RESUMEN
OBJECTIVE: Perivascular cells, including pericytes, macrophages, smooth muscle cells, and other specialized cell types, like podocytes, participate in various aspects of vascular function. However, aside from the well-established roles of smooth muscle cells and pericytes, the contributions of other vascular-associated cells are poorly understood. Our goal was to ascertain the function of perivascular macrophages in adult tissues under nonpathological conditions. APPROACH AND RESULTS: We combined confocal microscopy, in vivo cell depletion, and in vitro assays to investigate the contribution of perivascular macrophages to vascular function. We found that resident perivascular macrophages are associated with capillaries at a frequency similar to that of pericytes. Macrophage depletion using either clodronate liposomes or antibodies unexpectedly resulted in hyperpermeability. This effect could be rescued when M2-like macrophages, but not M1-like macrophages or dendritic cells, were reconstituted in vivo, suggesting subtype-specific roles for macrophages in the regulation of vascular permeability. Furthermore, we found that permeability-promoting agents elicit motility and eventual dissociation of macrophages from the vasculature. Finally, in vitro assays showed that M2-like macrophages attenuate the phosphorylation of VE-cadherin upon exposure to permeability-promoting agents. CONCLUSIONS: This study points to a direct contribution of macrophages to vessel barrier integrity and provides evidence that heterotypic cell interactions with the endothelium, in addition to those of pericytes, control vascular permeability.
Asunto(s)
Capilares/metabolismo , Permeabilidad Capilar , Comunicación Celular , Células Endoteliales/metabolismo , Macrófagos Peritoneales/metabolismo , Mesenterio/irrigación sanguínea , Peritoneo/irrigación sanguínea , Piel/irrigación sanguínea , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Movimiento Celular , Células Cultivadas , Técnicas de Cocultivo , Dextranos/metabolismo , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Ovalbúmina/metabolismo , Fenotipo , Fosforilación , Rodaminas/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
The ability of macrophages to promote vascular growth has been associated with the secretion and local delivery of classic proangiogenic factors (e.g., VEGF-A and proteases). More recently, a series of studies have also revealed that physical contact of macrophages with growing blood vessels coordinates vascular fusion of emerging sprouts. Interestingly, the interactions between macrophages and vascular endothelial cells (ECs) appear to be bidirectional, such that activated ECs also support the expansion and differentiation of proangiogenic macrophages from myeloid progenitors. Here, we discuss recent findings suggesting that dynamic angiogenic vascular niches might also exist in vivo, e.g. in tumors, where sprouting blood vessels and immature myeloid cells like monocytes engage in heterotypic interactions that are required for angiogenesis. Finally, we provide an account of emerging mechanisms of cell-to-cell communication that rely on secreted microvesicles, such as exosomes, which can offer a vehicle for the rapid exchange of molecules and genetic information between macrophages and ECs engaged in angiogenesis.
Asunto(s)
Comunicación Celular , Diferenciación Celular , Endotelio Vascular/citología , Macrófagos/citología , Neovascularización Patológica , Neovascularización Fisiológica , Animales , HumanosRESUMEN
Inhibitor of apoptosis-stimulating protein of p53 (iASPP) is the most ancient member of the ASPP family of proteins and an evolutionarily conserved inhibitor of p53. iASPP is also a binding partner and negative regulator of p65RelA. Because p65RelA and the p53 family members often have opposite effects in controlling cell fate, it is important to understand the cellular context in which iASPP can regulate their activities. To address this question and to study the biological importance of iASPP in vivo, we generated a transgenic mouse in which iASPP expression is controlled by the Cre/loxP recombination system. We observed that iASPP is able to prevent premature cellular senescence in mouse embryonic fibroblasts. iASPP loss resulted in increased differentiation of primary keratinocytes both in vitro and in vivo. In stratified epithelia, nuclear iASPP often colocalized with p63 in the nuclei of basal keratinocytes. Consistent with this, iASPP bound p63 and inhibited the transcriptional activity of both TAp63α and ΔNp63α in vitro and influenced the expression level of p63-regulated genes such as loricrin and involucrin in vivo. In contrast, under the same conditions, p65RelA was frequently expressed as a cytoplasmic protein in the suprabasal layers of stratified epithelia and rarely colocalized with nuclear iASPP. Thus, iASPP is likely to control epithelial stratification by regulating p63's transcriptional activity, rather than p65RelA's. This study identifies iASPP as an inhibitor of senescence and a key player in controlling epithelial stratification.
Asunto(s)
Senescencia Celular/genética , Epitelio/fisiología , Regulación de la Expresión Génica/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Queratinocitos/fisiología , Proteínas Represoras/metabolismo , Factor de Transcripción ReIA/metabolismo , Animales , Diferenciación Celular/fisiología , Senescencia Celular/fisiología , Regulación de la Expresión Génica/fisiología , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Precursores de Proteínas/metabolismo , Proteínas Represoras/genéticaRESUMEN
Cytotoxic chemotherapy is an effective treatment for invasive breast cancer. However, experimental studies in mice also suggest that chemotherapy has pro-metastatic effects. Primary tumours release extracellular vesicles (EVs), including exosomes, that can facilitate the seeding and growth of metastatic cancer cells in distant organs, but the effects of chemotherapy on tumour-derived EVs remain unclear. Here we show that two classes of cytotoxic drugs broadly employed in pre-operative (neoadjuvant) breast cancer therapy, taxanes and anthracyclines, elicit tumour-derived EVs with enhanced pro-metastatic capacity. Chemotherapy-elicited EVs are enriched in annexin A6 (ANXA6), a Ca2+-dependent protein that promotes NF-κB-dependent endothelial cell activation, Ccl2 induction and Ly6C+CCR2+ monocyte expansion in the pulmonary pre-metastatic niche to facilitate the establishment of lung metastasis. Genetic inactivation of Anxa6 in cancer cells or Ccr2 in host cells blunts the pro-metastatic effects of chemotherapy-elicited EVs. ANXA6 is detected, and potentially enriched, in the circulating EVs of breast cancer patients undergoing neoadjuvant chemotherapy.
Asunto(s)
Doxorrubicina/uso terapéutico , Vesículas Extracelulares/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Paclitaxel/uso terapéutico , Animales , Anexina A6/metabolismo , Línea Celular Tumoral , Quimiocina CCL2/metabolismo , Vesículas Extracelulares/metabolismo , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Ratones TransgénicosRESUMEN
Tumour-associated macrophages (TAMs) largely express an alternatively activated (or M2) phenotype, which entails immunosuppressive and tumour-promoting capabilities. Reprogramming TAMs towards a classically activated (M1) phenotype may thwart tumour-associated immunosuppression and unleash anti-tumour immunity. Here we show that conditional deletion of the microRNA (miRNA)-processing enzyme DICER in macrophages prompts M1-like TAM programming, characterized by hyperactive IFN-γ/STAT1 signalling. This rewiring abated the immunosuppressive capacity of TAMs and fostered the recruitment of activated cytotoxic T lymphocytes (CTLs) to the tumours. CTL-derived IFN-γ exacerbated M1 polarization of Dicer1-deficient TAMs and inhibited tumour growth. Remarkably, DICER deficiency in TAMs negated the anti-tumoral effects of macrophage depletion by anti-CSF1R antibodies, and enabled complete tumour eradication by PD1 checkpoint blockade or CD40 agonistic antibodies. Finally, genetic rescue of Let-7 miRNA activity in Dicer1-deficient TAMs partly restored their M2-like phenotype and decreased tumour-infiltrating CTLs. These findings suggest that DICER/Let-7 activity opposes IFN-γ-induced, immunostimulatory M1-like TAM activation, with potential therapeutic implications.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Interferón gamma/metabolismo , Activación de Macrófagos/inmunología , Macrófagos/inmunología , MicroARNs/genética , Ribonucleasa III/metabolismo , Microambiente Tumoral/genética , Animales , Células Cultivadas , ARN Helicasas DEAD-box/deficiencia , Humanos , Ratones , Neoplasias/genética , Neoplasias/inmunología , Ribonucleasa III/deficienciaRESUMEN
An intriguing biological question relating to cell signaling is how the inflammatory mediator NF-kB and the tumour suppressor protein p53 can be induced by similar triggers, like DNA damage or infection, yet have seemingly opposing or sometimes cooperative biological functions. For example, the NF-κB subunit RelA/p65 has been shown to inhibit apoptosis, whereas p53 induces apoptosis. One potential explanation may be their co-regulation by common cellular factors: inhibitor of Apoptosis Stimulating p53 Protein (iASPP) is one such common regulator of both RelA/p65 and p53. Here we show that iASPP is a novel substrate of caspases in response to apoptotic stimuli. Caspase cleaves the N-terminal region of iASPP at SSLD294 resulting in a prominent 80kDa fragment of iASPP. This caspase cleavage site is conserved in various species from zebrafish to Homo sapiens. The 80kDa fragment of iASPP translocates from the cytoplasm to the nucleus via the RaDAR nuclear import pathway, independent of p53. The 80kDa iASPP fragment can bind and inhibit p53 or RelA/p65 more efficiently than full-length iASPP. Overall, these data reveal a potential novel regulation of p53 and RelA/p65 activities in response to apoptotic stimuli.
Asunto(s)
Caspasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Represoras/metabolismo , Factor de Transcripción ReIA/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/fisiología , Línea Celular , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Inmunoprecipitación , Activación Transcripcional , TransfecciónRESUMEN
MicroRNA (miRNA) transfer via exosomes may mediate cell-to-cell communication. Interestingly, specific miRNAs are enriched in exosomes in a cell-type-dependent fashion. However, the mechanisms whereby miRNAs are sorted to exosomes and the significance of miRNA transfer to acceptor cells are unclear. We used macrophages and endothelial cells (ECs) as a model of heterotypic cell communication in order to investigate both processes. RNA profiling of macrophages and their exosomes shows that miRNA sorting to exosomes is modulated by cell-activation-dependent changes of miRNA target levels in the producer cells. Genetically perturbing the expression of individual miRNAs or their targeted transcripts promotes bidirectional miRNA relocation from the cell cytoplasm/P bodies (sites of miRNA activity) to multivesicular bodies (sites of exosome biogenesis) and controls miRNA sorting to exosomes. Furthermore, the use of Dicer-deficient cells and reporter lentiviral vectors (LVs) for miRNA activity shows that exosomal miRNAs are transferred from macrophages to ECs to detectably repress targeted sequences.
Asunto(s)
Células Endoteliales/metabolismo , Exosomas/metabolismo , Macrófagos/metabolismo , MicroARNs/metabolismo , Animales , Secuencia de Bases , Comunicación Celular , Células Cultivadas , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Ratones , Datos de Secuencia Molecular , Cuerpos Multivesiculares/metabolismo , Ribonucleasa III/genética , Ribonucleasa III/metabolismoRESUMEN
Nearly 90% of human melanomas contain inactivated wild-type p53, the underlying mechanisms for which are not fully understood. Here, we identify that cyclin B1/CDK1-phosphorylates iASPP, which leads to the inhibition of iASPP dimerization, promotion of iASPP monomer nuclear entry, and exposure of its p53 binding sites, leading to increased p53 inhibition. Nuclear iASPP is enriched in melanoma metastasis and associates with poor patient survival. Most wild-type p53-expressing melanoma cell lines coexpress high levels of phosphorylated nuclear iASPP, MDM2, and cyclin B1. Inhibition of MDM2 and iASPP phosphorylation with small molecules induced p53-dependent apoptosis and growth suppression. Concurrent p53 reactivation and BRAFV600E inhibition achieved additive suppression in vivo, presenting an alternative for melanoma therapy.