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1.
Nat Immunol ; 19(9): 986-1000, 2018 09.
Article in English | MEDLINE | ID: mdl-30127432

ABSTRACT

Gain-of-function mutations in the gene encoding the phosphatidylinositol-3-OH kinase catalytic subunit p110ƎĀ“ (PI3KƎĀ“) result in a human primary immunodeficiency characterized by lymphoproliferation, respiratory infections and inefficient responses to vaccines. However, what promotes these immunological disturbances at the cellular and molecular level remains unknown. We generated a mouse model that recapitulated major features of this disease and used this model and patient samples to probe how hyperactive PI3KƎĀ“ fosters aberrant humoral immunity. We found that mutant PI3KƎĀ“ led to co-stimulatory receptor ICOS-independent increases in the abundance of follicular helper T cells (TFH cells) and germinal-center (GC) B cells, disorganized GCs and poor class-switched antigen-specific responses to immunization, associated with altered regulation of the transcription factor FOXO1 and pro-apoptotic and anti-apoptotic members of the BCL-2 family. Notably, aberrant responses were accompanied by increased reactivity to gut bacteria and a broad increase in autoantibodies that were dependent on stimulation by commensal microbes. Our findings suggest that proper regulation of PI3KƎĀ“ is critical for ensuring optimal host-protective humoral immunity despite tonic stimulation from the commensal microbiome.


Subject(s)
B-Lymphocytes/physiology , Gastrointestinal Microbiome/immunology , Germinal Center/physiology , Mutation/genetics , Phosphatidylinositol 3-Kinases/genetics , T-Lymphocytes, Helper-Inducer/physiology , Animals , Autoantibodies/blood , Cells, Cultured , Class I Phosphatidylinositol 3-Kinases/genetics , Disease Models, Animal , Forkhead Box Protein O1/genetics , Forkhead Box Protein O1/metabolism , Humans , Immunity, Humoral/genetics , Immunoglobulin Class Switching/genetics , Immunologic Deficiency Syndromes/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism
2.
Nat Immunol ; 19(8): 838-848, 2018 08.
Article in English | MEDLINE | ID: mdl-29988091

ABSTRACT

Foxo transcription factors play an essential role in regulating specialized lymphocyte functions and in maintaining T cell quiescence. Here, we used a system in which Foxo1 transcription-factor activity, which is normally terminated upon cell activation, cannot be silenced, and we show that enforcing Foxo1 activity disrupts homeostasis of CD4 conventional and regulatory T cells. Despite limiting cell metabolism, continued Foxo1 activity is associated with increased activation of the kinase Akt and a cell-intrinsic proliferative advantage; however, survival and cell division are decreased in a competitive setting or growth-factor-limiting conditions. Via control of expression of the transcription factor Myc and the IL-2 receptor Ɵ-chain, termination of Foxo1 signaling couples the increase in cellular cholesterol to biomass accumulation after activation, thereby facilitating immunological synapse formation and mTORC1 activity. These data reveal that Foxo1 regulates the integration of metabolic and mitogenic signals essential for T cell competitive fitness and the coordination of cell growth with cell division.


Subject(s)
CD4-Positive T-Lymphocytes/physiology , Forkhead Box Protein O1/metabolism , T-Lymphocytes, Regulatory/physiology , Animals , Cell Proliferation , Cells, Cultured , Cholesterol/metabolism , Forkhead Box Protein O1/genetics , Gene Expression Profiling , Homeostasis , Immunological Synapses/metabolism , Interleukin-2 Receptor beta Subunit/genetics , Interleukin-2 Receptor beta Subunit/metabolism , Lymphocyte Activation , Mechanistic Target of Rapamycin Complex 1/metabolism , Mice , Mice, Knockout , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Signal Transduction
3.
Immunity ; 54(8): 1807-1824.e14, 2021 08 10.
Article in English | MEDLINE | ID: mdl-34380064

ABSTRACT

The transcription factor forkhead box O1 (FOXO1), which instructs the dark zone program to direct germinal center (GC) polarity, is typically inactivated by phosphatidylinositol 3-kinase (PI3K) signals. Here, we investigated how FOXO1 mutations targeting this regulatory axis in GC-derived B cell non-Hodgkin lymphomas (B-NHLs) contribute to lymphomagenesis. Examination of primary B-NHL tissues revealed that FOXO1 mutations and PI3K pathway activity were not directly correlated. Human B cell lines bearing FOXO1 mutations exhibited hyperactivation of PI3K and Stress-activated protein kinase (SAPK)/Jun amino-terminal kinase (JNK) signaling, and increased cell survival under stress conditions as a result of alterations in FOXO1 transcriptional affinities and activation of transcriptional programs characteristic of GC-positive selection. When modeled in mice, FOXO1 mutations conferred competitive advantage to B cells in response to key T-dependent immune signals, disrupting GC homeostasis. FOXO1 mutant transcriptional signatures were prevalent in human B-NHL and predicted poor clinical outcomes. Thus, rather than enforcing FOXO1 constitutive activity, FOXO1 mutations enable co-option of GC-positive selection programs during the pathogenesis of GC-derived lymphomas.


Subject(s)
B-Lymphocytes/cytology , Forkhead Box Protein O1/genetics , Germinal Center/immunology , Lymphoma, B-Cell/pathology , Animals , B-Lymphocytes/immunology , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Line , Cell Proliferation/genetics , Cell Survival/genetics , Gene Expression Regulation/genetics , HEK293 Cells , Humans , Lymphoma, B-Cell/genetics , MAP Kinase Kinase 4/metabolism , Mice , Mice, Inbred C57BL , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction/genetics , Signal Transduction/immunology
4.
Nature ; 630(8018): 976-983, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38867048

ABSTRACT

Interleukin (IL-)23 is a major mediator and therapeutic target in chronic inflammatory diseases that also elicits tissue protection in the intestine at homeostasis or following acute infection1-4. However, the mechanisms thatĀ shape these beneficial versus pathological outcomes remain poorly understood. To address this gap in knowledge, we performed single-cell RNA sequencing on all IL-23 receptor-expressing cells in the intestine and their acute response to IL-23, revealing a dominance of T cells and group 3 innate lymphoid cells (ILC3s). Unexpectedly, we identified potent upregulation of the immunoregulatory checkpoint molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) on ILC3s. This pathway was activated by gut microbes and IL-23 in a FOXO1- and STAT3-dependent manner. Mice lacking CTLA-4 on ILC3s exhibited reduced regulatory T cells, elevated inflammatory T cells and more-severe intestinal inflammation. IL-23 induction of CTLA-4+ ILC3s was necessary and sufficient to reduce co-stimulatory molecules and increase PD-L1 bioavailability on intestinal myeloid cells. Finally, human ILC3s upregulated CTLA-4 in response to IL-23 or gut inflammation and correlated with immunoregulation in inflammatory bowel disease. These results reveal ILC3-intrinsic CTLA-4 as an essential checkpoint that restrains the pathological outcomes of IL-23, suggesting that disruption of these lymphocytes, which occurs in inflammatory bowel disease5-7, contributes to chronic inflammation.


Subject(s)
Immunity, Innate , Inflammation , Interleukin-23 , Lymphocytes , Animals , Female , Humans , Male , Mice , CTLA-4 Antigen/metabolism , Forkhead Box Protein O1/metabolism , Forkhead Box Protein O1/genetics , Gastrointestinal Microbiome , Inflammation/immunology , Inflammation/pathology , Inflammation/metabolism , Interleukin-23/immunology , Intestines/immunology , Intestines/pathology , Lymphocytes/immunology , Lymphocytes/metabolism , Mice, Inbred C57BL , Myeloid Cells/metabolism , Single-Cell Gene Expression Analysis , STAT3 Transcription Factor/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism
5.
Nature ; 629(8010): 201-210, 2024 May.
Article in English | MEDLINE | ID: mdl-38600376

ABSTRACT

Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of haematological malignancies such as acute lymphoblastic leukaemia, B cell lymphoma and multiple myeloma1-4, but the efficacy of CAR T cell therapy in solid tumours has been limited5. This is owing to a number of factors, including the immunosuppressive tumour microenvironment that gives rise to poorly persisting and metabolically dysfunctional T cells. Analysis of anti-CD19 CAR T cells used clinically has shown that positive treatment outcomes are associated with a more 'stem-like' phenotype and increased mitochondrial mass6-8. We therefore sought to identify transcription factors that could enhance CAR T cell fitness and efficacy against solid tumours. Here we show that overexpression of FOXO1 promotes a stem-like phenotype in CAR T cells derived from either healthy human donors or patients, which correlates with improved mitochondrial fitness, persistence and therapeutic efficacy in vivo. This work thus reveals an engineering approach to genetically enforce a favourable metabolic phenotype that has high translational potential to improve the efficacy of CAR T cells against solid tumours.


Subject(s)
Forkhead Box Protein O1 , Immunotherapy, Adoptive , Neoplasms , Receptors, Chimeric Antigen , Stem Cells , T-Lymphocytes , Humans , Mice , Cell Line, Tumor , Forkhead Box Protein O1/metabolism , Forkhead Box Protein O1/genetics , Mitochondria/metabolism , Phenotype , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , T-Lymphocytes/cytology , Tumor Microenvironment/immunology , Stem Cells/cytology , Stem Cells/immunology , Stem Cells/metabolism , Neoplasms/immunology , Neoplasms/pathology , Neoplasms/therapy
6.
Development ; 151(18)2024 Sep 15.
Article in English | MEDLINE | ID: mdl-39166965

ABSTRACT

The microvascular system consists of two cell types: endothelial and mural (pericytes and vascular smooth muscle cells; VSMCs) cells. Communication between endothelial and mural cells plays a pivotal role in the maintenance of vascular homeostasis; however, in vivo molecular and cellular mechanisms underlying mural cell development remain unclear. In this study, we found that macrophages played a crucial role in TGFƟ-dependent pericyte-to-VSMC differentiation during retinal vasculature development. In mice with constitutively active Foxo1 overexpression, substantial accumulation of TGFƟ1-producing macrophages and pericytes around the angiogenic front region was observed. Additionally, the TGFƟ-SMAD pathway was activated in pericytes adjacent to macrophages, resulting in excess ectopic α-smooth muscle actin-positive VSMCs. Furthermore, we identified endothelial SEMA3C as an attractant for macrophages. In vivo neutralization of SEMA3C rescued macrophage accumulation and ectopic VSMC phenotypes in the mice, as well as drug-induced macrophage depletion. Therefore, macrophages play an important physiological role in VSMC development via the FOXO1-SEMA3C pathway.


Subject(s)
Forkhead Box Protein O1 , Macrophages , Muscle, Smooth, Vascular , Myocytes, Smooth Muscle , Semaphorins , Animals , Macrophages/metabolism , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/cytology , Mice , Semaphorins/metabolism , Semaphorins/genetics , Forkhead Box Protein O1/metabolism , Forkhead Box Protein O1/genetics , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/cytology , Pericytes/metabolism , Pericytes/cytology , Cell Differentiation , Signal Transduction , Retinal Vessels/metabolism , Forkhead Transcription Factors/metabolism , Forkhead Transcription Factors/genetics , Transforming Growth Factor beta1/metabolism , Mice, Inbred C57BL
7.
Nature ; 598(7881): 473-478, 2021 10.
Article in English | MEDLINE | ID: mdl-34646017

ABSTRACT

The progression of chronic liver disease to hepatocellular carcinoma is caused by the acquisition of somatic mutations that affect 20-30 cancer genes1-8. Burdens of somatic mutations are higher and clonal expansions larger in chronic liver disease9-13 than in normal liver13-16, which enables positive selection to shape the genomic landscape9-13. Here we analysed somatic mutations from 1,590 genomes across 34 liver samples, including healthy controls, alcohol-related liver disease and non-alcoholic fatty liver disease. Seven of the 29 patients with liver disease had mutations in FOXO1, the major transcription factor in insulin signalling. These mutations affected a single hotspot within the gene, impairing the insulin-mediated nuclear export of FOXO1. Notably, six of the seven patients with FOXO1S22W hotspot mutations showed convergent evolution, with variants acquired independently by up to nine distinct hepatocyte clones per patient. CIDEB, which regulates lipid droplet metabolism in hepatocytes17-19, and GPAM, which produces storage triacylglycerol from free fatty acids20,21, also had a significant excess of mutations. We again observed frequent convergent evolution: up to fourteen independent clones per patient with CIDEB mutations and up to seven clones per patient with GPAM mutations. Mutations in metabolism genes were distributed across multiple anatomical segments of the liver, increased clone size and were seen in both alcohol-related liver disease and non-alcoholic fatty liver disease, but rarely in hepatocellular carcinoma. Master regulators of metabolic pathways are a frequent target of convergent somatic mutation in alcohol-related and non-alcoholic fatty liver disease.


Subject(s)
Liver Diseases/genetics , Liver Diseases/metabolism , Liver/metabolism , Mutation/genetics , Active Transport, Cell Nucleus/genetics , Apoptosis Regulatory Proteins/genetics , Cell Line, Tumor , Chronic Disease , Cohort Studies , Fatty Acids, Nonesterified/metabolism , Female , Forkhead Box Protein O1/genetics , Forkhead Box Protein O1/metabolism , Humans , Insulin Resistance , Liver Diseases, Alcoholic/genetics , Liver Diseases, Alcoholic/metabolism , Male , Non-alcoholic Fatty Liver Disease/genetics , Non-alcoholic Fatty Liver Disease/metabolism , Triglycerides/metabolism
8.
Mol Cell ; 75(4): 807-822.e8, 2019 08 22.
Article in English | MEDLINE | ID: mdl-31442424

ABSTRACT

mTORC2 controls glucose and lipid metabolism, but the mechanisms are unclear. Here, we show that conditionally deleting the essential mTORC2 subunit Rictor in murine brown adipocytes inhibits de novo lipid synthesis, promotes lipid catabolism and thermogenesis, and protects against diet-induced obesity and hepatic steatosis. AKT kinases are the canonical mTORC2 substrates; however, deleting Rictor in brown adipocytes appears to drive lipid catabolism by promoting FoxO1 deacetylation independently of AKT, and in a pathway distinct from its positive role in anabolic lipid synthesis. This facilitates FoxO1 nuclear retention, enhances lipid uptake and lipolysis, and potentiates UCP1 expression. We provide evidence that SIRT6 is the FoxO1 deacetylase suppressed by mTORC2 and show an endogenous interaction between SIRT6 and mTORC2 in both mouse and human cells. Our findings suggest a new paradigm of mTORC2 function filling an important gap in our understanding of this more mysterious mTOR complex.


Subject(s)
Adipocytes, Brown/metabolism , Forkhead Box Protein O1/metabolism , Lipolysis , Mechanistic Target of Rapamycin Complex 2/metabolism , Sirtuins/metabolism , Adipocytes, Brown/cytology , Animals , Forkhead Box Protein O1/genetics , HEK293 Cells , HeLa Cells , Humans , Mechanistic Target of Rapamycin Complex 2/genetics , Mice , Mice, Transgenic , Rapamycin-Insensitive Companion of mTOR Protein/genetics , Rapamycin-Insensitive Companion of mTOR Protein/metabolism , Sirtuins/genetics
9.
Proc Natl Acad Sci U S A ; 121(41): e2408719121, 2024 Oct 08.
Article in English | MEDLINE | ID: mdl-39352930

ABSTRACT

As ambush-hunting predators that consume large prey after long intervals of fasting, Burmese pythons evolved with unique adaptations for modulating organ structure and function. Among these is cardiac hypertrophy that develops within three days following a meal (Andersen et al., 2005, Secor, 2008), which we previously showed was initiated by circulating growth factors (Riquelme et al., 2011). Postprandial cardiac hypertrophy in pythons also rapidly regresses with subsequent fasting (Secor, 2008); however, the molecular mechanisms that regulate the dynamic cardiac remodeling in pythons during digestion are largely unknown. In this study, we employed a multiomics approach coupled with targeted molecular analyses to examine remodeling of the python ventricular transcriptome and proteome throughout digestion. We found that forkhead box protein O1 (FoxO1) signaling was suppressed prior to hypertrophy development and then activated during regression, which coincided with decreased and then increased expression, respectively, of FoxO1 transcriptional targets involved in proteolysis. To define the molecular mechanistic role of FoxO1 in hypertrophy regression, we used cultured mammalian cardiomyocytes treated with postfed python plasma. Hypertrophy regression both in pythons and in vitro coincided with activation of FoxO1-dependent autophagy; however, the introduction of a FoxO1-specific inhibitor prevented both regression of cell size and autophagy activation. Finally, to determine whether FoxO1 activation could induce regression, we generated an adenovirus expressing a constitutively active FoxO1. FoxO1 activation was sufficient to prevent and reverse postfed plasma-induced hypertrophy, which was partially prevented by autophagy inhibition. Our results indicate that modulation of FoxO1 activity contributes to the dynamic ventricular remodeling in postprandial Burmese pythons.


Subject(s)
Boidae , Forkhead Box Protein O1 , Heart , Animals , Autophagy , Forkhead Box Protein O1/metabolism , Forkhead Box Protein O1/genetics , Myocytes, Cardiac/metabolism , Postprandial Period , Signal Transduction , Transcriptome , Heart/physiology
10.
Blood ; 144(12): 1314-1328, 2024 Sep 19.
Article in English | MEDLINE | ID: mdl-38848533

ABSTRACT

ABSTRACT: The liver plays a crucial role in maintaining systemic iron homeostasis by secreting hepcidin, which is essential for coordinating iron levels in the body. Imbalances in iron homeostasis are associated with various clinical disorders related to iron deficiency or iron overload. Despite the clinical significance, the mechanisms underlying how hepatocytes sense extracellular iron levels to regulate hepcidin synthesis and iron storage are not fully understood. In this study, we identified Foxo1, a well-known regulator of macronutrient metabolism, which translocates to the nucleus of hepatocytes in response to high-iron feeding, holo-transferrin, and bone morphogenetic protein 6 (BMP6) treatment. Furthermore, Foxo1 plays a crucial role in mediating hepcidin induction in response to both iron and BMP signals by directly interacting with evolutionally conserved Foxo binding sites within the hepcidin promoter region. These binding sites were found to colocalize with Smad-binding sites. To investigate the physiological relevance of Foxo1 in iron metabolism, we generated mice with hepatocyte-specific deletion of Foxo1. These mice exhibited reduced hepatic hepcidin expression and serum hepcidin levels, accompanied by elevated serum iron and liver nonheme iron concentrations. Moreover, high-iron diet further exacerbated these abnormalities in iron metabolism in mice lacking hepatic Foxo1. Conversely, hepatocyte-specific Foxo1 overexpression increased hepatic hepcidin expression and serum hepcidin levels, thereby ameliorating iron overload in a murine model of hereditary hemochromatosis (Hfe-/- mice). In summary, our study identifies Foxo1 as a critical regulator of hepcidin and systemic iron homeostasis. Targeting Foxo1 may offer therapeutic opportunities for managing conditions associated with aberrant iron metabolism.


Subject(s)
Forkhead Box Protein O1 , Hepatocytes , Hepcidins , Homeostasis , Iron , Animals , Forkhead Box Protein O1/metabolism , Forkhead Box Protein O1/genetics , Iron/metabolism , Hepcidins/metabolism , Hepcidins/genetics , Mice , Hepatocytes/metabolism , Humans , Mice, Knockout , Liver/metabolism , Mice, Inbred C57BL , Promoter Regions, Genetic , Gene Expression Regulation
11.
Immunity ; 46(4): 596-608, 2017 04 18.
Article in English | MEDLINE | ID: mdl-28410989

ABSTRACT

Understanding immunological memory formation depends on elucidating how multipotent memory precursor (MP) cells maintain developmental plasticity and longevity to provide long-term immunity while other effector cells develop into terminally differentiated effector (TE) cells with limited survival. Profiling active (H3K27ac) and repressed (H3K27me3) chromatin in naive, MP, and TE CD8+ TĀ cells during viral infection revealed increased H3K27me3 deposition at numerous pro-memory and pro-survival genes in TE relative to MP cells, indicative of fate restriction, but permissive chromatin at both pro-memory and pro-effector genes in MP cells, indicative of multipotency. Polycomb repressive complex 2 deficiency impaired clonal expansion and TE cell differentiation, but minimally impacted CD8+ memory TĀ cell maturation. Abundant H3K27me3 deposition at pro-memory genes occurred late during TE cell development, probably from diminished transcription factor FOXO1 expression. These results outline a temporal model for loss of memory cell potential through selective epigenetic silencing of pro-memory genes in effector TĀ cells.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cell Differentiation/immunology , Chromatin/immunology , Polycomb Repressive Complex 2/immunology , Animals , CD8-Positive T-Lymphocytes/metabolism , Cell Differentiation/genetics , Chromatin/genetics , Chromatin/metabolism , Enhancer of Zeste Homolog 2 Protein/genetics , Enhancer of Zeste Homolog 2 Protein/immunology , Enhancer of Zeste Homolog 2 Protein/metabolism , Flow Cytometry , Forkhead Box Protein O1/genetics , Forkhead Box Protein O1/immunology , Forkhead Box Protein O1/metabolism , Gene Expression/immunology , Histones/immunology , Histones/metabolism , Immunoblotting , Immunologic Memory/genetics , Immunologic Memory/immunology , Lysine/immunology , Lysine/metabolism , Methylation , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Models, Immunological , Multipotent Stem Cells/immunology , Multipotent Stem Cells/metabolism , Polycomb Repressive Complex 2/genetics , Polycomb Repressive Complex 2/metabolism , Reverse Transcriptase Polymerase Chain Reaction
12.
Immunity ; 47(6): 1067-1082.e12, 2017 12 19.
Article in English | MEDLINE | ID: mdl-29246441

ABSTRACT

Roquin proteins preclude spontaneous TĀ cell activation and aberrant differentiation of T follicular helper (Tfh) or T helper 17 (Th17) cells. Here we showed that deletion of Roquin-encoding alleles specifically in regulatory T (Treg) cells also caused the activation of conventional TĀ cells. Roquin-deficient Treg cells downregulated CD25, acquired a follicular Treg (Tfr) cell phenotype, and suppressed germinal center reactions but could not protect from colitis. Roquin inhibited the PI3K-mTOR signaling pathway by upregulation of Pten through interfering with miR-17Ć¢ĀˆĀ¼92 binding to an overlapping cis-element in the Pten 3'Ā UTR, and downregulated the Foxo1-specific E3 ubiquitin ligase Itch. Loss of Roquin enhanced Akt-mTOR signaling and protein synthesis, whereas inhibition of PI3K or mTOR in Roquin-deficient TĀ cells corrected enhanced Tfh and Th17 or reduced iTreg cell differentiation. Thereby, Roquin-mediated control of PI3K-mTOR signaling prevents autoimmunity by restraining activation and differentiation of conventional TĀ cells and specialization of Treg cells.


Subject(s)
Colitis/immunology , Phosphatidylinositol 3-Kinases/immunology , Repressor Proteins/immunology , TOR Serine-Threonine Kinases/immunology , Ubiquitin-Protein Ligases/immunology , Animals , B-Lymphocytes/immunology , B-Lymphocytes/pathology , Cell Differentiation , Colitis/genetics , Colitis/pathology , Disease Models, Animal , Female , Forkhead Box Protein O1/genetics , Forkhead Box Protein O1/immunology , Gene Expression Regulation , Germinal Center/immunology , Germinal Center/pathology , Interleukin-2 Receptor alpha Subunit/genetics , Interleukin-2 Receptor alpha Subunit/immunology , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Transgenic , MicroRNAs/genetics , MicroRNAs/immunology , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/immunology , Phosphatidylinositol 3-Kinases/genetics , Primary Cell Culture , Repressor Proteins/deficiency , Repressor Proteins/genetics , Signal Transduction , Spleen/immunology , Spleen/pathology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/pathology , TOR Serine-Threonine Kinases/genetics , Th17 Cells/immunology , Th17 Cells/pathology , Ubiquitin-Protein Ligases/deficiency , Ubiquitin-Protein Ligases/genetics
13.
J Biol Chem ; 300(4): 107209, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38519029

ABSTRACT

FOXO1 is a transcription factor and potential tumor suppressor that is negatively regulated downstream of PI3K-PKB/AKT signaling. Paradoxically, FOXO also promotes tumor growth, but the detailed mechanisms behind this role of FOXO are not fully understood. In this study, we revealed a molecular cascade by which the Thr24 residue of FOXO1 is phosphorylated by AKT and is dephosphorylated by calcineurin, which is a Ca2+-dependent protein phosphatase. Curiously, single nucleotide somatic mutations of FOXO1 in cancer occur frequently at and near Thr24. Using a calcineurin inhibitor and shRNA directed against calcineurin, we revealed that calcineurin-mediated dephosphorylation of Thr24 regulates FOXO1 protein stability. We also found that FOXO1 binds to the promoter region of MDM2 and activates transcription, which in turn promotes MDM2-mediated ubiquitination and degradation of p53. FOXO3a and FOXO4 are shown to control p53 activity; however, the significance of FOXO1 in p53 regulation remains largely unknown. Supporting this notion, FOXO1 depletion increased p53 and p21 protein levels in association with the inhibition of cell proliferation. Taken together, these results indicate that FOXO1 is stabilized by calcineurin-mediated dephosphorylation and that FOXO1 supports cancer cell proliferation by promoting MDM2 transcription and subsequent p53 degradation.


Subject(s)
Calcineurin , Cell Proliferation , Forkhead Box Protein O1 , Proteolysis , Proto-Oncogene Proteins c-mdm2 , Tumor Suppressor Protein p53 , Proto-Oncogene Proteins c-mdm2/metabolism , Proto-Oncogene Proteins c-mdm2/genetics , Humans , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Protein p53/genetics , Forkhead Box Protein O1/metabolism , Forkhead Box Protein O1/genetics , Calcineurin/metabolism , Calcineurin/genetics , Phosphorylation , Ubiquitination , Cell Line, Tumor , Neoplasms/metabolism , Neoplasms/pathology , Neoplasms/genetics , Forkhead Transcription Factors/metabolism , Forkhead Transcription Factors/genetics , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-akt/genetics , Protein Stability
14.
J Biol Chem ; 300(7): 107425, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38823639

ABSTRACT

Adenosine deaminase (ADA) catalyzes the irreversible deamination of adenosine (ADO) to inosine and regulates ADO concentration. ADA ubiquitously expresses in various tissues to mediate ADO-receptor signaling. A significant increase in plasma ADA activity has been shown to be associated with the pathogenesis of type 2 diabetes mellitus. Here, we show that elevated plasma ADA activity is a compensated response to high level of ADO in type 2 diabetes mellitus and plays an essential role in the regulation of glucose homeostasis. Supplementing with more ADA, instead of inhibiting ADA, can reduce ADO levels and decrease hepatic gluconeogenesis. ADA restores a euglycemic state and recovers functional islets in db/db and high-fat streptozotocin diabetic mice. Mechanistically, ADA catabolizes ADO and increases Akt and FoxO1 phosphorylation independent of insulin action. ADA lowers blood glucose at a slower rate and longer duration compared to insulin, delaying or blocking the incidence of insulinogenic hypoglycemia shock. Finally, ADA suppresses gluconeogenesis in fasted mice and insulin-deficient diabetic mice, indicating the ADA regulating gluconeogenesis is a universal biological mechanism. Overall, these results suggest that ADA is expected to be a new therapeutic target for diabetes.


Subject(s)
Adenosine Deaminase , Diabetes Mellitus, Experimental , Diabetes Mellitus, Type 2 , Gluconeogenesis , Animals , Male , Mice , Adenosine/metabolism , Adenosine Deaminase/metabolism , Blood Glucose/metabolism , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/pathology , Forkhead Box Protein O1/metabolism , Forkhead Box Protein O1/genetics , Insulin/metabolism , Liver/metabolism , Mice, Inbred C57BL , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-akt/genetics
15.
Eur J Immunol ; 54(10): e2350887, 2024 Oct.
Article in English | MEDLINE | ID: mdl-39072704

ABSTRACT

The migration is the key step for thymic T cells to enter circulation and then lymph nodes (LNs), essential for future immune surveillance. Although promoter-based transcriptional regulation through Foxo1, Klf2, Ccr7, and Sell regulates T-cell migration, it remains largely unexplored whether and how enhancers are involved in this process. Here we found that the conditional deletion of Med1, a component of the mediator complex and a mediator between enhancers and RNA polymerase II, caused a reduction of both CD4+ and CD8+ T cells in LNs, as well as a decrease of CD8+ T cells in the spleen. Importantly, Med1 deletion hindered the migration of thymic αƟT cells into the circulation and then into LNs, accompanied by the downregulation of KLF2, CCR7, and CD62L. Mechanistically, Med1 promotes Klf2 transcription by facilitating Foxo1 binding to the Klf2 enhancer. Furthermore, forced expression of Klf2 rescued Ccr7 and Sell expression, as well as αƟT-cell migration into LNs. Collectively, our study unveils a crucial role for Med1 in regulating the enhancer-based Foxo1-Klf2 transcriptional program and the migration of αƟT cells into LNs, providing valuable insights into the molecular mechanisms underlying T-cell migration.


Subject(s)
Cell Movement , Forkhead Box Protein O1 , Kruppel-Like Transcription Factors , Lymph Nodes , Mediator Complex Subunit 1 , Forkhead Box Protein O1/metabolism , Forkhead Box Protein O1/genetics , Animals , Mice , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Lymph Nodes/immunology , Lymph Nodes/cytology , Cell Movement/genetics , Cell Movement/immunology , Mediator Complex Subunit 1/genetics , Mediator Complex Subunit 1/metabolism , Transcription, Genetic , Enhancer Elements, Genetic/genetics , Thymus Gland/cytology , Thymus Gland/immunology , Thymus Gland/metabolism , Gene Expression Regulation , Mice, Knockout , Mice, Inbred C57BL
16.
Development ; 149(7)2022 04 01.
Article in English | MEDLINE | ID: mdl-35297995

ABSTRACT

Establishing a functional circulatory system is required for post-implantation development during murine embryogenesis. Previous studies in loss-of-function mouse models showed that FOXO1, a Forkhead family transcription factor, is required for yolk sac (YS) vascular remodeling and survival beyond embryonic day (E) 11. Here, we demonstrate that at E8.25, loss of Foxo1 in Tie2-cre expressing cells resulted in increased sprouty 2 (Spry2) and Spry4 expression, reduced arterial gene expression and reduced Kdr (also known as Vegfr2 and Flk1) transcripts without affecting overall endothelial cell identity, survival or proliferation. Using a Dll4-BAC-nlacZ reporter line, we found that one of the earliest expressed arterial genes, delta like 4, is significantly reduced in Foxo1 mutant YS without being substantially affected in the embryo proper. We show that FOXO1 binds directly to previously identified Spry2 gene regulatory elements (GREs) and newly identified, evolutionarily conserved Spry4 GREs to repress their expression. Furthermore, overexpression of Spry4 in transient transgenic embryos largely recapitulates the reduced expression of arterial genes seen in conditional Foxo1 mutants. Together, these data reveal a novel role for FOXO1 as a key transcriptional repressor regulating both pre-flow arterial specification and subsequent vessel remodeling within the murine YS.


Subject(s)
Nerve Tissue Proteins/metabolism , Vascular Remodeling , Yolk Sac , Animals , Arteries , Embryo, Mammalian/metabolism , Endothelial Cells/metabolism , Forkhead Box Protein O1/genetics , Forkhead Box Protein O1/metabolism , Mice , Vascular Remodeling/genetics , Yolk Sac/metabolism
17.
Immunity ; 44(6): 1284-98, 2016 06 21.
Article in English | MEDLINE | ID: mdl-27332731

ABSTRACT

T helper 17 (Th17) cells are key players in autoimmune diseases. However, the roles of non-coding RNAs in Th17 cell development and function are largely unknown. We found that deletion of the endoribonuclease-encoding Dicer1 specifically in Th17 cells protected mice from experimental autoimmune encephalomyelitis. We found that the Dicer1-regulated microRNA (miR)-183-96-182 cluster (miR-183C) was highly expressed in Th17 cells and was induced by cytokine IL-6-STAT3 signaling. miR-183C expression enhanced pathogenic cytokine production from Th17 cells during their development and promoted autoimmunity. Mechanistically, miR-183C in Th17 cells directly repressed expression of the transcription factor Foxo1. Foxo1 negatively regulated the pathogenicity of Th17 cells in part by inhibiting expression of cytokine receptor IL-1R1. These findings indicate that the miR-183C drives Th17 pathogenicity in autoimmune diseases via inhibition of Foxo1 and present promising therapeutic targets.


Subject(s)
DEAD-box RNA Helicases/metabolism , Encephalomyelitis, Autoimmune, Experimental/immunology , Forkhead Box Protein O1/metabolism , MicroRNAs/genetics , Multiple Sclerosis/immunology , Ribonuclease III/metabolism , Th17 Cells/physiology , Animals , Cells, Cultured , DEAD-box RNA Helicases/genetics , Forkhead Box Protein O1/genetics , Humans , Interleukin-6/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Interleukin-1 Type I/metabolism , Ribonuclease III/genetics , STAT3 Transcription Factor/metabolism
18.
Arterioscler Thromb Vasc Biol ; 44(9): 2004-2023, 2024 09.
Article in English | MEDLINE | ID: mdl-39087350

ABSTRACT

BACKGROUND: Lymphatic valves are specialized structures in collecting lymphatic vessels and are crucial for preventing retrograde lymph flow. Mutations in valve-forming genes have been clinically implicated in the pathology of congenital lymphedema. Lymphatic valves form when oscillatory shear stress from lymph flow signals through the PI3K/AKT pathway to promote the transcription of valve-forming genes that trigger the growth and maintenance of lymphatic valves. Conventionally, in many cell types, AKT is phosphorylated at Ser473 by the mTORC2 (mammalian target of rapamycin complex 2). However, mTORC2 has not yet been implicated in lymphatic valve formation. METHODS: In vivo and in vitro techniques were used to investigate the role of Rictor, a critical component of mTORC2, in lymphatic endothelium. RESULTS: Here, we showed that embryonic and postnatal lymphatic deletion of Rictor, a critical component of mTORC2, led to a significant decrease in lymphatic valves and prevented the maturation of collecting lymphatic vessels. RICTOR knockdown in human dermal lymphatic endothelial cells not only reduced the level of activated AKT and the expression of valve-forming genes under no-flow conditions but also abolished the upregulation of AKT activity and valve-forming genes in response to oscillatory shear stress. We further showed that the AKT target, FOXO1 (forkhead box protein O1), a repressor of lymphatic valve formation, had increased nuclear activity in Rictor knockout mesenteric lymphatic endothelial cells in vivo. Deletion of Foxo1 in Rictor knockout mice restored the number of valves to control levels in lymphatic vessels of the ear and mesentery. CONCLUSIONS: Our work identifies a novel role for RICTOR in the mechanotransduction signaling pathway, wherein it activates AKT and prevents the nuclear accumulation of the valve repressor, FOXO1, which ultimately enables the formation and maintenance of lymphatic valves.


Subject(s)
Carrier Proteins , Forkhead Box Protein O1 , Lymphangiogenesis , Lymphatic Vessels , Mechanistic Target of Rapamycin Complex 2 , Mechanotransduction, Cellular , Mice, Knockout , Proto-Oncogene Proteins c-akt , Rapamycin-Insensitive Companion of mTOR Protein , Signal Transduction , Animals , Rapamycin-Insensitive Companion of mTOR Protein/metabolism , Rapamycin-Insensitive Companion of mTOR Protein/genetics , Proto-Oncogene Proteins c-akt/metabolism , Forkhead Box Protein O1/metabolism , Forkhead Box Protein O1/genetics , Lymphatic Vessels/metabolism , Mechanistic Target of Rapamycin Complex 2/metabolism , Mechanistic Target of Rapamycin Complex 2/genetics , Humans , Carrier Proteins/metabolism , Carrier Proteins/genetics , Endothelial Cells/metabolism , Cells, Cultured , TOR Serine-Threonine Kinases/metabolism , Phosphorylation , Forkhead Transcription Factors/metabolism , Forkhead Transcription Factors/genetics , Mice , Multiprotein Complexes/metabolism , Multiprotein Complexes/genetics , Mice, Inbred C57BL , RNA Interference , Transfection
19.
Cell Mol Life Sci ; 81(1): 237, 2024 May 25.
Article in English | MEDLINE | ID: mdl-38795132

ABSTRACT

Ovarian endometriosis is a common gynecological disease, and one of its most significant symptoms is infertility. In patients with endometriosis, defects in endometrial decidualization lead to impaired endometrial receptivity and embryo implantation, thus affecting early pregnancy and women's desire to have children. However, the mechanisms underlying the development of endometriosis and its associated defective decidualization are unclear. We find that NEK2 expression is increased in the ectopic and eutopic endometrium of patients with endometriosis. Meanwhile, NEK2 interacts with FOXO1 and phosphorylates FOXO1 at Ser184, inhibiting the stability of the FOXO1 protein. Importantly, NEK2-mediated phosphorylation of FOXO1 at Ser184 promotes cell proliferation, migration, invasion and impairs decidualization. Furthermore, INH1, an inhibitor of NEK2, inhibits the growth of ectopic lesions in mouse models of endometriosis and promotes endometrial decidualization in mouse models of artificially induced decidualization. Taken together, these findings indicate that NEK2 regulates the development of endometriosis and associated disorders of decidualization through the phosphorylation of FOXO1, providing a new therapeutic target for its treatment.


Subject(s)
Cell Proliferation , Endometriosis , Endometrium , Forkhead Box Protein O1 , NIMA-Related Kinases , Female , Endometriosis/metabolism , Endometriosis/pathology , Forkhead Box Protein O1/metabolism , Forkhead Box Protein O1/genetics , Humans , Animals , Phosphorylation , Mice , NIMA-Related Kinases/metabolism , NIMA-Related Kinases/genetics , Endometrium/metabolism , Endometrium/pathology , Cell Movement , Decidua/metabolism , Decidua/pathology , Adult , Disease Models, Animal
20.
Cell Mol Life Sci ; 81(1): 360, 2024 Aug 19.
Article in English | MEDLINE | ID: mdl-39158700

ABSTRACT

Iron deficiency is a prevalent nutritional deficit associated with organ damage and dysfunction. Recent research increasingly associates iron deficiency with bone metabolism dysfunction, although the precise underlying mechanisms remain unclear. Some studies have proposed that iron-dependent methylation-erasing enzyme activity regulates cell proliferation and differentiation under physiological or pathological conditions. However, it remains uncertain whether iron deficiency inhibits the activation of quiescent mesenchymal stem cells (MSCs) by affecting histone demethylase activity. In our study, we identified KDM4D as a key player in the activation of quiescent MSCs. Under conditions of iron deficiency, the H3K9me3 demethylase activity of KDM4D significantly decreased. This alteration resulted in increased heterochromatin with H3K9me3 near the PIK3R3 promoter, suppressing PIK3R3 expression and subsequently inhibiting the activation of quiescent MSCs via the PI3K-Akt-Foxo1 pathway. Iron-deficient mice displayed significantly impaired bone marrow MSCs activation and decreased bone mass compared to normal mice. Modulating the PI3K-Akt-Foxo1 pathway could reverse iron deficiency-induced bone loss.


Subject(s)
Forkhead Box Protein O1 , Iron , Jumonji Domain-Containing Histone Demethylases , Mesenchymal Stem Cells , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Signal Transduction , Animals , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/cytology , Forkhead Box Protein O1/metabolism , Forkhead Box Protein O1/genetics , Proto-Oncogene Proteins c-akt/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Mice , Jumonji Domain-Containing Histone Demethylases/metabolism , Jumonji Domain-Containing Histone Demethylases/genetics , Iron/metabolism , Mice, Inbred C57BL , Cell Proliferation , Cell Differentiation , Male , Iron Deficiencies , Humans
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