ABSTRACT
RATIONALE: Forkhead box-O transcription factors (FoxOs) transduce a wide range of extracellular signals, resulting in changes in cell survival, cell cycle progression, and several cell type-specific responses. FoxO1 is expressed in many cell types, including endothelial cells (ECs). Previous studies have shown that Foxo1 knockout in mice results in embryonic lethality at E11 because of impaired vascular development. In contrast, somatic deletion of Foxo1 is associated with hyperproliferation of ECs. Thus, the precise role of FoxO1 in the endothelium remains enigmatic. OBJECTIVE: To determine the effect of endothelial-specific knockout and overexpression of FoxO1 on vascular homeostasis. METHODS AND RESULTS: We show that EC-specific disruption of Foxo1 in mice phenocopies the full knockout. Although endothelial expression of FoxO1 rescued otherwise Foxo1-null animals, overexpression of constitutively active FoxO1 resulted in increased EC size, occlusion of capillaries, elevated peripheral resistance, heart failure, and death. Knockdown of FoxO1 in ECs resulted in marked inhibition of basal and vascular endothelial growth factor-induced Akt-mammalian target of rapamycin complex 1 (mTORC1) signaling. CONCLUSIONS: Our findings suggest that in mice, endothelial expression of FoxO1 is both necessary and sufficient for embryonic development. Moreover, FoxO1-mediated feedback activation of Akt maintains growth factor responsive Akt/mTORC1 activity within a homeostatic range.
Subject(s)
Endothelial Cells/metabolism , Forkhead Transcription Factors/physiology , Heart Failure/genetics , Multiprotein Complexes/physiology , Neovascularization, Physiologic/physiology , Proto-Oncogene Proteins c-akt/physiology , TOR Serine-Threonine Kinases/physiology , Animals , Enzyme Induction , Forkhead Box Protein O1 , Forkhead Box Protein O3 , Forkhead Transcription Factors/antagonists & inhibitors , Forkhead Transcription Factors/deficiency , Forkhead Transcription Factors/genetics , Heart Failure/physiopathology , Homeostasis , Human Umbilical Vein Endothelial Cells , Mechanistic Target of Rapamycin Complex 1 , Mice , Mice, Knockout , Mice, Transgenic , Neovascularization, Physiologic/genetics , Nitric Oxide Synthase Type III/biosynthesis , Nitric Oxide Synthase Type III/genetics , Organ Specificity , RNA, Small Interfering/pharmacology , Recombinant Fusion Proteins , Signal Transduction/physiology , Yolk Sac/blood supplyABSTRACT
RNA editing by A-to-I modification is a widespread mechanism in complex organisms that leads to the posttranscriptional alteration of protein coding as well as noncoding sequences. MiRNA transcripts have been recognized as a major target for RNA editing enzymes, and single-nucleotide changes through editing can impact the biogenesis of mature miRNAs, as well as the target specificity of the regulatory RNA. Bona fide A-to-I RNA editing events are validated experimentally through parallel analysis of genomic DNA and transcribed sequences of miRNA genes isolated from the same specimen through gene-specific amplification and sequencing of endogenous transcripts.
Subject(s)
MicroRNAs , RNA Editing , Adenosine Deaminase/genetics , Adenosine Deaminase/metabolism , Animals , Base Sequence , Cells, Cultured , Humans , MicroRNAs/genetics , MicroRNAs/isolation & purification , MicroRNAs/metabolism , Molecular Sequence Data , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Processing, Post-Transcriptional , RNA-Binding Proteins , Reverse Transcriptase Polymerase Chain Reaction/instrumentation , Reverse Transcriptase Polymerase Chain Reaction/methodsABSTRACT
Single nucleotide polymorphisms (SNPs) are DNA sequence variations that can affect the expression or function of genes. As a result, they may lead to phenotypic differences between individuals, such as susceptibility to disease, response to medications, and disease progression. Millions of SNPs have been mapped within the human genome providing a rich resource for genetic variation studies. Adenosine-to-inosine RNA editing also leads to the production of RNA and protein sequence variants, but it acts on the level of primary gene transcripts. Sequence variations due to RNA editing may be misannotated as SNPs when relying solely on expressed sequence data instead of genomic material. In this study, we screened the human SNP database for potential cases of A-to-I RNA editing that cause amino acid changes in the encoded protein. Our search strategy applies five molecular features to score candidate sites. It identifies all previously known cases of editing present in the SNP database and successfully uncovers novel, bona fide targets of adenosine deamination editing. Our approach sets the stage for effective and comprehensive genome-wide screens for A-to-I editing targets.
