RESUMO
We have previously demonstrated the excellent bactericidal activity of josamycin against Staphylococcus aureus isolated from patients with atopic dermatitis (AD), with therapeutic efficacy equal to that of betamethasone. The present study was designed to evaluate the effectiveness of combination therapy with betamethasone and josamycin for AD. Betametasone (0.1%) and josamycin (0.1%) were topically administered to NC/Nga mice with severe AD-like skin lesions. Skin severity scores, histological changes in skin lesions, and serum immunoglobulin E (IgE) levels were assessed as indicators of therapeutic efficacy. Topical treatment with both drugs suppressed the skin severity score to a greater degree than betamethasone alone. This was associated with a reduction of epidermal thickening, a reduced density of dermal cellular infiltration, a decreased mast cell count in the dermis, and a reduced serum IgE level. In addition, both drugs in combination markedly reduced the expression of interferon (IFN)-γ and interleukin (IL)-4 in auricular lymph node cells, as well as the S. aureus count on the lesioned skin. These results show that simultaneous topical application of both drugs can ameliorate severe AD-like skin lesions in NC/Nga mice. It is suggested that combination therapy with betamethasone and josamycin would be beneficial for control of severe AD lesions colonized by S. aureus by inhibiting the development of both T helper (Th) type 1 (Th1) and Th2 cells and also through elimination of superficially located S. aureus.
Assuntos
Dermatite Atópica , Camundongos , Animais , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/patologia , Josamicina/uso terapêutico , Betametasona/uso terapêutico , Staphylococcus aureus , Pele/patologia , Modelos Animais de Doenças , Imunoglobulina ERESUMO
Hematopoietic stem cells (HSCs) are the most primitive cells in the hematopoietic system and are under tight regulation for self-renewal and differentiation. Notch signals are essential for the emergence of definitive hematopoiesis in mouse embryos and are critical regulators of lymphoid lineage fate determination. However, it remains unclear how Notch regulates the balance between HSC self-renewal and differentiation in the adult bone marrow (BM). Here we report a novel mechanism that prevents HSCs from undergoing premature lymphoid differentiation in BM. Using a series of in vivo mouse models and functional HSC assays, we show that leukemia/lymphoma related factor (LRF) is necessary for HSC maintenance by functioning as an erythroid-specific repressor of Delta-like 4 (Dll4) expression. Lrf deletion in erythroblasts promoted up-regulation of Dll4 in erythroblasts, sensitizing HSCs to T-cell instructive signals in the BM. Our study reveals novel cross-talk between HSCs and erythroblasts, and sheds a new light on the regulatory mechanisms regulating the balance between HSC self-renewal and differentiation.
Assuntos
Proteínas de Ligação a DNA/genética , Eritroblastos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Fatores de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Proteínas de Ligação ao Cálcio , Diferenciação Celular/genética , Proliferação de Células , Microambiente Celular/genética , Proteínas de Ligação a DNA/metabolismo , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Receptor Notch1/genética , Receptor Notch1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Linfócitos T/metabolismo , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transcriptoma/genéticaRESUMO
Clathrin-dependent endocytosis is an essential cellular process shared by all cell types. Despite this, precisely how endocytosis is regulated in a cell-type-specific manner and how this key pathway functions physiologically or pathophysiologically remain largely unknown. PICALM, which encodes the clathrin adaptor protein PICALM, was originally identified as a component of the CALM/AF10 leukemia oncogene. Here we show, by employing a series of conditional Picalm knockout mice, that PICALM critically regulates transferrin uptake in erythroid cells by functioning as a cell-type-specific regulator of transferrin receptor endocytosis. While transferrin receptor is essential for the development of all hematopoietic lineages, Picalm was dispensable for myeloid and B-lymphoid development. Furthermore, global Picalm inactivation in adult mice did not cause gross defects in mouse fitness, except for anemia and a coat color change. Freeze-etch electron microscopy of primary erythroblasts and live-cell imaging of murine embryonic fibroblasts revealed that Picalm function is required for efficient clathrin coat maturation. We showed that the PICALM PIP2 binding domain is necessary for transferrin receptor endocytosis in erythroblasts and absolutely essential for erythroid development from mouse hematopoietic stem/progenitor cells in an erythroid culture system. We further showed that Picalm deletion entirely abrogated the disease phenotype in a Jak2(V617F) knock-in murine model of polycythemia vera. Our findings provide new insights into the regulation of cell-type-specific transferrin receptor endocytosis in vivo. They also suggest a new strategy to block cellular uptake of transferrin-bound iron, with therapeutic potential for disorders characterized by inappropriate red blood cell production, such as polycythemia vera.
Assuntos
Hematopoese/genética , Proteínas Monoméricas de Montagem de Clatrina/genética , Policitemia Vera/genética , Anemia Hipocrômica/genética , Animais , Diferenciação Celular , Vesículas Revestidas por Clatrina/metabolismo , Vesículas Revestidas por Clatrina/ultraestrutura , Modelos Animais de Doenças , Endocitose , Eritroblastos/metabolismo , Eritroblastos/ultraestrutura , Eritropoese/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Imunofenotipagem , Linfopoese/genética , Camundongos , Camundongos Knockout , Proteínas Monoméricas de Montagem de Clatrina/química , Proteínas Monoméricas de Montagem de Clatrina/deficiência , Proteínas Monoméricas de Montagem de Clatrina/metabolismo , Mielopoese/genética , Fenótipo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Policitemia Vera/mortalidade , Domínios e Motivos de Interação entre Proteínas , Receptores da Transferrina/metabolismoRESUMO
BACKGROUND: Vascular pain is a common adverse drug reaction in colorectal cancer patients receiving peripheral venous administration of oxaliplatin. The aim of this work was to identify risk factors for vascular pain, and to examine whether currently used treatments reduce its incidence. METHODS: We conducted a multicenter retrospective study in Japanese colorectal cancer patients receiving peripheral venous administration of oxaliplatin. The effects of various treatments (administration of analgesics, addition of dexamethasone to the infusion solution for pH adjustment, dilution of the infusion solution, or use of hot gel for warming the injection site) on the incidence of vascular pain were assessed. Risk factors for vascular pain were identified by multiple logistic regression analysis. RESULTS: One hundred and ninety patients who had received an oxaliplatin-containing regimen via a peripheral venous route were analyzed. None of the preventive methods examined significantly reduced the incidence of vascular pain. BMI (BMI < 22), clinical stage (I-III) and oxaliplatin dosage (130 mg/m2 versus dose reduction) were identified as independent risk factors for development of vascular pain. The incidence of oxaliplatin-induced vascular pain was significantly higher in patients who had two or more risk factors. CONCLUSIONS: BMI, clinical stage and oxaliplatin dosage were identified as independent predictive markers for oxaliplatin-induced vascular pain. Existing treatments for vascular pain are not effective in reducing its incidence.
RESUMO
BACKGROUND: Recent large-scale clinical studies demonstrate that sodium-glucose cotransporter 2 (SGLT2) inhibitors protect the diabetic kidney. However, clinical and animal studies have not shown the changes of the total glomeruli in the whole kidney treated with SGLT2 inhibitors. METHODS: We performed computed tomography (CT) imaging on mice using synchrotron radiation to investigate the impact of luseogliflozin, a SGLT2 inhibitor, on the number and volume of glomeruli in the whole kidney. FINDINGS: We did not observe a significant difference in the total glomerular number (Nglom) among mice. Luseogliflozin redistributed the number of glomeruli in different regions, accompanied by the normalization of diabetes-augmented renal volume (Vkidney). Diabetic db/db mice had a larger glomerular volume in the mid-cortex than did control db/m mice, and luseogliflozin increased the glomerular volume in all renal cortical zones of the whole kidney in db/db mice. According to the multivariate regression analysis, hemoglobin A1c level was the most relevant determinant of Vkidney, not Nglom or mean glomerular volume (Vglom), indicating that hyperglycemia induced renal (tubular) hypertrophy, but not glomerular enlargement. Luseogliflozin increased hypoxia in the juxtamedullary region, sustained upregulated renal renin expression and plasma renin activity, and failed to decrease albuminuria by downregulating megalin in db/db mice. INTERPRETATION: Based on our findings, SGLT2 inhibitors may alter glomerular distribution and size in addition to their glucose-lowering effects, presumably by affecting oxygen metabolism and humoral factors. FUND: Funding for this research was provided by The Japan Society for the Promotion of Science, the Japan Diabetes Foundation, and Asahikawa Medical University.
Assuntos
Nefropatias Diabéticas/diagnóstico , Nefropatias Diabéticas/metabolismo , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Transportador 2 de Glucose-Sódio/metabolismo , Albuminúria , Animais , Biomarcadores , Modelos Animais de Doenças , Expressão Gênica , Hiperglicemia , Glomérulos Renais/patologia , Glomérulos Renais/ultraestrutura , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Masculino , Camundongos , Tamanho do Órgão , Renina/genética , Renina/metabolismo , Síncrotrons , Microtomografia por Raio-XRESUMO
To identify novel targets for acute myeloid leukemia (AML) therapy, we performed genome-wide CRISPR-Cas9 screening using AML cell lines, followed by a second screen in vivo. Here, we show that the mRNA decapping enzyme scavenger (DCPS) gene is essential for AML cell survival. The DCPS enzyme interacted with components of pre-mRNA metabolic pathways, including spliceosomes, as revealed by mass spectrometry. RG3039, a DCPS inhibitor originally developed to treat spinal muscular atrophy, exhibited anti-leukemic activity via inducing pre-mRNA mis-splicing. Humans harboring germline biallelic DCPS loss-of-function mutations do not exhibit aberrant hematologic phenotypes, indicating that DCPS is dispensable for human hematopoiesis. Our findings shed light on a pre-mRNA metabolic pathway and identify DCPS as a target for AML therapy.
Assuntos
Sistemas CRISPR-Cas/efeitos dos fármacos , Endorribonucleases/efeitos dos fármacos , Leucemia/tratamento farmacológico , Atrofia Muscular Espinal/tratamento farmacológico , Quinazolinas/farmacologia , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , Endorribonucleases/genética , Endorribonucleases/metabolismo , Humanos , Leucemia/genética , Masculino , Redes e Vias Metabólicas/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Atrofia Muscular Espinal/genética , Precursores de RNA/efeitos dos fármacos , Precursores de RNA/genética , Splicing de RNA/efeitos dos fármacos , Splicing de RNA/genética , RNA Mensageiro/genéticaRESUMO
Heme is required for survival of all cells, and in most eukaryotes, is produced through a series of eight enzymatic reactions. Although heme production is critical for many cellular processes, how it is coupled to cellular differentiation is unknown. Here, using zebrafish, murine, and human models, we show that erythropoietin (EPO) signaling, together with the GATA1 transcriptional target, AKAP10, regulates heme biosynthesis during erythropoiesis at the outer mitochondrial membrane. This integrated pathway culminates with the direct phosphorylation of the crucial heme biosynthetic enzyme, ferrochelatase (FECH) by protein kinase A (PKA). Biochemical, pharmacological, and genetic inhibition of this signaling pathway result in a block in hemoglobin production and concomitant intracellular accumulation of protoporphyrin intermediates. Broadly, our results implicate aberrant PKA signaling in the pathogenesis of hematologic diseases. We propose a unifying model in which the erythroid transcriptional program works in concert with post-translational mechanisms to regulate heme metabolism during normal development.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Eritropoetina/metabolismo , Fator de Transcrição GATA1/metabolismo , Heme/biossíntese , Transdução de Sinais , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Camundongos , Membranas Mitocondriais/metabolismo , Peixe-ZebraRESUMO
Genes encoding human ß-type globin undergo a developmental switch from embryonic to fetal to adult-type expression. Mutations in the adult form cause inherited hemoglobinopathies or globin disorders, including sickle cell disease and thalassemia. Some experimental results have suggested that these diseases could be treated by induction of fetal-type hemoglobin (HbF). However, the mechanisms that repress HbF in adults remain unclear. We found that the LRF/ZBTB7A transcription factor occupies fetal γ-globin genes and maintains the nucleosome density necessary for γ-globin gene silencing in adults, and that LRF confers its repressive activity through a NuRD repressor complex independent of the fetal globin repressor BCL11A. Our study may provide additional opportunities for therapeutic targeting in the treatment of hemoglobinopathies.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Hemoglobina Fetal/genética , Inativação Gênica , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , gama-Globinas/genética , Anemia Falciforme/genética , Animais , Proteínas de Transporte/genética , Linhagem Celular , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Eritroblastos/citologia , Eritropoese/genética , Humanos , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Deleção de Sequência , Talassemia/genética , Fatores de Transcrição/genéticaRESUMO
PICALM is a highly validated genetic risk factor for Alzheimer's disease (AD). We found that reduced expression of PICALM in AD and murine brain endothelium correlated with amyloid-ß (Aß) pathology and cognitive impairment. Moreover, Picalm deficiency diminished Aß clearance across the murine blood-brain barrier (BBB) and accelerated Aß pathology in a manner that was reversible by endothelial PICALM re-expression. Using human brain endothelial monolayers, we found that PICALM regulated PICALM/clathrin-dependent internalization of Aß bound to the low density lipoprotein receptor related protein-1, a key Aß clearance receptor, and guided Aß trafficking to Rab5 and Rab11, leading to Aß endothelial transcytosis and clearance. PICALM levels and Aß clearance were reduced in AD-derived endothelial monolayers, which was reversible by adenoviral-mediated PICALM transfer. Inducible pluripotent stem cell-derived human endothelial cells carrying the rs3851179 protective allele exhibited higher PICALM levels and enhanced Aß clearance. Thus, PICALM regulates Aß BBB transcytosis and clearance, which has implications for Aß brain homeostasis and clearance therapy.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Barreira Hematoencefálica/metabolismo , Córtex Cerebral/metabolismo , Proteínas Monoméricas de Montagem de Clatrina/metabolismo , Animais , Capilares/metabolismo , Endotélio Vascular/metabolismo , Homeostase , Humanos , Taxa de Depuração Metabólica , Camundongos , Camundongos Knockout , Proteínas Monoméricas de Montagem de Clatrina/deficiência , Células-Tronco Pluripotentes , TranscitoseRESUMO
B cells play a central role in immune system function. Deregulation of normal B cell maturation can lead to the development of autoimmune syndromes as well as B cell malignancies. Elucidation of the molecular features of normal B cell development is important for the development of new target therapies for autoimmune diseases and B cell malignancies. Employing B cell-specific conditional knockout mice, we have demonstrated here that the transcription factor leukemia/lymphoma-related factor (LRF) forms an obligate dimer in B cells and regulates mature B cell lineage fate and humoral immune responses via distinctive mechanisms. Moreover, LRF inactivation in transformed B cells attenuated their growth rate. These studies identify what we believe to be a new key factor for mature B cell development and provide a rationale for targeting LRF dimers for the treatment of autoimmune diseases and B cell malignancies.
Assuntos
Linfócitos B/imunologia , Proteínas de Ligação a DNA/imunologia , Centro Germinativo/imunologia , Fatores de Transcrição/imunologia , Animais , Linfócitos B/fisiologia , Diferenciação Celular/imunologia , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/imunologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Centro Germinativo/citologia , Centro Germinativo/fisiologia , Camundongos , Camundongos Knockout , Análise em Microsséries , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Baço/citologia , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
GATA-1-dependent transcription is essential for erythroid differentiation and maturation. Suppression of programmed cell death is also thought to be critical for this process; however, the link between these two features of erythropoiesis has remained elusive. Here, we show that the POZ-Krüppel family transcription factor, LRF (also known as Zbtb7a/Pokemon), is a direct target of GATA1 and plays an essential antiapoptotic role during terminal erythroid differentiation. We find that loss of Lrf leads to lethal anemia in embryos, due to increased apoptosis of late-stage erythroblasts. This programmed cell death is Arf and p53 independent and is instead mediated by upregulation of the proapoptotic factor Bim. We identify Lrf as a direct repressor of Bim transcription. In strong support of this mechanism, genetic Bim loss delays the lethality of Lrf-deficient embryos and rescues their anemia phenotype. Thus, our data define a key transcriptional cascade for effective erythropoiesis, whereby GATA-1 suppresses BIM-mediated apoptosis via LRF.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Apoptose , Proteínas de Ligação a DNA/fisiologia , Eritroblastos/citologia , Fator de Transcrição GATA1/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Fatores de Transcrição/fisiologia , Anemia/etiologia , Animais , Sequência de Bases , Proteína 11 Semelhante a Bcl-2 , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Células-Tronco Embrionárias/metabolismo , Eritroblastos/metabolismo , Células Precursoras Eritroides/metabolismo , Fator de Transcrição GATA1/genética , Proteínas de Ligação ao GTP/fisiologia , Perfilação da Expressão Gênica , Genes Letais , Integrases/metabolismo , Luciferases/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação , Proteínas de Resistência a Myxovirus , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Hematopoietic stem cells in the bone marrow give rise to lymphoid progenitors, which subsequently differentiate into B and T lymphocytes. Here we show that the proto-oncogene LRF plays an essential role in the B versus T lymphoid cell-fate decision. We demonstrate that LRF is key for instructing early lymphoid progenitors in mice to develop into B lineage cells by repressing T cell-instructive signals produced by the cell-fate signal protein, Notch. We propose a new model for lymphoid lineage commitment, in which LRF acts as a master regulator of the cell's determination of B versus T lineage.