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1.
J Biochem ; 2024 Jul 24.
Artículo en Inglés | MEDLINE | ID: mdl-39046461

RESUMEN

The maintenance of germ cells is critical for the prosperity of offspring. The amount of food consumption is known to be closely related to reproduction, i.e., the number of eggs decreases under calorie-restricted conditions in various organisms. Previous studies in Caenorhabditis elegans have reported that calorie restriction reduces the number of eggs and the reduction can be rescued by methionine. However, the effect of methionine on the reproductive process has not been fully understood. In this study, to assess the gonadal function of methionine metabolism, we firstly demonstrated that a depletion in dietary methionine resulted in reduced levels of S-adenosyl-l-methionine (SAM) and S-adenosyl homocysteine (SAH) in wild-type N2, but not in glp-1 mutants, which possess only a few germ cells. Second, we found no recovery in egg numbers upon methionine administration in SAM synthase (sams)-1 mutants. Furthermore, a reduced number of proliferative zone nuclei exhibited in the sams-1 mutants was not rescued via methionine. Thus, our results have shown that dietary methionine is required for the normal establishment of both the germline progenitor pool and fecundity, mediated by sams-1.

2.
MicroPubl Biol ; 20242024.
Artículo en Inglés | MEDLINE | ID: mdl-38571512

RESUMEN

Defecation is an ultradian rhythmic behavior in Caenorhabditis elegans . We investigated the involvement of sams family genes in regulating the defecation motor program. We found that sams-1 mutants exhibited longer cycles than wild-type animals. With aging, the sams-1 mutants also frequently skipped the expulsion (Exp) step of defecation behavior. The sams-1 knockdown is known to reduce phosphatidylcholine (PC) levels, which are reversed by choline supplementation. We examined the effect of choline supplementation on defecation cycle times and Exp steps from adult days 1-4. Although choline supplementation did not alter the longer defecation cycle times of sams-1 mutants, it restored the loss of the Exp step in sams-1 mutants on adult days 3 and 4, suggesting a link between the regulation of the Exp step in sams-1 mutants and PC production.

3.
EMBO J ; 40(14): e106434, 2021 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-34152017

RESUMEN

Alternative splicing of pre-mRNAs can regulate gene expression levels by coupling with nonsense-mediated mRNA decay (NMD). In order to elucidate a repertoire of mRNAs regulated by alternative splicing coupled with NMD (AS-NMD) in an organism, we performed long-read RNA sequencing of poly(A)+ RNAs from an NMD-deficient mutant strain of Caenorhabditis elegans, and obtained full-length sequences for mRNA isoforms from 259 high-confidence AS-NMD genes. Among them are the S-adenosyl-L-methionine (SAM) synthetase (sams) genes sams-3 and sams-4. SAM synthetase activity autoregulates sams gene expression through AS-NMD in a negative feedback loop. We furthermore find that METT-10, the orthologue of human U6 snRNA methyltransferase METTL16, is required for the splicing regulation in␣vivo, and specifically methylates the invariant AG dinucleotide at the distal 3' splice site (3'SS) in␣vitro. Direct RNA sequencing coupled with machine learning confirms m6 A modification of endogenous sams mRNAs. Overall, these results indicate that homeostasis of SAM synthetase in C. elegans is maintained by alternative splicing regulation through m6 A modification at the 3'SS of the sams genes.


Asunto(s)
Empalme Alternativo/genética , Homeostasis/genética , Ligasas/genética , Metionina Adenosiltransferasa/genética , Degradación de ARNm Mediada por Codón sin Sentido/genética , ARN Mensajero/genética , S-Adenosilmetionina/metabolismo , Animales , Caenorhabditis elegans/genética , Metiltransferasas/genética , Precursores del ARN/genética
4.
Biometals ; 34(5): 1207-1216, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34146190

RESUMEN

Cadmium is a well-known environmental toxicant. At the cellular level, exposure to cadmium results in cytotoxic effects through the elevation of reactive oxygen species (ROS) production. Although cadmium exposure leads to the dysfunction of various organs, the underlying mechanisms of the toxic effects of cadmium in vivo are still largely unknown. Caenorhabditis elegans (C. elegans) is a useful model animal and exhibits unique biological reactions in response to environmental toxicants. In this study, the toxic mechanisms of cadmium exposure in C. elegans were investigated using N-acetylcysteine (NAC), which has dual functions, i.e., as a chelator of metals and as an antioxidant. NAC did not inhibit the uptake of cadmium into nematodes, suggesting that NAC did not function as a chelator of cadmium under these experimental conditions. Based on this finding, we investigated the effect of NAC as an antioxidant on representative phenotypic traits caused by cadmium exposure-reduced body length, aversion behavior, and shortened lifespan. NAC did not reverse the decreased body size but did clearly restore the aversion behavior and the shortened lifespan. These data suggest that aversion behavior and shortened lifespan are mediated by oxidative stress in C. elegans.


Asunto(s)
Acetilcisteína , Caenorhabditis elegans , Acetilcisteína/farmacología , Animales , Antioxidantes/metabolismo , Antioxidantes/farmacología , Cadmio/toxicidad , Caenorhabditis elegans/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno
5.
J Biol Chem ; 294(9): 3091-3099, 2019 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-30606736

RESUMEN

The tricarboxylic acid (TCA) cycle (or citric acid cycle) is responsible for the complete oxidation of acetyl-CoA and formation of intermediates required for ATP production and other anabolic pathways, such as amino acid synthesis. Here, we uncovered an additional mechanism that may help explain the essential role of the TCA cycle in the early embryogenesis of Caenorhabditis elegans. We found that knockdown of citrate synthase (cts-1), the initial and rate-limiting enzyme of the TCA cycle, results in early embryonic arrest, but that this phenotype is not because of ATP and amino acid depletions. As a possible alternative mechanism explaining this developmental deficiency, we observed that cts-1 RNAi embryos had elevated levels of intracellular acetyl-CoA, the starting metabolite of the TCA cycle. Of note, we further discovered that these embryos exhibit hyperacetylation of mitochondrial proteins. We found that supplementation with acetylase-inhibiting polyamines, including spermidine and putrescine, counteracted the protein hyperacetylation and developmental arrest in the cts-1 RNAi embryos. Contrary to the hypothesis that spermidine acts as an acetyl sink for elevated acetyl-CoA, the levels of three forms of acetylspermidine, N1-acetylspermidine, N8-acetylspermidine, and N1,N8-diacetylspermidine, were not significantly increased in embryos treated with exogenous spermidine. Instead, we demonstrated that the mitochondrial deacetylase sirtuin 4 (encoded by the sir-2.2 gene) is required for spermidine's suppression of protein hyperacetylation and developmental arrest in the cts-1 RNAi embryos. Taken together, these results suggest the possibility that during early embryogenesis, acetyl-CoA consumption by the TCA cycle in C. elegans prevents protein hyperacetylation and thereby protects mitochondrial function.


Asunto(s)
Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismo , Ciclo del Ácido Cítrico , Desarrollo Embrionario , Proteínas Mitocondriales/metabolismo , Acetilación , Adenosina Trifosfato/metabolismo , Animales , Ácido Aspártico/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Citrato (si)-Sintasa/deficiencia , Citrato (si)-Sintasa/genética , Ácido Cítrico/metabolismo , Ácido Glutámico/metabolismo , Espacio Intracelular/metabolismo , Factores de Tiempo
6.
J Biochem ; 163(6): 465-474, 2018 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-29385568

RESUMEN

RNAs are post-transcriptionally modified in all kingdoms of life. Of these modifications, base methylations are highly conserved in eukaryote ribosomal RNA (rRNA). Recently, rRNA processing protein 8 (Rrp8) and nucleomethylin (NML) were identified as factors of N1-methyladenosine (m1A) modification in yeast 25 S and mammalian 28 S rRNA, respectively. However, m1A modification of rRNA is still poorly understood in Caenorhabditis elegans (C. elegans). Here, using the liquid chromatography/tandem mass spectrometry analysis and RNA immunoprecipitation assay, we have identified that the m1A modification is located around position 674 (A674) of 26 S rRNA in C. elegans. Furthermore, quantitative PCR-based analysis revealed that T07A9.8, a C. elegans homolog of yeast Rrp8 and human NML, is responsible for m1A modification at A674 of 26 S rRNA. This m1A modification site in C. elegans corresponds to those in yeast 25 S rRNA and human 28 S rRNA. Intriguingly, T07A9.8 is not associated with pre-rRNA transcription under normal nutrient conditions. Since the m1A modification of 26 S rRNA requires T07A9.8 in C. elegans, we designated the gene as rRNA adenine methyltransferase-1 (rram-1).


Asunto(s)
Adenina/metabolismo , Caenorhabditis elegans/genética , ARN Ribosómico/metabolismo , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismo , Animales , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/metabolismo , Metilación , ARN Ribosómico/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/genética
8.
J Biochem ; 163(5): 433-440, 2018 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-29361115

RESUMEN

Protein arginine methyltransferase 1 (PRMT1) catalyzes asymmetric arginine dimethylation of cellular proteins and thus modulates various biological processes, including gene regulation, RNA metabolism, cell signaling and DNA repair. Since prmt-1 null mutant completely abolishes asymmetric dimethylarginine in C. elegans, PRMT-1 is thought to play a crucial role in determining levels of asymmetric arginine dimethylation. However, the mechanism underlying the regulation of PRMT-1 activity remains largely unknown. Here, we explored for transcription factors that induce the expression of PRMT-1 by an RNAi screen using transgenic C. elegans harbouring prmt-1 promoter upstream of gfp. Of 529 clones, we identify a GATA transcription factor elt-2 as a positive regulator of Pprmt-1:: gfp expression and show that elt-2 RNAi decreases endogenous PRMT-1 expression at mRNA and protein levels. Nevertheless, surprisingly arginine methylation levels are increased when elt-2 is silenced, implying that erythroid-like transcription factor (ELT)-2 may also have ability to inhibit methyltransferase activity of PRMT-1. Supporting this idea, GST pull-down and co-immunoprecipitation assays demonstrate the interaction between ELT-2 and PRMT-1. Furthermore, we find that ELT-2 interferes with PRMT-1-induced arginine methylation in a dose-dependent manner. Collectively, our results illustrate the two modes of PRMT-1 regulation, which could determine the levels of asymmetric arginine dimethylation in C. elegans.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Factores de Transcripción GATA/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Animales , Caenorhabditis elegans/enzimología , Células Cultivadas , Células HEK293 , Humanos
9.
Genetics ; 208(2): 605-622, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29187506

RESUMEN

Ecdysteroids are steroid hormones that control many aspects of development and physiology. During larval development, ecdysone is synthesized in an endocrine organ called the prothoracic gland through a series of ecdysteroidogenic enzymes encoded by the Halloween genes. The expression of the Halloween genes is highly restricted and dynamic, indicating that their spatiotemporal regulation is mediated by their tight transcriptional control. In this study, we report that three zinc finger-associated domain (ZAD)-C2H2 zinc finger transcription factors-Séance (Séan), Ouija board (Ouib), and Molting defective (Mld)-cooperatively control ecdysone biosynthesis in the fruit fly Drosophila melanogaster Séan and Ouib act in cooperation with Mld to positively regulate the transcription of neverland and spookier, respectively, two Halloween genes. Remarkably, loss-of-function mutations in séan, ouib, or mld can be rescued by the expression of neverland, spookier, or both, respectively. These results suggest that the three transcription factors have distinct roles in coordinating the expression of just two genes in Drosophila Given that neverland and spookier are located in constitutive heterochromatin, Séan, Ouib, and Mld represent the first example of a transcription factor subset that regulates genes located in constitutive heterochromatin.


Asunto(s)
Drosophila/genética , Drosophila/metabolismo , Ecdisona/biosíntesis , Factores de Transcripción/metabolismo , Alelos , Animales , Regulación de la Expresión Génica , Larva , Mutación , Fenotipo , Regiones Promotoras Genéticas , Elementos de Respuesta , Dedos de Zinc
10.
J Biochem ; 161(2): 231-235, 2017 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-28173048

RESUMEN

The transmethylation to arginine residues of proteins is catalyzed by protein arginine methyltransferases (PRMTs) that form monomethylarginine (MMA), asymmetric (ADMA) and symmetric dimethylarginines (SDMA). Although we previously demonstrated that the generation of ADMA residues in whole proteins is driven by PRMT-1 in Caenorhabditis elegans, much less is known about MMA and SDMA in vivo. In this study, we measured the amounts of different methylarginines in whole protein extracts made from wild-type (N2) C. elegans and from prmt-1 and prmt-5 null mutants using liquid chromatography-tandem mass spectrometry. Interestingly, we found that the amounts of MMA and SDMA are about fourfold higher than those of ADMA in N2 protein lysates using acid hydrolysis. We were unable to detect SDMA residues in the prmt-5 null mutant. In comparison with N2, an increase in SDMA and decrease in MMA were observed in prmt-1 mutant worms with no ADMA, but ADMA and MMA levels were unchanged in prmt-5 mutant worms. These results suggest that PRMT-1 contributes, at least in part, to MMA production, but that PRMT-5 catalyzes the symmetric dimethylation of substrates containing MMA residues in vivo.


Asunto(s)
Arginina/análogos & derivados , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Animales , Arginina/análisis , Arginina/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/aislamiento & purificación , Cromatografía Liquida , Metilación , Mutación , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/aislamiento & purificación , Espectrometría de Masas en Tándem
11.
J Biochem ; 161(6): 521-527, 2017 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-28158808

RESUMEN

Protein arginine methyltransferases (PRMTs) catalyze the transfer of a methyl group from S-adenosylmethionine to arginine residues and are classified into two types: type I producing asymmetric dimethylarginine (ADMA) and type II producing symmetric dimethylarginine (SDMA). PRMTs have been shown to regulate many cellular processes, including signal transduction, transcriptional regulation and RNA processing. Since the loss-of-function mutation of PRMT1 and PRMT5, each of which is the predominant type I and II, respectively, causes embryonic lethality in mice, their physiological significance at the whole-body level remains largely unknown. Here, we show the morphological and functional phenotypes of single or double null alleles of prmt-1 and prmt-5 in Caenorhabditis elegans. The prmt-1;prmt-5 double mutants are viable, and exhibit short body length and small brood size compared to N2 and each of the single mutants. The liquid chromatography-tandem mass spectrometry analysis demonstrated that the levels of ADMA and SDMA were abolished in the prmt-1;prmt-5 double mutants. Both prmt-1 and prmt-5 were required for resistance to heat and oxidative stresses, whereas prmt-5 is not involved in lifespan regulation even when prmt-1 is ablated. This mutant strain would be a useful model animal for investigating the role of asymmetric and symmetric arginine dimethylation in vivo.


Asunto(s)
Arginina/metabolismo , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Animales , Metilación
12.
PLoS Genet ; 11(12): e1005712, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26658797

RESUMEN

Steroid hormones are crucial for many biological events in multicellular organisms. In insects, the principal steroid hormones are ecdysteroids, which play essential roles in regulating molting and metamorphosis. During larval and pupal development, ecdysteroids are synthesized in the prothoracic gland (PG) from dietary cholesterol via a series of hydroxylation and oxidation steps. The expression of all but one of the known ecdysteroid biosynthetic enzymes is restricted to the PG, but the transcriptional regulatory networks responsible for generating such exquisite tissue-specific regulation is only beginning to be elucidated. Here, we report identification and characterization of the C2H2-type zinc finger transcription factor Ouija board (Ouib) necessary for ecdysteroid production in the PG in the fruit fly Drosophila melanogaster. Expression of ouib is predominantly limited to the PG, and genetic null mutants of ouib result in larval developmental arrest that can be rescued by administrating an active ecdysteroid. Interestingly, ouib mutant animals exhibit a strong reduction in the expression of one ecdysteroid biosynthetic enzyme, spookier. Using a cell culture-based luciferase reporter assay, Ouib protein stimulates transcription of spok by binding to a specific ~15 bp response element in the spok PG enhancer element. Most remarkable, the developmental arrest phenotype of ouib mutants is rescued by over-expression of a functionally-equivalent paralog of spookier. These observations imply that the main biological function of Ouib is to specifically regulate spookier transcription during Drosophila development.


Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Ecdisteroides/genética , Factores de Transcripción/genética , Dedos de Zinc/genética , Animales , Sistema Enzimático del Citocromo P-450/metabolismo , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Ecdisteroides/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Larva/genética , Larva/crecimiento & desarrollo , Metamorfosis Biológica , Fenotipo , Unión Proteica
13.
J Recept Signal Transduct Res ; 33(1): 56-62, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23316847

RESUMEN

S-adenosyl-L-methionine (SAM) is an intermediate metabolite of methionine and serves as the methyl donor for many biological methylation reactions. The synthesis of SAM is catalyzed by SAM synthetase (SAMS), which transfers the adenosyl moiety of adenosine-5'-triphosphate to methionine. In the nematode Caenorhabditis elegans, four sams family genes, sams-1, -3, -4 and -5, are predicted to encode SAMS proteins. However, their physiological roles remain unclear. Here we show that the four predicted SAMS proteins in fact have the ability to catalyze the formation of SAM in vitro, and revealed that only sams-1 mutant animals among the family genes exhibited a significant reduction in egg-laying. Using transgenic animals carrying a transcriptional reporter for each sams gene promoter, we observed that each sams promoter confers a distinct expression pattern with respect to tissue, time of expression and expression level (i.e. promoter specificity). Promoter-swap experiments revealed that the ectopic expression of SAMS-3, -4 or -5 driven by the sams-1 promoter completely rescued egg-laying in sams-1 mutants. These data indicate that SAMS protein function is conserved throughout the entire family.


Asunto(s)
Animales Modificados Genéticamente/fisiología , Caenorhabditis elegans/fisiología , Metionina Adenosiltransferasa/metabolismo , Metionina/metabolismo , Oviposición/fisiología , Animales , Femenino
14.
Cell Metab ; 13(5): 505-16, 2011 May 04.
Artículo en Inglés | MEDLINE | ID: mdl-21531333

RESUMEN

Arginine methylation is a widespread posttranslational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMTs). It is well established that PRMTs are implicated in various cellular processes, but their physiological roles remain unclear. Using nematodes with a loss-of-function mutation, we show that prmt-1, the major asymmetric arginine methyltransferase, is a positive regulator of longevity in C. elegans. This regulation is dependent on both its enzymatic activity and DAF-16/FoxO transcription factor, which is negatively regulated by AKT-mediated phosphorylation downstream of the DAF-2/insulin signaling. prmt-1 is also required for stress tolerance and fat storage but not dauer formation in daf-2 mutants. Biochemical analyses indicate that PRMT-1 methylates DAF-16, thereby blocking its phosphorylation by AKT. Disruption of PRMT-1 induces phosphorylation of DAF-16 with a concomitant reduction in the expression of longevity-related genes. Thus, we provide a mechanism by which asymmetric arginine dimethylation acts as an antiaging modification in C. elegans.


Asunto(s)
Arginina/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Regulación de la Expresión Génica , Longevidad/genética , Metilación , Proteína-Arginina N-Metiltransferasas/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Arginina/metabolismo , Western Blotting , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/genética , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Inmunoprecipitación , Insulina/genética , Insulina/metabolismo , Datos de Secuencia Molecular , Mutación/genética , Fosforilación , Reacción en Cadena de la Polimerasa , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Factores de Transcripción/genética
15.
J Recept Signal Transduct Res ; 30(6): 403-9, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20735177

RESUMEN

Living organisms maintain energy homeostasis by constantly adjusting internal metabolic activities in response to nutritional states. Energy metabolism is regulated by the quality and quantity of the enzymes that catalyze metabolic reactions. Recruitment and regulation of enzymes responsible for transcriptional control, among others, play an important role in this process. Located downstream from intracellular signaling cascades, transcription factors receive information signals from multiple sources. Their primary function is to integrate and interpret this information in terms of transcriptional output. It was recently suggested that signal content is converted via post-transcriptional modifications of the transcription factors. Many studies have shown that multiple signaling pathways converge on single transcription factors. This review discusses the post-translational modifications of transcription factors involved in the regulation of glucose metabolism, as well as the signaling networks in which they play a role.


Asunto(s)
Metabolismo Energético/genética , Regulación de la Expresión Génica , Hígado/metabolismo , Hígado/fisiología , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Ayuno , Gluconeogénesis/fisiología , Glucosa/metabolismo , Procesamiento Proteico-Postraduccional , Transducción de Señal/genética , Factores de Transcripción/genética
16.
J Recept Signal Transduct Res ; 30(6): 484-92, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20684663

RESUMEN

Angiotensinogen (AGT), mainly produced in the liver, is the precursor of angiotensin II, an important regulator of blood pressure and electrolyte homeostasis. We previously showed, in hepatoma-derived HepG2 cells that a hepatocyte nuclear factor 4 (HNF4) potentiated human AGT (hAGT) promoter activity and identified its binding sites (termed regions C and J) in the hAGT promoter region. We also showed in transgenic mouse (TgM) that the hAGT is abundantly expressed in the kidney where the level of endogenous mouse AGT (mAGT) expression is low. To elucidate molecular mechanisms of the AGT gene activation in the kidney, we first investigated the HNF4 and AGT expression in the mouse kidney. Northern blot, in situ hybridization and immunohistochemical analyses revealed that the hAGT and HNF4 were both expressed in the proximal tubular (PT) cells of the kidney. We then transfected the hAGT reporter constructs into immortalized mouse PT (mProx) cells and found that regions C and J contributed additively to the HNF4-potentiated hAGT promoter activity. Curiously, no obvious HNF4 binding motif was found in the corresponding region of the mAGT promoter and co-transfected HNF4 failed to activate this promoter in neither HepG2 nor mProx cells. These results suggest that the high-level hAGT expression in the TgM kidney is, at least in part, due to a presence of high-affinity HNF4 binding sites in its promoter.


Asunto(s)
Angiotensinógeno/genética , Factor Nuclear 4 del Hepatocito/metabolismo , Regiones Promotoras Genéticas , Angiotensinógeno/metabolismo , Animales , Sitios de Unión/genética , Factor Nuclear 4 del Hepatocito/genética , Humanos , Riñón/metabolismo , Hígado/metabolismo , Ratones , Ratones Transgénicos
17.
J Biol Chem ; 283(47): 32432-41, 2008 Nov 21.
Artículo en Inglés | MEDLINE | ID: mdl-18805788

RESUMEN

Glucokinase (GK) and glucose-6-phosphatase (G6Pase) regulate rate-limiting reactions in the physiologically opposed metabolic cascades, glycolysis and gluconeogenesis, respectively. Expression of these genes is conversely regulated in the liver in response to fasting and feeding. We explored the mechanism of transcriptional regulation of these genes by nutritional condition and found that reciprocal function of HNF-4 and Foxo1 plays an important role in this process. In the GK gene regulation, Foxo1 represses HNF-4-potentiated transcription of the gene, whereas it synergizes with HNF-4 in activating the G6Pase gene transcription. These opposite actions of Foxo1 concomitantly take place in the cells under no insulin stimulus, and such gene-specific action was promoter context-dependent. Interestingly, HNF-4-binding elements (HBEs) in the GK and G6Pase promoters were required both for the insulin-stimulated GK gene activation and insulin-mediated G6Pase gene repression. Indeed, mouse in vivo imaging showed that mutating the HBEs in the GK and G6Pase promoters significantly impaired their reactivity to the nutritional states, even in the presence of intact Foxo1-binding sites (insulin response sequences). Thus, in the physiological response of the GK and G6Pase genes to fasting/feeding conditions, Foxo1 distinctly decodes the promoter context of these genes and differently modulates the function of HBE, which then leads to opposite outcomes of gene transcription.


Asunto(s)
Ayuno , Privación de Alimentos , Factores de Transcripción Forkhead/metabolismo , Regulación Enzimológica de la Expresión Génica , Glucoquinasa/metabolismo , Glucosa-6-Fosfatasa/metabolismo , Factor Nuclear 4 del Hepatocito/metabolismo , Transcripción Genética , Animales , Proteína Forkhead Box O1 , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL
18.
Int J Mol Med ; 19(5): 751-6, 2007 May.
Artículo en Inglés | MEDLINE | ID: mdl-17390079

RESUMEN

Bile acid homeostasis is tightly controlled by the feedback mechanism in which an atypical orphan nuclear receptor (NR), small heterodimer partner (SHP), inactivates several transcription factors. We previously demonstrated that bile acid represses the expression of gluconeogenic genes, including glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), and fructose-1,6-bisphosphatase (FBP1) in an SHP-dependent manner. Recently, peroxisome proliferator-activated receptor-gamma (PPAR-gamma) coactivator-1 (PGC-1) gene, a coactivator of NRs important for gluconeogenic gene expression, was also downregulated by bile acid in wild-type mice but not in farnesoid X receptor- or SHP-null mice. However, the molecular mechanism for the effect of bile acid on PGC-1 gene expression remains unknown. In the present study, a series of reporter assays demonstrated that the promoter activity of PGC-1 via a member of the forkhead transcription factors, Foxo1, FOXO3a, and Foxo4 was downregulated by treatment with chenodeoxicholic acid and with transfected SHP. These results revealed that bile acid inhibits the promoter activity of PGC-1 in an SHP-dependent manner.


Asunto(s)
Ácidos y Sales Biliares/farmacología , Regulación hacia Abajo/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/genética , Animales , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Gluconeogénesis/efectos de los fármacos , Gluconeogénesis/genética , Humanos , Ratones , Modelos Genéticos , Proteínas Proto-Oncogénicas c-akt/metabolismo
19.
Transl Res ; 148(2): 96-102, 2006 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16890150

RESUMEN

UNLABELLED: Cholesterol sulfate (CS) in the gastrointestinal tract exhibits a mucosal protective activity in mouse ulcer model. To clarify the possible role of CS for protection from the epithelial injury due to neutrophil elastase in the tracheobronchi, the authors determined the concentrations of CS and neutrophil elastase in bronchoalveolar lavage fluid (BALF) from patients under anesthesia, and they examined the inhibitory activity of CS toward neutrophil elastase. The concentrations of CS and neutrophil elastase were determined by thin-layer chromatography and enzyme-linked immunosorbent assaying, respectively, and the effect of CS on the activity of elastase was determined with a chromogenic substrate. CS was found to be present in human lung, tracheal mucosa, and BALF, and a high synthesis of it was detected in the tracheal mucosa, in which cellular cholesterol sulfotransferase was induced depending on the density of tracheal cells. Among lipids in the tracheal mucosa, only CS was demonstrated to exhibit inhibitory activity toward neutrophil elastase, a powerful erosive agent in inflammation. The secretion of elastase from neutrophils into BALF was stimulated during the course of general anesthesia. In contrast, the amount of CS in BALF gradually decreased during anesthesia. On immune-precipitation of neutrophil elastase in BALF, CS was detected in the immune precipitate, which indicates a possible association of CS with neutrophil elastase in BALF. CONCLUSION: CS, which is a major acidic lipid in the tracheobronchial epithelium, might function as an epithelial inhibitor toward neutrophil elastase secreted in response to several stimuli such as anesthesia.


Asunto(s)
Anestesia General/efectos adversos , Líquido del Lavado Bronquioalveolar/química , Ésteres del Colesterol/metabolismo , Ésteres del Colesterol/farmacología , Elastasa de Leucocito/antagonistas & inhibidores , Elastasa de Leucocito/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Inhibidores Enzimáticos/farmacología , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Metabolismo de los Lípidos , Lípidos/química , Masculino , Persona de Mediana Edad , Mucosa Respiratoria/metabolismo , Tráquea/metabolismo
20.
J Biol Chem ; 280(51): 42283-9, 2005 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-16230354

RESUMEN

Bile acids secreted in the small intestine are reabsorbed in the ileum where they activate the nuclear farnesoid X receptor (FXR), which in turn stimulates expression of the ileal bile acid-binding protein (I-BABP). We first hypothesized that I-BABP may negatively regulate the FXR activity by competing for the ligands, bile acids. Reporter assays using stable HEK293 cell lines expressing I-BABP revealed that I-BABP enhances rather than attenuates FXR activity. In these cells I-BABP localizes predominantly in the cytosol and partially in the nucleus, a distribution that does not shift in response to FXR expression. In vitro binding assays reveal that recombinant I-BABP is able to bind 35S-labeled FXR and that chenodeoxycholic acid (CDCA) stimulates this interaction modestly. When FLAG-tagged FXR was expressed in stable cells, the FXR.I-BABP complex in the nuclear extracts was more efficiently immunoprecipitable with anti-FLAG antibodies in the presence of CDCA. These results indicate that I-BABP stimulates FXR activity through a mutual interaction augmented by bile acids. When stable cells were transfected with an expression plasmid of the ileal bile acid transporter 14(IBAT) essential for the reabsorption of conjugated bile acids, the C-labeled conjugated bile acid, glycocholic acid, was more efficiently imported via IBAT in the presence than absence of I-BABP, whereas no change was observed in 14C-labeled CDCA uptake, which is independent of IBAT. Immunofluorescent staining analysis revealed that these two proteins co-localize in the vicinity of the plasma membrane in stable cells. Taken together, the current data provide the first evidence that I-BABP is functionally associated with FXR and IBAT in the nucleus and on the membrane, respectively, stimulating FXR transcriptional activity and the conjugated bile acid uptake mediated by IBAT in the ileum.


Asunto(s)
Ácidos y Sales Biliares/metabolismo , Proteínas de Unión al ADN/metabolismo , Íleon/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Factores de Transcripción/metabolismo , Northern Blotting , Western Blotting , Línea Celular , Núcleo Celular/metabolismo , Citosol/metabolismo , Expresión Génica , Humanos , Inmunohistoquímica , Proteínas de Transporte de Membrana/genética , Receptores Citoplasmáticos y Nucleares
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