RESUMEN
Global demand for animal protein is on the rise, but many practices common in conventional production are no longer scalable due to environmental impact, public health concerns, and fragility of food systems. For these reasons and more, a pressing need has arisen for sustainable, nutritious, and animal welfare-conscious sources of protein, spurring research dedicated to the production of cultivated meat. Meat mainly consists of muscle, fat, and connective tissue, all of which can be sourced and differentiated from pluripotent stem cells to resemble their nutritional values in muscle tissue. In this paper, we outline the approach that we took to derive bovine embryonic stem cell lines (bESCs) and to characterise them using FACS (fluorescence-activated cell sorting), real-time PCR and immunofluorescence staining. We show their cell growth profile and genetic stability and demonstrate their induced differentiation to mesoderm committed cells. In addition, we discuss our strategy for preparation of master and working cell banks, by which we can expand and grow cells in suspension in quantities suitable for mass production. Consequently, we demonstrate the potential benefits of harnessing bESCs in the production of cultivated meat.
Asunto(s)
Técnicas de Cultivo de Célula , Animales , Bovinos , Técnicas de Cultivo de Célula/veterinaria , Células Madre Embrionarias , Línea Celular , Oocitos , CarneRESUMEN
BACKGROUND/AIMS: The rapid nuclear translocation of signaling proteins upon stimulation is important for the regulation of de-novo gene expression. However, the molecular mechanisms of this translocation is not well understood, although some studies suggest that much of this translocation may be mediated by beta-like importins (Imps). Here we undertook to study the stimulated nuclear shuttling of JNK and p38 MAPKs. METHODS: For this purpose, we used coimmunoprecipitation, proximity ligation assay, gel filtration and immunostaining to examine the mechanism of nuclear translocation of these proteins. RESULTS: We found that JNK and p38 MAPKs translocate into the nucleus in a Ran dependent, but NLS- or NTS-independent manner, unrelated to their catalytic activity. We show that this translocation involves three ß-like Imps, 3, 7 and 9. Knockdown of these Imps inhibits the nuclear translocation of the MAPKs, and thereby, phosphorylation of their transcription factor targets. We further demonstrate that the translocation requires the stimulated formation of heterotrimers composed of Imp3/Imp7/MAPK or Imp3/Imp9/MAPK. JNK1/2 and p38α/ß bind to either Imp7 or Imp9 upon stimulated post-translational modifications of the two Imps, while Imp3 joins the complex after its stimulation-induced phosphorylation. Once formed, these heterotrimers move to the nuclear envelope where Imp3 remains, while Imp7 or Imp9 escort the MAPKs into the nucleus. CONCLUSION: These results suggest that ß-like Imps are central mediators of stimulated nuclear translocation of signaling proteins, providing a central level of regulation of the induction of cellular processes such as transcription upon stimulation.
Asunto(s)
Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Secuencia de Aminoácidos , Anisomicina/farmacología , Núcleo Celular/metabolismo , Células HeLa , Humanos , Células MCF-7 , Microscopía Fluorescente , Fosforilación , Unión Proteica , Transporte de Proteínas/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/genética , Alineación de SecuenciaRESUMEN
It is well established that the expression profiles of multiple and possibly redundant matrix-remodeling proteases (e.g., collagenases) differ strongly in health, disease, and development. Although enzymatic redundancy might be inferred from their close similarity in structure, their in vivo activity can lead to extremely diverse tissue-remodeling outcomes. We observed that proteolysis of collagen-rich natural extracellular matrix (ECM), performed uniquely by individual homologous proteases, leads to distinct events that eventually affect overall ECM morphology, viscoelastic properties, and molecular composition. We revealed striking differences in the motility and signaling patterns, morphology, and gene-expression profiles of cells interacting with natural collagen-rich ECM degraded by different collagenases. Thus, in contrast to previous notions, matrix-remodeling systems are not redundant and give rise to precise ECM-cell crosstalk. Because ECM proteolysis is an abundant biochemical process that is critical for tissue homoeostasis, these results improve our fundamental understanding its complexity and its impact on cell behavior.
Asunto(s)
Matriz Extracelular/metabolismo , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 1 de la Matriz/metabolismo , Proteolisis , Homología de Secuencia de Aminoácido , Animales , Uniones Célula-Matriz/metabolismo , Colágeno/metabolismo , Colágeno/ultraestructura , Elasticidad , Matriz Extracelular/ultraestructura , Fibroblastos/metabolismo , Humanos , Imagenología Tridimensional , Análisis de Componente Principal , Ratas , Reología , ViscosidadRESUMEN
Ineffective endometrial matrix remodeling, a key factor in infertility, impedes embryo implantation in the uterine wall. Our study reveals the cellular and molecular impact of human collagenase-1 administration in mouse uteri, demonstrating enhanced embryo implantation rates. Collagenase-1 promotes remodeling of the endometrial ECM, degrading collagen fibers and proteoglycans. This process releases matrix-bound bioactive factors (e.g., VEGF, decorin), facilitating vascular permeability and angiogenesis. Collagenase-1 elevates embryo implantation regulators, including NK cell infiltration and the key cytokine LIF. Remarkably, uterine tissue maintains structural integrity despite reduced endometrial collagen fiber tension. In-utero collagenase-1 application rescues implantation in heat stress and embryo transfer models, known for low implantation rates. Importantly, ex vivo exposure of human uterine tissue to collagenase-1 induces collagen de-tensioning and VEGF release, mirroring remodeling observed in mice. Our research highlights the potential of collagenases to induce and orchestrate cellular and molecular processes enhancing uterine receptivity for effective embryo implantation. This innovative approach underscores ECM remodeling mechanisms critical for embryo implantation.
Asunto(s)
Colagenasas , Implantación del Embrión , Útero , Femenino , Animales , Ratones , Colagenasas/metabolismo , Humanos , Útero/metabolismo , Matriz Extracelular/metabolismo , Endometrio/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Embarazo , Transferencia de Embrión/métodos , Colágeno/metabolismo , Ratones Endogámicos C57BLRESUMEN
The MAPK cascades are central signaling pathways that regulate a wide variety of stimulated cellular processes, including proliferation, differentiation, apoptosis and stress response. Therefore, dysregulation, or improper functioning of these cascades, is involved in the induction and progression of diseases such as cancer, diabetes, autoimmune diseases, and developmental abnormalities. Many of these physiological, and pathological functions are mediated by MAPK-dependent transcription of various regulatory genes. In order to induce transcription and the consequent functions, the signals transmitted via the cascades need to enter the nucleus, where they may modulate the activity of transcription factors and chromatin remodeling enzymes. In this review, we briefly cover the composition of the MAPK cascades, as well as their physiological and pathological functions. We describe, in more detail, many of the important nuclear activities of the MAPK cascades, and we elaborate on the mechanisms of ERK1/2 translocation into the nucleus, including the identification of their nuclear translocation sequence (NTS) binding to the shuttling protein importin7. Overall, the nuclear translocation of signaling components may emerge as an important regulatory layer in the induction of cellular processes, and therefore, may serve as targets for therapeutic intervention in signaling-related diseases such as cancer and diabetes. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Transporte Activo de Núcleo Celular/genética , Ensamble y Desensamble de Cromatina/fisiología , Regulación de la Expresión Génica , Genes Inmediatos-Precoces , Humanos , Sistema de Señalización de MAP Quinasas/genética , Modelos Biológicos , Señales de Localización Nuclear/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Estrés Fisiológico , Factores de Transcripción/fisiologíaRESUMEN
Topoisomerase I (topo I) is a nuclear enzyme which participates in most DNA transactions. It was shown to be inhibited in depolarized neurons by poly adenosine diphosphate (ADP)-ribosylation of the enzyme protein. We demonstrated previously an age and sex dependent topo I activity and enzyme protein level in the various regions of mouse brain. A specific distribution pattern of topo I was observed and the inhibitory neurons exhibited the highest enzyme activity and protein level in both the nucleus and the cytoplasm. Here, we show that neurotransmitters (glutamate and gamma-aminobutyric acid (GABA)) regulate the activity of topo I in mouse cerebellum sections. Glutamate exhibited a significant time-dependent inhibition of topo I activity but no effect of the enzyme protein level. GABA in contrary only slightly and transiently inhibited topo I activity. The inhibitory effect of glutamate was mediated by Ca(+2) and by ADP-ribosylation of topo I protein and the glutamate ionotropic receptors were involved. Glutamate also diminished the inhibitory effect of topotecan on topo I. These results point to distinct and highly specific effects of the major neurotransmitters on topo I activity in the cerebellum suggesting that topo I possesses a specific role in the brain which differs from its known biological functions.
Asunto(s)
Cerebelo/enzimología , Ácido Glutámico/metabolismo , Inhibidores de Topoisomerasa I , Ácido gamma-Aminobutírico/metabolismo , Animales , Western Blotting , Calcio/farmacología , Núcleo Celular/enzimología , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Citoplasma/enzimología , ADN-Topoisomerasas de Tipo I/metabolismo , Electroforesis en Gel de Agar , Técnica del Anticuerpo Fluorescente , Ácido Glutámico/farmacología , Inmunohistoquímica , Técnicas In Vitro , Ratones , Poli(ADP-Ribosa) Polimerasas/metabolismo , Topotecan/farmacología , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
The matrix metalloproteinases (MMPs) play a crucial role in irreversible remodeling of the extracellular matrix (ECM) in normal homeostasis and pathological states. Accumulating data from various studies strongly suggest that MMPs are tightly regulated, starting from the level of gene expression all the way to zymogen activation and endogenous inhibition, with each level controlled by multiple factors. Recent in vivo findings indicate that cell-ECM and cell-cell interactions, as well as ECM bio-active products, contribute an additional layer of regulation at all levels, indicating that individual MMP expression and activity in vivo are highly coordinated and tissue specific processes.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Metaloproteinasas de la Matriz/metabolismo , Comunicación Celular , Matriz Extracelular/metabolismo , Homeostasis , Humanos , Especificidad de Órganos , Especificidad por Sustrato , Inhibidores Tisulares de Metaloproteinasas/metabolismoRESUMEN
A hallmark of the ERK1/2 functioning is their nuclear translocation, which is mainly required for the induction of proliferation. Activated ERK1/2 molecules that remain in the cytoplasm initiate other activities, including immediate feedback loops. Prevention of the nuclear translocation should therefore inhibit proliferation, without affecting cytoplasm-induced cellular processes. Here we present an NTS-derived myristoylated phosphomimetic peptide, which blocks the interaction of importin7 and ERK1/2, and consequently the nuclear translocation of the latter. In culture, the peptide induces apoptosis of melanoma cells inhibits the viability of other cancer cells, but has no effect on non-transformed, immortalized cells. It even inhibits the viability of PLX4032- and U0126-resistant melanoma cells. In xenograft models, the peptide inhibits several cancers, and acts much better than PLX4032 in preventing melanoma recurrence. This study provides a proof of concept for using the nuclear translocation of ERK1/2 as a drug target for the combat of various ERK1/2-related cancers.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Péptidos/farmacología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Western Blotting , Células CHO , Línea Celular Tumoral , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Cricetulus , Células HCT116 , Células HeLa , Humanos , Inmunohistoquímica , Inmunoprecipitación , Etiquetado Corte-Fin in Situ , Ratones Desnudos , Ratones SCID , Microscopía Fluorescente , Terapia Molecular Dirigida , Trasplante de Neoplasias , Transporte de Proteínas/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The rapid nuclear translocation of signaling proteins upon stimulation is important for the regulation of de novo gene expression. We have studied the stimulated nuclear shuttling of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs) and found that they translocate into the nucleus in a Ran-dependent, but NLS- or NTS-independent, manner, unrelated to their catalytic activity. We show that this translocation involves three ß-like importins, importins 3, 7, and 9 (Imp3/7/9). Knockdown of these importins inhibits the nuclear translocation of the MAPKs and, thereby, activation of their transcription factor targets. We further demonstrate that the translocation requires the stimulated formation of heterotrimers composed of Imp3/Imp7/MAPK or Imp3/Imp9/MAPK. JNK1/2 and p38α/ß bind to either Imp7 or Imp9 upon stimulated posttranslational modification of the two Imps, while Imp3 joins the complex after its stimulation-induced phosphorylation. Once formed, these heterotrimers move to the nuclear envelope, where importin 3 remains, while importins 7 and 9 escort the MAPKs into the nucleus. These results suggest that ß-like importins are central mediators of stimulated nuclear translocation of signaling proteins and therefore add a central level of regulation to stimulated transcription.
Asunto(s)
Núcleo Celular/enzimología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Secuencia Conservada , Regulación de la Expresión Génica , Células HeLa , Humanos , Células MCF-7 , Proteínas Quinasas Activadas por Mitógenos/química , Datos de Secuencia Molecular , Señales de Localización Nuclear , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/metabolismo , Transcripción GenéticaRESUMEN
The kinase Mnk2 is a substrate of the MAPK pathway and phosphorylates the translation initiation factor eIF4E. In humans, MKNK2, the gene encoding for Mnk2, is alternatively spliced yielding two splicing isoforms with differing last exons: Mnk2a, which contains a MAPK-binding domain, and Mnk2b, which lacks it. We found that the Mnk2a isoform is downregulated in breast, lung, and colon tumors and is tumor suppressive. Mnk2a directly interacts with, phosphorylates, activates, and translocates p38α-MAPK into the nucleus, leading to activation of its target genes, increasing cell death and suppression of Ras-induced transformation. Alternatively, Mnk2b is pro-oncogenic and does not activate p38-MAPK, while still enhancing eIF4E phosphorylation. We further show that Mnk2a colocalization with p38α-MAPK in the nucleus is both required and sufficient for its tumor-suppressive activity. Thus, Mnk2a downregulation by alternative splicing is a tumor suppressor mechanism that is lost in some breast, lung, and colon tumors.
Asunto(s)
Empalme Alternativo , Núcleo Celular/metabolismo , Transformación Celular Neoplásica/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Transporte Activo de Núcleo Celular , Animales , Ratones , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteínas ras/metabolismoRESUMEN
The ERK cascade is a central signaling pathway that regulates a large number of intracellular processes including proliferation, differentiation, development and also survival or apoptosis. The induction of so many distinct and even opposing cellular processes raises the question as to how the signaling specificity of the cascade is regulated. In the past few years, subcellular localization of components of the ERK cascade was shown to play an important role in specificity determination. Here we describe the dynamic subcellular localization of Raf kinases, MEKs, and particularly ERKs, which translocate into the nucleus during many cellular processes to induce transcription. We also describe in details the recent identification of a novel nuclear translocation mechanism for ERKs, which is based on a nuclear translocation sequence (NTS) within their kinase insert domain (KID). Phosphorylation of this domain, mainly upon stimulation, allows ERKs to interact with the nuclear importing protein - importin7, which mediates the penetration of the interacting ERKs into the nucleus via nuclear pores. Interestingly, the NTS is not specific to ERKs, and seems to be a general signal for regulating nuclear accumulation of various proteins, including MEKs, upon their stimulation. Better understanding of this mechanism may clarify the role of the massive nuclear translocation of many regulatory proteins shortly after their stimulation.