ABSTRACT
AT-rich interactive domain 1A gene (ARID1A) loss is a frequent event in endometriosis-associated ovarian carcinomas. Endometriosis is a disease in which tissue that normally grows inside the uterus grows outside the uterus, and 50% of women with endometriosis are infertile. ARID1A protein levels were significantly lower in the eutopic endometrium of women with endometriosis compared to women without endometriosis. However, an understanding of the physiological effects of ARID1A loss remains quite poor, and the function of Arid1a in the female reproductive tract has remained elusive. In order to understand the role of Arid1a in the uterus, we have generated mice with conditional ablation of Arid1a in the PGR positive cells (Pgrcre/+Arid1af/f; Arid1ad/d). Ovarian function and uterine development of Arid1ad/d mice were normal. However, Arid1ad/d mice were sterile due to defective embryo implantation and decidualization. The epithelial proliferation was significantly increased in Arid1ad/d mice compared to control mice. Enhanced epithelial estrogen activity and reduced epithelial PGR expression, which impedes maturation of the receptive uterus, was observed in Arid1ad/d mice at the peri-implantation period. The microarray analysis revealed that ARID1A represses the genes related to cell cycle and DNA replication. We showed that ARID1A positively regulates Klf15 expression with PGR to inhibit epithelial proliferation at peri-implantation. Our results suggest that Arid1a has a critical role in modulating epithelial proliferation which is a critical requisite for fertility. This finding provides a new signaling pathway for steroid hormone regulation in female reproductive biology and furthers our understanding of the molecular mechanisms that underlie dysregulation of hormonal signaling in human reproductive disorders such as endometriosis.
Subject(s)
DNA-Binding Proteins/physiology , Nuclear Proteins/physiology , Transcription Factors/physiology , Animals , Case-Control Studies , DNA-Binding Proteins/genetics , Endometriosis/genetics , Female , Humans , Mice , Mice, Mutant Strains , Nuclear Proteins/genetics , Pregnancy , Transcription Factors/geneticsABSTRACT
Women with endometriosis can suffer from decreased fecundity or complete infertility via abnormal oocyte function or impaired placental-uterine interactions required for normal pregnancy establishment and maintenance. Although AT-rich interactive domain 1A (SWI-like) (ARID1A) is a putative tumor suppressor in human endometrial cancers and endometriosis-associated ovarian cancers, little is known about its role in normal uterine function. To study the potential function of ARID1A in the female reproductive tract, we generated mice with a conditional knockout of Arid1a using anti-Müllerian hormone receptor 2-Cre Female Arid1a conditional knockout mice exhibited a progressive decrease in number of pups per litter, with a precipitous decline after the second litter. We observed no tumors in virgin mice, although one knockout mouse developed a uterine tumor after pregnancy. Unstimulated virgin female knockout mice showed normal oviductal, ovarian, and uterine histology. Uteri of Arid1a knockout mice showed a normal decidualization response and appropriate responses to estradiol and progesterone stimulation. In vitro studies using primary cultures of human endometrial stromal fibroblasts revealed that small interfering RNA knockdown of ARID1A did not affect decidualization in vitro. Timed pregnancy studies revealed the significant resorption of embryos at Embryonic Day 16.5 in knockout mice in the third pregnancy. In addition to evidence of implantation site hemorrhage, pregnant Arid1a knockout mice showed abnormal placental morphology. These results suggest that Arid1a supports successful pregnancy through its role in placental function.
Subject(s)
DNA-Binding Proteins/metabolism , Fertility , Genitalia, Female/metabolism , Mesenchymal Stem Cells/metabolism , Nuclear Proteins/metabolism , Placentation , Animals , DNA-Binding Proteins/genetics , Embryo Implantation , Embryo Loss , Endometrium/physiology , Female , Gonadotropins/blood , Mice, Inbred C57BL , Mice, Knockout , Nuclear Proteins/genetics , Ovarian Neoplasms/etiology , Placenta/metabolism , Pregnancy , Transcription Factors , Uterine Neoplasms/etiologyABSTRACT
Pten inactivation promotes cell survival in leukemia cells by activating glycolytic metabolism. We found that targeting ribosomal protein S6 kinase 1 (S6K1) in Pten-deficient cells suppressed glycolysis and induced apoptosis. S6K1 knockdown decreased expression of HIF-1α, and HIF-1α was sufficient to restore glycolysis and survival of cells lacking S6K1. In the Pten(fl/fl) Mx1-Cre(+) mouse model of leukemia, S6K1 deletion delayed the development of leukemia. Thus, S6K1 is a critical mediator of glycolytic metabolism, cell survival, and leukemogenesis in Pten-deficient cells.
Subject(s)
Apoptosis , Glycolysis , Leukemia/enzymology , Neoplasm Proteins/metabolism , PTEN Phosphohydrolase/metabolism , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Animals , Cell Line, Tumor , Disease Models, Animal , Gene Knockdown Techniques , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Leukemia/genetics , Mice , Mice, Knockout , Neoplasm Proteins/genetics , PTEN Phosphohydrolase/genetics , Ribosomal Protein S6 Kinases, 90-kDa/geneticsABSTRACT
Akt signal transduction induces coordinated increases in glycolysis and apoptosis resistance in a broad spectrum of cancers. Downstream of Akt, the FoxO transcription factors regulate apoptosis via Bim, but the contributions of FoxOs in regulating Akt-induced glycolysis are not well described. We find that FoxO3a knockdown is sufficient to induce apoptosis resistance in conjunction with elevated glycolysis. Glycolysis in FoxO3a-deficient cells was associated with increased S6K1 phosphorylation and was sensitive to rapamycin, an inhibitor of the mTORC1 pathway that has been linked to glycolysis regulation. We show that mTORC1-dependent glycolysis is increased in FoxO3a knockdown cells due to decreased expression of the TSC1 tumor suppressor that opposes mTORC1 activation. FoxO3a binds to and transactivates the TSC1 promoter, indicating a key role for FoxO3a in regulating TSC1 expression. Together, these data demonstrate that FoxO3a regulates glycolysis downstream of Akt through transcriptional control of Tsc1.
Subject(s)
Forkhead Transcription Factors/metabolism , Glycolysis/physiology , Transcription, Genetic/physiology , Transcriptional Activation/physiology , Tumor Suppressor Proteins/biosynthesis , Animals , Cell Line , Forkhead Box Protein O3 , Forkhead Transcription Factors/genetics , Glycolysis/drug effects , Humans , Immunosuppressive Agents/pharmacology , Mechanistic Target of Rapamycin Complex 1 , Mice , Multiprotein Complexes , Phosphorylation/drug effects , Phosphorylation/physiology , Promoter Regions, Genetic/physiology , Proteins , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Ribosomal Protein S6 Kinases/genetics , Ribosomal Protein S6 Kinases/metabolism , Sirolimus/pharmacology , TOR Serine-Threonine Kinases , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic/drug effects , Transcriptional Activation/drug effects , Tuberous Sclerosis Complex 1 Protein , Tumor Suppressor Proteins/geneticsABSTRACT
Incorporation of the 21st amino acid, selenocysteine, into proteins is specified in all three domains of life by dynamic translational redefinition of UGA codons. In eukarya and archaea, selenocysteine insertion requires a cis-acting selenocysteine insertion sequence (SECIS) usually located in the 3'UTR of selenoprotein mRNAs. Here we present comparative sequence analysis and experimental data supporting the presence of a second stop codon redefinition element located adjacent to a selenocysteine-encoding UGA codon in the eukaryal gene, SEPN1. This element is sufficient to stimulate high-level (6%) translational redefinition of the SEPN1 UGA codon in human cells. Readthrough levels further increased to 12% when tested in the presence of the SEPN1 3'UTR SECIS. Directed mutagenesis and phylogeny of the sequence context strongly supports the importance of a stem loop starting six nucleotides 3' of the UGA codon. Sequences capable of forming strong RNA structures were also identified 3' adjacent to, or near, selenocysteine-encoding UGA codons in the Sps2, SelH, SelO, and SelT selenoprotein genes.
Subject(s)
Base Sequence , Codon , Eukaryotic Cells , Nucleic Acid Conformation , RNA , Selenocysteine/genetics , Sequence Analysis, DNA , Animals , Cell Line , Genes, Reporter , Humans , Molecular Sequence Data , Muscle Proteins/genetics , RNA/chemistry , RNA/genetics , Selenocysteine/metabolism , Selenoproteins , Sequence AlignmentABSTRACT
We report the translational readthrough levels induced by the aminoglycosides gentamicin, amikacin, tobramycin, and paromomycin for eight premature stop codon mutations identified in Duchenne's and Becker's muscular dystrophy patients. In a transient transfection reporter assay, aminoglycoside treatment results show that one stop codon mutation is suppressed significantly better (up to 10% stop codon readthrough) than the others; five show lower but statistically significant suppression (< 2% stop codon readthrough); and two appear refractory to aminoglycoside treatment. Readthrough levels do not substantially vary between different sources of gentamicin, and, for this set of mutations, the efficiency of termination at the premature stop codon mutation does not appear to correlate with disease severity.