ABSTRACT
Although polymerase chain reaction (PCR) amplification and sequencing of the bacterial 16S rDNA region has numerous scientific applications, it does not provide DNA methylation information. Herein, we propose a simple extension for bisulfite sequencing to investigate 5-methylcytosine residues in the bacterial 16S rDNA region from clinical isolates or flora. Multiple displacement amplification without DNA denaturation was used to preferentially pre-amplify single-stranded bacterial DNA after bisulfite conversion. Following the pre-amplification, the 16S rDNA region was analyzed using nested bisulfite PCR and sequencing, enabling the simultaneous identification of DNA methylation status and sequence data. We used this approach (termed sm16S rDNA PCR/sequencing) to identify novel methylation sites and a methyltransferase (M. MmnI) in Morganella morganii and different methylation motifs among Enterococcus faecalis strains from small volumes of clinical specimens. Further, our analysis suggested that M. MmnI may be correlated to erythromycin resistance. Thus, sm16S rDNA PCR/sequencing is a useful extension method for analyzing the DNA methylation of 16S rDNA regions in a microflora, providing additional information not provided by conventional PCR. Given the relationship between DNA methylation status and drug resistance in bacteria, we believe this technique can be effectively applied in clinical sample testing.
Subject(s)
Bacteria , DNA Methylation , DNA, Ribosomal/genetics , DNA, Ribosomal/analysis , DNA, Bacterial/chemistry , Bacteria/genetics , Sequence Analysis, DNA , RNA, Ribosomal, 16S/geneticsABSTRACT
The microRNA (miR) miR-874, a potential tumour suppressor, causes cell death via target gene suppression in various cancer types. Mevalonate pathway inhibition also causes cell death in breast cancer. However, the relationship between the mevalonate pathway and miR-874-induced apoptosis or its association with the tumour suppressor p53 has not been elucidated. We identified phosphomevalonate kinase (PMVK), a key mevalonate pathway enzyme, and sterol regulatory element-binding factor 2 (SREBF2), the master cholesterol biosynthesis regulator, as direct miR874 targets. Next-generation sequencing analysis revealed a significant miR-874-mediated downregulation of PMVK and SREBF2 gene expression and p53 pathway enrichment. Luciferase reporter assays showed that miR-874 directly regulated PMVK and SREBF2. miR-874-induced apoptosis was p53 dependent, and single-cell RNA sequencing analysis demonstrated that miR-874 transfection resulted in apoptosis and p53 pathway activation. Downregulation of PMVK expression also caused cell cycle arrest and p53 pathway activation, which was rescued by geranylgeranyl pyrophosphate (GGPP) supplementation. Analysis of The Cancer Genome Atlas (TCGA) database indicated a negative correlation between miR-874 and PMVK expression and between miR-874 and SREBF2 expression. These findings suggest that miR-874 suppresses the mevalonate pathway by targeting SREBF2 and PMVK, resulting in GGPP depletion, which activates the p53 pathway and promotes cycle arrest or apoptosis.
Subject(s)
MicroRNAs , Tumor Suppressor Protein p53 , Humans , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Mevalonic Acid/metabolism , Cell Line, Tumor , MicroRNAs/metabolism , Apoptosis/genetics , Cell Transformation, Neoplastic/genetics , Cell Proliferation/genetics , Gene Expression Regulation, NeoplasticABSTRACT
The clinical characteristics of growth hormone (GH)-producing pituitary adenomas/somatotroph pituitary neuroendocrine tumors (GHomas/somatotroph PitNETs) vary across patients. In this study, we aimed to integrate the genetic alterations, protein expression profiles, transcriptomes, and clinical characteristics of GHomas/somatotroph PitNETs to identify molecules associated with acromegaly characteristics. Targeted capture sequencing and copy number analysis of 36 genes and nontargeted proteomics analysis were performed on fresh-frozen samples from 121 sporadic GHomas/somatotroph PitNETs. Targeted capture sequencing revealed GNAS as the only driver gene, as previously reported. Classification by consensus clustering using both RNA sequencing and proteomics revealed many similarities between the proteome and the transcriptome. Gene ontology analysis was performed for differentially expressed proteins between wild-type and mutant GNAS samples identified by nontargeted proteomics and involved in G protein-coupled receptor (GPCR) pathways. The results suggested that GNAS mutations impact endocrinological features in acromegaly through GPCR pathway induction. ATP2A2 and ARID5B correlated with the GH change rate in the octreotide loading test, and WWC3, SERINC1, and ZFAND3 correlated with the tumor volume change rate after somatostatin analog treatment. These results identified a biological connection between GNAS mutations and the clinical and biochemical characteristics of acromegaly, revealing molecules associated with acromegaly that may affect medical treatment efficacy.
Subject(s)
Acromegaly , Adenoma , Growth Hormone-Secreting Pituitary Adenoma , Neuroendocrine Tumors , Pituitary Neoplasms , Proteogenomics , Somatotrophs , Humans , Somatotrophs/metabolism , Somatotrophs/pathology , Acromegaly/complications , Acromegaly/metabolism , Acromegaly/pathology , Pituitary Neoplasms/genetics , Pituitary Neoplasms/metabolism , Pituitary Neoplasms/pathology , Neuroendocrine Tumors/genetics , Neuroendocrine Tumors/pathology , Adenoma/genetics , Adenoma/metabolism , Adenoma/pathology , Growth Hormone-Secreting Pituitary Adenoma/genetics , Growth Hormone-Secreting Pituitary Adenoma/complications , Growth Hormone-Secreting Pituitary Adenoma/pathologyABSTRACT
The liver stores glycogen and releases glucose into the blood upon increased energy demand. Group 2 innate lymphoid cells (ILC2) in adipose and pancreatic tissues are known for their involvement in glucose homeostasis, but the metabolic contribution of liver ILC2s has not been studied in detail. Here we show that liver ILC2s are directly involved in the regulation of blood glucose levels. Mechanistically, interleukin (IL)-33 treatment induces IL-13 production in liver ILC2s, while directly suppressing gluconeogenesis in a specific Hnf4a/G6pc-high primary hepatocyte cluster via Stat3. These hepatocytes significantly interact with liver ILC2s via IL-13/IL-13 receptor signaling. The results of transcriptional complex analysis and GATA3-ChIP-seq, ATAC-seq, and scRNA-seq trajectory analyses establish a positive regulatory role for the transcription factor GATA3 in IL-13 production by liver ILC2s, while AP-1 family members are shown to suppress IL-13 release. Thus, we identify a regulatory role and molecular mechanism by which liver ILC2s contribute to glucose homeostasis.
Subject(s)
Gluconeogenesis , Interleukin-13 , Blood Glucose/metabolism , GATA3 Transcription Factor/genetics , GATA3 Transcription Factor/metabolism , Glycogen/metabolism , Immunity, Innate , Interleukin-13/metabolism , Liver/metabolism , Lymphocytes/metabolism , Receptors, Interleukin-13/metabolism , Transcription Factor AP-1/metabolismABSTRACT
TAS4464, a potent, selective small molecule NEDD8-activating enzyme (NAE) inhibitor, leads to inactivation of cullin-RING E3 ubiquitin ligases (CRLs) and consequent accumulations of its substrate proteins. Here, we investigated the antitumor properties and action mechanism of TAS4464 in acute myeloid leukemia (AML). TAS4464 induced apoptotic cell death in various AML cell lines. TAS4464 treatments resulted in the activation of both the caspase-9-mediated intrinsic apoptotic pathway and caspase-8-mediated extrinsic apoptotic pathway in AML cells; combined treatment with inhibitors of these caspases markedly diminished TAS4464-induced apoptosis. In each apoptotic pathway, TAS4464 induced the mRNA transcription of the intrinsic proapoptotic factor NOXA and decreased that of the extrinsic antiapoptotic factor c-FLIP. RNA-sequencing analysis showed that the signaling pathway of the CRL substrate c-Myc was enriched after TAS4464 treatment. Chromatin immunoprecipitation (ChIP) assay revealed that TAS4464-induced c-Myc bound to the PMAIP1 (encoding NOXA) and CFLAR (encoding c-FLIP) promoter regions, and siRNA-mediated c-Myc knockdown neutralized both TAS4464-mediated NOXA induction and c-FLIP downregulation. TAS4464 activated both caspase-8 and caspase-9 along with an increase in NOXA and a decrease in c-FLIP, resulting in complete tumor remission in a human AML xenograft model. These findings suggest that NAE inhibition leads to anti-AML activity via a novel c-Myc-dependent apoptosis induction mechanism.
Subject(s)
Leukemia, Myeloid, Acute/drug therapy , NEDD8 Protein/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-myc/genetics , Pyrimidines/pharmacology , Pyrroles/pharmacology , Animals , Apoptosis/drug effects , CASP8 and FADD-Like Apoptosis Regulating Protein/genetics , Caspase 8/genetics , Cell Line, Tumor , Enzyme Inhibitors/pharmacology , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Mice , NEDD8 Protein/antagonists & inhibitors , RNA, Small Interfering/genetics , RNA, Small Interfering/pharmacology , RNA-Seq , Signal Transduction/drug effects , Ubiquitin-Protein Ligases/genetics , Xenograft Model Antitumor AssaysABSTRACT
Sphingolipids represent a family of cellular lipid-molecules that regulate physiological and pathophysiological processes. Glucosylceramide (GlcCer), the simplest glycosphingolipid (GSL), is synthesized from ceramide and UDP-glucose by GlcCer synthase (GCS). Both GlcCer (and resulting GSLs) and ceramide regulate various cellular functions including cell death and multiple drug resistance. Src family tyrosine kinases are up-regulated in various human cancer cells. We examined the effect of v-Src expression on GCS activity, the formation of 4-nitrobenzo-2-oxa-1,3-diazole (NBD)-labeled GlcCer from NBD-ceramide, and the effect of tyrosine132 mutation in GCS on ceramide-induced cytotoxicity in HeLa cells. Expression of v-Src increased the formation of NBD-GlcCer in both intact cells without marked changes in other sphingolipid metabolites and cell homogenates without changing affinities of NBD-ceramide and UDP-glucose. Expression of v-Src also increased tyrosine-phosphorylated levels in GCS proteins in HeLa and HEK293T cells. In HEK293T cells transiently expressing the GCS mutant, GCS-Y132F-HA, showing replacement of the tyrosine132 residue with phenylalanine, tyrosine-phosphorylated levels in GCS proteins were significantly lower than those in control cells expressing the GCS-wild-type-HA. The formation of NBD-GlcCer in HeLa cells stably expressing GCS-Y132F-HA was significantly lower than that in the control. Ceramide-induced cytotoxicity in HeLa-GCS-Y132F-HA cells was significantly greater than in the control. In this study, we showed for the first time that expression of v-Src up-regulated GCS activity via tyrosine phosphorylation of the enzyme in a post-translational manner. Mechanisms of Src-induced resistance to ceramide-induced cytotoxicity are discussed in relation to the Src-induced up-regulation of GCS activity.
Subject(s)
Glucosylceramides/pharmacology , Glucosyltransferases/genetics , Oncogene Protein pp60(v-src)/genetics , Phenylalanine/metabolism , Tyrosine/metabolism , 4-Chloro-7-nitrobenzofurazan/analogs & derivatives , 4-Chloro-7-nitrobenzofurazan/metabolism , Cell Survival/drug effects , Ceramides/metabolism , Enzyme Activation/drug effects , Glucosyltransferases/metabolism , HEK293 Cells , HeLa Cells , Humans , Mutation , Oncogene Protein pp60(v-src)/metabolism , Phenylalanine/genetics , Phosphorylation/drug effects , Tyrosine/genetics , Uridine Diphosphate Glucose/metabolismABSTRACT
Ceramide kinase (CerK) phosphorylates ceramide to ceramide-1-phosphate (C1P), a bioactive sphingolipid. Since the mechanisms responsible for regulating the proliferation and migration/metastasis of cancer cells by the CerK/C1P pathway remain unclear, we conducted the present study. The knockdown of CerK in A549 lung and MCF-7 breast cancer cells (shCerK cells) increased the formation of lamellipodia, which are membrane protrusions coupled with cell migration. Mouse embryonic fibroblasts prepared from CerK-null mice also showed an enhanced formation of lamellipodia. The overexpression of CerK inhibited lamellipodium formation in A549 cells. The knockdown of CerK increased the number of cells having lamellipodia with Rac1 and the levels of active Rac1-GTP form, whereas the overexpression of CerK decreased them. CerK was located in lamellipodia after the epidermal growth factor treatment, indicating that CerK functioned there to inhibit Rac1. The migration of A549 cells was negatively regulated by CerK. An intravenous injection of A549-shCerK cells into nude mice resulted in markedly stronger metastatic responses in the lungs than an injection of control cells. The in vitro growth of A549 cells and in vivo expansion after the injection into mouse flanks were not affected by the CerK knockdown. These results suggest that the activation of CerK/C1P pathway has inhibitory roles on lamellipodium formation, migration, and metastasis of A549 lung cancer cells.
Subject(s)
Lung Neoplasms/pathology , Neoplasm Metastasis/pathology , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Pseudopodia/metabolism , rac1 GTP-Binding Protein/metabolism , A549 Cells , Animals , Cell Movement , Ceramides/metabolism , Gene Knockdown Techniques , Humans , MCF-7 Cells , Male , Mice , Neoplasm Invasiveness/pathology , Phosphotransferases (Alcohol Group Acceptor)/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Xenograft Model Antitumor AssaysABSTRACT
Next-generation sequencing (NGS) is a revolutionary sequencing technology for analyzing genomes. However, preprocessing methods for mitochondrial DNA (mtDNA) sequencing remain complex, and it is required to develop an authenticated preprocessing method. Here, we developed a simple and easy preprocessing method based on isothermal rolling circle mtDNA amplification using commercially available reagents. Isothermal amplification of mtDNA was successfully performed using both nanoliter quantities of plasma directly and 25 ng of total DNA extracted from blood or tissue samples. Prior to mtDNA amplification, it was necessary to treat the extracted total DNA with Exonuclease V, but it was not required to treat plasma. The NGS libraries generated from the amplified mtDNA provided sequencing coverage of the entire human mitochondrial genome. Furthermore, the sequencing results successfully detected heteroplasmy in patient samples, with called mutations and variants matching those from previous, independent, Sanger sequencing analysis. Additionally, a novel single nucleotide variant was detected in a healthy volunteer. The successful analysis of mtDNA using very small samples from patients is likely to be valuable in clinical medicine, as it could reduce patient discomfort by reducing sampling-associated damage to tissues. Overall, the simple and convenient preprocessing method described herein may facilitate the future development of NGS-based clinical and forensic mtDNA tests.
Subject(s)
Genetic Testing , Genome, Mitochondrial , Genomics , Whole Genome Sequencing , Alleles , Chromosome Mapping , Gene Frequency , Genetic Testing/methods , Genetic Variation , Genomics/methods , Humans , Mitochondrial Diseases/diagnosis , Mitochondrial Diseases/genetics , Molecular Diagnostic Techniques , Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
Sphingosine kinases (SphKs) and ceramide kinase (CerK) phosphorylate sphingosine to sphingosine-1-phosphate (S1P) and ceramide to ceramide-1-phosphate (C1P), respectively. S1P and C1P are bioactive lipids that regulate cell fate/function and human health/diseases. The translocation and activity of SphK1 are regulated by its phosphorylation of Ser 225 and by anionic lipids such as phosphatidic acid and phosphatidylserine. However, the roles of another anionic lipid C1P on SphK1 functions have not yet been elucidated, thus, we here investigated the regulation of SphK1 by CerK/C1P. C1P concentration dependently bound with and activated recombinant human SphK1. The inhibition of CerK reduced the phorbol 12-myristate 13-acetate-induced translocation of SphK1 to the plasma membrane (PM) and activation of the enzyme in membrane fractions of cells. A treatment with C1P translocated wild-type SphK1, but not the SphK1-S225A mutant, to the PM without affecting phosphorylation signaling. A cationic RxRH sequence is proposed to be a C1P-binding motif in α-type cytosolic phospholipase A 2 and tumor necrosis factor α-converting enzyme. The mutation of four cationic amino acids to Ala in the 56-RRNHAR-61 domain in SphK1 reduced the phorbol 12-myristate 13-acetate- and C1P-induced translocation of SphK1 to the PM, however, the capacity of C1P to bind with and activate SphK1 was not affected by this mutation. In conclusion, C1P modulates SphK1 functions by interacting with multiple sites in SphK1.
Subject(s)
Ceramides/genetics , Phosphotransferases (Alcohol Group Acceptor)/genetics , Amino Acids/genetics , Cell Membrane/genetics , Cell Membrane/metabolism , Ceramides/metabolism , Cytosol/metabolism , Humans , Lysophospholipids/genetics , Lysophospholipids/metabolism , Mutation/genetics , Phospholipases A2/genetics , Phosphorylation/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Protein Transport/drug effects , Protein Transport/genetics , Signal Transduction/genetics , Sphingosine/analogs & derivatives , Sphingosine/genetics , Sphingosine/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Tumor Necrosis Factor-alpha/geneticsABSTRACT
The up-regulated expression of E-type prostanoid (EP) 4 receptors has been implicated in carcinogenesis; however, the expression of EP4 receptors has also been reported to be weaker in tumor tissues than in normal tissues. Indeed, EP4 receptors have been suggested to play a role in the maintenance of colorectal homeostasis. This study aimed to examine the underlying mechanisms/reasons for why inconsistent findings have been reported regarding EP4 receptor expression levels in homeostasis and carcinogenesis by focusing on cellular densities. Thus, the human colon cancer HCA-7 cells, which retain some functional features of normal epithelia, and luciferase reporter genes containing wild-type or mutated EP4 receptor promoters were used for elucidating the cellular density-dependent mechanisms about the regulation of EP4 receptor expression. In silico analysis was also utilized for confirming the relevance of the findings with respect to colon cancer development. We here demonstrated that the expression of EP4 receptors was up-regulated by c-Myc by binding to Sp-1 under low cellular density conditions, but was down-regulated under high cellular density conditions via the increase in the expression levels of HIF-1α protein, which may pull out c-Myc and Sp-1 from DNA-binding. The tightly regulated EP4 receptor expression mechanism may be a critical system for maintaining homeostasis in normal colorectal epithelial cells. Therefore, once the system is altered, possibly due to the transient overexpression of EP4 receptors, it may result in aberrant cellular proliferation and transformation to cancerous phenotypes. However, at the point, EP4 receptors themselves and their mediated homeostasis would be no longer required.
Subject(s)
Colonic Neoplasms/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Promoter Regions, Genetic/genetics , Proto-Oncogene Proteins c-myc/metabolism , Receptors, Prostaglandin E, EP4 Subtype/genetics , Carcinogenesis/genetics , Cell Count , Cell Line, Tumor , Colonic Neoplasms/pathology , Computational Biology , Down-Regulation , Gene Expression Regulation, Neoplastic , Humans , Receptors, Prostaglandin E, EP4 Subtype/metabolism , Sp1 Transcription Factor/metabolism , Up-RegulationABSTRACT
A new ceramide analog, 1, containing two fluorescent dyes, NBD in the N-acyl part and KFL5 in the alkyl part, was synthesized. The fluorescence from both NBD and KFL5 was detected in living cells in a time-dependent manner. A multi-wavelength fluorescence detector was used to detect ceramide metabolites including sphingosine, sphingosine-1-phosphate, glucosylceramide, and sphingomyelin, which are connected to the fluorescent dyes, simultaneously in a single TLC plate.
Subject(s)
Ceramides/metabolism , Fluorescent Dyes/chemistry , Sphingolipids/metabolism , Animals , Chromatography, Thin Layer , Cricetinae , FluorescenceABSTRACT
Missense mutations in Ten-eleven translocation 2 (TET2) gene are frequently found in leukaemia patients. Although mutations span the entire coding region, they tend to cluster in the C-terminal enzymatic domain and a cysteine-rich (CR) domain of unknown function. Herein, we found the CR domain binds chromatin preferentially at the histone H3 tail by recognising H3 lysine 36 mono- and dimethylation (H3K36me1/2). Importantly, missense mutations in the CR domain perturbed TET2 recruitment to the target locus and its enzymatic activities. Our findings identify a novel H3K36me recognition domain and uncover a critical link between histone modification and DNA hydroxylation in leukaemogenesis.
Subject(s)
Cysteine/metabolism , DNA-Binding Proteins/metabolism , Histones/metabolism , Lysine/metabolism , Proto-Oncogene Proteins/metabolism , Binding Sites/genetics , Chromatin/metabolism , Cysteine/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Dioxygenases , HEK293 Cells , Histones/chemistry , Humans , Immunoblotting , Lysine/chemistry , Methylation , Microscopy, Confocal , Models, Molecular , Mutation, Missense , Protein Binding , Protein Domains , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/geneticsABSTRACT
Upper-layer (UL) neocortical neurons are the most prominent distinguishing features of the mammalian neocortex compared with those of the avian dorsal cortex and are vastly expanded in primates. However, little is known about the identities of the genes that control the specification of UL neurons. Here, we found that Prdm8, a member of the PR (PRDI-BF1 and RIZ homology) domain protein family, was specifically expressed in the postnatal UL neocortex, particular those in late-born RORß-positive layer IV neurons. We generated homozygous Prdm8 knockout (Prdm8 KO) mice and found that the deletion of Prdm8 causes growth retardation and a reduced brain weight, although the brain weight-to-body weight ratio is unchanged at postnatal day 8 (P8). Immunohistochemistry showed that the relative UL thickness, but not the thickness of the deep layer (DL), was significantly reduced in Prdm8 KO mice compared with wild-type (WT) mice. In addition, we found that a number of late-born Brn2-positive UL neurons were significantly decreased in Prdm8 KO mice. To identify genes regulated by Prdm8 during neocortical development, we compared expression profiling analysis in Prdm8 KO and WT mice, and identified some candidate genes. These results suggest that the proper expression of Prdm8 is required for the normal development and construction of UL neurons in the mammalian neocortex.
Subject(s)
Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Neocortex/growth & development , Neurons/metabolism , Animals , DNA-Binding Proteins , Gene Deletion , Histone Methyltransferases , Mice , Mice, Knockout , Neocortex/cytology , Neurons/cytologyABSTRACT
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.
Subject(s)
Arginine/genetics , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Gene Expression Regulation , Longevity/genetics , Methylation , Protein-Arginine N-Methyltransferases/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Arginine/metabolism , Blotting, Western , Caenorhabditis elegans/genetics , Caenorhabditis elegans/growth & development , Caenorhabditis elegans Proteins/genetics , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Immunoprecipitation , Insulin/genetics , Insulin/metabolism , Molecular Sequence Data , Mutation/genetics , Phosphorylation , Polymerase Chain Reaction , Protein-Arginine N-Methyltransferases/genetics , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Receptor, Insulin/genetics , Receptor, Insulin/metabolism , Sequence Homology, Amino Acid , Signal Transduction , Transcription Factors/geneticsABSTRACT
Protein arginine methylation is a common posttranslational modification catalyzed by a family of the protein arginine methyltransferases (PRMTs). We have previously reported that PRMT1 methylates Forkhead box O transcription factors at two arginine residues within an Akt consensus phosphorylation motif (RxRxxS/T), and that this methylation blocks Akt-mediated phosphorylation of the transcription factors. These findings led us to hypothesize that the functional crosstalk between arginine methylation and phosphorylation could be extended to other Akt target proteins as well as Forkhead box O proteins. Here we identify BCL-2 antagonist of cell death (BAD) as an additional substrate for PRMT1 among several Akt target proteins. We show that PRMT1 specifically binds and methylates BAD at Arg-94 and Arg-96, both of which comprise the Akt consensus phosphorylation motif. Consistent with the hypothesis, PRMT1-mediated methylation of these two arginine residues inhibits Akt-mediated phosphorylation of BAD at Ser-99 in vitro and in vivo. We also demonstrate that the complex formation of BAD with 14-3-3 proteins, which occurs subsequent to Akt-mediated phosphorylation, is negatively regulated by PRMT1. Furthermore, PRMT1 knockdown prevents mitochondrial localization of BAD and its binding to the antiapoptotic BCL-X(L) protein. BAD overexpression causes an increase in apoptosis with concomitant activation of caspase-3, whereas PRMT1 knockdown significantly suppresses these apoptotic processes. Taken together, our results add a new dimension to the complexity of posttranslational BAD regulation and provide evidence that arginine methylation within an Akt consensus phosphorylation motif functions as an inhibitory modification against Akt-dependent survival signaling.
Subject(s)
Arginine/metabolism , Protein Processing, Post-Translational , Protein-Arginine N-Methyltransferases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Repressor Proteins/metabolism , bcl-Associated Death Protein/metabolism , Apoptosis , Arginine/genetics , Gene Knockdown Techniques , HEK293 Cells , Humans , Methylation , Phosphorylation , Protein-Arginine N-Methyltransferases/genetics , Repressor Proteins/genetics , bcl-Associated Death Protein/antagonists & inhibitors , bcl-Associated Death Protein/geneticsSubject(s)
Arginine/metabolism , Cell Physiological Phenomena , Methylation , Protein-Arginine N-Methyltransferases , Animals , Cell Differentiation , DNA Damage/physiology , Hematopoietic Stem Cells/cytology , Histones/physiology , Humans , Insulin/physiology , Interferons/physiology , Oxidative Stress/physiology , Protein-Arginine N-Methyltransferases/physiology , Receptors, Cytoplasmic and Nuclear/physiology , Signal Transduction/physiology , Transforming Growth Factor beta/physiologyABSTRACT
Forkhead box O (FOXO) transcription factors, the key regulators of cell survival, are negatively controlled through the PI3K-Akt signaling pathway. Phosphorylation of FOXO by Akt leads to cytoplasmic localization and subsequent degradation via the ubiquitin-proteasome system. Here we show a paradigm of FOXO1 regulation by the protein arginine methyltransferase PRMT1. PRMT1 methylated FOXO1 at conserved Arg248 and Arg250 within a consensus motif for Akt phosphorylation; this methylation directly blocked Akt-mediated phosphorylation of FOXO1 at Ser253 in vitro and in vivo. Silencing of PRMT1 by small interfering RNA enhanced nuclear exclusion, polyubiquitination, and proteasomal degradation of FOXO1. PRMT1 knockdown led to a decrease in oxidative-stress-induced apoptosis depending on the PI3K-Akt signaling pathway. Furthermore, stable expression of enzymatic inactive PRMT1 mutant increased resistance to apoptosis, whereas this effect was reversed by expression of phosphorylation-deficient FOXO1. Our findings predict a role for arginine methylation as an inhibitory modification against Akt-mediated phosphorylation.
Subject(s)
Arginine/metabolism , Forkhead Transcription Factors/metabolism , Proto-Oncogene Proteins c-akt/physiology , Active Transport, Cell Nucleus , Amino Acid Sequence , Apoptosis , Consensus Sequence , Forkhead Box Protein O1 , Gene Silencing , Humans , Methylation , Oxidative Stress , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Proteasome Endopeptidase Complex/metabolism , Protein-Arginine N-Methyltransferases/genetics , Protein-Arginine N-Methyltransferases/physiology , Repressor Proteins/genetics , Repressor Proteins/physiology , Serine/metabolism , Transcriptional Activation/physiology , UbiquitinationABSTRACT
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.
Subject(s)
Bile Acids and Salts/pharmacology , Down-Regulation/drug effects , Promoter Regions, Genetic/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription Factors/genetics , Animals , Forkhead Box Protein O1 , Forkhead Transcription Factors/metabolism , Gluconeogenesis/drug effects , Gluconeogenesis/genetics , Humans , Mice , Models, Genetic , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
Bile acid homeostasis is tightly controlled by the feedback mechanism in which an atypical orphan nuclear receptor (NR) small heterodimer partner (SHP) inactivates several NRs such as liver receptor homologue-1 and hepatocyte nuclear factor 4. Although NRs have been implicated in the transcriptional regulation of gluconeogenic genes, the effect of bile acids on gluconeogenic gene expression remained unknown. Here, we report that bile acids inhibit the expression of gluconeogenic genes, including glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase, and fructose 1,6-bis phosphatase in an SHP-dependent fashion. Cholic acid diet decreased the mRNA levels of these gluconeogenic enzymes, whereas those of SHP were increased. Reporter assays demonstrated that the promoter activity of phosphoenolpyruvate carboxykinase and fructose 1,6-bis phosphatase via hepatocyte nuclear factor 4, or that of G6Pase via the forkhead transcription factor Foxo1, was down-regulated by treatment with chenodeoxicholic acid and with transfected SHP. Remarkably, Foxo1 interacted with SHP in vivo and in vitro, which led to the repression of Foxo1-mediated G6Pase transcription by competition with a coactivator cAMP response element-binding protein-binding protein. These findings reveal a novel mechanism by which bile acids regulate gluconeogenic gene expression via an SHP-dependent regulatory pathway.
Subject(s)
Bile Acids and Salts/metabolism , DNA-Binding Proteins/antagonists & inhibitors , Gluconeogenesis/genetics , Phosphoproteins/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Animals , Cell Line , Feedback , Forkhead Box Protein O1 , Forkhead Transcription Factors , Gene Expression Regulation , Hepatocyte Nuclear Factor 4 , Male , Mice , Mice, Inbred C57BL , Receptors, Cytoplasmic and Nuclear/genetics , Transcription Factors/geneticsABSTRACT
Bile acids function as transcriptional regulators for the genes important in bile acid synthesis and cholesterol homeostasis. In this study, we identified angiotensinogen (ANG), the precursor of vasoactive octapeptide angiotensin II, as a novel target gene of bile acids. In human ANG transgenic mice, administration of cholic acid resulted in the down-regulation of human ANG gene expression in the liver. ANG gene expression in HepG2 cells was also repressed by chenodeoxycholic acid. Because the expression of small heterodimer partner (SHP) mRNA was induced by chenodeoxycholic acid in HepG2 cells, we analyzed the effects of SHP on the human ANG promoter. Promoter mutation analysis demonstrated that SHP repressed human ANG promoter activity through the element, which has been previously determined as a binding site for hepatocyte nuclear factor-4 (HNF-4). SHP repressed human ANG promoter activity only when the HNF-4 expression vector was cotransfected in HeLa cells. Furthermore, we found that SHP bound to the HNF-4 N-terminal region including the DNA-binding domain and activation function-1 and that SHP prevented HNF-4 from binding to the human ANG promoter. These results suggest that bile acids negatively regulate the human ANG gene through the inhibitory effect of SHP on HNF-4.