RESUMEN
Autoimmune disorders are the third most common diseases in the United States, and affect the daily lives of millions of people. In this study, we analyzed patient samples, utilized a transgenic mouse model and human B cells to reveal Natural Killer Cell Transcript 4 (NK4) as a novel regulator that promotes the development of autoimmune disorders. NK4 was significantly elevated in samples from patients with SjÓ§gren's Syndrome (SS). SS patients show elevated NK4 levels. There is a strong and positive correlation between the increased levels of NK4 and the duration of SS. Interestingly, transgenic expression of NK4 in a mouse model led to the development of autoantibodies and lymphocytic infiltration in salivary glands similar to those in SS patients. Those phenotypes were associated with increased B1a cells in the peritoneum, plasma cells in the spleen, and increased IgM, IgA, and IgG2a in serum of the NK4 transgenic mice. The autoimmune phenotypes became more severe in older mice. Moreover, after NK4 transfection, human naïve B cells were activated and memory B cells differentiation into IgG and IgA-plasmablasts, resulting in an increased production of autoantibodies.NK4 regulated the differentiation and activation of B cells through activating Rap1 activity. NK4 also promoted B cell migration in a paracrine fashion through an induction of CXCL13 in endothelial cells. Collectively, these findings identify NK4 as a promoter of the development of autoimmune disorders through its roles on B cells. Therefore, NK4 may be a novel therapeutic target for the treatment of autoimmune diseases.
Asunto(s)
Linfocitos B/inmunología , Interleucinas/inmunología , Síndrome de Sjögren/inmunología , Proteínas de Unión al GTP rap1/inmunología , Adulto , Anciano , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular Tumoral , Movimiento Celular/genética , Movimiento Celular/inmunología , Células Cultivadas , Quimiocina CXCL13/genética , Quimiocina CXCL13/inmunología , Quimiocina CXCL13/metabolismo , Células Endoteliales/citología , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Femenino , Citometría de Flujo/métodos , Humanos , Interleucinas/genética , Interleucinas/metabolismo , Masculino , Ratones Transgénicos , Persona de Mediana Edad , Glándulas Salivales/inmunología , Glándulas Salivales/metabolismo , Síndrome de Sjögren/genética , Síndrome de Sjögren/metabolismo , Adulto Joven , Proteínas de Unión al GTP rap1/genética , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
Vav1 is a Rho/Rac (Ras-related C3 botulinum toxin substrate) guanine nucleotide exchange factor expressed in hematopoietic and endothelial cells that are involved in a wide range of cellular functions. It is also stabilized under hypoxic conditions when it regulates the accumulation of the transcription factor HIF (Hypoxia Inducible Factor)-1α, which activates the transcription of target genes to orchestrate a cellular response to low oxygen. One of the genes induced by HIF-1α is GLUT (Glucose Transporter)-1, which is the major glucose transporter expressed in vessels that supply energy to the brain. Here, we identify a role for Vav1 in providing glucose to the brain. We found that Vav1 deficiency downregulates HIF-1α and GLUT-1 levels in endothelial cells, including blood-brain barrier cells. This downregulation of GLUT-1, in turn, reduced glucose uptake to endothelial cells both in vitro and in vivo, and reduced glucose levels in the brain. Furthermore, endothelial cell-specific Vav1 knock-out in mice, which caused glucose uptake deficiency, also led to a learning delay in fear conditioning experiments. Our results suggest that Vav1 promotes learning by activating HIF-1α and GLUT-1 and thereby distributing glucose to the brain. We further demonstrate the importance of glucose transport by endothelial cells in brain functioning and reveal a potential new axis for targeting GLUT-1 deficiency syndromes and other related brain diseases.
Asunto(s)
Glucemia/metabolismo , Encéfalo/metabolismo , Regulación de la Expresión Génica , Transportador de Glucosa de Tipo 1/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Proteínas Proto-Oncogénicas c-vav/genética , Animales , Femenino , Transportador de Glucosa de Tipo 1/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Proteínas Proto-Oncogénicas c-vav/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , TransfecciónRESUMEN
RATIONALE: Cryptogenic strokes, those of unknown cause, have been estimated as high as 30% to 40% of strokes. Inflammation has been suggested as a critical etiologic factor. However, there is lack of experimental evidence. OBJECTIVE: In this study, we investigated inflammation-associated stroke using a mouse model that developed spontaneous stroke because of myeloid deficiency of TGF-ß (transforming growth factor-ß) signaling. METHODS AND RESULTS: We report that mice with deletion of Tgfbr2 in myeloid cells (Tgfbr2Myeko) developed cerebrovascular inflammation in the absence of significant pathology in other tissues, culminating in stroke and severe neurological deficits with 100% penetrance. The stroke phenotype can be transferred to syngeneic wild-type mice via Tgfbr2Myeko bone marrow transplant and can be rescued in Tgfbr2Myeko mice with wild-type bone marrow. The underlying mechanisms involved an increased type 1 inflammation and cerebral endotheliopathy, characterized by elevated NF-κB (nuclear factor-κB) activation and TNF (tumor necrosis factor) production by myeloid cells. A high-fat diet accelerated stroke incidence. Anti-TNF treatment, as well as metformin and methotrexate, which are associated with decreased stroke risk in population studies, delayed stroke occurrence. CONCLUSIONS: Our studies show that TGF-ß signaling in myeloid cells is required for maintenance of vascular health and provide insight into inflammation-mediated cerebrovascular disease and stroke.
Asunto(s)
Células Mieloides/metabolismo , Transducción de Señal , Accidente Cerebrovascular/metabolismo , Factor de Crecimiento Transformador beta/genética , Animales , Línea Celular , Inmunosupresores/uso terapéutico , Inflamación/complicaciones , Inflamación/metabolismo , Metformina/uso terapéutico , Metotrexato/uso terapéutico , Ratones , Ratones Endogámicos C57BL , FN-kappa B/genética , FN-kappa B/metabolismo , Penetrancia , Accidente Cerebrovascular/etiología , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/prevención & control , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Treatment of cancer metastasis has been largely ineffective. It is paramount to understand the mechanisms underlying the metastatic process, of which the tumor microenvironment is an indispensable participant. What are the critical cellular and molecular players at the primary tumor site where metastatic cascade initiates? How is tumor-associated inflammation regulated? How do altered vasculatures contribute to metastasis? What is the dynamic nature or heterogeneity of primary tumors and what are the challenges to catch a moving target? This review summarizes recent progress, mechanistic understanding, and options for metastasis-targeted therapy.
Asunto(s)
Inflamación/patología , Neoplasias/metabolismo , Neoplasias/patología , Microambiente Tumoral , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Biomarcadores de Tumor , Evolución Clonal , Progresión de la Enfermedad , Humanos , Inflamación/etiología , Inflamación/metabolismo , Terapia Molecular Dirigida , Metástasis de la Neoplasia , Neoplasias/tratamiento farmacológico , Neoplasias/etiología , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
KEY POINTS: Combining nitric oxide (NO)-mediated increased blood flow with angiopoietin-1-Tie2 receptor signalling induces arteriolargenesis - the formation of arterioles from capillaries - in a model of physiological angiogenesis. This NO-Tie-mediated arteriolargenesis requires endogenous vascular endothelial growth factor (VEGF) signalling. Inhibition of VEGF signalling increases pericyte coverage in microvessels. Together these findings indicate that generation of functional neovasculature requires close titration of NO-Tie2 signalling and localized VEGF induction, suggesting that the use of exogenous VEGF expression as a therapeutic for neovascularization may not be successful. ABSTRACT: Signalling through vascular endothelial growth factor (VEGF) receptors and the tyrosine kinase with IgG and EGF domains-2 (Tie2) receptor by angiopoietins is required in combination with blood flow for the formation of a functional vascular network. We tested the hypothesis that VEGF and angiopoietin-1 (Ang1) contribute differentially to neovascularization induced by nitric oxide (NO)-mediated vasodilatation, by comparing the phenotype of new microvessels in the mesentery during induction of vascular remodelling by over-expression of endothelial nitric oxide synthase in the fat pad of the adult rat mesentery during inhibition of angiopoietin signalling with soluble Tie2 (sTie2) and VEGF signalling with soluble Fms-like tyrosine kinase receptor-1 (sFlt1). We found that NO-mediated angiogenesis was blocked by inhibition of VEGF with sFlt1 (from 881 ± 98% increase in functional vessel area to 279 ± 72%) and by inhibition of angiopoietin with sTie2 (to 337 ± 67%). Exogenous angiopoietin-1 was required to induce arteriolargenesis (8.6 ± 1.3% of vessels with recruitment of vascular smooth muscle cells; VSMCs) in the presence of enhanced flow. sTie2 and sFlt1 both inhibited VSMC recruitment (both 0%), and VEGF inhibition increased pericyte recruitment to newly formed vessels (from 27 ± 2 to 54 ± 3% pericyte ensheathment). We demonstrate that a fine balance of VEGF and angiopoietin signalling is required for the formation of a functional vascular network. Endogenous VEGF signalling prevents excess neovessel pericyte coverage, and is required for VSMC recruitment during increased nitric oxide-mediated vasodilatation and angiopoietin signalling (NO-Tie-mediated arteriogenesis). Therapeutic vascular remodelling paradigms may therefore require treatments that modulate blood flow to utilize endogenous VEGF, in combination with exogenous Ang1, for effective neovascularization.
Asunto(s)
Angiopoyetina 1/fisiología , Neovascularización Fisiológica/fisiología , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Masculino , Mesenterio/irrigación sanguínea , Mesenterio/fisiología , Ratas Wistar , Receptor TIE-2/fisiología , Flujo Sanguíneo Regional , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 1 de Factores de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
Mesenchymal stem cells (MSCs) are multipotent stromal cells residing in the bone marrow. MSCs have the potential to differentiate to adipocytes, chondrocytes, and other types of cells. In this study, we investigated the molecular mechanism that controls MSC cell fate decisions for differentiation. We found that Vav1, a guanine nucleotide exchange factor for Rho GTPase, was highly expressed in MSCs. Interestingly, loss of Vav1 in MSCs led to spontaneous adipogenic but impaired chondrogenic differentiation, and accordingly Vav1 null mice displayed an increase in fat content and a decrease in cartilage. Conversely, ectopic expression of Vav1 in MSCs reversed this phenotype, and led to enhanced MSC differentiation into chondrocyte but retarded adipogenesis. Mechanistically, loss of Vav1 reduced the level of Sirt1, which was responsible for an increase of acetylated PPARγ. As acetylation activates PPARγ, it increased C/EBPα expression and promoted adipogenesis. On the other hand, loss of Vav1 resulted in an increase of acetylated Sox9, a target of Sirt1. As acetylation represses Sox9 activity, it led to a dramatic reduction of collagen 2α1, a key regulator in chondrocyte differentiation. Finally, we found that Vav1 regulates Sirt1 in MSCs through Creb. Together this study reveals a novel function of Vav1 in regulating MSC cell fate decisions for differentiation through Sirt1. Sirt1 deacetylates PPARγ and Sox9, two key mediators that control adipocyte and chondrocyte differentiation. The acetylation status of PPARγ and Sox9 has opposite effects on its activity, thereby controlling cell fate decision. Stem Cells 2016;34:1934-1946.
Asunto(s)
Adipocitos/citología , Diferenciación Celular , Condrocitos/citología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Proteínas Proto-Oncogénicas c-vav/metabolismo , Sirtuina 1/metabolismo , Adipocitos/metabolismo , Adipogénesis/genética , Adiposidad , Animales , Diferenciación Celular/genética , Condrocitos/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Femenino , Eliminación de Gen , Ratones Endogámicos C57BL , Factores de Transcripción/metabolismoRESUMEN
OBJECTIVE: The IκB kinase (IKK) is an enzyme complex that initiates the nuclear factor κB transcription factor cascade, which is important in regulating multiple cellular responses. IKKα is directly associated with 2 major prosurvival pathways, PI3K/Akt and nuclear factor κB, but its role in cell survival is not clear. Macrophages play critical roles in the pathogenesis of atherosclerosis, yet the impact of IKKα signaling on macrophage survival and atherogenesis remains unclear. APPROACH AND RESULTS: Here, we demonstrate that genetic IKKα deficiency, as well as pharmacological inhibition of IKK, in mouse macrophages significantly reduces Akt S(473) phosphorylation, which is accompanied by suppression of mTOR complex 2 signaling. Moreover, IKKα null macrophages treated with lipotoxic palmitic acid exhibited early exhaustion of Akt signaling compared with wild-type cells. This was accompanied by a dramatic decrease in the resistance of IKKα(-/-) monocytes and macrophages to different proapoptotic stimuli compared with wild-type cells. In vivo, IKKα deficiency increased macrophage apoptosis in atherosclerotic lesions and decreased early atherosclerosis in both female and male low-density lipoprotein receptor (LDLR)(-/-) mice reconstituted with IKKα(-/-) hematopoietic cells and fed with the Western diet for 8 weeks compared with control LDLR(-/-) mice transplanted with wild-type cells. CONCLUSIONS: Hematopoietic IKKα deficiency in mouse suppresses Akt signaling, compromising monocyte/macrophage survival and this decreases early atherosclerosis.
Asunto(s)
Aterosclerosis/prevención & control , Quinasa I-kappa B/deficiencia , Macrófagos Peritoneales/enzimología , Proteínas Proto-Oncogénicas c-akt , Animales , Apoptosis , Aterosclerosis/enzimología , Aterosclerosis/genética , Aterosclerosis/patología , Supervivencia Celular , Células Cultivadas , Dieta Occidental , Modelos Animales de Enfermedad , Femenino , Quinasa I-kappa B/antagonistas & inhibidores , Quinasa I-kappa B/genética , Mediadores de Inflamación/metabolismo , Hígado/embriología , Hígado/enzimología , Trasplante de Hígado , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/patología , Masculino , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones Endogámicos C57BL , Ratones Noqueados , Complejos Multiproteicos/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Receptores de LDL/deficiencia , Receptores de LDL/genética , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Factores de TiempoRESUMEN
Aberrant TGFbeta signaling is common in human cancers and contributes to tumor metastasis. Here, we demonstrate that Gr-1+CD11b+ myeloid cells are recruited into mammary carcinomas with type II TGF beta receptor gene (Tgfbr2) deletion and directly promote tumor metastasis. Gr-1+CD11b+ cells infiltrate into the invasive front of tumor tissues and facilitate tumor cell invasion and metastasis through a process involving metalloproteinase activity. This infiltration of Gr-1+CD11b+ cells also results in increased abundance of TGF beta 1 in tumors with Tgfbr2 deletion. The recruitment of Gr-1+CD11b+ cells into tumors with Tgfbr2 deletion involves two chemokine receptor axes, the SDF-1/CXCR4 and CXCL5/CXCR2 axes. Together, these data indicate that Gr-1+CD11b+ cells contribute to TGFbeta-mediated metastasis through enhancing tumor cell invasion and metastasis.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Antígeno CD11b/metabolismo , Células Mieloides/patología , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Animales , Neoplasias de la Mama/enzimología , Línea Celular Tumoral , Femenino , Eliminación de Gen , Humanos , Metaloproteinasas de la Matriz/biosíntesis , Ratones , Modelos Biológicos , Células Mieloides/enzimología , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismoRESUMEN
Platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) are prototypic growth factors and receptor tyrosine kinases which have critical functions in development. We show that PDGFs share a conserved region in their prodomain sequences which can remain noncovalently associated with the mature cystine-knot growth factor domain after processing. The structure of the PDGF-A/propeptide complex reveals this conserved, hydrophobic association mode. We also present the structure of the complex between PDGF-B and the first three Ig domains of PDGFRbeta, showing that two PDGF-B protomers clamp PDGFRbeta at their dimerization seam. The PDGF-B:PDGFRbeta interface is predominantly hydrophobic, and PDGFRs and the PDGF propeptides occupy overlapping positions on mature PDGFs, rationalizing the need of propeptides by PDGFs to cover functionally important hydrophobic surfaces during secretion. A large-scale structural organization and rearrangement is observed for PDGF-B upon receptor binding, in which the PDGF-B L1 loop, disordered in the structure of the free form, adopts a highly specific conformation to form hydrophobic interactions with the third Ig domain of PDGFRbeta. Calorimetric data also shows that the membrane-proximal homotypic PDGFRalpha interaction, albeit required for activation, contributes negatively to ligand binding. The structural and biochemical data together offer insights into PDGF-PDGFR signaling, as well as strategies for PDGF-antagonism.
Asunto(s)
Factor de Crecimiento Derivado de Plaquetas/química , Animales , Cristalografía por Rayos X/métodos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Conformación Molecular , Péptidos/química , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Unión Proteica , Conformación Proteica , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/química , Proteínas Recombinantes/química , Transducción de Señal , TermodinámicaRESUMEN
Through integration of a MOSFET-based microfluidic Coulter counter with a dc-dielectrophoretic cell sorter, we demonstrate simultaneous on-chip cell separation and sizing with three different samples including 1) binary mixtures of polystyrene beads, 2) yeast cells of continuous size distribution, and 3) mixtures of 4T1 tumor cells and murine bone marrow cells. For cells with continuous size distribution, it is found that the receiver operator characteristic analysis is an ideal method to characterize the separation performance. The characterization results indicate that dc-DEP separation performance degrades as the sorting throughput (cell sorting rate) increases, which provides insights into the design and operation of size-based microfluidic cell separation.
Asunto(s)
Células de la Médula Ósea/citología , Separación Celular/instrumentación , Electroforesis por Microchip/instrumentación , Neoplasias Mamarias Experimentales/química , Técnicas Analíticas Microfluídicas , Saccharomyces cerevisiae/química , Animales , Células Cultivadas , Femenino , Citometría de Flujo , Ratones , Microesferas , Análisis de Secuencia por Matrices de Oligonucleótidos , Poliestirenos/químicaRESUMEN
Myeloid-derived suppressor cells (MDSCs) are significantly increased in cancer patients and tumor bearing-animals. MDSCs infiltrate into tumors and promote tumor invasion and metastasis. To identify the mediator responsible for the prometastatic property of MDSCs, we used proteomics. We found neutrophilic granule protein (NGP) was decreased >2-fold in MDSCs from metastatic 4T1 tumor-bearing mice compared to nonmetastatic 67NR controls. NGP mRNA levels were decreased in bone marrow and in tumor-infiltrating MDSCs by 45 and 66%, respectively, in 4T1 tumor-bearing mice compared to 67NR controls. Interestingly, 4T1-conditioned medium reduced myeloid cell NGP expression by â¼ 40%, suggesting that a secreted factor mediates gene reduction. Sequence analysis shows a putative cystatin domain in NGP, and biochemical analysis confirms NGP a novel cathepsin inhibitor. It inhibited cathepsin B activity by nearly 40% in vitro. NGP expression in 4T1 tumor cells suppressed cell invasion, delayed primary tumor growth, and greatly reduced lung metastasis in vivo. A 2.8-fold reduction of cathepsin activity was found in tumors expressing NGP compared to controls. NGP significantly reduced tumor angiogenesis to 12.6 from 19.6 and lymphangiogenesis to 4.6 from 9.1 vessels/field. Necrosis was detectable only in NGP-expressing tumors, and the number of apoptotic cells increased to 22.4 from 8.3 in controls. Taken together, this study identifies a negative regulator of tumor metastasis in MDSCs, NGP, which is down-regulated in metastatic conditions. The finding suggests that malignant tumors promote invasion/metastasis not only through up-regulation of proteases but also down-regulation of protease inhibitors.
Asunto(s)
Cistatinas/fisiología , Células Mieloides/fisiología , Metástasis de la Neoplasia/prevención & control , Animales , Catepsina B/genética , Catepsina B/metabolismo , Línea Celular Tumoral , Proliferación Celular , Cistatinas/genética , Femenino , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Animales/irrigación sanguínea , Neoplasias Mamarias Animales/patología , Neoplasias Mamarias Animales/fisiopatología , Ratones , Ratones Endogámicos BALB C , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Invasividad Neoplásica/fisiopatología , Invasividad Neoplásica/prevención & control , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Metástasis de la Neoplasia/fisiopatología , Neovascularización Patológica , Inhibidores de Proteasas/metabolismo , ProteómicaRESUMEN
Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells with suppressive function on immune response. In this review, we discuss recent studies about mechanisms of expansion and suppressive function of MDSCs during inflammation, infection and autoimmune diseases, as well as pro-angiogenic and pro-metastatic functions of these cells in tumor development. Further, we focus on novel studies of MDSCs and therapeutic approaches to eliminate these cells in cancer.
Asunto(s)
Células Dendríticas/metabolismo , Inflamación/patología , Células Progenitoras Mieloides/metabolismo , Proteínas de Neoplasias/inmunología , Neoplasias/patología , Linfocitos T/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Comunicación Celular/genética , Comunicación Celular/inmunología , Proliferación Celular , Células Dendríticas/inmunología , Células Dendríticas/patología , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Ratones , Células Progenitoras Mieloides/inmunología , Células Progenitoras Mieloides/patología , Proteínas de Neoplasias/genética , Neoplasias/inmunología , Neoplasias/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Linfocitos T/inmunología , Linfocitos T/patología , Escape del Tumor/genéticaRESUMEN
We demonstrate a novel tumor-promoting role of myeloid immune suppressor Gr+CD11b+ cells, which are evident in cancer patients and tumor-bearing animals. These cells constitute approximately 5% of total cells in tumors. Tumors coinjected with Gr+CD11b+ cells exhibited increased vascular density, vascular maturation, and decreased necrosis. These immune cells produce high levels of MMP9. Deletion of MMP9 in these cells completely abolishes their tumor-promoting ability. Gr+CD11b+ cells were also found to directly incorporate into tumor endothelium. Consistent with this observation, Gr+CD11b+ cells acquire endothelial cell (EC) properties in tumor microenvironment and proangiogenic culture conditions. Our data provide evidence that Gr+CD11b+ cells of immune origin induced by tumors directly contribute to tumor growth and vascularization by producing MMP9 and differentiating into ECs.
Asunto(s)
Antígeno CD11b/metabolismo , Neoplasias/irrigación sanguínea , Neoplasias/inmunología , Neovascularización Patológica , Animales , Apoptosis , Médula Ósea/metabolismo , Antígeno CD11b/inmunología , Diferenciación Celular , Células Cultivadas , Progresión de la Enfermedad , Endotelio/metabolismo , Endotelio/patología , Femenino , Humanos , Metaloproteinasa 9 de la Matriz/biosíntesis , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Noqueados , Necrosis , Trasplante de Neoplasias , Neoplasias/metabolismo , Neoplasias/patología , Factor de Células Madre/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
A versatile microfluidic platform allowing co-culture of multiple cell populations in close proximity with separate control of their microenvironments would be extremely valuable for many biological applications. Here, we report a simple and compact microfluidic platform that has these desirable features and allows for real-time, live-cell imaging of cell-cell interactions. Using a pneumatically/hydraulically controlled poly(dimethylsiloxane) (PDMS) valve barrier, distinct cell types can be cultured in side-by-side microfluidic chambers with their optimum culture media and treated separately without affecting the other cell population. The platform is capable of both two-dimensional and three-dimensional cell co-culture and through variations of the valve barrier design, the platform allows for cell-cell interactions through either direct cell contact or soluble factors alone. The platform has been used to perform dynamic imaging of synapse formation in hippocampal neurons by separate transfection of two groups of neurons with fluorescent pre- and post-synaptic protein markers. In addition, cross-migration of 4T1 tumor cells and endothelial cells has been studied under normoxic and hypoxic conditions, which revealed different migration patterns, suggesting the importance of the microenvironments in cell-cell interactions and biological activities.
Asunto(s)
Técnicas de Cocultivo/instrumentación , Células Endoteliales/citología , Técnicas Analíticas Microfluídicas/instrumentación , Neuronas/citología , Animales , Comunicación Celular , Línea Celular Tumoral , Movimiento Celular , Dimetilpolisiloxanos/química , Humanos , Hidrodinámica , Ratones , Presión , Sefarosa/químicaRESUMEN
The vascular response to hypoxia and ischemia is essential for maintaining homeostasis during stressful conditions and is particularly critical for vital organs such as the heart. Hypoxia-inducible factor-1 (HIF-1) is a central regulator of the response to hypoxia by activating transcription of numerous target genes, including vascular endothelial growth factor (VEGF). Here we identify the guanine nucleotide exchange factor (GEF) Vav1, a regulator of the small Rho-GTPase and cell signaling in endothelial cells, as a key vascular regulator of hypoxia. We show that Vav1 is present in the vascular endothelium and is essential for HIF-1 activation under hypoxia. So, we hypothesized that Vav1 could be a key regulator of HIF-1 signaling. In our findings, Vav1 regulates HIF-1α stabilization through the p38/Siah2/PHD3 pathway. In normoxia, Vav1 binds to vascular endothelial growth factor receptor 1 (VEGFR1), which directs Vav1 to lysosomes for degradation. In contrast, hypoxia upregulates Vav1 protein levels by inhibiting lysosomal degradation, which is analogous to HIF-1α regulation by hypoxia: both proteins are constitutively produced and degraded in normoxia allowing for a rapid response when stress occurs. Consequently, hypoxia rapidly stabilizes Vav1, which is required for HIF-1α accumulation. This shows that Vav1 is the key mediator controlling the stabilization of HIF1α in hypoxic conditions. With this finding, we report a novel pathway to stabilize HIF-1, which shows a possible reason why clinical trials targeting HIF-1 has not been effective. Targeting Vav1 can be the new approach to overcome hypoxic tumors.
RESUMEN
IL-32 is a newly discovered protein found in human and certain primates, but absent in rodent. Various reports suggest its role as a proinflammatory mediator. Since vascular endothelium is critical in inflammation, we investigate IL-32 in endothelial cells. We found that the gene is expressed in human endothelial cells and Akt strongly induces its expression. Sequence analysis indicates IL-32 beta as the major isoform in endothelial cells. Surprisingly, we did not detect any secretion of IL-32 beta in human endothelial cells; instead we observed co-localization of IL-32 beta with endoplasmic reticulum, suggesting IL-32 beta is an intracellular protein in these cells. Promoter analysis identified a minimum required region for IL-32 transcription at -0.1 to +0.5 kb around the initially identified transcription start site. We also defined a transcriptional suppressor-binding site at -2.0 to -1.5 kb. Importantly, RNA ligase mediated rapid amplification of cDNA ends in endothelial cells determined the transcription start site at the 328 bp downstream from the original identified site. Finally, we found a positive correlation of IL-32 levels with human breast cancer and glioblastoma multiforme (GBM). These findings improve our understanding of IL-32 in vascular endothelium. IL-32 expression might be valuable as a biomarker for cancer.
Asunto(s)
Células Endoteliales/inmunología , Interleucinas/inmunología , Isoformas de Proteínas/inmunología , Animales , Secuencia de Bases , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Bovinos , Células Cultivadas , Células Endoteliales/citología , Femenino , Regulación de la Expresión Génica , Humanos , Interleucinas/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Isoformas de Proteínas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Sitio de Iniciación de la TranscripciónRESUMEN
Angiogenesis, the formation of new blood vessels from existing vessels, is tightly linked to chronic inflammation and cancer. Angiogenesis is one of the molecular events bridging the gap between inflammation and cancer. One of the events linking inflammation and cancer is an increase in cellular adhesion molecules that are expressed on the luminal surface of endothelium upon inflammation. Cellular adhesion molecules are involved in leukocyte recruitment and subsequently lead to extravasation of leukocytes to the injury site. These adhesion molecules are known to be shared by some cancer cells and have the ability to contribute to metastasis. Thus, an elevation of these molecules in chronic inflammation may be a risk factor for metastasis. In this chapter, we discuss the method used to determine the adhesion molecules expressed on endothelium, and leukocyte adhesion to endothelium.
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Bioensayo , Inflamación/patología , Neoplasias/patología , Neovascularización Patológica , Bioensayo/instrumentación , Bioensayo/métodos , Adhesión Celular , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Enfermedad Crónica , Humanos , Leucocitos/metabolismoRESUMEN
Interleukin-33 (IL-33) is one of the members of the IL-1 family of cytokines and a ligand of ST2 and IL-1 receptor accessory protein (IL-1RAcP) that is known to affect Th2 inflammatory response with partial effects on Th1 responses. This cytokine is released by epithelial and smooth muscle cells of the airway system during their injury by several environmental stimuli, such as allergens, viruses, helminths, and pollutants. IL-33 is an alarmin that acts as an endogenous danger signal, and it has been known to affect various types of cells, such as mast cells, basophils, eosinophils, T cells, and specific subsets of innate lymphoid cells (ILCs). In recent findings, this cytokine is believed to have a critical role in several types of cancers, such as lung cancer, liver cancer, and head and neck squamous cell cancer. The expression of IL-33/ST2 in cancer tissues shows a close association with tumor growth and tumor progression in several types of cancer, suggesting the IL-33/ST2 pathway as a potential target for therapy.
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Proteína 1 Similar al Receptor de Interleucina-1/inmunología , Interleucina-33/inmunología , Neoplasias/inmunología , Transducción de Señal/inmunología , Animales , Humanos , Neoplasias/patología , Microambiente TumoralRESUMEN
Oxygen sensing is crucial for adaptation to variable habitats and physiological conditions. Low oxygen tension, or hypoxia, is a common feature of solid tumors, and hypoxic tumors are often more aggressive and resistant to therapy. Here we show that, in cultured mammalian cells, hypoxia suppressed lysosomal acidification/activation and receptor tyrosine kinase (RTK) degradation. Hypoxia down-regulated mTORc1, reducing its ability to activate transcription factor EB (TFEB), a master regulator of V-ATPase, the lysosomal proton pump. Hypoxia prevented epidermal growth factor receptor (EGFR) degradation in tumor tissues, whereas activation of lysosomes enhanced tumor cell response to anti-EGFR treatment. Our results link oxygen tension and lysosomal activity, provide a molecular explanation of the malignant phenotype associated with hypoxic tumors, and suggest activation of lysosomes may provide therapeutic benefit in RTK-targeted cancer therapy.
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Neutrophilic granule protein (NGP) was previously reported as a granular protein of neutrophils in mouse, but the function has not been known clearly. We found the presence of the possible signal peptide in NGP and validated this protein is circulating in the bloodstream. In our findings, NGP is being modified post-translationally in Golgi apparatus and endoplasmic reticulum, which is a universal character of secretory molecules with a signal peptide. The secreted NGP protein could be detected both in vitro and in vivo. NGP has sequence similarity with an antimicrobial protein cathelicidin, and we observed the aspect of inflammation of NGP. Interestingly, NGP interacts with the complex of LPS and LPS binding protein (LBP). This interaction blocks the binding of the complex of LPS and LBP to TLR4 and the downstream inflammatory signals. Furthermore, the inhibitory function of NGP against the inflammatory effect of LPS could be observed in both in vitro and in vivo. With these findings, we report NGP is a novel secretory protein to mask LPS and inhibit its function.