RESUMO
BACKGROUND: Syndecans regulate cell migration thus having key roles in scarring and wound healing processes. Our previous results have shown that Thy-1/CD90 can engage both αvß3 integrin and Syndecan-4 expressed on the surface of astrocytes to induce cell migration. Despite a well-described role of Syndecan-4 during cell movement, information is scarce regarding specific Syndecan-4 partners involved in Thy-1/CD90-stimulated cell migration. METHODS: Mass spectrometry (MS) analysis of complexes precipitated with the Syndecan-4 cytoplasmic tail peptide was used to identify potential Syndecan-4-binding partners. The interactions found by MS were validated by immunoprecipitation and proximity ligation assays. The conducted research employed an array of genetic, biochemical and pharmacological approaches, including: PAR-3, Syndecan-4 and Tiam1 silencing, active Rac1 GEFs affinity precipitation, and video microscopy. RESULTS: We identified PAR-3 as a Syndecan-4-binding protein. Its interaction depended on the carboxy-terminal EFYA sequence present on Syndecan-4. In astrocytes where PAR-3 expression was reduced, Thy-1-induced cell migration and focal adhesion disassembly was impaired. This effect was associated with a sustained Focal Adhesion Kinase activation in the siRNA-PAR-3 treated cells. Our data also show that Thy-1/CD90 activates Tiam1, a PAR-3 effector. Additionally, we found that after Syndecan-4 silencing, Tiam1 activation was decreased and it was no longer recruited to the membrane. Syndecan-4/PAR-3 interaction and the alteration in focal adhesion dynamics were validated in mouse embryonic fibroblast (MEF) cells, thereby identifying this novel Syndecan-4/PAR-3 signaling complex as a general mechanism for mesenchymal cell migration involved in Thy-1/CD90 stimulation. CONCLUSIONS: The newly identified Syndecan-4/PAR-3 signaling complex participates in Thy-1/CD90-induced focal adhesion disassembly in mesenchymal cells. The mechanism involves focal adhesion kinase dephosphorylation and Tiam1 activation downstream of Syndecan-4/PAR-3 signaling complex formation. Additionally, PAR-3 is defined here as a novel adhesome-associated component with an essential role in focal adhesion disassembly during polarized cell migration. These novel findings uncover signaling mechanisms regulating cell migration, thereby opening up new avenues for future research on Syndecan-4/PAR-3 signaling in processes such as wound healing and scarring.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Adesões Focais/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Sindecana-4/metabolismo , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Adesão Celular , Linhagem Celular , Movimento Celular , Polaridade Celular , Fibroblastos/metabolismo , Inativação Gênica , Camundongos , Microtúbulos/metabolismo , Ligação Proteica , Ratos , Antígenos Thy-1/metabolismoRESUMO
MicroRNAs are small non-coding RNAs involved in post-transcriptional regulation of gene expression related to many cellular functions. We performed a small-RNAseq analysis of cardiac differentiation from pluripotent stem cells. Our analyses identified some new aspects about microRNA expression in this differentiation process. First, we described a dynamic expression profile of microRNAs where some of them are clustered according to their expression level. Second, we described the extensive network of isomiRs and ADAR modifications. Third, we identified the microRNAs families and clusters involved in the establishment of cardiac lineage and define the mirRNAome based on these groups. Finally, we were able to determine a more accurate miRNAome associated with cardiomyocytes by comparing the expressed microRNAs with other mature cells. MicroRNAs exert their effect in a complex and interconnected way, making necessary a global analysis to better understand their role. Our data expands the knowledge of microRNAs and their implications in cardiomyogenesis.
Assuntos
Biomarcadores/metabolismo , Linhagem da Célula/genética , Regulação da Expressão Gênica , Mesoderma/metabolismo , MicroRNAs/genética , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular , Células Cultivadas , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mesoderma/citologia , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologiaRESUMO
Primordial odontogenic tumor (POT) is composed of variably cellular myxoid connective tissue, surrounded by cuboidal to columnar odontogenic epithelium resembling the inner epithelium of the enamel organ, which often invaginates into the underlying connective tissue. The tumor is delimited at least partially by a thin fibrous capsule. It derives from the early stages of tooth development. Syndecan-1 is a heparan sulfate proteoglycan that has a physiological role in several cellular functions, including maintenance of the epithelial architecture, cell-to-cell adhesion and interaction of cells with extracellular matrix, and with diverse growth factors, stimulating cell proliferation. Ki-67 is considered the gold standard as a cell proliferation marker. The aim of this study was to examine the expression of Syndecan-1 and Ki-67 proliferation index in POT and normal tooth germs to better understand the biological behavior of this tumor. Results showed that Syndecan-1 was more intensely expressed in subepithelial mesenchymal areas of POT, in a pattern that resembles the early stages of tooth development. The cell proliferation index (4.1%) suggests that POT is a slow growing tumor. Syndecan-1 expression in tooth germs in late cap and early bell stages was similar to POT, showing immunopositivity in subepithelial mesenchymal condensed areas. The immunohistochemical findings showed a pattern in which the population of subepithelial mesenchymal cells exhibited greater proliferative activity than the central portion of the dental papilla.
Assuntos
Antígeno Ki-67/metabolismo , Odontogênese , Tumores Odontogênicos/metabolismo , Sindecana-1/metabolismo , Germe de Dente/metabolismo , Proliferação de Células , Humanos , Mesoderma/metabolismo , Tumores Odontogênicos/fisiopatologia , Estudos Retrospectivos , Germe de Dente/fisiologiaRESUMO
Hedgehog signaling (Hh) has been shown to be hyper-activated in several cancers. However, active Hh signaling can promote or inhibit tumor growth; thus identification of markers beyond main canonical Hh target genes is needed to improve patient selection and clinical outcome in response to Hh inhibitors. Cancer-associated fibroblasts (CAFs) have been linked with tumor progression and beneficial response to Hh inhibitors. Thus, we hypothesized that genes associated with Hh-activated CAFs can be used for stratification of tumors that will benefit from Hh inhibitors. In this work, we evaluated a 15-gene fingerprint that combines Hh and mesenchymal genes associated with CAF phenotype to profile breast cancer sub-types based on gene expression patterns among clustered groups. About 3800 cancer samples were evaluated using random forest models and linear discriminant analysis to sort breast cancer by subtypes and therapeutic approach. The results showed that the Hh-mesenchyme gene fingerprint has a highly sensitive and differential expression pattern among basal and luminal A sub-groups. Basal samples with high levels of Hh target genes had better prognosis than luminal A samples. Luminal A samples with a tendency towards Hh signaling suppression had higher overall and disease-free survival rates particularly if deprived of hormone therapy. Hh transcriptional repressor GLI3 and signaling activator SMO were the top 2 genes for discriminating among samples with active Hh signaling in human breast cancer subtypes and Hh-inhibitor resistant tumors. Caveolin-1 (CAV1), a gene with low expression in CAFs, shows strong correlation with active Hh signaling and discrimination among survival curves in luminal A patients with active or inactive Hh signaling. Our data suggest that CAV1 is an important gene for monitoring Hh inhibition in tumors and support further stratification by hormone therapy status prior to use of Hh inhibitors.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas Hedgehog/metabolismo , Caveolina 1/metabolismo , Proliferação de Células/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Mesoderma/metabolismo , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: During the initial stages zebrafish neurulation, neural plate cells undergo highly coordinated movements before they assemble into a multicellular solid neural rod. We have previously identified that the underlying mesoderm is critical to ensure such coordination and generate correct neural tube organization. However, how intertissue coordination is achieved in vivo during zebrafish neural tube morphogenesis is unknown. RESULTS: In this work, we use quantitative live imaging to study the coordinated movements of neural ectoderm and mesoderm during dorsal tissue convergence. We show the extracellular matrix components laminin and fibronectin that lie between mesoderm and neural plate act to couple the movements of neural plate and mesoderm during early stages of neurulation and to maintain the close apposition of these two tissues. CONCLUSIONS: Our study highlights the importance of the extracellular matrix proteins laminin and libronectin in coupling the movements and spatial proximity of mesoderm and neuroectoderm during the morphogenetic movements of neurulation. Developmental Dynamics 245:580-589, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Matriz Extracelular/fisiologia , Mesoderma/metabolismo , Placa Neural/metabolismo , Neurulação , Animais , Embrião não Mamífero , Fibronectinas/fisiologia , Laminina/fisiologia , Morfogênese , Tubo Neural , Peixe-ZebraRESUMO
Substances that mimic endogenous hormones may alter the cell signaling that govern prostate development and predispose it to developing lesions in adult and senile life. Bisphenol A is able to mimic estrogens, and studies have demonstrated that low levels of exposure to this compound have caused alterations during prostate development. The aim of this study was to describe the prostate development in both male and female neonatal gerbils in normal conditions and under exposure to BPA during intrauterine life, and also to analyze whether the effects of intrauterine exposure to BPA remain in adulthood. Morphological, stereological, three-dimensional reconstruction, and immunohistochemical methods were employed. The results demonstrated that in 1-day-old normal gerbils, the female paraurethral glands and the male ventral lobe are morphologically similar, although its tissue components-epithelial buds (EB), periurethral mesenchyme (PeM), paraurethral mesenchyme (PaM) or ventral mesenchymal pad (VMP), and smooth muscle (SM)-have presented different immunolabeling pattern for androgen receptor (AR), and for proliferating cell nuclear antigen (PCNA). Moreover, we observed a differential response of male and female prostate to intrauterine BPA exposure. In 1-day-old males, the intrauterine exposure to BPA caused a decrease of AR-positive cells in the PeM and SM, and a decrease of the proliferative status in the EB. In contrast, no morphological alterations were observed in ventral prostate of adult males. In 1-day-old females, BPA exposure promoted an increase of estrogen receptor alpha (ERα) positive cells in PeM and PaM, a decrease of AR-positive cells in EB and PeM, besides a reduction of cell proliferation in EB. Additionally, the adult female prostate of BPA-exposed animals presented an increase of AR- and PCNA-positive cells. These results suggest that the prostate of female gerbils were more susceptible to the intrauterine BPA effects, since they became more proliferative in adult life. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1740-1750, 2016.
Assuntos
Compostos Benzidrílicos/toxicidade , Disruptores Endócrinos/toxicidade , Fenóis/toxicidade , Sistema Urogenital/efeitos dos fármacos , Fatores Etários , Animais , Animais Recém-Nascidos , Proliferação de Células/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Receptor alfa de Estrogênio/metabolismo , Feminino , Gerbillinae , Masculino , Exposição Materna/efeitos adversos , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Próstata/citologia , Próstata/efeitos dos fármacos , Próstata/embriologia , Próstata/crescimento & desenvolvimento , Receptores Androgênicos/metabolismo , Fatores Sexuais , Sistema Urogenital/citologia , Sistema Urogenital/embriologia , Sistema Urogenital/crescimento & desenvolvimentoRESUMO
Antioxidant cellular mechanisms are essential for cell redox homeostasis during animal development and in adult life. Previous in situ hybridization analyses of antioxidant enzymes in zebrafish have indicated that they are ubiquitously expressed. However, spatial information about the protein distribution of these enzymes is not available. Zebrafish embryos are particularly suitable for this type of analysis due to their small size, transparency and fast development. The main objective of the present work was to analyze the spatial and temporal gene expression pattern of the two reported zebrafish glutathione peroxidase 4 (GPx4) genes during the first day of zebrafish embryo development. We found that the gpx4b gene shows maternal and zygotic gene expression in the embryo proper compared to gpx4a that showed zygotic gene expression in the periderm covering the yolk cell only. Following, we performed a GPx4 protein immunolocalization analysis during the first 24-h of development. The detection of this protein suggests that the antibody recognizes GPx4b in the embryo proper during the first 24 h of development and GPx4a at the periderm covering the yolk cell after 14-somite stage. Throughout early cleavages, GPx4 was located in blastomeres and was less abundant at the cleavage furrow. Later, from the 128-cell to 512-cell stages, GPx4 remained in the cytoplasm but gradually increased in the nuclei, beginning in marginal blastomeres and extending the nuclear localization to all blastomeres. During epiboly progression, GPx4b was found in blastoderm cells and was excluded from the yolk cell. After 24 h of development, GPx4b was present in the myotomes particularly in the slow muscle fibers, and was excluded from the myosepta. These results highlight the dynamics of the GPx4 localization pattern and suggest its potential participation in fundamental developmental processes.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Glutationa Peroxidase/genética , Proteínas de Peixe-Zebra/genética , Animais , Blastoderma/metabolismo , Citocinese , Embrião não Mamífero , Desenvolvimento Embrionário , Glutationa Peroxidase/biossíntese , Glutationa Peroxidase/metabolismo , Hibridização In Situ , Isoenzimas , Mesoderma/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Peixe-Zebra , Proteínas de Peixe-Zebra/biossínteseRESUMO
In vertebrates, the embryonic dorsal midline is a crucial signalling centre that patterns the surrounding tissues during development. Members of the FoxA subfamily of transcription factors are expressed in the structures that compose this centre. Foxa2 is essential for dorsal midline development in mammals, since knock-out mouse embryos lack a definitive node, notochord and floor plate. The related gene foxA4 is only present in amphibians. Expression begins in the blastula -chordin and -noggin expressing centre (BCNE) and is later restricted to the dorsal midline derivatives of the Spemann's organiser. It was suggested that the early functions of mammalian foxa2 are carried out by foxA4 in frogs, but functional experiments were needed to test this hypothesis. Here, we show that some important dorsal midline functions of mammalian foxa2 are exerted by foxA4 in Xenopus. We provide new evidence that the latter prevents the respecification of dorsal midline precursors towards contiguous fates, inhibiting prechordal and paraxial mesoderm development in favour of the notochord. In addition, we show that foxA4 is required for the correct regionalisation and maintenance of the central nervous system. FoxA4 participates in constraining the prospective rostral forebrain territory during neural specification and is necessary for the correct segregation of the most anterior ectodermal derivatives, such as the cement gland and the pituitary anlagen. Moreover, the early expression of foxA4 in the BCNE (which contains precursors of the whole forebrain and most of the midbrain and hindbrain) is directly required to restrict anterior neural development.
Assuntos
Sistema Nervoso Central/embriologia , Sistema Nervoso Central/metabolismo , Embrião não Mamífero/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Mesoderma/embriologia , Notocorda/embriologia , Proteínas de Xenopus/metabolismo , Xenopus/embriologia , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Blástula/efeitos dos fármacos , Blástula/metabolismo , Padronização Corporal/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Glicoproteínas/metabolismo , Cabeça/anormalidades , Cabeça/embriologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Modelos Biológicos , Morfogênese/efeitos dos fármacos , Morfolinos/farmacologia , Placa Neural/embriologia , Placa Neural/metabolismo , Neurogênese/efeitos dos fármacos , Notocorda/efeitos dos fármacos , Notocorda/metabolismo , Fenótipo , Xenopus/metabolismoRESUMO
BACKGROUND: Quantitative real time polymerase chain reaction (qPCR) is an extremely powerful technique for monitoring gene expression. The quantity of the messenger ribonucleic acids (mRNA) of interest should be normalized using a reference gene, in order to avoid unreliable results originated by the obtained RNA quality and quantity, manipulation errors and inhibitory contaminants. A reference gene is any gene that is stably and consistently expressed under the conditions being studied. Completely false data can be generated if a reference gene is not chosen adequately. RESULTS: In the present study, we compared expression levels of five putative reference genes (HPRT1, ACTB, GAPDH, RPL13A and B2M) in primary cultures of four different human cells: mesenchymal stromal cells obtained from bone marrow, adipose tissue or umbilical cord Whartons Jelly, and dermal fibroblasts, under different expansion and differentiation conditions. We observed that reference genes are not the same for different cells under the same culture conditions. CONCLUSION: Most stable reference genes under our experimental conditions were: RPL13A for adipose tissue- and Whartons Jelly-derived mesenchymal stromal cells, and HPRT1 for bone marrow-derived mesenchymal stromal cells and dermal fibroblasts. ACTB was the most unstable gene when evaluating adipose tissue- and Whartons Jelly-derived mesenchymal stromal cells, whilst GAPDH and B2M were the most unstable genes for bone marrow-derived mesenchymal stromal cells and dermal fibroblasts, respectively.
Assuntos
Diferenciação Celular , Mesoderma/citologia , Mesoderma/metabolismo , Reação em Cadeia da Polimerase/normas , Técnicas de Cultura de Células , Proliferação de Células , Humanos , Padrões de Referência , Estatística como AssuntoRESUMO
BACKGROUND: Neural tube defects (NTD) involve disruptions in the axial mesenchyme, and are related to an imbalance between folic acid (FA) and homocysteine (Hcy). This study evaluated the effects of FA/Hcy imbalance on cell proliferation and expression of the Pax 1/9 and Sox 9 gene products in the axial mesenchyme of chickens. METHODS: Embryos were incubated (38°C) and pretreated at 24 h and treated at 46 h of incubation. The experimental groups were: FA-pretreated with saline and treated with 0.5 µg FA/saline; Hcy-pretreated with 50 µl saline and treated with 20 µmol D,L-Hcy/50 µl saline; FA+Hcy-pretreated with 0.5 µg FA/50 µl saline and treated with 20 µmol D,L-Hcy/50 µl saline; and the control embryos were pretreated and treated with saline. Embryos were analyzed at E4 and E6. Immunohistochemistry was performed to identify proliferating cells and the expression of the gene products of Pax 1/9 and Sox 9. Total RNA of the E4 embryos was extracted and a RT-qPCR assay was performed to quantify Pax 1/9 mRNA expression. RESULTS: Hcy treatment caused spinal NTD and abnormalities in axial mesenchyme development, affecting the distribution of sclerotomal cells and chondrification. Hcy also reduced cell proliferation and changed the expression of Pax 1/9 and Sox 9 in the mesenchyme. CONCLUSIONS: Our data clarified the relationship between spinal NTD genesis and disruptions of Pax 1/9 and Sox 9 gene products in the axial mesenchyme caused by the FA/Hcy imbalance.