RESUMEN
Various human diseases are triggered by molecular alterations influencing the fine-tuned expression and activity of transcription factors, usually due to imbalances in targets including protein-coding genes and non-coding RNAs, such as microRNAs (miRNAs). The transcription factor EB (TFEB) modulates human cellular networks, overseeing lysosomal biogenesis and function, plasma-membrane trafficking, autophagic flux, and cell cycle progression. In endothelial cells (ECs), TFEB is essential for the maintenance of endothelial integrity and function, ensuring vascular health. However, the comprehensive regulatory network orchestrated by TFEB remains poorly understood. Here, we provide novel mechanistic insights into how TFEB regulates the transcriptional landscape in primary human umbilical vein ECs (HUVECs), using an integrated approach combining high-throughput experimental data with dedicated bioinformatics analysis. By analyzing HUVECs ectopically expressing TFEB using ChIP-seq and examining both polyadenylated mRNA and small RNA sequencing data from TFEB-silenced HUVECs, we have developed a bioinformatics pipeline mapping the different gene regulatory interactions driven by TFEB. We show that TFEB directly regulates multiple miRNAs, which in turn post-transcriptionally modulate a broad network of target genes, significantly expanding the repertoire of gene programs influenced by this transcription factor. These insights may have significant implications for vascular biology and the development of novel therapeutics for vascular disease.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Biología Computacional , Redes Reguladoras de Genes , Células Endoteliales de la Vena Umbilical Humana , MicroARNs , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Biología Computacional/métodos , Regulación de la Expresión Génica , Células Endoteliales/metabolismoRESUMEN
Transcription factor TFEB is thought to control cellular functions-including in the vascular bed-primarily via regulation of lysosomal biogenesis and autophagic flux. Here, we report that TFEB also orchestrates a non-canonical program that controls the cell cycle/VEGFR2 pathway in the developing vasculature. In endothelial cells, TFEB depletion halts proliferation at the G1-S transition by inhibiting the CDK4/Rb pathway. TFEB-deficient cells attempt to compensate for this limitation by increasing VEGFR2 levels at the plasma membrane via microRNA-mediated mechanisms and controlled membrane trafficking. TFEB stimulates expression of the miR-15a/16-1 cluster, which limits VEGFR2 transcript stability and negatively modulates expression of MYO1C, a regulator of VEGFR2 trafficking to the cell surface. Altered levels of miR-15a/16-1 and MYO1C in TFEB-depleted cells cause increased expression of plasma membrane VEGFR2, but in a manner associated with low signaling strength. An endothelium-specific Tfeb-knockout mouse model displays defects in fetal and newborn mouse vasculature caused by reduced endothelial proliferation and by anomalous function of the VEGFR2 pathway. These previously unrecognized functions of TFEB expand its role beyond regulation of the autophagic pathway in the vascular system.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/fisiología , Proliferación Celular , Embrión de Mamíferos/citología , Endotelio Vascular/citología , Regulación del Desarrollo de la Expresión Génica , Neovascularización Fisiológica , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Células Cultivadas , Embrión de Mamíferos/fisiología , Endotelio Vascular/fisiología , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
The dynamic integrin-mediated adhesion of endothelial cells (ECs) to the surrounding ECM is fundamental for angiogenesis both in physiological and pathological conditions, such as embryonic development and cancer progression. The dynamics of EC-to-ECM adhesions relies on the regulation of the conformational activation and trafficking of integrins. Here, we reveal that oncogenic transcription factor EB (TFEB), a known regulator of lysosomal biogenesis and metabolism, also controls a transcriptional program that influences the turnover of ECM adhesions in ECs by regulating cholesterol metabolism. We show that TFEB favors ECM adhesion turnover by promoting the transcription of genes that drive the synthesis of cholesterol, which promotes the aggregation of caveolin-1, and the caveolin-dependent endocytosis of integrin ß1. These findings suggest that TFEB might represent a novel target for the pharmacological control of pathological angiogenesis and bring new insights in the mechanism sustaining TFEB control of endocytosis.
Asunto(s)
Células Endoteliales , Integrinas , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Caveolina 1/metabolismo , Adhesión Celular/genética , Colesterol , Células Endoteliales/metabolismo , Humanos , Integrina beta1/metabolismo , Integrinas/metabolismo , Neovascularización Patológica/metabolismoRESUMEN
BACKGROUND: In non-small cell lung cancer (NSCLC) the efficacy of chemo-immunotherapy is affected by the high expression of drug efflux transporters as ABCC1 and by the low expression of ABCA1, mediating the isopentenyl pyrophosphate (IPP)-dependent anti-tumor activation of Vγ9Vδ2 T-lymphocytes. In endothelial cells ABCA1 is a predicted target of the transcription factor EB (TFEB), but no data exists on the correlation between TFEB and ABC transporters involved in the chemo-immuno-resistance in NSCLC. METHODS: The impact of TFEB/ABCC1/ABCA1 expression on NSCLC patients' survival was analyzed in the TCGA-LUAD cohort and in a retrospective cohort of our institution. Human NSCLC cells silenced for TFEB (shTFEB) were analyzed for ABC transporter expression, chemosensitivity and immuno-killing. The chemo-immuno-sensitizing effects of nanoparticles encapsulating zoledronic acid (NZ) on shTFEB tumors and on tumor immune-microenvironment were evaluated in Hu-CD34+ mice by single-cell RNA-sequencing. RESULTS: TFEBlowABCA1lowABCC1high and TFEBhighABCA1highABCC1low NSCLC patients had the worst and the best prognosis, respectively, in the TCGA-LUAD cohort and in a retrospective cohort of patients receiving platinum-based chemotherapy or immunotherapy as first-line treatment. By silencing shTFEB in NSCLC cells, we demonstrated that TFEB was a transcriptional inducer of ABCA1 and a repressor of ABCC1. shTFEB cells had also a decreased activity of ERK1/2/SREBP2 axis, implying reduced synthesis and efflux via ABCA1 of cholesterol and its intermediate IPP. Moreover, TFEB silencing reduced cholesterol incorporation in mitochondria: this event increased the efficiency of OXPHOS and the fueling of ABCC1 by mitochondrial ATP. Accordingly, shTFEB cells were less immuno-killed by the Vγ9Vδ2 T-lymphocytes activated by IPP and more resistant to cisplatin. NZ, which increased IPP efflux but not OXPHOS and ATP production, sensitized shTFEB immuno-xenografts, by reducing intratumor proliferation and increasing apoptosis in response to cisplatin, and by increasing the variety of anti-tumor infiltrating cells (Vγ9Vδ2 T-lymphocytes, CD8+T-lymphocytes, NK cells). CONCLUSIONS: This work suggests that TFEB is a gatekeeper of the sensitivity to chemotherapy and immuno-killing in NSCLC, and that the TFEBlowABCA1lowABCC1high phenotype can be predictive of poor response to chemotherapy and immunotherapy. By reshaping both cancer metabolism and tumor immune-microenvironment, zoledronic acid can re-sensitize TFEBlow NSCLCs, highly resistant to chemo- and immunotherapy.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , Animales , Femenino , Inmunoterapia/métodos , Línea Celular Tumoral , Masculino , Estudios RetrospectivosRESUMEN
Obesity is characterized by poor collateral vessel formation, a process involving vascular endothelial growth factor (VEGF) action on vascular smooth muscle cells (VSMC). Free fatty acids are involved in the pathogenesis of obesity vascular complications, and we have aimed to clarify whether oleic acid (OA) enhances VEGF synthesis/secretion in VSMC, and whether this effect is impaired in obesity. In cultured aortic VSMC from lean and obese Zucker rats (LZR and OZR, respectively) we measured the influence of OA on VEGF-A synthesis/secretion, signaling molecules and reactive oxygen species (ROS). In VSMC from LZR we found the following: (a) OA increases VEGF-A synthesis/secretion by a mechanism blunted by inhibitors of Akt, mTOR, ERK-1/2, PKC-beta, NADPH-oxidase and mitochondrial electron transport chain complex; (b) OA activates the above mentioned signaling pathways and increases ROS; (c) OA-induced activation of PKC-beta enhances oxidative stress, which activates signaling pathways responsible for the increased VEGF synthesis/secretion. In VSMC from OZR, which present enhanced baseline oxidative stress, the above mentioned actions of OA on VEGF-A, signaling pathways and ROS are impaired: this impairment is reproduced in VSMC from LZR by incubation with hydrogen peroxide. Thus, in OZR chronically elevated oxidative stress causes a resistance to the action on VEGF that OA exerts in LZR by increasing ROS.
Asunto(s)
Músculo Liso Vascular/efectos de los fármacos , Ácido Oléico/farmacología , Estrés Oxidativo/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Células Cultivadas , Peróxido de Hidrógeno/farmacología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , NADPH Oxidasas/metabolismo , Obesidad/metabolismo , Obesidad/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Proteína Quinasa C beta/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Zucker , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Endothelial cells (ECs) that line vascular and lymphatic vessels are being increasingly recognized as important to organ function in health and disease. ECs participate not only in the trafficking of gases, metabolites, and cells between the bloodstream and tissues but also in the angiocrine-based induction of heterogeneous parenchymal cells, which are unique to their specific tissue functions. The molecular mechanisms regulating EC heterogeneity between and within different tissues are modeled during embryogenesis and become fully established in adults. Any changes in adult tissue homeostasis induced by aging, stress conditions, and various noxae may reshape EC heterogeneity and induce specific transcriptional features that condition a functional phenotype. Heterogeneity is sustained via specific genetic programs organized through the combinatory effects of a discrete number of transcription factors (TFs) that, at the single tissue-level, constitute dynamic networks that are post-transcriptionally and epigenetically regulated. This review is focused on outlining the TF-based networks involved in EC specialization and physiological and pathological stressors thought to modify their architecture.
Asunto(s)
Células Endoteliales , Factores de Transcripción , Células Endoteliales/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The introduction of targeted therapies represented one of the most significant advances in the treatment of BRAFV600E melanoma. However, the onset of acquired resistance remains a challenge. Previously, we showed in mouse xenografts that vascular endothelial growth factor (VEGFA) removal enhanced the antitumor effect of BRAF inhibition through the recruitment of M1 macrophages. In this work, we explored the strategy of VEGFA/BRAF inhibition in immunocompetent melanoma murine models. In BRAF mutant D4M melanoma tumors, VEGFA/BRAF targeting reshaped the tumor microenvironment, largely by stimulating infiltration of M1 macrophages and CD8+ T cells, and sensitized tumors to immune checkpoint blockade (ICB). Furthermore, we reported that the association of VEGFA/BRAF targeting with anti-PD-1 antibody (triple therapy) resulted in a durable response and enabled complete tumor eradication in 50% of the mice, establishing immunological memory. Neutralization and CRISPR-Cas-mediated editing of granulocyte-macrophage colony-stimulating factor (GM-CSF) abrogated antitumor response prompted by triple therapy and identified GM-CSF as the cytokine instrumental in M1-macrophage recruitment. Our data suggest that VEGFA/BRAF targeting in melanoma induces the activation of innate and adaptive immunity and prepares tumors for ICB. Our study contributes to understanding the tumor biology of BRAFV600E melanoma and suggests VEGFA as therapeutic target.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos , Melanoma , Humanos , Animales , Ratones , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Linfocitos T CD8-positivos/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Melanoma/metabolismo , Macrófagos/metabolismo , Microambiente TumoralRESUMEN
Innate immunity may activate paracrine circuits able to entail vascular system in the onset and progression of several chronic degenerative diseases. In particular, interleukin (IL)-12 triggers a genetic program in lymphomononuclear cells characterized by the production of interferon-γ and specific chemokines resulting in an angiostatic activity. The aim of this study is to identify molecules involved in the regulation of cell cycle in endothelial cells co-cultured with IL-12-stimulated lymphomonuclear cells. By using a transwell mediated co-culture system we demonstrated that IL-12-stimulated lymphomonuclear cells induce an arrest of endothelial cells cycle in G1, which is mainly mediated by the up-regulation of p21(Cip1/Waf1), an inhibitor of cyclin kinases. This effect requires the activation of STAT1, PKCδ and p38 MAPK, while p53 is ineffective. In accordance, siRNA-dependent silencing of these molecules in endothelial cells inhibited the increase of p21(Cip1/Waf1) and the modification in cell cycle promoted by IL-12-stimulated lymphomonuclear cells. These results indicate that the angiostatic action of IL-12-stimulated lymphomononuclear cells may lie in the capability to arrest endothelial cells in G1 phase through a mechanisms mainly based on the specific up-regulation of p21(Cip1/Waf1) induced by the combined activity of STAT1, PKCδ and p38 MAPK.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Fase G1 , Inmunidad Innata , Interleucina-12/fisiología , Fase de Descanso del Ciclo Celular , Técnicas de Cocultivo , Humanos , ARN Interferente PequeñoRESUMEN
Type 1 diabetes is characterized by insulin deficiency, type 2 by both insulin deficiency and insulin resistance: in both conditions, hyperglycaemia is accompanied by an increased cardiovascular risk, due to increased atherosclerotic plaque formation/instabilization and impaired collateral vessel formation. An important factor in these phenomena is the Vascular Endothelial Growth Factor (VEGF), a molecule produced also by Vascular Smooth Muscle Cells (VSMC). We aimed at evaluating the role of high glucose on VEGF-A(164) synthesis and secretion in VSMC from lean insulin-sensitive and obese insulin-resistant Zucker rats (LZR and OZR). In cultured aortic VSMC from LZR and OZR incubated for 24 h with d-glucose (5.5, 15 and 25 mM) or with the osmotic controls l-glucose and mannitol, we measured VEGF-A(164) synthesis (western, blotting) and secretion (western blotting and ELISA). We observed that: (i) d-glucose dose-dependently increases VEGF-A(164) synthesis and secretion in VSMC from LZR and OZR (n = 6, ANOVA p = 0.002-0.0001); (ii) all the effects of 15 and 25 mM d-glucose are attenuated in VSMC from OZR vs. LZR (p = 0.0001); (iii) l-glucose and mannitol reproduce the VEGF-A(164) modulation induced by d-glucose in VSMC from both LZR and OZR. Thus, glucose increases via an osmotic mechanism VEGF synthesis and secretion in VSMC, an effect attenuated in the presence of insulin resistance.
Asunto(s)
Aorta/metabolismo , Glucosa/farmacología , Músculo Liso Vascular/metabolismo , Obesidad/metabolismo , Delgadez/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Aorta/efectos de los fármacos , Aorta/patología , Hiperglucemia/fisiopatología , Hipoglucemiantes/farmacología , Insulina/farmacología , Resistencia a la Insulina , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Obesidad/tratamiento farmacológico , Obesidad/patología , Presión Osmótica , Ratas , Ratas Zucker , Edulcorantes/farmacología , Delgadez/tratamiento farmacológico , Delgadez/patología , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacosRESUMEN
Transcription factor EB (TFEB) belongs to the microphthalmia family of bHLH-leucine zipper transcription factors and was first identified as an oncogene in a subset of renal cell carcinomas. In addition to exhibiting oncogenic activity, TFEB coordinates genetic programs connected with the cellular response to stress conditions, including roles in lysosome biogenesis, autophagy, and modulation of metabolism. As is the case for other transcription factors, the activities of TFEB are not limited to a specific cellular condition such as the response to stress, and recent findings indicate that TFEB has more widespread functions. Here, we review the emerging roles of TFEB in regulating cellular proliferation and motility. The well-established and emerging roles of TFEB suggest that this protein serves as a hub of signaling networks involved in many non-communicable diseases, such as cancer, ischaemic diseases and immune disorders, drug resistance mechanisms, and tissue generation.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Neoplasias , Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Puntos de Control del Ciclo Celular , Humanos , Lisosomas , Factores de TranscripciónRESUMEN
Epithelial-mesenchymal transition (EMT) is a complex and pivotal process involved in organogenesis and is related to several pathological processes, including cancer and fibrosis. During heart development, EMT mediates the conversion of epicardial cells into vascular smooth muscle cells and cardiac interstitial fibroblasts. Here, we show that the oncogenic transcription factor EB (TFEB) is a key regulator of EMT in epicardial cells and that its genetic overexpression in mouse epicardium is lethal due to heart defects linked to impaired EMT. TFEB specifically orchestrates the EMT-promoting function of transforming growth factor (TGF) ß, and this effect results from activated transcription of thymine-guanine-interacting factor (TGIF)1, a TGFß/Smad pathway repressor. The Tgif1 promoter is activated by TFEB, and in vitro and in vivo findings demonstrate its increased expression when Tfeb is overexpressed. Furthermore, Tfeb overexpression in vitro prevents TGFß-induced EMT, and this effect is abolished by Tgif1 silencing. Tfeb loss of function, similar to that of Tgif1, sensitizes cells to TGFß, inducing an EMT response to low doses of TGFß. Together, our findings reveal an unexpected function of TFEB in regulating EMT, which might provide insights into injured heart repair and control of cancer progression.
Asunto(s)
Transición Epitelial-Mesenquimal , Factor de Crecimiento Transformador beta , Animales , Células Cultivadas , Transición Epitelial-Mesenquimal/fisiología , Ratones , Organogénesis , Pericardio/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Transcription factor EB (TFEB) represents an emerging player in vascular biology. It belongs to the bHLH-leucine zipper transcription factor microphthalmia family, which includes microphthalmia-associated transcription factor, transcription factor E3 and transcription factor EC, and is known to be deregulated in cancer. The canonical transcriptional pathway orchestrated by TFEB adapts cells to stress in all kinds of tissues by supporting lysosomal and autophagosome biogenesis. However, emerging findings highlight that TFEB activates other genetic programs involved in cell proliferation, metabolism, inflammation and immunity. Here, we first summarize the general principles and mechanisms by which TFEB activates its transcriptional program. Then, we analyze the current knowledge of TFEB in the vascular system, placing particular emphasis on its regulatory role in angiogenesis and on the involvement of the vascular unit in inflammation and atherosclerosis.
RESUMEN
The oncogenic Transcription Factor EB (TFEB), a member of MITF-TFE family, is known to be the most important regulator of the transcription of genes responsible for the control of lysosomal biogenesis and functions, autophagy, and vesicles flux. TFEB activation occurs in response to stress factors such as nutrient and growth factor deficiency, hypoxia, lysosomal stress, and mitochondrial damage. To reach the final functional status, TFEB is regulated in multimodal ways, including transcriptional rate, post-transcriptional regulation, and post-translational modifications. Post-transcriptional regulation is in part mediated by miRNAs. miRNAs have been linked to many cellular processes involved both in physiology and pathology, such as cell migration, proliferation, differentiation, and apoptosis. miRNAs also play a significant role in autophagy, which exerts a crucial role in cell behaviour during stress or survival responses. In particular, several miRNAs directly recognise TFEB transcript or indirectly regulate its function by targeting accessory molecules or enzymes involved in its post-translational modifications. Moreover, the transcriptional programs triggered by TFEB may be influenced by the miRNA-mediated regulation of TFEB targets. Finally, recent important studies indicate that the transcription of many miRNAs is regulated by TFEB itself. In this review, we describe the interplay between miRNAs with TFEB and focus on how these types of crosstalk affect TFEB activation and cellular functions.
Asunto(s)
Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Regulación de la Expresión Génica , MicroARNs/metabolismo , Transducción de Señal , Apoptosis , Diferenciación Celular , Movimiento Celular , HumanosRESUMEN
Transcription factor EB (TFEB) represents an emerging player in cancer biology. Together with microphthalmia-associated transcription factor, transcription factor E3 and transcription factor EC, TFEB belongs to the microphthalmia family of bHLH-leucine zipper transcription factors that may be implicated in human melanomas, renal and pancreatic cancers. TFEB was originally described as being translocated in a juvenile subset of pediatric renal cell carcinoma; however, whole-genome sequencing reported that somatic mutations were sporadically found in many different cancers. Besides its oncogenic activity, TFEB controls the autophagy-lysosomal pathway by recognizing a recurrent motif present in the promoter regions of a set of genes that participate in lysosome biogenesis; furthermore, its dysregulation was found to have a crucial pathogenic role in different tumors by modulating the autophagy process. Other than regulating cancer cell-autonomous responses, recent findings indicate that TFEB participates in the regulation of cellular functions of the tumor microenvironment. Here, we review the emerging role of TFEB in regulating cancer cell behavior and choreographing tumor-microenvironment interaction. Recognizing TFEB as a hub of network of signals exchanged within the tumor between cancer and stroma cells provides a fresh perspective on the molecular principles of tumor self-organization, promising to reveal numerous new and potentially druggable vulnerabilities.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Microambiente Tumoral , Humanos , Melanoma/patologíaRESUMEN
Visceral obesity is a relevant pathological condition closely associated with high risk of atherosclerotic vascular disease including myocardial infarction and stroke. The increased vascular risk is related also to peculiar dysfunction in the endocrine activity of adipose tissue responsible of vascular impairment (including endothelial dysfunction), prothrombotic tendency, and low-grade chronic inflammation. In particular, increased synthesis and release of different cytokines, including interleukins and tumor necrosis factor-alpha (TNF-alpha), and adipokines-such as leptin-have been reported as associated with future cardiovascular events. Since vascular cell dysfunction plays a major role in the atherothrombotic complications in central obesity, this paper aims at focusing, in particular, on the relationship between platelets and vascular smooth muscle cells, and the impaired secretory pattern of adipose tissue.
Asunto(s)
Adipoquinas/fisiología , Aterosclerosis/etiología , Plaquetas/fisiología , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/fisiología , Trombosis/etiología , Animales , Humanos , Inflamación/etiología , Resistencia a la Insulina , Interleucina-6/fisiología , Nicotinamida Fosforribosiltransferasa/fisiología , Obesidad/sangre , Obesidad/complicaciones , Obesidad/metabolismo , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
OBJECTIVE: The bacteriostatic preservative sodium azide (NaN(3)) activates soluble guanylate cyclase (sGC) in vascular tissues, thus elevating cellular 3',5'-cyclic guanosine monophosphate (cGMP). Because the sGC/cGMP pathway is involved in the control of platelet aggregation, we investigated whether in human platelets NaN(3) influences the responses to agonists, cGMP levels and cGMP-regulated pathways. DESIGN AND METHOD: Concentration- and time-dependent effects of NaN(3) (1-100 micromol/L; 5-60 min incubation) on ADP- and collagen-induced aggregation, NO synthase (NOS) activity, cGMP synthesis and vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser239 were investigated in platelets from 21 healthy individuals. RESULTS: NaN(3) exerted concentration- and time-dependent antiaggregatory effects starting from 1 micromol/L (IC(50) with 5-min incubation: 2.77+/-0.35 micromol/L with ADP and 4.64+/-0.48 micromol/L with collagen) and significantly increased intraplatelet cGMP levels and phosphorylation of VASP at Ser239 at 1-100 micromol/L; these effects were prevented by sGC inhibition, but not by NOS inhibition. CONCLUSIONS: NaN(3) exerts antiaggregatory effects in human platelets via activation of the sGC/cGMP/VASP pathway. This biological effect must be considered when azide-containing reagents are used for in vitro studies on platelet function.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Azida Sódica/farmacología , Adulto , Femenino , Humanos , Masculino , Óxido Nítrico/metabolismo , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
In this study we aimed to test the hypothesis that in human vascular smooth muscle cells (VSMC) homocysteine influences synthesis and release of matrix metalloproteinase-2 (MMP-2), which is deeply involved in vascular remodeling and atherosclerotic plaque instabilization. Experiments were carried out in cultured human VSMC exposed to 50-500 micromol/l homocysteine after a 24-hour culture with MEM containing 0.1% BSA. Both in supernatants and cell lysates we evaluated MMP-2 activity (gelatin zimography), MMP-2 andTIMP-2 protein synthesis (Western immunoblotting). Homocysteine effects were investigated also after cell exposure to i) specific MEK inhibitor PD98059 (30 micromol/l) to evaluate the involvement of Mitogen-Activated Protein Kinase (MAPK) and ii) specific phosphatidylinositol 3-kinase (P13-K) inhibitor LY294002 (100 micromol/l) to evaluate the involvement of P13-K pathway. Gelatin zimography evidenced that MMP-2 activity is increased both in conditioned media and in cell lysates starting from 8-hour incubation with 100 micromol/l homocysteine. Western blot analysis evidenced increased MMP-2 levels in both conditioned media and cell lysates. Cell exposure to PD98059 and LY294002 prevented homocysteine effects on MMP-2 synthesis. Homocysteine, at concentrations associated with increased risk of cardiovascular events, increases MMP-2 activity, synthesis and secretion in VSMC through a mechanism involving the activation of MAPK and P13-K pathways. These data suggest that homocysteine is directly involved in mechanisms leading to remodelling and instabilization of atherosclerotic plaques.
Asunto(s)
Homocisteína/farmacología , Metaloproteinasa 2 de la Matriz/biosíntesis , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , Aorta/citología , Aorta/enzimología , Movimiento Celular , Células Cultivadas , Cromonas/farmacología , Dactinomicina/farmacología , Relación Dosis-Respuesta a Droga , Flavonoides/farmacología , Humanos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Morfolinas/farmacología , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de la Síntesis de la Proteína/farmacología , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
The aim was to evaluate whether high glucose influences the nitric oxide (NO)/cyclic nucleotide pathway in human platelets via osmotic stress and to clarify the role of protein kinase C (PKC) in this phenomenon. The study was carried out on 33 healthy lean male volunteers, aged 28.3+/-1.3 years. NO synthesis was detected as L-citrulline production after L-arginine incubation in platelets incubated for 6 min with 22.0 mM D-glucose and iso-osmolar concentrations of mannitol, L-glucose and fructose. To evaluate the influence of PKC, experiments with D-glucose and mannitol were repeated in the presence of the PKC-beta selective inhibitor LY379196, and NO synthesis was detected after a 6-min incubation with phorbol 12-myristate 13-acetate (PMA), a non-selective PKC activator. Platelet content of guanosine-3',5'-cyclic monophosphate (cGMP) and adenosine-3',5'-cyclic monophosphate (cAMP) was measured by radioimmunoassay in platelets incubated with iso-osmolar concentrations of D-glucose, mannitol, L-glucose and fructose. NO-dependence of cyclic nucleotide enhancements was evaluated by inhibiting NO synthase and guanylate cyclase. Platelet aggregation to ADP and collagen was evaluated in Platelet-Rich Plasma (PRP) in the presence of a 6-min incubation with D-glucose and mannitol, both without and with LY379196 and the guanylate cyclase inhibitor (H-[1,2,4]Oxadiazolo [4,3-a]quinoxaline-1-one)(ODQ). Iso-osmolar concentrations of D-glucose, mannitol, L-glucose and fructose, and PMA increased NO production (p=0.0001). Effects of D-glucose and mannitol were blunted by LY379196. D-glucose and mannitol enhanced platelet cGMP and cAMP (p=0.0001) with a mechanism blunted by NO synthase and guanylate-cyclase inhibition, but did not modify platelet aggregation. In conclusion, glucose activates the NO/cyclic nucleotide pathway in human platelets with an osmotic mechanism mediated by PKC-beta.
Asunto(s)
Glucemia/metabolismo , Plaquetas/metabolismo , Óxido Nítrico/metabolismo , Nucleótidos Cíclicos/metabolismo , Presión Osmótica , Adulto , Glucemia/fisiología , AMP Cíclico/análisis , GMP Cíclico/análisis , Humanos , Masculino , Óxido Nítrico/biosíntesis , Agregación Plaquetaria , Proteína Quinasa C/fisiologíaRESUMEN
AIMS: We have evaluated, in cultured human cavernosal smooth muscle cells, the expression and activity of calcium-dependent constitutive nitric oxide synthase (cNOS) and the ability of insulin to induce nitric oxide (NO) production and to increase intracellular cyclic nucleotides guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP). METHODS: cNOS mRNA was detected by RT-PCR amplification, cNOS protein by immunofluorescence, cNOS activity as l-[3H]-citrulline production from l-[3H]-arginine and cyclic nucleotides by radioimmunoassay. RESULTS: cNOS mRNA and cNOS protein were found in cultured cells; cNOS activity was increased by 5-min exposure to 1 micro mol/l calcium ionophore ionomycin (from 0.1094+/-0.0229 to 0.2685+/-0.0560 pmol/min per mg cell protein, P=0.011) and to 2 nmol/l insulin (from 0.1214+/-0.0149 to 0.2045+/-0.0290 pmol/min per mg cell protein, P=0.041). Insulin increased both cGMP and cAMP in a dose- and time-dependent manner (i.e. with 2 nmol/l insulin, cGMP rose from 2.71+/-0.10 to 6.80+/-0.40 pmol/10(6) cells at 30 min, P=0.0001; cAMP from 1.26+/-0.06 to 3.02+/-0.30 pmol/10(6) cells at 60 min, P=0.0001). NOS inhibitor N(G)-monomethyl-l-arginine and phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors wortmannin and LY 294002 blunted these effects of insulin. The action of insulin on cyclic nucleotides persisted in the presence of phosphodiesterase inhibition, guanylate cyclase activation by NO donors and adenylate cyclase activation by Iloprost or forskolin. CONCLUSION: Human cavernosal smooth muscle cells, by expressing cNOS activity, are a source of NO and not only its target; in these cells, insulin rapidly activates cNOS through a PI 3-kinase pathway, with a consequent increase of both cyclic nucleotides, thus directly influencing the mechanisms involved in penile vascular tone and interplaying with classical haemodynamic mediators.
Asunto(s)
Insulina/farmacología , Músculo Liso/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/metabolismo , Nucleótidos Cíclicos/metabolismo , Pene/metabolismo , Adulto , Calcio/fisiología , División Celular , Células Cultivadas , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática/fisiología , Guanilato Ciclasa/metabolismo , Humanos , Insulina/administración & dosificación , Insulina/fisiología , Masculino , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Óxido Nítrico/biosíntesis , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/genética , Pene/citología , Pene/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , ARN Mensajero/metabolismo , Transducción de Señal/fisiología , Factores de TiempoRESUMEN
Human platelets synthesize nitric oxide (NO) through an endothelial-type NO synthase (ecNOS) activated also by substances enhancing 3',5'-cyclic adenosine monophosphate (cAMP) concentrations, such as catecholamines, beta-adrenoceptor agonists and adenosine. To verify whether cAMP directly activates ecNOS through the cAMP-dependent protein kinase A (PKA), we evaluated (i) the influence of 8-Br-cAMP, adenosine and forskolin on ecNOS activity and phosphorylation at Ser(1177) and (ii) the effect of PKA inhibition on ecNOS activity. Platelets from 10 healthy male volunteers were used for aggregation studies and measurement of NOS activity (conversion of L-[(3)H]-arginine to L-[(3)H]-citrulline) following exposure to 8-Br-cAMP, adenosine and forskolin, both in the absence and in the presence of the PKA inhibitor Rp-cAMPS (100 micromol/l). The phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein (VASP) at Ser(157) and Ser(239) and of ecNOS at Ser(1177) was evaluated by Western blot. NOS activity (pmol L-citrulline/10(8) platelets) increased from 0.090+/-0.002 to 0.148+/-0.013 with 500 micromol/l 8-Br-cAMP (p<0.0001), to 0.140+/-0.008 with 30 micromol/l adenosine (p<0.0001) and to 0.140+/-0.009 with 10 micromol/l forskolin (p<0.0001). Rp-cAMPS decreased baseline NOS activity from 0.093+/-0.001 to 0.075+/-0.006 (p<0.02) and prevented the stimulation by 8-Br-cAMP, adenosine and forskolin. Platelet exposure to 8-Br-cAMP and forskolin, beside the phosphorylation of the specific PKA substrate VASP, markedly increased the expression of ecNOS protein phosphorylated at Ser(1177). The study shows that NOS activity of human platelets is increased by the cAMP/PKA pathway which is involved in NO synthesis induced by adenosine, forskolin and potentially by every antiaggregating substance enhancing intraplatelet cAMP via receptor-dependent and -independent mechanisms.