ABSTRACT
ABSTRACT: Platelet α-granules have numerous proteins, some synthesized by megakaryocytes (MK) and others not synthesized but incorporated by endocytosis, an incompletely understood process in platelets/MK. Germ line RUNX1 haplodeficiency, referred to as familial platelet defect with predisposition to myeloid malignancies (FPDMMs), is associated with thrombocytopenia, platelet dysfunction, and granule deficiencies. In previous studies, we found that platelet albumin, fibrinogen, and immunoglobulin G (IgG) were decreased in a patient with FPDMM. We now show that platelet endocytosis of fluorescent-labeled albumin, fibrinogen, and IgG is decreased in the patient and his daughter with FPDMM. In megakaryocytic human erythroleukemia (HEL) cells, small interfering RNA RUNX1 knockdown (KD) increased uptake of these proteins over 24 hours compared with control cells, with increases in caveolin-1 and flotillin-1 (2 independent regulators of clathrin-independent endocytosis), LAMP2 (a lysosomal marker), RAB11 (a marker of recycling endosomes), and IFITM3. Caveolin-1 downregulation in RUNX1-deficient HEL cells abrogated the increased uptake of albumin, but not fibrinogen. Albumin, but not fibrinogen, partially colocalized with caveolin-1. RUNX1 KD resulted in increased colocalization of albumin with flotillin and fibrinogen with RAB11, suggesting altered trafficking of both proteins. The increased uptake of albumin and fibrinogen, as well as levels of caveolin-1, flotillin-1, LAMP2, and IFITM3, were recapitulated by short hairpin RNA RUNX1 KD in CD34+-derived MK. To our knowledge, these studies provide first evidence that platelet endocytosis of albumin and fibrinogen is impaired in some patients with RUNX1-haplodeficiency and suggest that megakaryocytes have enhanced endocytosis with defective trafficking, leading to loss of these proteins by distinct mechanisms. This study provides new insights into mechanisms governing endocytosis and α-granule deficiencies in RUNX1-haplodeficiency.
Subject(s)
Blood Coagulation Disorders, Inherited , Blood Platelet Disorders , Hemostatics , Leukemia, Erythroblastic, Acute , Leukemia, Myeloid, Acute , Humans , Megakaryocytes/metabolism , Caveolin 1/metabolism , Fibrinogen/metabolism , Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor Alpha 2 Subunit/metabolism , Endocytosis , Albumins/metabolism , Immunoglobulin G , Membrane Proteins/metabolism , RNA-Binding Proteins/metabolismABSTRACT
Heterozygous defects in runt-related transcription factor 1 (RUNX1) are causative of a familial platelet disorder with associated myeloid malignancy (FPDMM). Because RUNX1-deficient animal models do not mimic bleeding disorder or leukemic risk associated with FPDMM, development of a proper model system is critical to understanding the underlying mechanisms of the observed phenotype and to identifying therapeutic interventions. We previously reported an in vitro megakaryopoiesis system comprising human CD34+ hematopoietic stem and progenitor cells that recapitulated the FPDMM quantitative megakaryocyte defect through a decrease in RUNX1 expression via a lentiviral short hairpin RNA strategy. We now show that shRX-megakaryocytes have a marked reduction in agonist responsiveness. We then infused shRX-megakaryocytes into immunocompromised NOD scid gamma (NSG) mice and demonstrated that these megakaryocytes released fewer platelets than megakaryocytes transfected with a nontargeting shRNA, and these platelets had a diminished half-life. The platelets were also poorly responsive to agonists, unable to correct thrombus formation in NSG mice homozygous for a R1326H mutation in von Willebrand Factor (VWFR1326H), which switches the species-binding specificity of the VWF from mouse to human glycoprotein Ibα. A small-molecule inhibitor RepSox, which blocks the transforming growth factor ß1 (TGFß1) pathway and rescued defective megakaryopoiesis in vitro, corrected the thrombopoietic defect, defects in thrombus formation and platelet half-life, and agonist response in NSG/VWFR1326H mice. Thus, this model recapitulates the defects in FPDMM megakaryocytes and platelets, identifies previously unrecognized defects in thrombopoiesis and platelet half-life, and demonstrates for the first time, reversal of RUNX1 deficiency-induced hemostatic defects by a drug.
Subject(s)
Megakaryocytes , Thrombopoiesis , Humans , Mice , Animals , Megakaryocytes/metabolism , Thrombopoiesis/genetics , Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor Alpha 2 Subunit/metabolism , Half-Life , Blood Platelets/metabolismABSTRACT
Transcription factor RUNX1 is a master regulator of hematopoiesis and megakaryopoiesis. RUNX1 haplodeficiency (RHD) is associated with thrombocytopenia and platelet granule deficiencies and dysfunction. Platelet profiling of our study patient with RHD showed decreased expression of RAB31, a small GTPase whose cell biology in megakaryocytes (MKs)/platelets is unknown. Platelet RAB31 messenger RNA was decreased in the index patient and in 2 additional patients with RHD. Promoter-reporter studies using phorbol 12-myristate 13-acetate-treated megakaryocytic human erythroleukemia cells revealed that RUNX1 regulates RAB31 via binding to its promoter. We investigated RUNX1 and RAB31 roles in endosomal dynamics using immunofluorescence staining for markers of early endosomes (EEs; early endosomal autoantigen 1) and late endosomes (CD63)/multivesicular bodies. Downregulation of RUNX1 or RAB31 (by small interfering RNA or CRISPR/Cas9) showed a striking enlargement of EEs, partially reversed by RAB31 reconstitution. This EE defect was observed in MKs differentiated from a patient-derived induced pluripotent stem cell line (RHD-iMKs). Studies using immunofluorescence staining showed that trafficking of 3 proteins with distinct roles (von Willebrand factor [VWF], a protein trafficked to α-granules; epidermal growth factor receptor; and mannose-6-phosphate) was impaired at the level of EE on downregulation of RAB31 or RUNX1. There was loss of plasma membrane VWF in RUNX1- and RAB31-deficient megakaryocytic human erythroleukemia cells and RHD-iMKs. These studies provide evidence that RAB31 is downregulated in RHD and regulates megakaryocytic vesicle trafficking of 3 major proteins with diverse biological roles. EE defect and impaired vesicle trafficking is a potential mechanism for the α-granule defects observed in RUNX1 deficiency.
Subject(s)
Leukemia, Erythroblastic, Acute , Megakaryocytes , Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor Alpha 2 Subunit/metabolism , ErbB Receptors/metabolism , Humans , Megakaryocytes/metabolism , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolism , von Willebrand Factor/metabolismABSTRACT
IDO2 is one of two closely related tryptophan catabolizing enzymes induced under inflammatory conditions. In contrast to the immunoregulatory role defined for IDO1 in cancer models, IDO2 has a proinflammatory function in models of autoimmunity and contact hypersensitivity. In humans, two common single-nucleotide polymorphisms have been identified that severely impair IDO2 enzymatic function, such that <25% of individuals express IDO2 with full catalytic potential. This, together with IDO2's relatively weak enzymatic activity, suggests that IDO2 may have a role outside of its function in tryptophan catabolism. To determine whether the enzymatic activity of IDO2 is required for its proinflammatory function, we used newly generated catalytically inactive IDO2 knock-in mice together with established models of contact hypersensitivity and autoimmune arthritis. Contact hypersensitivity was attenuated in catalytically inactive IDO2 knock-in mice. In contrast, induction of autoimmune arthritis was unaffected by the absence of IDO2 enzymatic activity. In pursuing this nonenzymatic IDO2 function, we identified GAPDH, Runx1, RANbp10, and Mgea5 as IDO2-binding proteins that do not interact with IDO1, implicating them as potential mediators of IDO2-specific function. Taken together, our findings identify a novel function for IDO2, independent of its tryptophan catabolizing activity, and suggest that this nonenzymatic function could involve multiple signaling pathways. These data show that the enzymatic activity of IDO2 is required only for some inflammatory immune responses and provide, to our knowledge, the first evidence of a nonenzymatic role for IDO2 in mediating autoimmune disease.
Subject(s)
Arthritis/immunology , Autoimmunity/immunology , Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Animals , Antigens, Neoplasm/metabolism , Cell Line , Core Binding Factor Alpha 2 Subunit/metabolism , Gene Knock-In Techniques , Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/metabolism , Guanine Nucleotide Exchange Factors/metabolism , HEK293 Cells , Humans , Inflammation/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Microtubule-Associated Proteins/metabolism , Polymorphism, Single Nucleotide/geneticsSubject(s)
Core Binding Factor Alpha 2 Subunit/genetics , DNA-Binding Proteins/genetics , Gene Expression Regulation, Leukemic , Histone-Lysine N-Methyltransferase/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Oncogene Proteins, Fusion/genetics , Osteopontin/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Transcriptional Elongation Factors/genetics , Alternative Splicing , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Child , Child, Preschool , Core Binding Factor Alpha 2 Subunit/metabolism , DNA-Binding Proteins/metabolism , Female , Histone-Lysine N-Methyltransferase/metabolism , Humans , Infant , Male , Myeloid-Lymphoid Leukemia Protein/metabolism , Oncogene Proteins, Fusion/metabolism , Osteopontin/antagonists & inhibitors , Osteopontin/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Prognosis , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Risk Factors , Transcriptional Elongation Factors/metabolismABSTRACT
Germline RUNX1 mutations lead to thrombocytopenia and platelet dysfunction in familial platelet disorder with predisposition to acute myelogenous leukemia (AML). Multiple aspects of platelet function are impaired in these patients, associated with altered expression of genes regulated by RUNX1 We aimed to identify RUNX1-targets involved in platelet function by combining transcriptome analysis of patient and shRUNX1-transduced megakaryocytes (MK). Down-regulated genes included TREM-like transcript (TLT)-1 (TREML1) and the integrin subunit alpha (α)-2 (ITGA2) of collagen receptor α2-beta (ß)-1, which are involved in platelet aggregation and adhesion, respectively. RUNX1 binding to regions enriched for H3K27Ac marks was demonstrated for both genes using chromatin immunoprecipitation. Cloning of these regions upstream of the respective promoters in lentivirus allowing mCherry reporter expression showed that RUNX1 positively regulates TREML1 and ITGA2, and this regulation was abrogated after deletion of RUNX1 sites. TLT-1 content was reduced in patient MK and platelets. A blocking anti-TLT-1 antibody was able to block aggregation of normal but not patient platelets, whereas recombinant soluble TLT-1 potentiated fibrinogen binding to patient platelets, pointing to a role for TLT-1 deficiency in the platelet function defect. Low levels of α2 integrin subunit were demonstrated in patient platelets and MK, coupled with reduced platelet and MK adhesion to collagen, both under static and flow conditions. In conclusion, we show that gene expression profiling of RUNX1 knock-down or mutated MK provides a suitable approach to identify novel RUNX1 targets, among which downregulation of TREML1 and ITGA2 clearly contribute to the platelet phenotype of familial platelet disorder with predisposition to AML.
Subject(s)
Blood Platelet Disorders/genetics , Blood Platelet Disorders/metabolism , Core Binding Factor Alpha 2 Subunit/metabolism , Gene Expression Regulation , Integrin alpha2/genetics , Leukemia, Myeloid, Acute/etiology , Receptors, Immunologic/genetics , Blood Platelet Disorders/blood , Blood Platelets/metabolism , Disease Susceptibility , Gene Expression Profiling , Humans , Leukemia, Myeloid, Acute/diagnosis , Megakaryocytes/metabolism , Mutation , Platelet Aggregation , Platelet Function Tests , Protein BindingABSTRACT
Introducción: el gen de fusión RUNX1-RUNX1T codifica para una proteína quimérica con múltiples efectos en la proliferación, diferenciación y viabilidad de las células leucémicas. Objetivo: describir el comportamiento del RUNX1-RUNX1T1 en pacientes cubanos con dicha enfermedad. Método: Para ello se estudió el gen de fusión RUNX1-RUNX1T1 en 251 pacientes con leucemia mieloide aguda, mediante la reacción en cadena de la polimerasa, en el Instituto de Hematología e Inmunología de La Habana, entre los años 2000 y 2016. Resultados: El 20,3 por ciento (51 pacientes) fue positivo para el gen de fusión RUNX1-RUNX1T1, con una edad comprendida entre los 11 meses y los 80 años, media de 26 años. En los pacientes pediátricos la frecuencia del transcrito fue casi el doble de la de los adultos (29,2 por ciento y 15,3 por ciento, respectivamente) (p= 0,009). Mayor cantidad de pacientes masculinos presentaron el gen quimérico. En menores de 25 años hubo una mayor frecuencia del transcrito (p=0,019) con predominio significativo de la mutación en los adolescentes (p=0,027). Cinco pacientes fueron positivos al RUNX1-RUNX1T1 y a la duplicación interna en tándem del gen FLT3 (12,2 por ciento). Ningún paciente positivo al RUNX1-RUNX1T1 presentó el gen de fusión CBFB-MYH11. La mayor asociación estuvo con la mutación A del gen NPM1 para un 25 por ciento. El debut de la enfermedad se caracterizó por anemia moderada (p= 0,024), trombocitopenia severa (p= 0,004) y gran infiltración medular. La mayor discrepancia entre diagnósticos se concentró entre las variantes morfológicas M2 y M3 (p= 0,000). Conclusiones: En pacientes cubanos la leucemia mieloide aguda con gen de fusión RUNX1-RUNX1T1 positivo, tiene un comportamiento similar a lo descrito internacionalmente con algunas particularidadesen las características hematológicas de presentación de la enfermedad. El estudio molecular es imprescindible para definir el diagnóstico, y la estrategia terapéutica en estos pacientes(AU)
Introduction: The RUNX1-RUNX1T fusion gene codes for a chimeric protein with multiple effects on the proliferation, differentiation and viability of leukemic cells. Objective: To describe the behavior of RUNX1-RUNX1T1 in Cuban patients with this disease. Method: The RUNX1-RUNX1T1 fusion gene was studied in 251 patients with acute myeloid leukemia, through the polymerase chain reaction, at the Institute of Hematology and Immunology of Havana, between 2000 and 2016. Results: The 20.3 percent (51 patients) were positive for the RUNX1-RUNX1T1 fusion gene, with an age between 11 months and 80 years, average of 26 years.In pediatric patients, the transcript frequency was almost twice that of adults (29.2 percent and 15.3 percent , respectively) (p= 0.009). More male patients presented the chimeric gene. There was a higher frequency of the transcript in children under 25 years of age (p= 0.019) with a significant predominance of the mutation in adolescents (p= 0.027).Five patients were positive for RUNX1-RUNX1T1 and for internal tandem duplication of the FLT3 gene (12.2 percent ).No patient positive for RUNX1-RUNX1T1 presented the CBFB-MYH11 fusion gene. The greatest association was with the A mutation of the NPM1 gene for 25 percent . The onset of the disease was characterized by moderate anemia (p= 0.024), severe thrombocytopenia (p= 0.004) and extensive bone marrow infiltration. The greatest discrepancy between diagnoses was concentrated between the morphological variants M2 and M3 (p= 0.000). Conclusions: In Cuban patients, acute myeloid leukemia with a positive RUNX1-RUNX1T1 fusion gene has a behavior similar to that described internationally with some peculiarities in the hematological characteristics of the disease presentation.The molecular study is essential to define the diagnosis, and the therapeutic strategy in these patients(AU)
Subject(s)
Humans , Core Binding Factor Alpha 2 Subunit/metabolism , Pathology, Molecular/methods , RUNX1 Translocation Partner 1 Protein/metabolism , Epidemiology, Descriptive , Retrospective Studies , Longitudinal StudiesABSTRACT
The purinergic receptor P2X3 (P2X3-R) plays important roles in molecular pathways of pain, and reduction of its activity or expression effectively reduces chronic inflammatory and neuropathic pain sensation. Inflammation, nerve injury, and cancer-induced pain can increase P2X3-R mRNA and/or protein levels in dorsal root ganglia (DRG). However, P2X3-R expression is unaltered or even reduced in other pain studies. The reasons for these discrepancies are unknown and might depend on the applied traumatic intervention or on intrinsic factors such as age, gender, genetic background, and/or epigenetics. In this study, we sought to get insights into the molecular mechanisms responsible for inflammatory hyperalgesia by determining P2X3-R expression in DRG neurons of juvenile male rats that received a Complete Freund's Adjuvant (CFA) bilateral paw injection. We demonstrate that all CFA-treated rats showed inflammatory hyperalgesia, however, only a fraction (14-20%) displayed increased P2X3-R mRNA levels, reproducible across both sides. Immunostaining assays did not reveal significant increases in the percentage of P2X3-positive neurons, indicating that increased P2X3-R at DRG somas is not critical for inducing inflammatory hyperalgesia in CFA-treated rats. Chromatin immunoprecipitation (ChIP) assays showed a correlated (R2 = 0.671) enrichment of the transcription factor Runx1 and the epigenetic active mark histone H3 acetylation (H3Ac) at the P2X3-R gene promoter in a fraction of the CFA-treated rats. These results suggest that animal-specific increases in P2X3-R mRNA levels are likely associated with the genetic/epigenetic context of the P2X3-R locus that controls P2X3-R gene transcription by recruiting Runx1 and epigenetic co-regulators that mediate histone acetylation.
Subject(s)
Freund's Adjuvant/adverse effects , Ganglia, Spinal/metabolism , Gene Expression Regulation/drug effects , Hyperalgesia/chemically induced , Hyperalgesia/metabolism , Receptors, Purinergic P2X3/biosynthesis , Transcription, Genetic/drug effects , Animals , Core Binding Factor Alpha 2 Subunit/metabolism , Freund's Adjuvant/pharmacology , Ganglia, Spinal/pathology , Hyperalgesia/pathology , Male , Rats , Rats, Sprague-DawleyABSTRACT
RUNX1 a member of the family of runt related transcription factors (RUNX), is essential for hematopoiesis. The expression of RUNX1 gene is controlled by two promoters; the distal P1 promoter and the proximal P2 promoter. Several isoforms of RUNX1 mRNA are generated through the use of both promoters and alternative splicing. These isoforms not only differs in their temporal expression pattern but also exhibit differences in tissue specificity. The RUNX1 isoforms derived from P2 are expressed in a variety of tissues, but expression of P1-derived isoform is restricted to cells of hematopoietic lineage. However, the control of hematopoietic-cell specific expression is poorly understood. Here we report regulation of P1-derived RUNX1 mRNA by RUNX1 protein. In silico analysis of P1 promoter revealed presence of two evolutionary conserved RUNX motifs, 0.6kb upstream of the transcription start site, and three RUNX motifs within 170bp of the 5'UTR. Transcriptional contribution of these RUNX motifs was studied in myeloid and T-cells. RUNX1 genomic fragment containing all sites show very low basal activity in both cell types. Mutation or deletion of RUNX motifs in the UTR enhances basal activity of the RUNX1 promoter. Chromatin immunoprecipitation revealed that RUNX1 protein is recruited to these sites. Overexpression of RUNX1 in non-hematopoietic cells results in a dose dependent activation of the RUNX1 P1 promoter. We also demonstrate that RUNX1 protein regulates transcription of endogenous RUNX1 mRNA in T-cell. Finally we show that SCL transcription factor is recruited to regions containing RUNX motifs in the promoter and the UTR and regulates activity of the RUNX1 P1 promoter in vitro. Thus, multiple lines of evidence show that RUNX1 protein regulates its own gene transcription.
Subject(s)
Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor Alpha 2 Subunit/metabolism , Gene Expression Regulation , Promoter Regions, Genetic , Transcription, Genetic , 5' Untranslated Regions , Base Sequence , Basic Helix-Loop-Helix Transcription Factors/metabolism , Binding Sites , Cell Line, Tumor , Humans , Molecular Sequence Data , Mutation , Nucleotide Motifs , Protein Binding , Proto-Oncogene Proteins/metabolism , RNA, Messenger , Sequence Alignment , T-Cell Acute Lymphocytic Leukemia Protein 1 , Transcriptional ActivationABSTRACT
Runx1 participation in epithelial mammary cells is still under review. Emerging data indicates that Runx1 could be relevant for breast tumor promotion. However, to date no studies have specifically evaluated the functional contribution of Runx1 to control gene expression in mammary epithelial tumor cells. It has been described that Runx1 activity is defined by protein context interaction. Interestingly, Foxp3 is a breast tumor suppressor gene. Here we show that endogenous Runx1 and Foxp3 physically interact in normal mammary cells and this interaction blocks Runx1 transcriptional activity. Furthermore we demonstrate that Runx1 is able to bind to R-spondin 3 (RSPO3) and Gap Junction protein Alpha 1 (GJA1) promoters. This binding upregulates Rspo3 oncogene expression and downregulates GJA1 tumor suppressor gene expression in a Foxp3-dependent manner. Moreover, reduced Runx1 transcriptional activity decreases tumor cell migration properties. Collectively, these data provide evidence of a new mechanism for breast tumor gene expression regulation, in which Runx1 and Foxp3 physically interact to control mammary epithelial cell gene expression fate. Our work suggests for the first time that Runx1 could be involved in breast tumor progression depending on Foxp3 availability.
Subject(s)
Adenocarcinoma/metabolism , Breast Neoplasms/metabolism , Connexin 43/metabolism , Core Binding Factor Alpha 2 Subunit/metabolism , Forkhead Transcription Factors/metabolism , Gene Expression Regulation, Neoplastic , Thrombospondins/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Animals , Apoptosis , Blotting, Western , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Movement , Cell Proliferation , Chromatin Immunoprecipitation , Female , Fluorescent Antibody Technique , Humans , Immunoenzyme Techniques , Immunoprecipitation , Mice , Mice, Inbred BALB C , Microscopy, Confocal , Microscopy, Fluorescence , Promoter Regions, Genetic/genetics , Tumor Cells, Cultured , Wound Healing , Xenograft Model Antitumor AssaysABSTRACT
We investigated the expression differences of the TEL-AML1 fusion gene in a leukemia glucocorticoid (GC)-sensitive cell line (CEM) and a GC-resistant cell line (Jurkat). Changes in TEL-AML1 expression before and after GC exposure were analyzed. Expression of GC-sensitive and GC-resistant leukemia cells following initial diagnosis and during treatment was simulated. Leukemia cells were divided into a GC-unexposed or a GC-exposed group. A methyl thiazolyl tetrazolium assay was used to detect cell proliferation inhibition, flow cytometry was used to observe cell apoptosis, reverse transcription-polymerase chain reaction was used to detect the mRNA expression of TEL-AML1 before and after exposure, and western blotting was used to analyze protein levels of TEL-AML1 before and after exposure. Inhibitory concentrations of 50% of cells in the Jurkat and CEM cells at 24 h were 382 and 9 mM, respectively, and at 48 h they were 216 and 2 mM. The proliferation inhibition effect of dexamethasone sodium phosphate on Jurkat cells was much lower than that on CEM cells. Jurkat cells showed obvious apoptosis after exposure to 100 mM dexamethasone sodium phosphate for 48 h. In the exposed group, Jurkat cells showed higher TEL-AML1 expression than did CEM cells (P < 0.05). In the unexposed group, TEL-AML1 gene expression in Jurkat cells was not affected by GC exposure (P > 0.05), while the CEM cells presented significant differences before and after exposure (P < 0.05). Sustained high expression of TEL-AML1 participated in and maintained the occurrence of GC resistance. Inhibition of TEL-AML1 may provide a new therapeutic approach to reverse GC resistance.
Subject(s)
Core Binding Factor Alpha 2 Subunit/genetics , Drug Resistance, Neoplasm/drug effects , Gene Expression Regulation, Leukemic/drug effects , Glucocorticoids/pharmacology , Oncogene Proteins, Fusion/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Apoptosis/drug effects , Blotting, Western , Cell Proliferation/drug effects , Core Binding Factor Alpha 2 Subunit/metabolism , Dexamethasone/pharmacology , Drug Resistance, Neoplasm/genetics , Flow Cytometry , Humans , Jurkat Cells , Oncogene Proteins, Fusion/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The majority of endometrioid endometrial carcinomas (EEC) is diagnosed at stage I. Among these, 30% present myometrial invasion (stage IB), which is associated with tumor spread and relapse after primary treatment. Although an increased expression of RUNX1/AML1 and ERM/ETV5 in EEC have been suggested to be associated with early events of myometrial infiltration, there is no data regarding its expression along the evolution of EEC and possible associations with other clinicopathological parameters. Therefore, ERM/ETV5 and RUNX1/AML1 protein and gene expression profiles were assessed in different EEC stages to evaluate their role in endometrial carcinogenesis. RUNX1/AML1 and ERM/ETV5 proteins were analyzed by immunohistochemistry in 219 formalin fixed paraffin embedded endometrioid tumors and in 12 normal atrophic and proliferative endometrium samples. RUNX1/AML1 and ERM/ETV5 genes expression were analyzed by RT-qPCR. RUNX1/AML1 and ERM/ETV5 expression were decreased with increasing EEC stage, with a positive correlation between protein and gene expression for ERM/ETV5, but not for RUNX1/AML1. Both proteins were present in the nucleus of the tumor cells, whereas RUNX1/AML1, but not ERM/ETV5, was expressed in 7 out of 12 normal endometrial samples, with its expression being restricted to the cytoplasm of the positive cells. We concluded that there is a higher expression of ERM/ETV5 in early stages of EEC, whereas there seems to be a RUNX1/AML1 translocation from cytoplasm to nucleus in EEC neoplastic transformation.
Subject(s)
Carcinoma, Endometrioid/metabolism , Core Binding Factor Alpha 2 Subunit/metabolism , DNA-Binding Proteins/metabolism , Endometrial Neoplasms/metabolism , Transcription Factors/metabolism , Adult , Aged , Aged, 80 and over , Carcinoma, Endometrioid/pathology , Core Binding Factor Alpha 2 Subunit/genetics , DNA-Binding Proteins/genetics , Disease Progression , Endometrial Neoplasms/pathology , Endometrium/metabolism , Endometrium/pathology , Female , Humans , Middle Aged , Transcription Factors/genetics , TranscriptomeABSTRACT
BACKGROUND: Familial platelet disorder with a predisposition to acute myelogenous leukemia (FPD/AML) is an inherited platelet disorder caused by a germline RUNX1 mutation and characterized by thrombocytopenia, a platelet function defect, and leukemia predisposition. The mechanisms underlying FPD/AML platelet dysfunction remain incompletely clarified. We aimed to determine the contribution of platelet structural abnormalities and defective activation pathways to the platelet phenotype. In addition, by using a candidate gene approach, we sought to identify potential RUNX1-regulated genes involved in these defects. METHODS: Lumiaggregometry, α-granule and dense granule content and release, platelet ultrastructure, αIIb ß3 integrin activation and outside-in signaling were assessed in members of one FPD/AML pedigree. Expression levels of candidate genes were measured and luciferase reporter assays and chromatin immunoprecipitation were performed to study NF-E2 regulation by RUNX1. RESULTS: A severe decrease in platelet aggregation, defective αIIb ß3 integrin activation and combined αδ storage pool deficiency were found. However, whereas the number of dense granules was markedly reduced, α-granule content was heterogeneous. A trend towards decreased platelet spreading was found, and ß3 integrin phosphorylation was impaired, reflecting altered outside-in signaling. A decrease in the level of transcription factor p45 NF-E2 was shown in platelet RNA and lysates, and other deregulated genes included RAB27B and MYL9. RUNX1 was shown to bind to the NF-E2 promoter in primary megakaryocytes, and wild-type RUNX1, but not FPD/AML mutants, was able to activate NF-E2 expression. CONCLUSIONS: The FPD/AML platelet function defect represents a complex trait, and RUNX1 orchestrates platelet function by regulating diverse aspects of this process. This study highlights the RUNX1 target NF-E2 as part of the molecular network by which RUNX1 regulates platelet biogenesis and function.
Subject(s)
Blood Platelet Disorders/blood , Blood Platelet Disorders/complications , Blood Platelets/cytology , Core Binding Factor Alpha 2 Subunit/metabolism , Leukemia, Myeloid, Acute/blood , Leukemia, Myeloid, Acute/complications , Adenosine Triphosphate/metabolism , Adult , Family Health , Female , Gene Expression Profiling , Humans , Integrin beta3/metabolism , Male , NF-E2 Transcription Factor, p45 Subunit/metabolism , Pedigree , Phenotype , Phosphorylation , Platelet Aggregation , Platelet Function Tests , Platelet Membrane Glycoprotein IIb/metabolism , Signal Transduction , Tyrosine/metabolism , Young AdultABSTRACT
Peroxisome proliferator-activated receptor-gamma (PPARγ) regulates both glucose metabolism and bone mass. Evidence suggests that the therapeutic modulation of PPARγ with synthetic agonists activity may elicit undesirable effects on bone. However, there is no information regarding its natural agonist 15d-PGJ2, besides its excellent anti-inflammatory action. In the present study the effects of 15d-PGJ2 on osteoblastic cells were determined. Osteoblastic cells (MC3T3) were cultured in an osteogenic medium in the presence of 1, 3 or 10 µM of 15d-PGJ2 during 21 days and alizarin and Von Kossa staining were employed. The protein expression (type-I collagen, osteonectin, osteopontin, RANKL, osteoprotegerin, HDAC-9c and PPAR-γ) was evaluated after 3 days in the presence of 15d-PGJ2 by western blotting and indirect immunofluorescence methods. The production of mineralized extracellular matrix was observed by transmission electron microscopy. After 72 h of culture, the mRNA was extracted for RT-qPCR analysis of RUNX expression. In the presence of all 3 tested 15d-PGJ2 doses, alizarin red and Von kossa staining were positive demonstrating the ability to the osteoblast differentiation. Type-I collagen and osteonectin proteins expression were up-regulated (p < 0.05) after 72 h in the presence of the smaller doses of 15d-PGJ2. In contrast, osteopontin, RANKL and OPG expression did not significantly alter. In the presence of 15d-PGJ2 it was possible to visualize mineralized nodules in the extracellular matrix confirmed with the increased RUNX mRNA expression. 15d-PGJ2 at small doses increased the osteoblast activity and the bone-related proteins expression.
Subject(s)
Osteoblasts/drug effects , Prostaglandin D2/analogs & derivatives , Animals , Cell Line , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Collagen Type I/metabolism , Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor Alpha 2 Subunit/metabolism , Histone Deacetylases/metabolism , Mice , Osteoblasts/metabolism , Osteonectin/metabolism , Osteopontin/metabolism , Osteoprotegerin/metabolism , PPAR gamma , Prostaglandin D2/pharmacology , RANK Ligand/metabolism , Repressor Proteins/metabolismABSTRACT
Transient Receptor Potential Vanilloid type 1 channel (TRPV1) is an important endogenous transducer of noxious heat and chemical stimuli and is required during development of inflammatory hypersensitivity. The transcription factor Runx1 is known to play a relevant role in sensory neuron differentiation as it controls the expression of several sensory nociceptive receptors, including TRPV1. Here, we show that Runx1 up-regulates TRPV1 transcription activity by interacting directly with the proximal TRPV1 gene promoter sequence. Importantly, C/EBPß a well-established heterodimer partner of Runx1 also binds to the TRPV1 promoter and cooperates with Runx1 to further stimulate TRPV1 transcription. Our results support a mechanism where Runx1-C/EBPß-containing transcription regulatory complexes are recruited to the TRPV1 gene promoter to modulate TRPV1 expression in dorsal root ganglia neurons.
Subject(s)
CCAAT-Enhancer-Binding Protein-beta/genetics , Core Binding Factor Alpha 2 Subunit/genetics , Pain/genetics , TRPV Cation Channels/genetics , Animals , CCAAT-Enhancer-Binding Protein-beta/metabolism , Core Binding Factor Alpha 2 Subunit/metabolism , Ganglia, Spinal/metabolism , Neurons/metabolism , PC12 Cells , Pain/metabolism , Promoter Regions, Genetic , Rats , TRPV Cation Channels/biosynthesis , TRPV Cation Channels/metabolism , Transcription, Genetic , Transcriptional Activation , Up-RegulationABSTRACT
Bone marrow samples from children with acute lymphoblastic leukemia were analyzed for the expression of RUNX1a/b/c isoforms. Obtained patterns were associated with genetic abnormalities and the expression of the RUNX1 regulated gene BLK. RUNX1c was present in all patients, but the expected over-expression of RUNX1a was not observed. Over-expression of total RUNT domain isoforms was detected in patients with extra RUNX1 copies, and unexpectedly, in those with t(4;11). Only expression of the total RUNT domain-containing isoforms and BLK presented positive correlation. Results suggest a more complex role of RUNX1 in leukemogenesis than the proposed antagonism between the isoforms.
Subject(s)
Core Binding Factor Alpha 2 Subunit/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Protein-Tyrosine Kinases/genetics , Adolescent , Amino Acid Sequence , Base Sequence , Child , Child, Preschool , Core Binding Factor Alpha 2 Subunit/metabolism , Female , Gene Expression Regulation, Leukemic , Humans , Infant , Infant, Newborn , Male , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein-Tyrosine Kinases/metabolismABSTRACT
Recent evidence indicates that transcription factor Runx1 modulates the expression of several phenotypic markers in dorsal root ganglia (DRGs) neurons, including the pain-related P2X3 receptor. In several cell lineages C/EBP transcription factors interact with the Runx factor family members to jointly bind and activate transcription of target genes. Here, we examine whether these two transcription factors directly regulate P2X3 gene expression. Through in silico analyses of the first 2 kb of the P2X3 gene promoter we identified putative consensus-binding sites for both Runx1 and C/EBPß transcription factors. Transient over-expression in PC12 cells of either Runx1 or C/EBPß increases P2X3 gene promoter activity and co-expression of both factors results in an additive stimulatory effect on the promoter function. Accordingly, chromatin immunoprecipitation assays demonstrate that both Runx1 and C/EBPß bind to the P2X3 promoter in PC12 cells expressing this gene. Site-directed mutagenesis of the proximal Runx1 and C/EBPß consensus elements in the P2X3 promoter decrease Runx1- and C/EBPß-mediated transcriptional activity. Moreover, C/EBPß-mediated enhancement of the P2X3 promoter requires a functional Runx1 binding site. Altogether our results support a functional and coordinated role for Runx1 and C/EBPß transcription factors during activation of P2X3 gene transcription.