RESUMO
DNA-binding protein-A (DbpA; gene: Ybx3) belongs to the cold shock protein family with known functions in cell cycling, transcription, translation, and tight junction communication. In chronic nephritis, DbpA is upregulated. However, its activities in acute injury models, such as kidney ischemia/reperfusion injury (IRI), are unclear. To study this, mice harboring Ybx3+/+, Ybx3+/- or the Ybx3-/- genotype were characterized over 24 months and following experimental kidney IRI. Mitochondrial function, number and integrity were analyzed by mitochondrial stress tests, MitoTracker staining and electron microscopy. Western Blot, immunohistochemistry and flow cytometry were performed to quantify tubular cell damage and immune cell infiltration. DbpA was found to be dispensable for kidney development and tissue homeostasis under healthy conditions. Furthermore, endogenous DbpA protein localizes within mitochondria in primary tubular epithelial cells. Genetic deletion of Ybx3 elevates the mitochondrial membrane potential, lipid uptake and metabolism, oxygen consumption rates and glycolytic activities of tubular epithelial cells. Ybx3-/- mice demonstrated protection from IRI with less immune cell infiltration, endoplasmic reticulum stress and tubular cell damage. A presumed protective mechanism was identified via upregulated antioxidant activities and reduced ferroptosis, when Ybx3 was deleted. Thus, our studies reveal DbpA acts as a mitochondrial protein with profound adverse effects on cell metabolism and highlights a protective effect against IRI when Ybx3 is genetically deleted. Hence, preemptive DbpA targeting in situations with expected IRI, such as kidney transplantation or cardiac surgery, may preserve post-procedure kidney function.
Assuntos
Camundongos Knockout , Mitocôndrias , Traumatismo por Reperfusão , Animais , Masculino , Camundongos , Modelos Animais de Doenças , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/deficiência , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Rim/patologia , Rim/metabolismo , Potencial da Membrana Mitocondrial , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologiaRESUMO
Glomerular-tubular crosstalk within the kidney has been proposed, but the paracrine signals enabling this remain largely unknown. The cold-shock protein Y-box binding protein 1 (YBX1) is known to regulate inflammation and kidney diseases but its role in podocytes remains undetermined. Therefore, we analyzed mice with podocyte specific Ybx1 deletion (Ybx1ΔPod). Albuminuria was increased in unchallenged Ybx1ΔPod mice, which surprisingly was associated with reduced glomerular, but enhanced tubular damage. Tubular toll-like receptor 4 (TLR4) expression, node-like receptor protein 3 (NLRP3) inflammasome activation and kidney inflammatory cell infiltrates were all increased in Ybx1ΔPod mice. In vitro, extracellular YBX1 inhibited NLRP3 inflammasome activation in tubular cells. Co-immunoprecipitation, immunohistochemical analyses, microscale cell-free thermophoresis assays, and blunting of the YBX1-mediated TLR4-inhibition by a unique YBX1-derived decapeptide suggests a direct interaction of YBX1 and TLR4. Since YBX1 can be secreted upon post-translational acetylation, we hypothesized that YBX1 secreted from podocytes can inhibit TLR4 signaling in tubular cells. Indeed, mice expressing a non-secreted YBX1 variant specifically in podocytes (Ybx1PodK2A mice) phenocopied Ybx1ΔPod mice, demonstrating a tubular-protective effect of YBX1 secreted from podocytes. Lipopolysaccharide-induced tubular injury was aggravated in Ybx1ΔPod and Ybx1PodK2A mice, indicating a pathophysiological relevance of this glomerular-tubular crosstalk. Thus, our data show that YBX1 is physiologically secreted from podocytes, thereby negatively modulating sterile inflammation in the tubular compartment, apparently by binding to and inhibiting tubular TLR4 signaling. Hence, we have uncovered an YBX1-dependent molecular mechanism of glomerular-tubular crosstalk.
Assuntos
Nefropatias , Podócitos , Camundongos , Animais , Inflamassomos/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Resposta ao Choque Frio , Rim/metabolismo , Podócitos/metabolismo , Nefropatias/metabolismo , Inflamação/metabolismoRESUMO
In aging kidneys, a decline of function resulting from extracellular matrix (ECM) deposition and organ fibrosis is regarded as "physiological." Whether a direct link between high salt intake and fibrosis in aging kidney exists autonomously from arterial hypertension is unclear. This study explores kidney intrinsic changes (inflammation, ECM derangement) induced by a high-salt diet (HSD) in a murine model lacking arterial hypertension. The contribution of cold shock Y-box binding protein (YB-1) as a key orchestrator of organ fibrosis to the observed differences is determined by comparison with a knockout strain (Ybx1ΔRosaERT+TX). Comparisons of tissue from mice fed with normal-salt diet (NSD, standard chow) or high-salt diet (HSD, 4% NaCl in chow; 1% NaCl in water) for up to 16 mo revealed that with HSD tubular cell numbers decrease and tubulointerstitial scarring [periodic acid-Schiff (PAS), Masson's trichrome, Sirius red staining] prevails. In Ybx1ΔRosaERT+TX animals tubular cell damage, a loss of cell contacts with profound tubulointerstitial alterations, and tubular cell senescence was seen. A distinct tubulointerstitial distribution of fibrinogen, collagen type VI, and tenascin-C was detected under HSD, transcriptome analyses determined patterns of matrisome regulation. Temporal increase of immune cell infiltration was seen under HSD of wild type, but not Ybx1ΔRosaERT+TX animals. In vitro Ybx1ΔRosaERT+TX bone marrow-derived macrophages exhibited a defect in polarization (IL-4/IL-13) and abrogated response to sodium chloride. Taken together, HSD promotes progressive kidney fibrosis with premature cell aging, ECM deposition, and immune cell recruitment that is exacerbated in Ybx1ΔRosaERT+TX animals.NEW & NOTEWORTHY Short-term experimental studies link excessive sodium ingestion with extracellular matrix accumulation and inflammatory cell recruitment, yet long-term data are scarce. Our findings with a high-salt diet over 16 mo in aging mice pinpoints to a decisive tipping point after 12 mo with tubular stress response, skewed matrisome transcriptome, and immune cell infiltration. Cell senescence was aggravated in knockout animals for cold shock Y-box binding protein (YB-1), suggesting a novel protective protein function.
Assuntos
Hipertensão , Nefropatias , Camundongos , Animais , Cloreto de Sódio , Rim/metabolismo , Nefropatias/induzido quimicamente , Nefropatias/genética , Nefropatias/patologia , Inflamação/metabolismo , Envelhecimento , Hipertensão/metabolismo , Cloreto de Sódio na Dieta/efeitos adversos , Fibrose , Ingestão de AlimentosRESUMO
High salt diet (HSD) is a hallmark of blood pressure elevations, weight gain and diabetes onset in the metabolic syndrome. In kidney, compensatory mechanisms are activated to balance salt turnover and maintain homeostasis. Data on the long-term effects of HSD with respect to tubular cell functions and kidney architecture that exclude confounding indirect blood pressure effects are scarce. Additionally we focus on cold shock Y-box binding protein-1 as a tubular cell protective factor. A HSD model (4% NaCl in chow; 1% NaCl in water) was compared to normal salt diet (NSD, standard chow) over 16 months using wild type mice and an inducible conditional whole body knockout for cold shock Y-box binding protein-1 (BL6J/N, Ybx1). HSD induced no difference in blood pressure over 16 months, comparing NSD/HSD and Ybx1 wild type/knockout. Nevertheless, marked phenotypic changes were detected. Glucosuria and subnephrotic albuminuria ensued in wild type animals under HSD, which subsided in Ybx1-deficient animals. At the same time megalin receptors were upregulated. The sodium-glucose cotransporter-2 (SGLT2) was completely downregulated in wild type HSD animals that developed glucosuria. In Ybx1 knockouts, expression of AQP1 and SGLT2 was maintained under HSD; proximal tubular widening and glomerular tubularization developed. Concurrently, amino aciduria of neutral and hydrophobic amino acids was seen. In vitro translation confirmed that YB-1 translationally represses Sglt2 transcripts. Our data reveal profound effects of HSD primarily within glomeruli and proximal tubular segments. YB-1 is regulated by HSD and orchestrates HSD-dependent changes; notably, sets reabsorption thresholds for amino acids, proteins and glucose.
Assuntos
Resposta ao Choque Frio/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Sódio na Dieta/farmacologia , Transportador 2 de Glucose-Sódio/genética , Fatores de Transcrição/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Feminino , Túbulos Renais Proximais/citologia , Leucócitos/citologia , Macrófagos/citologia , Masculino , Fenótipo , Podócitos/efeitos dos fármacos , Renina/biossíntese , Renina/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Kidney injuries that result in chronic inflammation initiate crosstalk between stressed resident cells and infiltrating immune cells. In animal models, whole-body receptor Notch3 deficiency protects from leukocyte infiltration and organ fibrosis. However, the relative contribution of Notch3 expression in tissue versus infiltrating immune cells is unknown. METHODS: Chimeric mice deficient for Notch3 in hematopoietic cells and/or resident tissue cells were generated, and kidney fibrosis and inflammation after unilateral ureteral obstruction (UUO) were analyzed. Adoptive transfer of labeled bone marrow-derived cells validated the results in a murine Leishmania ear infection model. In vitro adhesion assays, integrin activation, and extracellular matrix production were analyzed. RESULTS: Fibrosis follows UUO, but inflammatory cell infiltration mostly depends upon Notch3 expression in hematopoietic cells, which coincides with an enhanced proinflammatory milieu (e.g., CCL2 and CCL5 upregulation). Notch3 expression on CD45+ leukocytes plays a prominent role in efficient cell transmigration. Functionally, leukocyte adhesion and integrin activation are abrogated in the absence of receptor Notch3. Chimeric animal models also reveal that tubulointerstitial fibrosis develops, even in the absence of prominent leukocyte infiltrates after ureteral obstruction. Deleting Notch3 receptors on resident cells blunts kidney fibrosis, ablates NF-κB signaling, and lessens matrix deposition. CONCLUSIONS: Cell-specific receptor Notch3 signaling independently orchestrates leukocyte infiltration and organ fibrosis. Interference with Notch3 signaling may present a novel therapeutic approach in inflammatory as well as fibrotic diseases.
Assuntos
Rim/patologia , Leucócitos/fisiologia , Macrófagos/fisiologia , Nefrite/patologia , Receptor Notch3/genética , Receptor Notch3/metabolismo , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/fisiologia , Adesão Celular , Proliferação de Células , Células Cultivadas , Quimera , Matriz Extracelular/metabolismo , Feminino , Fibrose , Integrinas/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Leucócitos/metabolismo , Macrófagos/metabolismo , Camundongos , NF-kappa B/metabolismo , Nefrite/etiologia , Transdução de Sinais , Transcriptoma , Migração Transendotelial e Transepitelial , Obstrução Ureteral/complicaçõesRESUMO
Cold shock Y-box binding protein-1 (YB-1) coordinates several molecular processes between the nucleus and the cytoplasm and plays a crucial role in cell function. Moreover, it is involved in cancer progression, invasion, and metastasis. As trophoblast cells share similar characteristics with cancer cells, we hypothesized that YB-1 might also be necessary for trophoblast functionality. In samples of patients with intrauterine growth restriction, YB-1 mRNA levels were decreased, while they were increased in preeclampsia and unchanged in spontaneous abortions when compared to normal pregnant controls. Studies with overexpression and downregulation of YB-1 were performed to assess the key trophoblast processes in two trophoblast cell lines HTR8/SVneo and JEG3. Overexpression of YB-1 or exposure of trophoblast cells to recombinant YB-1 caused enhanced proliferation, while knockdown of YB-1 lead to proliferative disadvantage in JEG3 or HTR8/SVneo cells. The invasion and migration properties were affected at different degrees among the trophoblast cell lines. Trophoblast expression of genes mediating migration, invasion, apoptosis, and inflammation was altered upon YB-1 downregulation. Moreover, IL-6 secretion was excessively increased in HTR8/SVneo. Ultimately, YB-1 directly binds to NF-κB enhancer mark in HTR8/SVneo cells. Our data show that YB-1 protein is important for trophoblast cell functioning and, when downregulated, leads to trophoblast disadvantage that at least in part is mediated by NF-κB.
Assuntos
Complicações na Gravidez/metabolismo , Trofoblastos/metabolismo , Aborto Espontâneo/genética , Aborto Espontâneo/metabolismo , Aborto Espontâneo/patologia , Adulto , Apoptose , Estudos de Casos e Controles , Linhagem Celular , Movimento Celular , Proliferação de Células , Regulação para Baixo , Feminino , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/metabolismo , Retardo do Crescimento Fetal/patologia , Técnicas de Silenciamento de Genes , Humanos , Técnicas In Vitro , Masculino , NF-kappa B/metabolismo , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/patologia , Gravidez , Complicações na Gravidez/genética , Complicações na Gravidez/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Trofoblastos/patologia , Regulação para Cima , Proteína 1 de Ligação a Y-Box/antagonistas & inibidores , Proteína 1 de Ligação a Y-Box/genética , Proteína 1 de Ligação a Y-Box/metabolismo , Adulto JovemRESUMO
BACKGROUND: Phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (PAG1) is a transmembrane adaptor protein that affects immune receptor signaling in T and B cells. Evidence from genome-wide association studies of asthma suggests that genetic variants that regulate the expression of PAG1 are associated with asthma risk. However, it is not known whether PAG1 expression is causally related to asthma pathophysiology. Here, we investigated the role of PAG1 in a preclinical mouse model of house dust mite (HDM)-induced allergic sensitization and allergic airway inflammation. METHODS: Pag1-deficient (Pag1-/- ) and wild-type (WT) mice were sensitized or sensitized/challenged to HDM, and hallmark features of allergic inflammation were assessed. The contribution of T cells was assessed through depletion (anti-CD4 antibody) and adoptive transfer studies. RESULTS: Type 2 inflammation (eosinophilia, eotaxin-2 expression, IL-4/IL-5/IL-13 production, mucus production) in the airways and lungs was significantly increased in HDM sensitized/challenged Pag1-/- mice compared to WT mice. The predisposition to allergic sensitization was associated with increased airway epithelial high-mobility group box 1 (HMGB1) translocation and release, increased type 2 innate lymphoid cells (ILC2s) and monocyte-derived dendritic cell numbers in the mediastinal lymph nodes, and increased T-helper type 2 (TH 2)-cell differentiation. CD4+ T-cell depletion studies or the adoptive transfer of WT OVA-specific CD4+ T cells to WT or Pag1-/- recipients demonstrated that the heightened propensity for TH 2-cell differentiation was both T cell intrinsic and extrinsic. CONCLUSION: PAG1 deficiency increased airway epithelial activation, ILC2 expansion, and TH 2 differentiation. As a consequence, PAG1 deficiency predisposed toward allergic sensitization and increased the severity of experimental asthma.
Assuntos
Alérgenos/imunologia , Asma/imunologia , Pulmão/imunologia , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Pyroglyphidae/imunologia , Células Th2/imunologia , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/genética , Citocinas/metabolismo , Células Dendríticas/imunologia , Modelos Animais de Doenças , Proteína HMGB1/metabolismo , Imunidade Inata , Inflamação/imunologia , Pulmão/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfoproteínas/deficiência , Fosfoproteínas/genéticaRESUMO
Inflammation and an influx of macrophages are common elements in many diseases. Among pro-inflammatory cytokines, tumor necrosis factor α (TNFα) plays a central role by amplifying the cytokine network. Progranulin (PGRN) is a growth factor that binds to TNF receptors and interferes with TNFα-mediated signaling. Extracellular PGRN is processed into granulins by proteases released from immune cells. PGRN exerts anti-inflammatory effects, whereas granulins are pro-inflammatory. The factors coordinating these ambivalent functions remain unclear. In our study, we identify Y-box binding protein-1 (YB-1) as a candidate for this immune-modulating activity. Using a yeast-2-hybrid assay with YB-1 protein as bait, clones encoding for progranulin were selected using stringent criteria for strong interaction. We demonstrate that at physiological concentrations, YB-1 interferes with the binding of TNFα to its receptors in a dose-dependent manner using a flow cytometry-based binding assay. We show that YB-1 in combination with progranulin interferes with TNFα-mediated signaling, supporting the functionality with an NF-κB luciferase reporter assay. Together, we show that YB-1 displays immunomodulating functions by affecting the binding of TNFα to its receptors and influencing TNFα-mediated signaling via its interaction with progranulin.
Assuntos
Macrófagos/imunologia , Progranulinas/imunologia , Receptores do Fator de Necrose Tumoral/imunologia , Transdução de Sinais/imunologia , Fatores de Transcrição/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Macrófagos/patologia , Camundongos , Progranulinas/genética , Células RAW 264.7 , Receptores do Fator de Necrose Tumoral/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
Cold shock proteins are multifunctional RNA/DNA binding proteins, characterized by the presence of one or more cold shock domains. In humans, the best characterized members of this family are denoted Y-box binding proteins, such as Y-box binding protein-1 (YB-1). Biological activities range from the regulation of transcription, splicing and translation, to the orchestration of exosomal RNA content. Indeed, the secretion of YB-1 from cells via exosomes has opened the door to further potent activities. Evidence links a skewed cold shock protein expression pattern with cancer and inflammatory diseases. In this review the evidence for a causative involvement of cold shock proteins in disease development and progression is summarized. Furthermore, the potential application of cold shock proteins for diagnostics and as targets for therapy is elucidated.
Assuntos
Proteínas e Peptídeos de Choque Frio/metabolismo , Doença , Retroalimentação Fisiológica , Humanos , Estresse FisiológicoRESUMO
Established therapies for diabetic nephropathy (dNP) delay but do not prevent its progression. The shortage of established therapies may reflect the inability to target the tubular compartment. The chemical chaperone tauroursodeoxycholic acid (TUDCA) ameliorates maladaptive endoplasmic reticulum (ER) stress signaling and experimental dNP. Additionally, TUDCA activates the farnesoid X receptor (FXR), which is highly expressed in tubular cells. We hypothesized that TUDCA ameliorates maladaptive ER signaling via FXR agonism specifically in tubular cells. Indeed, TUDCA induced expression of FXR-dependent genes (SOCS3 and DDAH1) in tubular cells but not in other renal cells. In vivo, TUDCA reduced glomerular and tubular injury in db/db and diabetic endothelial nitric oxide synthase-deficient mice. FXR inhibition with Z-guggulsterone or vivo-morpholino targeting of FXR diminished the ER-stabilizing and renoprotective effects of TUDCA. Notably, these in vivo approaches abolished tubular but not glomerular protection by TUDCA. Combined intervention with TUDCA and the angiotensin-converting enzyme inhibitor enalapril in 16-week-old db/db mice reduced albuminuria more efficiently than did either treatment alone. Although both therapies reduced glomerular damage, only TUDCA ameliorated tubular damage. Thus, interventions that specifically protect the tubular compartment in dNP, such as FXR agonism, may provide renoprotective effects on top of those achieved by inhibiting angiotensin-converting enzyme.
Assuntos
Nefropatias Diabéticas/prevenção & controle , Túbulos Renais , Receptores Citoplasmáticos e Nucleares/agonistas , Ácido Tauroquenodesoxicólico/uso terapêutico , Animais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Tubular cells recruit monocytic cells in inflammatory tubulointerstitial kidney diseases. The cell-cell communication that establishes pro- or anti-inflammatory activities is mainly influenced by cytokines, reactive oxygen species, nitric oxide, and phagocytosis. Key proteins orchestrating these processes such as cold-shock proteins linked with chemoattraction and cell maturation have been identified. The prototypic member of the cold-shock protein family, Y-box binding protein (YB)-1, governs specific phenotypic alterations in monocytic cells and was explored in the present study. Following tubulointerstitial injury by unilateral ureteral obstruction, increased inflammatory cell infiltration and tubular cell CCL5 expression was found in conditional Ybx1 knockout animals with specific depletion in monocytes/macrophages (YB-1ΔLysM). Furthermore, YB-1ΔLysM mice exhibit enhanced tissue damage, myofibroblast activation, and fibrosis. To investigate relevant molecular mechanism(s), we utilized bone marrow-derived macrophage cultures and found that YB-1-deficient macrophages display defects in cell polarization and function, including reduced proliferation and nitric oxide production, loss of phagocytic activity, and failure to upregulate IL-10 and CCL5 expression in response to inflammatory stimuli. Co-culture with primary tubular cells confirmed these findings. Thus, monocytic YB-1 has prominent and distinct roles for cellular feed-forward crosstalk and resolution of inflammatory processes by its ability to regulate cell differentiation and cytokine/chemokine synthesis.
Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Túbulos Renais/patologia , Monócitos/patologia , Nefrite Intersticial/patologia , Animais , Comunicação Celular , Quimiocina CCL5/metabolismo , Técnicas de Cocultura , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Progressão da Doença , Feminino , Fibrose , Humanos , Interleucina-10/metabolismo , Túbulos Renais/citologia , Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Cultura Primária de CélulasRESUMO
DNA binding protein A (DbpA) is a member of the human cold shock domain-containing protein superfamily, with known functions in cell proliferation, differentiation, and stress responses. DbpA mediates tight junction-associated activities in tubular epithelial cells, but the function of DbpA in mesangial cells is unknown. Here, we found DbpA protein expression restricted to vascular smooth muscle cells in healthy human kidney tissue but profound induction of DbpA protein expression within the glomerular mesangial compartment in mesangioproliferative nephritis. In vitro, depletion or overexpression of DbpA using lentiviral constructs led to inhibition or promotion, respectively, of mesangial cell proliferation. Because platelet-derived growth factor B (PDGF-B) signaling has a pivotal role in mesangial cell proliferation, we examined the regulatory effect of PDGF-B on DbpA. In vitro studies of human and rat mesangial cells confirmed a stimulatory effect of PDGF-B on DbpA transcript numbers and protein levels. Additional in vivo investigations showed DbpA upregulation in experimental rat anti-Thy1.1 nephritis and murine mesangioproliferative nephritis models. To interfere with PDGF-B signaling, we injected nephritic rats with PDGF-B neutralizing aptamers or the MEK/ERK inhibitor U0126. Both interventions markedly decreased DbpA protein expression. Conversely, continuous PDGF-B infusion in healthy rats induced DbpA expression predominantly within the mesangial compartment. Taken together, these results indicate that DbpA is a novel target of PDGF-B signaling and a key mediator of mesangial cell proliferation.
Assuntos
Proteínas e Peptídeos de Choque Frio/fisiologia , Proteínas de Ligação a DNA/fisiologia , Mesângio Glomerular/patologia , Mesângio Glomerular/fisiologia , Glomerulonefrite/etiologia , Células Mesangiais/patologia , Animais , Proliferação de Células , Células Cultivadas , Humanos , Nefrite Lúpica/etiologia , RatosRESUMO
BACKGROUND: Antigenic stimulation of the T cell receptor (TCR) initiates a change from a resting state into an activated one, which ultimately results in proliferation and the acquisition of effector functions. To accomplish this task, T cells require dramatic changes in metabolism. Therefore, we investigated changes of metabolic intermediates indicating for crucial metabolic pathways reflecting the status of T cells. Moreover we analyzed possible regulatory molecules required for the initiation of the metabolic changes. RESULTS: We found that proliferation inducing conditions result in an increase in key glycolytic metabolites, whereas the citric acid cycle remains unaffected. The upregulation of glycolysis led to a strong lactate production, which depends upon AKT/PKB, but not mTOR. The observed upregulation of lactate dehydrogenase results in increased lactate production, which we found to be dependent on IL-2 and to be required for proliferation. Additionally we observed upregulation of Glucose-transporter 1 (GLUT1) and glucose uptake upon stimulation, which were surprisingly not influenced by AKT inhibition. CONCLUSIONS: Our findings suggest that AKT plays a central role in upregulating glycolysis via induction of lactate dehydrogenase expression, but has no impact on glucose uptake of T cells. Furthermore, under apoptosis inducing conditions, T cells are not able to upregulate glycolysis and induce lactate production. In addition maintaining high glycolytic rates strongly depends on IL-2 production.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Ativação Linfocitária , Metabolômica , Trifosfato de Adenosina/metabolismo , Animais , Anticorpos/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Antígeno CTLA-4/metabolismo , Proliferação de Células/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Humanos , Interleucina-2/metabolismo , L-Lactato Desidrogenase/metabolismo , Lactatos/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
INTRODUCTION: The cold shock Y-box binding protein-1 (YB-1) fulfills important roles in regulating cell proliferation and differentiation. Overexpression occurs in various tumor cells. Given the existence of extracellular YB-1 we set out to determine the diagnostic, predictive and prognostic role of serum YB-1/p18 for patients with primary epithelial ovarian cancer (EOC). METHODS: The protein fragment YB-1/p18 was quantified by sandwich ELISA in serum samples from 132 healthy female volunteers and 206 patients with histological diagnosis of primary EOC. The ELISA sensitivity and specificity to detect EOC were calculated using receiver operating curves. Survival data were calculated using Kaplan Maier curves. RESULTS: Median age at the time of diagnosis was 60years and follow-up ended with a mean of 44.8month. 188 (91%) patients were diagnosed at advanced stages (FIGO III/IV) and 188 patients (91%) suffered from high-grade serous ovarian carcinoma. YB-1/p18 levels were significantly decreased in older patients (p=0.021). Significantly lower serum levels of YB-1/p18 were detected in the EOC cohort when compared to the control group (p<0.0001, AUC=0.827; 95% CI, 0.787-0.867). Using the expression of serum YB-1/p18 in early stages I and II cases these could be differentiated from control cases (p<0.0001, AUC=0.816; 95% CI 0.704-0.929). No other significant associations between clinical prognostic factors and YB-1/p18 serum levels were detected. Immunoblotting results with serum samples suggest that masking of epitopes by the YB-1/p18 fragment in multiprotein-complexes under non reducing conditions leads to the observed reduced ELISA readings in the EOC cohort. CONCLUSIONS: The quantification of fragment YB-1/p18 derived from cold shock protein YB-1 in serum samples could be useful for the early diagnosis of EOC.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias Epiteliais e Glandulares/sangue , Neoplasias Epiteliais e Glandulares/diagnóstico , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/diagnóstico , Proteína 1 de Ligação a Y-Box/sangue , Idoso , Carcinoma Epitelial do Ovário , Estudos de Casos e Controles , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologia , Prognóstico , Sensibilidade e EspecificidadeRESUMO
Ischemia-reperfusion injury (IRI) is the leading cause of ARF. A pathophysiologic role of the coagulation system in renal IRI has been established, but the functional relevance of thrombomodulin (TM)-dependent activated protein C (aPC) generation and the intracellular targets of aPC remain undefined. Here, we investigated the role of TM-dependent aPC generation and therapeutic aPC application in a murine renal IRI model and in an in vitro hypoxia and reoxygenation (HR) model using proximal tubular cells. In renal IRI, endogenous aPC levels were reduced. Genetic or therapeutic reconstitution of aPC efficiently ameliorated renal IRI independently of its anticoagulant properties. In tubular cells, cytoprotective aPC signaling was mediated through protease activated receptor-1- and endothelial protein C receptor-dependent regulation of the cold-shock protein Y-box binding protein-1 (YB-1). The mature 50 kD form of YB-1 was required for the nephro- and cytoprotective effects of aPC in vivo and in vitro, respectively. Reduction of mature YB-1 and K48-linked ubiquitination of YB-1 was prevented by aPC after renal IRI or tubular HR injury. aPC preserved the interaction of YB-1 with the deubiquitinating enzyme otubain-1 and maintained expression of otubain-1, which was required to reduce K48-linked YB-1 ubiquitination and to stabilize the 50 kD form of YB-1 after renal IRI and tubular HR injury. These data link the cyto- and nephroprotective effects of aPC with the ubiquitin-proteasome system and identify YB-1 as a novel intracellular target of aPC. These insights may provide new impetus for translational efforts aiming to restrict renal IRI.
Assuntos
Rim/patologia , Proteína C/metabolismo , Traumatismo por Reperfusão/patologia , Fatores de Transcrição/metabolismo , Ubiquitinação , Alelos , Animais , Anticoagulantes/química , Cruzamentos Genéticos , Cisteína Endopeptidases/genética , Modelos Animais de Doenças , Éxons , Hipóxia/patologia , Túbulos Renais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oxigênio/química , Transdução de Sinais , Trombose/metabolismoRESUMO
BACKGROUND: Expression of the cold shock protein Y-box protein 1 (YB-1) is associated with deleterious outcome in various malignant diseases. Our group recently showed that the detection of an 18 kDa YB-1 fragment (YB-1/p18) in human plasma identifies patients with malignant diseases. We now tested the prevalence, clinical, and diagnostic value of YB-1/p18 detection in common tumors. METHODS: A newly established monoclonal YB-1 antibody was used to detect YB-1/p18 by immunoblotting in plasma samples from 151 unselected tumor patients, alongside established tumor markers and various diagnostic measures, during evaluation for a cancerous disease and in follow-up studies after therapeutic interventions. RESULTS: Circulating YB-1/p18 was detected in 78% of patients having a tumor disease. YB-1/p18 positivity was highly prevalent in all examined malignancies, including lung cancer (32/37; 87%), breast cancer (7/10; 70%), cancer of unknown primary (CUP; 5/5, 100%) or hematological malignancies (42/62; 68%). Positivity for YB-1/p18 was independent of other routine laboratory parameters, tumor stage, or histology. In comparison to 13 established tumor markers (cancer antigens 15-3, 19-9, 72-4, and 125; carcinoembryonic antigen; cytokeratin fragments 21-1; neuron-specific enolase; alpha-fetoprotein; beta-2-microglobulin; squamous cell carcinoma antigen; thymidine kinase; tissue polypeptide antigen; pro-gastrin-releasing peptide), YB-1/p18 detection within serum samples was the most sensitive general parameter identifying malignant disorders. YB-1/p18 concentrations altered during therapeutic interventions, but did not predict prognosis. CONCLUSIONS: Plasma YB-1/p18 detection has a high specific prevalence in malignancies, thereby providing a novel tool for cancer screening independent of the tumor origin.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias/sangue , Fragmentos de Peptídeos/sangue , Proteína 1 de Ligação a Y-Box/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais , Biomarcadores Tumorais/imunologia , Western Blotting , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/terapia , Fragmentos de Peptídeos/imunologia , Valor Preditivo dos Testes , Prognóstico , Fatores de Tempo , Proteína 1 de Ligação a Y-Box/imunologia , Adulto JovemRESUMO
BACKGROUND: Fibrosis is characterized by excessive extracellular matrix formation in solid organs, disrupting tissue architecture and function. The Y-box binding protein-1 (YB-1) regulates fibrosis-related genes (e.g., Col1a1, Mmp2, and Tgfß1) and contributes significantly to disease progression. This study aims to identify fibrogenic signatures and the underlying signaling pathways modulated by YB-1. METHODS: Transcriptomic changes associated with matrix gene patterns in human chronic kidney diseases and murine acute injury models were analyzed with a focus on known YB-1 targets. Ybx1-knockout mouse strains (Ybx1ΔRosaERT+TX and Ybx1ΔLysM) were subjected to various kidney injury models. Fibrosis patterns were characterized by histopathological staining, transcriptome analysis, qRT-PCR, methylation analysis, zymography, and Western blotting. RESULTS: Integrative transcriptomic analyses revealed that YB-1 is involved in several fibrogenic signatures related to the matrisome, the WNT, YAP/TAZ, and TGFß pathways, and regulates Klotho expression. Changes in the methylation status of the Klotho promoter by specific methyltransferases (DNMT) are linked to YB-1 expression, extending to other fibrogenic genes. Notably, kidney-resident cells play a significant role in YB-1-modulated fibrogenic signaling, whereas infiltrating myeloid immune cells have a minimal impact. CONCLUSIONS: YB-1 emerges as a master regulator of fibrogenesis, guiding DNMT1 to fibrosis-related genes. This highlights YB-1 as a potential target for epigenetic therapies interfering in this process.
Assuntos
Injúria Renal Aguda , Proteínas e Peptídeos de Choque Frio , Humanos , Camundongos , Animais , Proteínas e Peptídeos de Choque Frio/metabolismo , Rim/patologia , Injúria Renal Aguda/metabolismo , Metilação , Fibrose , Camundongos KnockoutRESUMO
BACKGROUND: Signaling through the TCR is crucial for the generation of different cellular responses including proliferation, differentiation, and apoptosis. A growing body of evidence indicates that differences in the magnitude and the duration of the signal are critical determinants in eliciting cellular responses. RESULTS: Here, we have analyzed signaling dynamics correlating with either unresponsiveness or proliferation induced upon TCR/CD28 ligation in primary human T cells. We used two widely employed methods to stimulate T cells in vitro, antibodies either cross-linked in solution (sAbs) or immobilized on microbeads (iAbs). A comparative analysis of the signaling properties of iAbs and sAbs revealed that, under proliferation-inducing conditions, feedback regulation is markedly different from that leading to an unresponsive state. In fact, upon iAbs stimulation TCR-mediated signaling is prolonged by a positive feedback loop involving Erk, whereas sAbs strongly activate inhibitory molecules that likely terminate signaling. We additionally found that, by enhancing the phosphorylation of Src family kinases under proliferation-inducing conditions, signaling and T-cell activation are terminated. CONCLUSIONS: In summary, our analysis documents TCR signaling kinetics and feedback regulation under conditions of stimulation inducing either unresponsiveness or proliferation.
Assuntos
Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Anticorpos/farmacologia , Proliferação de Células , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Retroalimentação Fisiológica , Humanos , Proteínas Imobilizadas/farmacologia , Células Jurkat , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Fosforilação , Transdução de Sinais , Quinases da Família src/metabolismoRESUMO
BACKGROUND: PAG/Cbp represents a ubiquitous mechanism for regulating Src family kinases by recruiting Csk to the plasma membrane, thereby controlling cellular activation. Since Src kinases are known oncogenes, we used RNA interference in primary human T cells to test whether the loss of PAG resulted in lymphocyte transformation. RESULTS: PAG-depletion enhanced Src kinase activity and augmented proximal T-cell receptor signaling; exactly the phenotype expected for loss of this negative regulator. Surprisingly, rather than becoming hyper-proliferative, PAG-suppressed T cells became unresponsive. This was mediated by a Fyn-dependent hyper-phosphorylation of the inhibitory receptor CTLA-4, which recruited the protein tyrosine phosphatase Shp-1 to lipid rafts. Co-suppression of CTLA-4 abrogates this inhibition and restores proliferation to T cells. CONCLUSION: We have identified a fail-safe mechanism as well as a novel contribution of CTLA-4 to setting the activation threshold in T cells.
RESUMO
BACKGROUND: The Y-box protein-1 (YB-1) fulfills pleiotropic functions relating to gene transcription, mRNA processing, and translation. It remains elusive how YB-1 shuttling into the nuclear and cytoplasmic compartments is regulated and whether limited proteolysis by the 20S proteasome releases fragments with distinct function(s) and subcellular distribution(s). RESULTS: To address these questions, mapping of domains responsible for subcellular targeting was performed. Three nuclear localization signals (NLS) were identified. NLS-1 (aa 149-156) and NLS-2 (aa 185-194) correspond to residues with unknown function(s), whereas NLS-3 (aa 276-292) matches with a designated multimerization domain. Nuclear export signal(s) were not identified. Endoproteolytic processing by the 20S proteasome before glycine 220 releases a carboxy-terminal fragment (CTF), which localized to the nucleus, indicating that NLS-3 is operative. Genotoxic stress induced proteolytic cleavage and nuclear translocation of the CTF. Co-expression of the CTF and full-length YB-1 resulted in an abrogated transcriptional activation of the MMP-2 promoter, indicating an autoregulatory inhibitory loop, whereas it fulfilled similar trans-repressive effects on the collagen type I promoter. CONCLUSION: Compartmentalization of YB-1 protein derivatives is controlled by distinct NLS, one of which targets a proteolytic cleavage product to the nucleus. We propose a model for an autoregulatory negative feedback loop that halts unlimited transcriptional activation.