RESUMO
Diabetic rat embryos have increased cortical neurogenesis and neuron maturation, and their offspring presented altered neuron polarity, lamination, and diminished neuron excitability. The FOXP2 overexpression results in higher cortical neurogenesis by increasing the transition of radial glia to the intermediate progenitor. Similarly, histamine through H1-receptor activation increases cortical neuron differentiation. Indeed, blocking the H1-receptor by the systemic administration of chlorpheniramine to diabetic pregnant rats prevents increased neurogenesis. Here, we explore the relationship between the H1-receptor and FOXP2 on embryo neurogenesis from diabetic dams. Through qRT-PCR, Western blot, immunohistofluorescence, and flow cytometry, we showed an increased FOXP2 expression and nuclear localization, a reduced Nestin expression and -positive cells number, and a higher PKCα expression in the cortical neuroepithelium of fourteen-day-old embryos from diabetic rats. Interestingly, this scenario was prevented by the chlorpheniramine systemic administration to diabetic pregnant rats at embryo day twelve. These data, together with the bioinformatic analysis, suggest that higher H1-receptor activity in embryos under high glucose increases FOXP2 nuclear translocation, presumably through PKCα phosphorylation, impairing the transition of radial glia to intermediate progenitor and increasing neuron differentiation in embryos of diabetic rats.
Assuntos
Diabetes Mellitus Experimental , Células-Tronco Neurais , Animais , Feminino , Gravidez , Ratos , Clorfeniramina/metabolismo , Diabetes Mellitus Experimental/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Histamina/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Proteína Quinase C-alfa/metabolismo , Telencéfalo/metabolismo , Receptores Histamínicos H1RESUMO
Anaesthesia with propofol is frequently associated with hypotension, which is at least partially attributable to increased nitric oxide (NO) formation derived from the activation of protein kinase C (PKC)/endothelial NO synthase (NOS3) axis. In this cross-sectional study, we tested whether PRKCA (which encodes PKCα) polymorphisms, or haplotypes, and interactions among PRKCA and NOS3 polymorphisms affect the hypotensive responses to propofol. We collected venous blood samples from 164 patients before and 10 min after propofol administration. Genotypes were determined by PCR and haplotype frequencies were estimated. Nitrite and NOx (nitrites+nitrates) levels were measured by using an ozone-based chemiluminescence assay and the Griess reaction, respectively. We used multifactor dimensionality reduction to test interactions among PRKCA and NOS3 polymorphisms. Propofol promoted enhanced blood pressure-lowering effects and increased nitrite levels in subjects carrying GA + AA genotypes for the rs16960228 and TC + CC genotypes for the rs1010544 PRKCA polymorphisms, and the CCG haplotype. Moreover, genotypes for the rs1010544 PRKCA polymorphism were associated with higher or lower blood pressure decreases in response to propofol depending on the genotypes for the rs2070744 NOS3 polymorphism. Our findings suggest that PRKCA genotypes and haplotypes impact the hypotensive responses to propofol, possibly by modifying NO bioavailability, and that PRKCA-NOS3 interactions modify the blood pressure-lowering effects of propofol.
Assuntos
Hipotensão/induzido quimicamente , Óxido Nítrico Sintase Tipo III/genética , Propofol/efeitos adversos , Proteína Quinase C-alfa/genética , Adulto , Idoso , Anestésicos Intravenosos/administração & dosagem , Anestésicos Intravenosos/efeitos adversos , Estudos Transversais , Feminino , Genótipo , Haplótipos , Humanos , Hipotensão/genética , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/metabolismo , Propofol/administração & dosagemRESUMO
Hijacking the autophagic machinery is a key mechanism through which invasive pathogens such as Staphylococcus aureus replicate in their host cells. We have previously demonstrated that the bacteria replicate in phagosomes labeled with the autophagic protein LC3, before escaping to the cytoplasm. Here, we show that the Ca2+-dependent PKCα binds to S. aureus-containing phagosomes and that α-hemolysin, secreted by S. aureus, promotes this recruitment of PKCα to phagosomal membranes. Interestingly, the presence of PKCα prevents the association of the autophagic protein LC3. Live cell imaging experiments using the PKC activity reporter CKAR reveal that treatment of cells with S. aureus culture supernatants containing staphylococcal secreted factors transiently activates PKC. Functional studies reveal that overexpression of PKCα causes a marked inhibition of bacterial replication. Taken together, our data identify enhancing PKCα activity as a potential approach to inhibit S. aureus replication in mammalian cells.
Assuntos
Autofagia , Interações Hospedeiro-Patógeno , Fagossomos/metabolismo , Proteína Quinase C-alfa/metabolismo , Infecções Estafilocócicas/etiologia , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/fisiologia , Animais , Autofagia/imunologia , Células CHO , Linhagem Celular , Células Cultivadas , Cricetulus , Suscetibilidade a Doenças , Imunofluorescência , Genes Reporter , Interações Hospedeiro-Patógeno/imunologia , Modelos Biológicos , Fagossomos/imunologia , Proteína Quinase C-alfa/genéticaRESUMO
Lysophosphatidic acid (LPA) induces a wide range of cellular processes and its signaling is increased in several cancers including glioblastoma (GBM), a high-grade astrocytoma, which is the most common malignant brain tumor. LPA1 receptor is expressed in GBM cells and its signaling pathways activate protein kinases C (PKCs). A downstream target of PKC, involved in GBM progression, is the intracellular progesterone receptor (PR), which can be phosphorylated by this enzyme, increasing its transcriptional activity. Interestingly, in GBM cells, PKCα isotype translocates to the nucleus after LPA stimulation, resulting in an increase in PR phosphorylation. In this study, we determined that LPA1 receptor activation induces protein-protein interaction between PKCα and PR in human GBM cells; this interaction increased PR phosphorylation in serine400. Moreover, LPA treatment augmented VEGF transcription, a known PR target. This effect was blocked by the PR selective modulator RU486; also, the activation of LPA1/PR signaling promoted migration of GBM cells. Interestingly, using TCGA data base, we found that mRNA expression of LPAR1 increases according to tumor malignancy and correlates with a lower survival in grade III astrocytomas. These results suggest that LPA1/PR pathway regulates GBM progression.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Proteína Quinase C-alfa/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Receptores de Progesterona/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Lisofosfolipídeos/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sobrevida , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Breast cancer is the leading cause of cancer death among women worldwide. Blocking a single signaling pathway is often an ineffective therapy, especially in the case of aggressive or drug-resistant tumors. Since we have previously described the mechanism involved in the crosstalk between Retinoic Acid system and protein kinase C (PKC) pathway, the rationale of our study was to evaluate the effect of combining all-trans-retinoic acid (ATRA) with a classical PCK inhibitor (Gö6976) in preclinical settings. Employing hormone-independent mammary cancer models, Gö6976 and ATRA combined treatment induced a synergistic reduction in proliferative potential that correlated with an increased apoptosis and RARs modulation towards an anti-oncogenic profile. Combined treatment also impairs growth, self-renewal and clonogenicity potential of cancer stem cells and reduced tumor growth, metastatic spread and cancer stem cells frequency in vivo. An in-silico analysis of "Kaplan-Meier plotter" database indicated that low PKCα together with high RARα mRNA expression is a favorable prognosis factor for hormone-independent breast cancer patients. Here we demonstrate that a classical PKC inhibitor potentiates ATRA antitumor effects also targeting cancer stem cells growth, self-renewal and frequency.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Mamárias Experimentais , Proteínas de Neoplasias , Células-Tronco Neoplásicas/enzimologia , Proteína Quinase C beta , Proteína Quinase C-alfa , Animais , Linhagem Celular Tumoral , Feminino , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Proteína Quinase C beta/antagonistas & inibidores , Proteína Quinase C beta/metabolismo , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-alfa/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Tretinoína/farmacologiaRESUMO
PURPOSE: Retinoids have proved to be effective for hematologic malignancies treatment but till nowadays, their use as single agent for the solid tumor's management is still controversial. All-trans retinoic acid (ATRA), the main active metabolite of vitamin A, exerts non-genomic interactions with different members of the protein kinase C (PKC) family, recognized modulators of different tumor progression pathways. To determine whether a group of patients could become benefited employing a retinoid therapy, in this study we have evaluated whether PKCα expression (a poor prognosis marker in breast cancer) could sensitizes mammary cells to ATRA treatment. METHODS: PKCα overexpression was achieved by stable transfection and confirmed by western blot. Transfected PKC functionality was determined by nuclear translocation-induction and confocal microscopy. In vitro proliferation was evaluated by cell counting and cell cycle distribution was analyzed by flow cytometry. In vivo studies were performed to evaluate orthotopic tumor growth and experimental lung colonization. Retinoic acid response elements (RARE) and AP1 sites-dependent activity was studied by gene reporter assays and retinoic acid receptors (RARs) were measured by RT-qPCR. RESULTS: Our findings suggest that high PKCα levels improve the differentiation response to ATRA in a RAR signaling-dependent manner. Moreover, RARß expression appears to be critical to induce ATRA sensitization, throughout AP1 trans-repression. CONCLUSION: Here we propose that retinoids could lead a highly personalized anticancer treatment, bringing benefits to patients with aggressive breast tumors resulting from high PKCα expression but, an adequate expression of the RARß receptor is required to ensure the effect on this process.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Proteína Quinase C-alfa/genética , Receptores do Ácido Retinoico/genética , Tretinoína/farmacologia , Animais , Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Diferenciação Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Xenoenxertos , Humanos , Células MCF-7 , Camundongos , Retinoides/farmacologia , Transdução de Sinais/efeitos dos fármacos , Vitamina A/genéticaRESUMO
Neutrophil extracellular traps (NETs) emerge from the cell as a DNA scaffold associated with cytoplasmic and granular proteins, able to immobilize and kill pathogens. This association occurs following nuclear and granular membrane disintegration, allowing contact with the decondensed chromatin. Thus, it is reasonable to speculate that the DNA can also mix with miRNAs and carry them in NETs. Here, we report for the first time the presence of the miRNA carriers associated with NETs and miRNAs present in NET-enriched supernatants (NET-miRs), thus adding a novel class of molecules and new proteins that can be released and transported in the NET platform. We observed that the majority of NET-miRs were common to all four stimuli used (PMA, interleukin-8, amyloid fibrils and Leishmania), and that miRNA-142-3p carried by NETs down-modulates protein kinase Cα and regulates TNF-α production in macrophages upon NET interaction with these cells. Our findings unveil a novel role for NETs in the cell communication processes, allowing the conveyance of miRNA from neutrophils to neighboring cells.
Assuntos
Comunicação Celular/imunologia , Armadilhas Extracelulares/imunologia , MicroRNAs/genética , Neutrófilos/imunologia , Fator de Necrose Tumoral alfa/genética , Amiloide/farmacologia , Antagomirs/genética , Antagomirs/metabolismo , Meios de Cultivo Condicionados/farmacologia , Armadilhas Extracelulares/metabolismo , Regulação da Expressão Gênica , Humanos , Interleucina-8/farmacologia , Leishmania braziliensis , MicroRNAs/antagonistas & inibidores , MicroRNAs/imunologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/microbiologia , Cultura Primária de Células , Proteína Quinase C-alfa/genética , Proteína Quinase C-alfa/imunologia , Transdução de Sinais , Células THP-1 , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Neurons are continuously produced at different rates and locations in the teleost retina. Goldfish rods are homogeneously distributed and maintain a stable density throughout growth, whereas little is known about their postsynaptic partners. We examined the distribution and density of mixed-input ON bipolar cells (ON mBCs) in 57 goldfish of various sizes by immunolabeling their retinas with an antibody against PKCα and counting PKCα-positive neurons in wholemounts. Cell densities were correlated with morphometric data for the same animals, and the spatial resolution of the ON mBC mosaic was calculated in each case. The distribution of ON mBCs is homogeneous throughout growth. For a 10-fold change in body size (i.e., from 20 to 200 mm), the total number of ON mBCs increases 2.8 times, while retinal area expands around 10 times. As a consequence, the density of ON mBCs in large fish falls to â¼1/3 of that of small animals, and intercellular spacing doubles. The eye and the lens become around three times larger from small to large fish. This causes the retinal magnification factor (and thereby the image projected onto retina) to augment by the same amount. Because the retinal magnification factor rises more than the intercellular spacing in the same animals, the spatial resolution of the ON mBC mosaic improves from 0.8 to 1.4 cycles/degree as the body size increases from 20 to 200 mm. As ON mBCs are mostly rod-driven, our results suggest that the scotopic acuity of the goldfish may improve as the animal grows.
Assuntos
Carpa Dourada/anatomia & histologia , Carpa Dourada/crescimento & desenvolvimento , Neurônios Retinianos/citologia , Animais , Tamanho Corporal , Contagem de Células , Proteínas de Peixes/metabolismo , Carpa Dourada/metabolismo , Imuno-Histoquímica , Tamanho do Órgão , Proteína Quinase C-alfa/metabolismo , Neurônios Retinianos/metabolismoRESUMO
17ß-Estradiol (E2) has been shown to modulate the renin-angiotensin system in hydromineral and blood pressure homeostasis mainly by attenuating angiotensin II (ANGII) actions. However, the cellular mechanisms of the interaction between E2 and angiotensin II (ANGII) and its physiological role are largely unknown. The present experiments were performed to better understand the interaction between ANGII and E2 in body fluid control in female ovariectomized (OVX) rats. The present results are the first to demonstrate that PKC/p38 MAPK signaling is involved in ANGII-induced water and sodium intake and oxytocin (OT) secretion in OVX rats. In addition, previous data from our group revealed that the ANGII-induced vasopressin (AVP) secretion requires ERK1/2 signaling. Therefore, taken together, the present observations support a novel concept that distinct intracellular ANGII signaling gives rise to distinct neurohypophyseal hormone release. Furthermore, the results show that E2 attenuates p38 MAPK phosphorylation in response to ANGII but not PKC activity in the hypothalamus and the lamina terminalis, suggesting that E2 modulates ANGII effects through the attenuation of the MAPK pathway. In conclusion, this work contributes to the further understanding of the interaction between E2 and ANGII signaling in hydromineral homeostasis, as well as it contributes to further elucidate the physiological relevance of PKC/p38 MAPK signaling on the fluid intake and neurohypophyseal release induced by ANGII.
Assuntos
Angiotensina II/farmacologia , Encéfalo/efeitos dos fármacos , Estradiol/farmacologia , Proteína Quinase C-alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Benzofenantridinas/farmacologia , Encéfalo/enzimologia , Ingestão de Líquidos/efeitos dos fármacos , Interações Medicamentosas , Feminino , Homeostase/efeitos dos fármacos , Imidazóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Ovariectomia , Ocitocina/metabolismo , Proteína Quinase C-alfa/antagonistas & inibidores , Piridinas/farmacologia , Ratos Wistar , Vasopressinas/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Although PTHrP is implicated in several cancers, its role in chemoresistance is not fully elucidated. We found that in CRC cells, PTHrP exerts proliferative and protective effects and induces cell migration. The aim of this work was to further study the effects of PTHrP in CRC cells. Herein we evidenced, for the first time, that PTHrP induces resistance to CPT-11 in Caco-2 and HCT116â¯cells; although both cell lines responded to the drug through different molecular mechanisms, the chemoresistance by PTHrP in these models is mediated through ERK, which in turn is activated by PCK, Src and Akt. Moreover, continue administration of PTHrP in nude mice xenografts increased the protein levels of this MAPK and of other markers related to tumorigenic events. The understanding of the molecular mechanisms leading to ERK 1/2 activation and the study of ERK targets may facilitate the development of new therapeutic strategies for CRC treatment.