholipase D (lysoPLD), which hydrolyzes lysophosphatidylcholine (lysoPC) to lysophosphatidic acid (LPA), from rat brain and identified the heterotrimeric G protein subunits Gαq and Gß1 in the lysoPLD active fractions. Tag-affinity purified Gαq exhibits lysoPLD activity but a mutant that affected cellular localization or interaction with the Gß subunit reduced lysoPLD activity. Size exclusion chromatography revealed that active lysoPLD is a much higher molecular mass complex than is heterotrimeric G protein, suggesting the presence of other components. Liquid chromatography-tandem mass spectrometry of lysoPLD purified from rat brain identified glycerophosphodiesterase 4 (GDE4), recently reported as lysoPLD, in the same fraction as G proteins. The overexpressed and tag-purified Gαq fractions, which exhibit lysoPLD activity, contained GDE4. Exogenously expressed GDE4 was co-immunoprecipitated with endogenous Gαq and Gß and exhibited high lysoPLD activity. The results of confocal microscopy and cell fractionation experiments indicated that exogenously expressed GDE4 in cells mainly localized at the endoplasmic reticulum and partially co-localized with Gαq protein at the plasma membrane. Proteinase K protection assay results suggested that the catalytic domain of GDE4 faces the lumen/extracellular space. Mutations at the conserved amino acids in the C-terminus cytoplasmic regions amongst GDE1, 4 and 7, dramatically suppressed GDE4 enzyme activities. When both the Gαq and Gα11 genes in Neuro2A cells were disrupted using the CRISPR-Cas9 system, endogenous lysoPLD activity was partially reduced but rescued by overexpression of Gαq. These results suggest that GDE4 is a new effector of G protein signaling that produces bioactive phospholipid LPA and/or modulates membrane homeostasis.
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Cromograninas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Animales , Línea Celular Tumoral , Membrana Celular , Cromograninas/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Eliminación de Gen , Regulación Enzimológica de la Expresión Génica , Ratones , Hidrolasas Diéster Fosfóricas/genéticaRESUMEN
Ethanolamine phosphotransferase (EPT)1, also known as selenoprotein 1 (SELENOI), is an enzyme that transfers phosphoethanolamine from cytidine diphosphate-ethanolamine to lipid acceptors to produce ethanolamine glycerophospholipids, such as diacyl-linked phosphatidylethanolamine (PE) and ether-linked plasmalogen [1-alkenyl-2-acyl-glycerophosphoethanolamine (plasmenyl-PE)]. However, to date there has been no analysis of the metabolomic consequences of the mutation of EPT1 on the concentration of ethanolamine glycerophospholipids in mammalian cells. We studied a patient with severe complicated hereditary spastic paraplegia, sensorineural-deafness, blindness, and seizures. Neuroimaging revealed hypomyelination, followed by brain atrophy mainly in the cerebellum and brainstem. Using whole exome sequencing, we identified a novel EPT1 mutation (exon skipping). In vitro EPT activity, as well as the rate of biosynthesis of ethanolamine glycerophospholipids, was markedly reduced in cultures of the patient's skin fibroblasts. Quantification of phospholipids by LC-MS/MS demonstrated reduced levels of several PE species with polyunsaturated fatty acids, such as 38:6, 38:4, 40:6, 40:5, and 40:4. Notably, most plasmenyl-PE species were significantly decreased in the patient's cells, whereas most plasmanylcholine [1-alkyl-2-acyl-glycerophosphocholine (plasmanyl-PC)] species were increased. Similar findings regarding decreased plasmenyl-PE and increased plasmanyl-PC were obtained using EPT1-KO HeLa cells. Our data demonstrate for the first time the indispensable role of EPT1 in the myelination process and neurodevelopment, and in the maintenance of normal homeostasis of ether-linked phospholipids in humans.
Asunto(s)
Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Etanolaminofosfotransferasa/metabolismo , Plasmalógenos/metabolismo , Encéfalo/enzimología , Preescolar , Etanolaminofosfotransferasa/deficiencia , Etanolaminofosfotransferasa/genética , Femenino , Fibroblastos/metabolismo , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Lactante , Recién Nacido , Vaina de Mielina/metabolismo , Fosfolípidos/metabolismo , Embarazo , Piel/citologíaRESUMEN
Phosphatidylcholine (PC) is a major phospholipid of mitochondria, comprising 40-50% of both the outer and the inner membranes. However, PC must be imported from its production organelles because mitochondria lack the enzymes essential for PC biosynthesis. In a previous study, we found that StarD7 mediates the intracellular transfer of PC to mitochondria. Therefore, in this study, we analyzed the contribution of StarD7 to the maintenance of mitochondrial phospholipid content and function using siRNA-mediated knockdown and knock-out (KO) of the StarD7 gene in HEPA-1 cells. Real time analysis of respiratory activity demonstrated that the oxygen consumption rate and activity of mitochondrial complexes were impaired in StarD7-KD cells. To confirm these results, we established StarD7-KO HEPA-1 cells by double nicking using CRISPR/Cas9n. As expected, StarD7-KD and -KO cells showed a significant reduction in mitochondrial PC content. The ATP level and growth rate of KO cells were notably lower compared with wild-type cells when cultured in glucose-free galactose-containing medium to force cells to rely on mitochondrial ATP production. In KO cells, the level of the MTCO1 protein, a primary subunit of complex IV, was reduced without a concomitant decrease in its mRNA, but the level was restored when StarD7-I was overexpressed. StarD7-KO cells showed impaired formation of the mitochondrial supercomplexes and exhibited a disorganized cristae structure, with no changes in optic atrophy 1 protein. These findings indicate that StarD7 plays important roles in maintaining the proper composition of mitochondrial phospholipids as well as mitochondrial function and morphogenesis.
Asunto(s)
Proteínas Portadoras/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Consumo de Oxígeno/fisiología , Fosfatidilcolinas/metabolismo , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Animales , Proteínas Portadoras/genética , Línea Celular Tumoral , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Técnicas de Silenciamiento del Gen , Ratones , Mitocondrias/genética , Proteínas Mitocondriales/genética , Fosfatidilcolinas/genéticaRESUMEN
Induced pluripotent stem cells (iPSCs) are opening up new possibilities for medicine. Understanding the regulation of iPSC biology is important when attempting to apply these cells to disease models or therapy. Changes of lipid metabolism in iPSCs were investigated by matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-TOF-IMS). Analysis revealed changes of the intensity and distribution of peaks at m/z 782.5 and 798.5 in iPSC colonies during spontaneous differentiation. Two phosphatidylcholines (PCs) were identified: C44H81NO8P, PC(36:4)[M+H]+ at m/z 782.5 and C42H82NO8P, PC(34:1)[M+K]+ at m/z 798.5. The intensity of PC(36:4) showed an inverse relation between undifferentiated and differentiated iPSC colonies. PC(34:1) displayed a diffuse distribution in undifferentiated iPSC colonies, while it showed a concentric distribution in differentiated iPSC colonies, and was localized at the border of the differentiated and undifferentiated areas or the border between undifferentiated iPSC and feeder cells. These findings suggested that the distribution of lipids changes during the growth and differentiation of iPSCs and that MALDI-TOF-IMS was useful for analyzing these changes. PC(36:4) might play a role in maintaining pluripotency, while PC(34:1) might play a role in the differentiation and spread of iPSCs. Graphical Abstract MALDI Imaging for phosphatidylcholine distribution changes during sponteneous differentiaton of induced pluiripotent stem cells colonies.
Asunto(s)
Diferenciación Celular , Células Madre Pluripotentes Inducidas/metabolismo , Fosfatidilcolinas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Línea Celular , Cromatografía Líquida de Alta Presión/métodos , Cromatografía de Fase Inversa/métodos , Inmunohistoquímica , Células Madre Pluripotentes Inducidas/citología , RatonesRESUMEN
Pcyt2 (CTP:phosphoethanolamine cytidylyltransferase) is the rate-limiting enzyme in mammalian PE (phosphatidylethanolamine) biosynthesis. Previously, we reported that Pcyt2 mRNA levels increased in several types of cells after serum starvation, an effect that could be suppressed by supplementation with low-density lipoprotein or 25-HC (25-hydroxycholesterol). Transcription of Hmgcr, which encodes 3-hydroxy-3-methylglutaryl-CoA reductase, is also suppressed by 25-HC in the same dose-dependent manner. Nevertheless, a sterol-regulatory element was not detected in the Pcyt2 promoter region. The important element for transcriptional control of Pcyt2 by 25-HC (1.25 µM) was determined to reside between -56 and -36 on the basis of analysis with several Pcyt2 promoter deletion-luciferase reporters in NIH 3T3 cells. Using the yeast one-hybrid system, we found that NF-Y (nuclear factor-Y) binds at C(-37)CAAT(-41) and YY1 (Yin Yang1) binds at C(-42)AT(-40) in the Pcyt2 promoter. Endogenous NF-Y and YY1 bind clearly and competitively to these sites and are important for basal Pcyt2 transcription. Moreover, NF-Y binds to the Hmgcr promoter at C(-14)CA(-12) in gel-shift analysis, and suppression of the basal luciferase activity of the Hmgcr promoter-reporter construct (-30/+61) by 25-HC was abolished when C(-14)CA(-12) was mutated. Furthermore, transcriptional suppression of Pcyt2 by 25-HC was reduced following knockdown targeting of NF-YA or YY1. ChIP analysis revealed that 25-HC inhibited the interaction between NF-Y and RNA polymerase II on the Pcyt2 and Hmgcr promoters. On the basis of these results, we conclude that NF-Y and YY1 are important for the basal transcription of Pcyt2 and that NF-Y is involved in the inhibitory effects of 25-HC on Pcyt2 transcription.
Asunto(s)
Factor de Unión a CCAAT/metabolismo , ARN Nucleotidiltransferasas/genética , Transcripción Genética/efectos de los fármacos , Factor de Transcripción YY1/metabolismo , Animales , Factor de Unión a CCAAT/genética , Proteínas de Unión al ADN/genética , Humanos , Hidroxicolesteroles/administración & dosificación , Hidroxicolesteroles/metabolismo , Hidroximetilglutaril-CoA Reductasas/biosíntesis , Ratones , Células 3T3 NIH , Regiones Promotoras Genéticas , Factor de Transcripción YY1/genéticaRESUMEN
Acyl-CoA thioesterase 12 (ACOT12) is the major enzyme known to hydrolyze the thioester bond of acetyl-CoA in the cytosol in the liver. ACOT12 contains a catalytic thioesterase domain at the N terminus and a steroidogenic acute regulatory protein-related lipid transfer (START) domain at the C terminus. We investigated the effects of lipids (phospholipids, sphingolipids, fatty acids, and sterols) on ACOT12 thioesterase activity and found that the activity was inhibited by phosphatidic acid (PA) in a noncompetitive manner. In contrast, the enzymatic activity of a mutant form of ACOT12 lacking the START domain was not inhibited by the lipids. These results suggest that the START domain is important for regulation of ACOT12 activity by PA. We also found that PA could bind to thioesterase domain, but not to the START domain, and had no effect on ACOT12 dissociation. ACOT12 is detectable in the liver but not in hepatic cell lines such as HepG2, Hepa-1, and Fa2N-4. ACOT12 mRNA and protein levels in rat primary hepatocytes decreased following treatment with insulin. These results suggest that cytosolic acetyl-CoA levels in the liver are controlled by lipid metabolites and hormones, which result in allosteric enzymatic and transcriptional regulation of ACOT12.
Asunto(s)
Acetil-CoA Hidrolasa/genética , Acetil-CoA Hidrolasa/metabolismo , Citoplasma/enzimología , Transcripción Genética/genética , Acetil-CoA Hidrolasa/antagonistas & inhibidores , Acetil-CoA Hidrolasa/deficiencia , Animales , Insulina/farmacología , Lípidos/biosíntesis , Hígado/enzimología , Hígado/metabolismo , Datos de Secuencia Molecular , Ácidos Fosfatidicos/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Transcripción Genética/efectos de los fármacosRESUMEN
In a previous study we purified a novel lysoPLD (lysophospholipase D) which converts LPC (lysophosphatidylcholine) into a bioactive phospholipid, LPA (lysophosphatidic acid), from the rat brain. In the present study, we identified the purified 42 and 35 kDa proteins as the heterotrimeric G protein subunits Gα(q) and Gß(1) respectively. When FLAG-tagged Gα(q) or Gß(1) was expressed in cells and purified, significant lysoPLD activity was observed in the microsomal fractions. Levels of the hydrolysed product choline increased over time, and the Mg(2+) dependency and substrate specificity of Gα(q) were similar to those of lysoPLD purified from the rat brain. Mutation of Gα(q) at amino acids Lys(52), Thr(186) or Asp(205), residues that are predicted to interact with nucleotide phosphates or catalytic Mg(2+), dramatically reduced lysoPLD activity. GTP does not compete with LPC for the lysoPLD activity, indicating that these substrate-binding sites are not identical. Whereas the enzyme activity of highly purified FLAG-tagged Gα(q) overexpressed in COS-7 cells was ~4 nmol/min per mg, the activity from Neuro2A cells was 137.4 nmol/min per mg. The calculated K(m) and V(max) values for lysoPAF (1-O-hexadecyl-sn-glycero-3-phosphocholine) obtained from Neuro2A cells were 21 µM and 0.16 µmol/min per mg respectively, similar to the enzyme purified from the rat brain. These results reveal a new function for Gα(q) and Gß(1) as an enzyme with lysoPLD activity. Tag-purified Gα(11) also exhibited a high lysoPLD activity, but Gα(i) and Gα(s) did not. The lysoPLD activity of the Gα subunit is strictly dependent on its subfamily and might be important for cellular responses. However, treatment of Hepa-1 cells with Gα(q) and Gα(11) siRNAs (small interfering RNAs) did not change lysoPLD activity in the microsomal fraction. Clarification of the physiological relevance of lysoPLD activity of these proteins will need further studies.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Animales , Células COS , Chlorocebus aethiops , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades beta de la Proteína de Unión al GTP/genética , Lisofosfolípidos/biosíntesis , Ratones , ARN Interferente Pequeño/farmacología , RatasRESUMEN
BACKGROUND: Silencing Mediator of Retinoid and Thyroid hormone receptors (SMRT; NCoR2) is a transcriptional corepressor (CoR) which has been recognized as an important player in the regulation of hepatic lipogenesis and in somatic development in mouse embryo. SMRT protein is also widely expressed in mouse connective tissues, for example adipocytes and muscle. We recently reported that mice with global deletion of SMRT develop significant obesity and muscle wasting which are independent from thyroid hormone (TH) signaling and thermogenesis. However, the tissue specific role of SMRT in skeletal muscle is still not clear. METHODS: To clarify role of SMRT in muscle differentiation, we made myogenic C2C12 clones which lack SMRT protein (C2C12-SKO) by using CRISPR-Cas9. Wild-type C2C12 (C2C12-WT) and C2C12-SKO cells were cultured in differentiation medium, and the resulting gene and protein profiles were compared between the two cell lines both before and after differentiation. We also analyzed muscle tissues which were dissected from whole body SMRT knockout (KO) mice and their controls. RESULTS: We found significant up-regulation of muscle specific ß-oxidation markers; Peroxisome proliferator-activated receptor δ (PPARδ) and PPARγ coactivator-1α (PGC-1α) in the C2C12-SKO cells, suggesting that the cells had a similar gene profile to what is found in exercised rodent skeletal muscle. On the other hand, confocal microscopic analysis showed the significant loss of myotubes in C2C12-SKO cells similar to the morphology found in immature myoblasts. Proteomics analysis also confirmed that the C2C12-SKO cells had higher expression of markers of fibrosis (ex. Collagen1A1; COL1A1 and Fibroblast growth factor-2; FGF-2), indicating the up-regulation of Transforming growth factor-ß (TGF-ß) receptor signaling. Consistent with this, treatment with a specific TGF-ß receptor inhibitor ameliorated both the defects in myotube differentiation and fibrosis. CONCLUSION: Taken together, we demonstrate that SMRT functions as a pivotal transcriptional mediator for both ß-oxidation and the prevention for the fibrosis via TGF-ß receptor signaling in the differentiation of C2C12 myoblasts. In contrast to the results from C2C12 cells, SMRT does not appear to play a role in adult skeletal muscle of whole body SMRT KO mice. Thus, SMRT plays a significant role in the differentiation of myoblasts.
Asunto(s)
Fibras Musculares Esqueléticas , Co-Represor 2 de Receptor Nuclear , PPAR delta , Animales , Ratones , Diferenciación Celular , Factor 2 de Crecimiento de Fibroblastos , Fibrosis , Músculo Esquelético , Co-Represor 2 de Receptor Nuclear/genéticaRESUMEN
Extracellular administration of side-chain oxysterols, such as 24S-hydroxycholesterol (24S-HC), 27-hydroxycholesterol (27-HC) and 25-hydroxycholesterol (25-HC) to cells suppresses HMG-CoA reductase (Hmgcr) and CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) mRNA levels. Oxysterols are enzymatically produced in cells from cholesterol by cytochrome P450 46A1 (Cyp46A1), Cyp27A1, Cyp3A11 and cholesterol 25-hydroxylase (Ch25h). We analyzed which of these oxysterol-producing enzymes are expressed in NIH3T3 cells and found that only Cyp46A1 was expressed. When Cyp46A1 was overexpressed in NIH3T3 cells, intrinsic oxysterols increased in the order 24S-HC > 25-HC > 27-HC. We investigated the mechanism regulating the production of endogenous oxysterols in NIH3T3 cells by Cyp46A1 and found that the mRNA, relative protein levels and enzymatic activity of Cyp46A1, and the amounts of 24S-HC, 25-HC and 27-HC significantly increased under serum-starved conditions, and these increases were suppressed by FBS supplementation. The aqueous phase of FBS obtained by the Bligh & Dyer method significantly suppressed Cyp46A1 mRNA levels. Fractionation of the aqueous phase by HPLC and analysis of the inhibiting fractions by nanoLC and TripleTOF MS/MS identified insulin-like factor-II (IGF-II). Cyp46A1 mRNA levels in serum-starved NIH3T3 cells were significantly suppressed by the addition of IGFs and insulin and endogenous oxysterol levels were decreased. CYP46A1 mRNA levels in the T98G human glioblastoma cell line were also increased by serum starvation but not by FBS supplementation, and the aqueous phase did not inhibit the increase. These results suggest that mRNA levels of Cyp46A1 are regulated by factors in FBS.
Asunto(s)
Insulinas , Espectrometría de Masas en Tándem , Animales , Colesterol 24-Hidroxilasa , Humanos , Ratones , Células 3T3 NIH , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Steroidogenic acute regulatory protein-related lipid transfer (START) domains, found in 15 mammalian proteins termed StarD1-StarD15, are lipid-binding domains implicated in the intracellular lipid transport systems. In the present study, we analyzed the lipid ligand and function of StarD7. We found two variable forms of mammalian StarD7, termed StarD7-I and StarD7-II. Unlike StarD7-II, StarD7-I contained a mitochondrial-targeting sequence in its N terminus. Overexpressed StarD7-I tagged with V5/His in HEPA-1 cells was mainly observed in the mitochondria of cells prepared at low cellular density, but it was distributed in the cytoplasm of high density cells. StarD7-II was constantly distributed in the cytoplasm at any cellular density. Endogenous StarD7 in HEPA-1 cells and rat liver was also distributed in both the cytoplasm and the mitochondria. A protease K protection assay indicated that the mitochondrial StarD7 was associated with the outer mitochondrial membrane. The purified recombinant StarD7 specifically catalyzed the transfer of PC between lipid vesicles in vitro. Furthermore, the intracellular transport of fluorescent PC that was exogenously incorporated into the mitochondria was increased in cells that overexpressed StarD7-I. These results suggest that StarD7 facilitates the delivery of PC to mitochondria in non-vesicular system.
Asunto(s)
Proteínas Portadoras/metabolismo , Mitocondrias/metabolismo , Fosfatidilcolinas/metabolismo , Isoformas de Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico/fisiología , Proteínas Portadoras/clasificación , Proteínas Portadoras/genética , Línea Celular , Vesículas Citoplasmáticas/metabolismo , Hígado/metabolismo , Datos de Secuencia Molecular , Filogenia , Isoformas de Proteínas/genética , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Fracciones Subcelulares/metabolismoRESUMEN
The rate-limiting step in phosphatidylethanolamine (PE) synthesis by the CDP-ethanolamine pathway is the second step, which is catalyzed by CTP:phosphoethanolamine cytidylyltransferase (ET). The rate-limiting step for phosphatidylcholine biosynthesis by the CDP-choline pathway is also the second step, which is catalyzed by CTP:phosphocholine cytidylyltransferase (CT). The transcription of the most active form of CT, CTalpha, in serum-starved cells was stimulated by fetal bovine serum (FBS). Therefore, we were interested in the effects of FBS on the transcription of ET. Unexpectedly, the ET mRNA levels were significantly increased after NIH3T3 cells were cultured in serum-starved medium (0.5% FBS) longer than 8h, and the increase was suppressed by the addition of FBS. Actinomycin-D inhibited the increased ET mRNA levels in serum-starved cells. ET enzyme activities and protein amounts were also increased after serum starvation. These results suggest that FBS contains substances that inhibit the transcription of ET. To identify these substances, cells were incubated with several fractions of FBS separated by molecular sizes. As expected from the results, low-density lipoprotein, 25-hydroxycholesterol (25-OHC), 24-OHC, 27-OHC, 24(S),25-epoxycholesterol and mevalonolactate suppressed the ET mRNA levels in serum-starved cells, similar to 3-hydroxy-3-methylglutaryl-CoA reductase but not CTalpha. These results suggest that oxysterols are important regulating lipids for the suppression of ET transcription and may help maintain the contents of PE and cholesterol at the same ratio in the cellular membrane.
Asunto(s)
Colesterol/análogos & derivados , Lipoproteínas LDL/farmacología , ARN Nucleotidiltransferasas/genética , Transcripción Genética/efectos de los fármacos , Animales , Western Blotting , Colesterol/farmacología , Medio de Cultivo Libre de Suero/farmacología , Dactinomicina/farmacología , Etanolaminas/metabolismo , Células HeLa , Humanos , Técnicas In Vitro , Ratones , Células 3T3 NIH , ARN Nucleotidiltransferasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
PC is made via the CDP-choline pathway, in which CTP:phosphocholine cytidylyltransferase alpha (CTalpha), encoded by Pcyt1a, is the rate-limiting enzyme whose mRNA expression is strictly regulated. Previously, we reported that Ets1 enhanced and Net repressed CTalpha transcription by binding at the Ets binding site (-49/-47) in the Pcyt1a promoter. In this study, we asked if an Ets1 analogue, Ets2, also regulates CTalpha transcription and investigated the importance of its nuclear localization signal (NLS) and nuclear export signal (NES). Ets2 is primarily detected in the nucleus. Various mutated Ets2 proteins fused with enhanced green fluorescent protein were constructed to identify the NLS and NES in Ets2. Mutation of Ets2 at amino acids 404-410 results in a protein that is evenly distributed in the cell. Interestingly, an Ets2 protein deleted at the C-terminus (amino acids 1-392 present) was localized to the cytoplasm and site-specific mutation in the region 364-372 of this construct resulted in cytoplasmic and nuclear distribution. These results suggest that the NLS in Ets2 is between amino acids 404 and 410, and that the NES is between amino acids 364 and 372. Ets2 enhanced, but the mutant forms of Ets2 had little effects on the transcription of a CTalpha-reporter construct. When RAW264 cells, murine macrophage cell-line, were stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA) or macrophage-colony stimulating factor, the transcription of CTalpha was enhanced accompanied by increased mRNA of Ets2. These results suggest that the induction of Ets2 is important for CTalpha transcription by TPA and macrophage-colony stimulating factor.
Asunto(s)
Citidililtransferasa de Colina-Fosfato/genética , Regulación de la Expresión Génica/efectos de los fármacos , Factor Estimulante de Colonias de Macrófagos/farmacología , Señales de Exportación Nuclear/genética , Señales de Localización Nuclear/genética , Proteína Proto-Oncogénica c-ets-2/genética , Acetato de Tetradecanoilforbol/farmacología , Animales , Células COS , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Citidililtransferasa de Colina-Fosfato/metabolismo , Luciferasas/metabolismo , Ratones , Mutación/genética , Señales de Localización Nuclear/metabolismo , Regiones Promotoras Genéticas , Proteína Proto-Oncogénica c-ets-1/genética , Proteína Proto-Oncogénica c-ets-1/metabolismo , Proteína Proto-Oncogénica c-ets-2/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción Genética , TransfecciónRESUMEN
StarD7 is a phosphatidylcholine (PC)-specific lipid transfer protein essential for the maintenance of mitochondrial PC composition, morphogenesis, and respiration. Here, we studied the role of StarD7 in skeletal myoblast differentiation using mouse myoblast C2C12 cells and human primary myoblasts. Immunofluorescence and immuno-electron microscopy revealed that StarD7 was distributed in the cytosol, inner mitochondria space, and outer leaflet of the outer mitochondrial membrane in C2C12 cells. Unlike human kidney embryonic cell line HEK293 cells, the mitochondrial proteinase PARL was not involved in the processing and maturation of StarD7 in C2C12 cells. StarD7 was constantly expressed during myogenic differentiation of C2C12 cells. The siRNA-mediated knockdown of StarD7 in C2C12 cells and human primary myoblasts significantly impaired myogenic differentiation and reduced the expression of myomaker, myomerger and PGC-1α. The reduction in mitochondrial PC levels and oxygen consumption rates, decreased expression of myomaker, myomerger and PGC-1α, as well as impaired myogenic differentiation, were completely restored when the protein was reintroduced into StarD7-knockout C2C12 cells. These results suggest that StarD7 is important for skeletal myogenesis in mammals.
Asunto(s)
Proteínas Portadoras/genética , Músculo Esquelético/crecimiento & desarrollo , Mioblastos/metabolismo , Animales , Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica/genética , Técnicas de Inactivación de Genes , Humanos , Ratones , Desarrollo de Músculos/genética , Músculo Esquelético/metabolismo , Mioblastos/patología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Cultivo Primario de CélulasRESUMEN
INTRODUCTION: Vascular endothelial cell disorders are closely related to cardiovascular disease (CVD) and pulmonary diseases. Abnormal lipid metabolism in the endothelium leads to changes in cell signalling, and the expression of genes related to immunity and inflammation. It is therefore important to investigate the pathophysiology of vascular endothelial disorders in terms of lipid metabolism, using a disease model of endothelium. METHODS: Human induced pluripotent stem cell-derived endothelial cells (iECs) were cultured on a matrigel to form an iEC network. Lipids in the iEC network were investigated by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) imaging mass spectrometry (IMS) analysis. Ion fragments obtained by mass spectrometry were analysed using an infusion method, involving precursor ion scanning with fragment ion. RESULTS: The MALDI TOF IMS analysis revealed co-localized intensity of peaks at m/z 592.1 and 593.1 in the iEC network. Tandem mass spectrometry (MS/MS) analysis by MALDI-imaging, in conjunction with precursor ion scanning using an infusion method with lipid extracts, identified that these precursor ions were lysophosphatidylcholine (LPC) (22:5) and its isotype. CONCLUSION: The MALDI-imaging analysis showed that LPC (22:5) was abundant in an iEC network. As an in vitro test model for disease and potential therapy, present analysis methods using MALDI-imaging combined with, for example, mesenchymal stem cells (MSC) to a disease derived iEC network may be useful in revealing the changes in the amount and distribution of lipids under various stimuli.