RESUMEN
Mastitis is one the most widespread and serious diseases in dairy cattle. Recurrent and chronic infections are often attributable to certain pathogenicity mechanisms in mastitis-causing pathogens such as Staphylococcus spp. These include growing in biofilm and invading cells, both of which make it possible to resist or evade antimicrobial therapies and the host's immune system. This study tested the effects of active vitamin D3 (i.e., calcitriol or 1,25-dihydroxyvitamin D3) on the internalization and phagocytosis of biofilm-forming Staphylococcus spp. isolated from animals with mastitis. Two established bovine cell lines were used: MAC-T (mammary epithelial cells) and BoMac (macrophages). Calcitriol (0-200â¯nM) did not affect the viability of MAC-T cells nor that of BoMac cells after 24 and 72â¯h. Concentrations of 0-100â¯mM for 24â¯h upregulated the expression of 24-hydroxylase in MAC-T cells, but did not alter that of VDR. Pre-treatment of the cells with calcitriol for 24â¯h decreased the internalization of S. aureus V329 into MAC-T cells (0-100â¯nM), and stimulated the phagocytosis of the same strain and of S. xylosus 4913 (0-10â¯nM). Calcitriol and two conditioned media, obtained by treating the cells with 25-200â¯nM of the metabolite for 24â¯h, were also assessed in terms of their antimicrobial and antibiofilm activity. Neither calcitriol by itself nor the conditioned media affected staphylococcal growth or biofilm formation (0-200â¯nM for 12 and 24â¯h, respectively). In contrast, the conditioned media (0-100â¯nM for 24â¯h) decreased the biomass of preformed non-aureus staphylococcal biofilms and killed the bacteria within them, without affecting metabolic activity. These effects may be mediated by reactive oxygen species and proteins with antimicrobial and/or antibiofilm activity. In short, calcitriol could make pathogens more accessible to antimicrobial therapies and enhance bacterial clearance by professional phagocytes. Moreover, it may modulate the host's endogenous defenses in the bovine udder and help combat preformed non-aureus staphylococcal biofilms (S. chromogenes 40, S. xylosus 4913, and/or S. haemolyticus 6). The findings confirm calcitriol's potential as an adjuvant to prevent and/or treat intramammary infections caused by Staphylococcus spp., which would in turn contribute to reducing antibiotic use on dairy farms.
Asunto(s)
Biopelículas , Inmunidad Innata , Mastitis Bovina , Fagocitosis , Staphylococcus , Animales , Bovinos , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Femenino , Mastitis Bovina/microbiología , Mastitis Bovina/inmunología , Inmunidad Innata/efectos de los fármacos , Staphylococcus/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Calcitriol/farmacología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/veterinaria , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/tratamiento farmacológico , Línea Celular , Glándulas Mamarias Animales/microbiología , Glándulas Mamarias Animales/inmunología , Macrófagos/microbiología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismoRESUMEN
Staphylococcus aureus is the most frequent causal agent of bovine mastitis, which is largely responsible for milk production losses worldwide. The pathogen's ability to form stable biofilms facilitates intramammary colonization and may explain disease persistence. This virulence factor is also highly influential in the development of chronic intramammary infections refractory to antimicrobial therapy, which is why novel therapies that can tackle multiple targets are necessary. Since udder microbiota have important implications in mastitis pathogenesis, they offer opportunities to develop alternative prophylactic and therapeutic strategies. Here, we observed that a Bacillus strain from the teat apex of lactating cows was associated to reduce colonization by S. aureus. The strain, identified as Bacillus sp. H21, was able to antagonize in-formation or mature S. aureus biofilms associated to intramammary infections without affecting cell viability. When exploring the metabolite responsible for this activity, we found that a widespread class of Bacillus exopolysaccharide, levan, eliminated the pathogenic biofilm under evaluated conditions. Moreover, levan had no cytotoxic effects on bovine cellular lines at the biologically active concentration range, which demonstrates its potential for pathogen control. Our results indicate that commensal Bacillus may counteract S. aureus-induced mastitis, and could therefore be used in novel biotechnological strategies to prevent and/or treat this disease.
Asunto(s)
Bacillus , Enfermedades de los Bovinos , Mastitis Bovina , Infecciones Estafilocócicas , Femenino , Bovinos , Animales , Staphylococcus aureus , Lactancia , Glándulas Mamarias Animales/patología , Infecciones Estafilocócicas/prevención & control , Infecciones Estafilocócicas/veterinaria , Biopelículas , Mastitis Bovina/prevención & control , Leche , Enfermedades de los Bovinos/patologíaRESUMEN
Glioblastoma multiforme is the most aggressive type of tumor of the CNS with an overall survival rate of approximately one year. Since this rate has not changed significantly over the last 20 years, the development of new therapeutic strategies for the treatment of these tumors is peremptory. The over-expression of the proto-oncogene c-Fos has been observed in several CNS tumors including glioblastoma multiforme and is usually associated with a poor prognosis. Besides its genomic activity as an AP-1 transcription factor, this protein can also activate phospholipid synthesis by a direct interaction with key enzymes of their metabolic pathways. Given that the amino-terminal portion of c-Fos (c-Fos-NA: amino acids 1-138) associates to but does not activate phospholipid synthesizing enzymes, we evaluated if c-Fos-NA or some shorter derivatives are capable of acting as dominant-negative peptides of the activating capacity of c-Fos. The over-expression or the exogenous administration of c-Fos-NA to cultured T98G cells hampers the interaction between c-Fos and PI4K2A, an enzyme activated by c-Fos. Moreover, it was observed a decrease in tumor cell proliferation rates in vitro and a reduction in tumor growth in vivo when a U87-MG-generated xenograft on nude mice is intratumorally treated with recombinant c-Fos-NA. Importantly, a smaller peptide of 92 amino acids derived from c-Fos-NA retains the capacity to interfere with tumor proliferation in vitro and in vivo. Taken together, these results support the use of the N-terminal portion of c-Fos, or shorter derivatives as a novel therapeutic strategy for the treatment of glioblastoma multiforme.
Asunto(s)
Proliferación Celular , Glioblastoma/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Fosfolípidos/biosíntesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Línea Celular Tumoral , Activación Enzimática , Glioblastoma/genética , Glioblastoma/patología , Humanos , Antígenos de Histocompatibilidad Menor/genética , Fosfolípidos/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-fos/genética , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismoRESUMEN
Differentiation of neuronal cells is crucial for the development and function of the nervous system. This process involves high rates of membrane expansion, during which the synthesis of membrane lipids must be tightly regulated. In this work, using a variety of molecular and biochemical assays and approaches, including immunofluorescence microscopy and FRET analyses, we demonstrate that the proto-oncogene c-Fos (c-Fos) activates cytoplasmic lipid synthesis in the central nervous system and thereby supports neuronal differentiation. Specifically, in hippocampal primary cultures, blocking c-Fos expression or its activity impairs neuronal differentiation. When examining its subcellular localization, we found that c-Fos co-localizes with endoplasmic reticulum markers and strongly interacts with lipid-synthesizing enzymes, whose activities were markedly increased in vitro in the presence of recombinant c-Fos. Of note, the expression of c-Fos dominant-negative variants capable of blocking its lipid synthesis-activating activity impaired neuronal differentiation. Moreover, using an in utero electroporation model, we observed that neurons with blocked c-Fos expression or lacking its AP-1-independent activity fail to initiate cortical development. These results highlight the importance of c-Fos-mediated activation of lipid synthesis for proper nervous system development.
Asunto(s)
Corteza Cerebral/embriología , Neurogénesis , Neuronas/citología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Animales , Células Cultivadas , Femenino , Regulación del Desarrollo de la Expresión Génica , Ratones Endogámicos C57BL , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Ratas , Ratas WistarRESUMEN
The mechanisms that coordinately regulate lipid synthesis in the nervous system together with the high rates of membrane biogenesis needed to support cell growth are largely unknown as are their subcellular site of synthesis. c-Fos, a well-known AP-1 transcription factor, has emerged as a unique protein with the capacity to associate to specific enzymes of the pathway of synthesis of phospholipids at the endoplasmic reticulum and activate their synthesis to accompany genomic decisions of growth. Herein, we discuss this effect of c-Fos in the context of neuronal differentiation and also with respect to pathologies of the nervous system such as the development and growth of tumors. We also provide insights into the sub-cellular sites where this regulation occurs at the endoplasmic reticulum membranes and the molecular mechanism by which c-Fos exerts this activity.
RESUMEN
PURPOSE: BRCA1 and BRCA2 deficiencies are widespread drivers of human cancers that await the development of targeted therapies. We aimed to identify novel synthetic lethal relationships with therapeutic potential using BRCA-deficient isogenic backgrounds. EXPERIMENTAL DESIGN: We developed a phenotypic screening technology to simultaneously search for synthetic lethal (SL) interactions in BRCA1- and BRCA2-deficient contexts. For validation, we developed chimeric spheroids and a dual-tumor xenograft model that allowed the confirmation of SL induction with the concomitant evaluation of undesired cytotoxicity on BRCA-proficient cells. To extend our results using clinical data, we performed retrospective analysis on The Cancer Genome Atlas (TCGA) breast cancer database. RESULTS: The screening of a kinase inhibitors library revealed that Polo-like kinase 1 (PLK1) inhibition triggers strong SL induction in BRCA1-deficient cells. Mechanistically, we found no connection between the SL induced by PLK1 inhibition and PARP inhibitors. Instead, we uncovered that BRCA1 downregulation and PLK1 inhibition lead to aberrant mitotic phenotypes with altered centrosomal duplication and cytokinesis, which severely reduced the clonogenic potential of these cells. The penetrance of PLK1/BRCA1 SL interaction was validated using several isogenic and nonisogenic cellular models, chimeric spheroids, and mice xenografts. Moreover, bioinformatic analysis revealed high-PLK1 expression in BRCA1-deficient tumors, a phenotype that was consistently recapitulated by inducing BRCA1 deficiency in multiple cell lines as well as in BRCA1-mutant cells. CONCLUSIONS: We uncovered an unforeseen addiction of BRCA1-deficient cancer cells to PLK1 expression, which provides a new means to exploit the therapeutic potential of PLK1 inhibitors in clinical trials, by generating stratification schemes that consider this molecular trait in patient cohorts.
Asunto(s)
Proteína BRCA1/deficiencia , Proteínas de Ciclo Celular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Mutaciones Letales Sintéticas/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteína BRCA2/deficiencia , Proteína BRCA2/genética , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Células Cultivadas , Aberraciones Cromosómicas , Daño del ADN , Modelos Animales de Enfermedad , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasa Tipo Polo 1RESUMEN
c-Fos is a well-recognized member of the AP-1 (activator protein-1) family of transcription factors. In addition to this canonical activity, we previously showed that cytoplasmic c-Fos activates phospholipid synthesis through a mechanism independent of its genomic AP-1 activity. c-Fos associates with particular enzymes of the lipid synthesis pathway at the endoplasmic reticulum and increases the Vmax of the reactions without modifying the Km values. This lipid synthesis activation is associated with events of differentiation and proliferation that require high rates of membrane biogenesis. Since lipid synthesis also occurs in the nucleus, and different phospholipids have been assigned transcription regulatory functions, in the present study we examine if c-Fos also acts as a regulator of phospholipid synthesis in the nucleus. Furthermore, we examine if c-Fos modulates transcription through its phospholipid synthesis activator capacity. We show that nuclear-localized c-Fos associates with and activates PI4P5K (phosphatidylinositol-4-monophosphate 5-kinase), but not with PI4KIIIß (type IIIß phosphatidylinositol 4-kinase) thus promoting PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) formation, which, in turn, promotes transcriptional changes. We propose c-Fos as a key regulator of nuclear PtdIns(4,5)P2 synthesis in response to growth signals that results in c-Fos-dependent transcriptional changes promoted by the newly synthesized lipids.