133 research outputs found
A novel Yap/Taz zebrafish reporter reveals a role of Hippo pathway transducers in angiogenesis
No full textYAP and TAZ, by orchestrating cell proliferation, cell death and cell-fate decisions, are key players of a complex network of signaling pathways acting during development. Deregulation of YAP/TAZ signaling causes robust organ overgrowth during organogenesis, which translates to loss of tissue homeostasis in the adult and consequent cancer development. YAP/TAZ are transcriptional co-activators that interact with TEAD transcription factors to promote cell proliferation and survival. Their transcriptional activity is regulated by nucleocytoplasmic shuttling and nuclear accumulation, which are controlled by the Hippo kinase cascade, but also by mechanical cues sensed by the cell and by other pathways. Among these, Wnt/β-catenin takes on a particular relevance, since it was recently shown to regulate YAP/TAZ activity through AXIN-mediated sequestration of YAP/TAZ in the β-catenin destruction complex.
Here, we describe the generation, validation and characterization of a novel biosensor zebrafish reporting the activity of Yap/Taz. It expresses nuclear mCherry, eGFP or the destabilized green fluorescent protein VenusPEST under the control of a promoter fragment of the human YAP/TAZ target gene CTGF, that contains 3 TEAD DNA-binding sites. Several independent founder fish transmitting the transgene to the germline were identified and used to establish the stable reporter lines. All stable transgenic fish shared a similar expression pattern, which was maintained in subsequent generations. Knockdown and overexpression approaches were used to validate the reporter. Co-injection of two morpholinos targeting Yap and Taz pre-mRNAs reduced the reporter signal, whereas injection of mRNAs coding for a constitutively active form of Yap, Taz and Tead (YAP-5SA, TAZ-4SA, TEAD-VP16) increased it. The CTGF-based transgenic lines represent therefore bona fide Yap/Taz reporters.
During development, strong reporter signal is visible mainly in the lens and otic vesicles, the pharyngeal arches, the heart, the pectoral fin and the vasculature, but the reporter protein expression is also detected in many other tissues and organs. The almost ubiquitous activation of Yap/Taz observed during early embryogenesis, consistent with the general role of YAP/TAZ in promoting cell proliferation and organ growth, is largely silenced in the adult fish, where the reporter signal is restricted to the lens, the ovary, the heart and the whole vasculature. We also showed that the CTGF-based biosensor zebrafish is able to report Yap/Taz activation during larval and adult fin regeneration, as expected from the role that YAP/TAZ signaling plays in the regenerative processes.
The zebrafish CTGF-based reporter permitted to show in a living organism during development the regulation that the Wnt/β-catenin pathway exerts on Yap/Taz activity. Our results in terms of variations of the reporter signal, after both genetic and pharmacological modulation of the Wnt pathway activity, are in accordance with the model recently depicted in vitro.
The general and sustained reporter activity we observed in the endothelium during embryogenesis suggested a functional involvement of Yap/Taz signaling in developmental angiogenesis. Yap/Taz knockdown impaired the intersegmental vessels (ISVs) growth, while the overactivation of Yap/Taz-mediated transcription caused an aberrant sprouting from the ISVs. The vessel sprouting-promoting capacity of Yap/Taz is cell-autonomous, as the same phenomenon was observed by expressing TAZ-4SA under the control of an endothelium-specific promoter.
The CTGF-based zebrafish reporter is a new powerful tool to study in vivo Yap/Taz pathway activation, with possible applications in drug screening, regeneration and cancer biology. It permitted to confirm in vivo during development the crosstalk between Wnt/β-catenin and Yap/Taz pathways and to discover a novel role of Yap/Taz in vessel sprouting, suggesting a pro-angiogenic function of YAP/TAZ transcriptional activity
Time to fight: targeting the circadian clock molecular machinery in cancer therapy
No full textThe circadian clock regulates a wide range of molecular pathways and biological processes. The expression of clock genes is often altered in cancer, fostering tumor initiation and progression. Inhibition and activation of core circadian clock genes, as well as treatments that restore circadian rhythmicity, have been successful in counteracting tumor growth in different experimental models. Here, we provide an up-to-date overview of studies that show the therapeutic effects of targeting the clock molecular machinery in cancer, both genetically and pharmacologically. We also highlight future areas for progress that offer a promising path towards innovative anticancer strategies. Substantial limitations in the current understanding of the complex interplay between the circadian clock and cancer in vivo need to be addressed in order to allow clock-targeting therapies in cancer
“No logo”: verso un Warenfetisch transzendental contemporaneo?
No full textIn this article, commenting the journalistic investigation of “No logo” by Naomi Klein (2000), we’ll argue for a re-definition of the concept of “fetichism” of the Marxist tradition and the urge of update it. Starting from the assumption of the author concerning a more “conceptual” economy, based on the care of image and style of contemporary industries, we would like to refer at a syncretic kind of mixture between “culture” and abstract contents of what is sold and searched by the post-modern consumer. We’ll assume the claim that successes of brands are fundamental, also, in the development of individuality and in the process of recognition; the end of the article will inquire about the anti-marketing movements and its consequences. For protesters there would be some advantages to ruin the enemy industries or the challenge itself indicates the way for a sufficient supervision of customers on their products
The circadian protein BMAL1 supports endothelial cell cycle during angiogenesis
No full textAims The circadian clock is an internal biological timer that co-ordinates physiology and gene expression with the 24-h solar day. Circadian clock perturbations have been associated to vascular dysfunctions in mammals, and a function of the circadian clock in angiogenesis has been suggested. However, the functional role of the circadian clock in endothelial cells (ECs) and in the regulation of angiogenesis is widely unexplored. Methods and results Here, we used both in vivo and in vitro approaches to demonstrate that ECs possess an endogenous molecular clock and show robust circadian oscillations of core clock genes. By impairing the EC-specific function of the circadian clock transcriptional activator basic helix-loop-helix ARNT like 1 (BMAL1) in vivo, we detect angiogenesis defects in mouse neonatal vascular tissues, as well as in adult tumour angiogenic settings. We then investigate the function of circadian clock machinery in cultured EC and show evidence that BMAL and circadian locomotor output cycles protein kaput knock-down impair EC cell cycle progression. By using an RNA- and chromatin immunoprecipitation sequencing genome-wide approaches, we identified that BMAL1 binds the promoters of CCNA1 and CDK1 genes and controls their expression in ECs. Conclusion(s) Our findings show that EC display a robust circadian clock and that BMAL1 regulates EC physiology in both developmental and pathological contexts. Genetic alteration of BMAL1 can affect angiogenesis in vivo and in vitro settings
Wnt/β-Catenin Signaling Regulates Yap/Taz Activity during Embryonic Development in Zebrafish
No full text: Hippo-YAP/TAZ and Wnt/β-catenin signaling pathways, by controlling proliferation, migration, cell fate, stemness, and apoptosis, are crucial regulators of development and tissue homeostasis. We employed zebrafish embryos as a model system to elucidate in living reporter organisms the crosstalk between the two signaling pathways. Co-expression analysis between the Wnt/β-catenin Tg(7xTCF-Xla.Siam:GFP)ia4 and the Hippo-Yap/Taz Tg(Hsa.CTGF:nlsmCherry)ia49 zebrafish reporter lines revealed shared spatiotemporal expression profiles. These patterns were particularly evident in key developmental regions such as the midbrain-hindbrain boundary (MHB), epidermis, muscles, neural tube, notochord, floorplate, and otic vesicle. To investigate the relationship between the Wnt/β-catenin pathway and Hippo-Yap/Taz signaling in vivo, we conducted a series of experiments employing both pharmacological and genetic strategies. Modulation of the Wnt/β-catenin pathway with IWR-1, XAV939, or BIO resulted in a significant regulation of the Yap/Taz reporter signal, highlighting a clear correlation between β-catenin and Yap/Taz activities. Furthermore, genetic perturbation of the Wnt/β-catenin pathway, by APC inhibition or DKK1 upregulation, elicited evident and robust alteration of Yap/Taz activity. These findings revealed the intricate regulatory mechanisms underlying the crosstalk between the Wnt/β-catenin and Hippo-Yap/Taz signaling, shedding light on their roles in orchestrating developmental processes in vivo
Triple modulation of 5-fluorouracil with methotrexate, folinic acid and cisplatin in the treatment of advanced gastric cancer: a phase II study.
No full textTripla modulazione del 5-Fluorouracile (5-FU) con Methotrexate (MTX), Acido Folinico (FA) e Cisplatino (CDDP) nella terapia dell’adenocarcinoma gastrico in fase avanzata: studio di fase II
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Generation and characterization of a novel transgenic line reporting the activity Hif1alpha, the central component of hypoxia sensing in the cell.
No full textThe availability of new tools to study the activation of specific cells signaling regulating cell survival and death, proliferation and differentiation are the new frontiers for understanding in-vivo cell development, tissue homeostasis and tumor growth.
Here we describe the generation and validation of a transgenic reporter line to study the hypoxia signaling pathway regulated by Hif1alpha, the central component of oxygen sensing in the cell. Hif1alpha activity is deregulated in many human cancers, especially those that are highly hypoxic. For these reasons, understanding the hypoxia signaling pathway in vivo could be a big step in the study the molecular mechanisms of solid tumors. To investigate Hif1alpha activity we developed two novel zebrafish transgenic lines named Tg(4xHRE:GFP) and Tg(4xHRE:nlsmCherry), expressing GFP or mCherry under the control of a synthetic promoter containing Hif1alpha DNA binding sites. At first, we observed that Tg(4xHRE) zebrafish transgenic lines are responsive to changes in environmental oxygen levels and to chemical compounds mimicking hypoxic condition. A strong Hif1alpha activation was observed in embryos from early development stages. We detect Hif1alpha activity in several tissues of developing embryos; particularly evident districts are retina, heart, eye lens, notochord and muscle cells. GFP or mCherry signals were present in different areas of the head, such as the cerebral vascular system and the craniofacial skeleton. Under physiological condition the activity of Hif1alpha was also maintained in adult fish, namely in the kidney, ovary, heart, eye lens, cerebral vascular system and the swim bladder.
In conclusion, we demonstrated that Tg(4xHRE) lines are a reliable and sensitive biosensor to monitor Hif1alpha activity in vivo and to study at the whole organism level the hypoxia signaling pathway both in physiological and pathological conditions
Neoadjuvant treatment of metastatic liver disease with irinotecan and 5-fluorouracil (5-FU) plus folinic acid (L-FA) in colorectal cancer patients.
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