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1.
World Neurosurg ; 136: e469-e475, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31953100

RESUMO

OBJECTIVE: The present study aimed to characterize the mechanism of fluid shear stress (FSS)-induced endothelial cell (EC) injury via protein kinase C alpha (PKCα)-mediated vascular endothelial cadherin (VE-cadherin) and p120-catenin (p120ctn) expression. METHODS: We designed a T chamber system that produced stable FSS on ECs in vitro. Human umbilical vein endothelial cells (HUVECs) in which PKCα was knocked down and normal HUVECs were cultured on the coverslips. FSS was impinged on these 2 types of ECs for 0 hours and 6 hours. The morphology and density of HUVECs were evaluated, and expression levels of phosphorylated PKCα, p120-catenin (p120ctn), VE-cadherin, phosphorylated p120ctn at S879 (p-S879p120ctn), and nuclear factor kappa B (NF-κB) were analyzed by Western blot. RESULTS: HUVECs exposed to FSS were characterized by a polygonal shape and decreased cell density. The phosphorylated PKCα level was increased under FSS at 6 hours (P < 0.05). In normal HUVECs during FSS, p120ctn and VE-cadherin were decreased, whereas p-S879p120ctn and NF-κB were increased, at 6 hours (P < 0.05). In HUVECs after PKCα knockdown, p120ctn and VE-cadherin were not significantly changed (P > 0.05), p-S879p120ctn was undetectable, but NF-κB was decreased (P < 0.05) at 6 hours. CONCLUSIONS: The possible mechanism of FSS-induced EC injury may be as follows: 1) PKCα induces low expression of p120ctn, which leads to activation of NF-κB and degradation of VE-cadherin; 2) PKCα-mediated phosphorylation of p120ctn at S879 disrupts p120ctn binding to VE-cadherin.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Cateninas/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Proteína Quinase C-alfa/metabolismo , Estresse Fisiológico/fisiologia , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Técnicas de Silenciamento de Genes , Humanos
2.
Chemosphere ; 241: 125086, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31627110

RESUMO

Triclosan (TCS) is widely used in personal care products, and its chronic exposure leads to severely toxic effects in zebrafish (Danio rerio). PKCα, Nrf2 and p53 are three important signaling pathways concerned with cell development. Herein, we speculated on and verified a novel TCS regulatory pathway: (1) TCS acted on GPER (G-protein-coupled estrogen receptor) to activate MAPK/ERK pathway, further resulting in the expression changes of protein kinase C (PKC) family; (2) PKC participated in Nrf2 phosphorylation; (3) The expression of miR-125b was regulated by Nrf2; and (4) The expression changes of related genes in the PKCs-Nrf2-ARE pathway showed the specificity of zebrafish tissue and organ. TCS exposure led to down-regulation of the Nrf2 and phosphorylated Nrf2(Ser40) protein in diencephalon nucleus, stratum marginale and stratum centrale areas in adult zebrafish brain. The phosphorylated Nrf2(Ser40) was mainly expressed in PGz area, while it was not the case for Nrf2. Both Nrf2 and phosphorylated Nrf2 were activated by TCS exposure; however, the changing trend of PKCs was opposite to that of Nrf2 in the liver. Both DAPI staining and Merge images demonstrated that TCS induced oxidative phosphorylation, and phosphorylated Nrf2 is translocated into the nucleus as the transcription factor to regulate gene transcription in liver and brain. Nrf2 over-expression increased accumulation of lipid droplets in yolk, brain and liver, resulting from the upregulation of pri-miR-125b1, pri-miR-125b3, but not pri-miR-125b2. These findings reveal the upstream regulation mechanism of miR-125b for TCS-induced fat-metabolism disorder from the regulatory perspective of the pri-miR-125b promoter region.


Assuntos
Encéfalo/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , MicroRNAs , Fator 2 Relacionado a NF-E2/metabolismo , Triclosan/toxicidade , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra , Animais , Encéfalo/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fosforilação/efeitos dos fármacos , Proteína Quinase C-alfa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética
3.
Acta Biochim Biophys Sin (Shanghai) ; 52(1): 72-83, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31844893

RESUMO

Type 2 diabetes increases the risk for cancer. Centrosome amplification can initiate tumorigenesis. We have described that type 2 diabetes increases the centrosome amplification of peripheral blood mononuclear cells, with high glucose, insulin, and palmitic acid as the triggers, which suggests that centrosome amplification is a candidate biological mechanism linking diabetes to cancer. In this study, we aimed to further investigate the signaling pathways of the diabetes-associated centrosome amplification and to examine whether and how resveratrol inhibits the centrosome amplification. The results showed that treatment with high glucose, insulin, and palmitic acid, alone or in combination, could increase the protein levels of phospho-protein kinase C alpha (p-PKCα), phospho-p38 mitogen-activated protein kinases (p-p38), c-myc, and c-jun, as well as the mRNA levels of c-myc and c-jun. PKCα inhibitor could inhibit the treatment-induced increase in the protein levels of p-p38, c-myc, and c-jun. Inhibitor or siRNA of p38 was also able to inhibit the treatment-induced increase in the levels of p-p38, c-myc, and c-jun. Meanwhile, knockdown of c-myc or c-jun did not alter the treatment-induced increase in the phosphorylation of PKCα or p38. Importantly, inhibition of the phosphorylation of PKCα or p38 and knockdown of c-myc or c-jun could attenuate the centrosome amplification. In diabetic mice, the levels of p-PKCα, p-p38, c-myc, and c-jun were all increased in the colon tissues. Interestingly, resveratrol, but not metformin, was able to attenuate the treatment-induced increase in the levels of p-PKCα, p-p38, c-myc, and c-jun, as well as the centrosome amplification. In conclusion, our results suggest that PKCα-p38 to c-myc/c-jun is the signaling pathway of the diabetes-associated centrosome amplification, and resveratrol attenuates the centrosome amplification by inhibiting this signaling pathway.


Assuntos
Centrossomo/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Proteína Quinase C-alfa/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Resveratrol/farmacologia , Resveratrol/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Centrossomo/metabolismo , Colo/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Técnicas de Silenciamento de Genes , Glucose/farmacologia , Células HCT116 , Humanos , Insulina/farmacologia , Camundongos , Ácido Palmítico/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Quinase C-alfa/genética , Proteínas Proto-Oncogênicas c-myc/genética , RNA Interferente Pequeno/genética , Estreptozocina/efeitos adversos , Estreptozocina/farmacologia , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/genética
4.
Int J Mol Sci ; 20(17)2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31480672

RESUMO

Cardiovascular diseases have a high prevalence worldwide and constitute the leading causes of mortality. Recently, malfunctioning of ß-catenin signaling has been addressed in hypertensive heart condition. Ang-II is an important mediator of cardiovascular remodeling processes which not only regulates blood pressure but also leads to pathological cardiac changes. However, the contribution of Ang-II/ß-catenin axis in hypertrophied hearts is ill-defined. Employing in vitro H9c2 cells and in vivo spontaneously hypertensive rats (SHR) cardiac tissue samples, western blot analysis, luciferase assays, nuclear-cytosolic protein extracts, and immunoprecipitation assays, we found that under hypertensive condition ß-catenin gets abnormally induced that co-activated LEF1 and lead to cardiac hypertrophy changes by up-regulating the IGF-IIR signaling pathway. We identified putative LEF1 consensus binding site on IGF-IIR promoter that could be regulated by ß-catenin/LEF1 which in turn modulate the expression of cardiac hypertrophy agents. This study suggested that suppression of ß-catenin expression under hypertensive condition could be exploited as a clinical strategy for cardiac pathological remodeling processes.


Assuntos
Cardiomegalia/induzido quimicamente , Cardiomegalia/metabolismo , Receptor IGF Tipo 2/metabolismo , Transdução de Sinais , beta Catenina/metabolismo , Angiotensina II , Animais , Biomarcadores/metabolismo , Cardiomegalia/patologia , Núcleo Celular/metabolismo , Fator de Transcrição GATA4/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Miócitos Cardíacos/metabolismo , Fatores de Transcrição NFATC/metabolismo , Regiões Promotoras Genéticas/genética , Proteína Quinase C-alfa/metabolismo , Ratos Endogâmicos SHR
5.
J Pharmacol Sci ; 140(3): 263-272, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31474557

RESUMO

Atypical antipsychotics improve positive and negative symptoms but are not effective for treating cognitive impairments in patients with schizophrenia. We previously reported that cognitive impairments in neonatal ventral hippocampus (NVH)-lesioned rats show resistance to atypical antipsychotics risperidone and are associated with reduced calcium/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) signaling in memory-related regions. The cognitive enhancer ST101 (spiro[imi-dazo[1,2-a]pyridine-3,2-indan]-2(3H)-one) stimulates CaMKII activity in the hippocampus and medial prefrontal cortex (mPFC). We thus tested ST101 on cognitive impairments in NVH-lesioned rats. Chronic ST101 administration (0.1 and/or 0.5 mg/kg, p.o.) significantly improved deficits in prepulse inhibition (PPI), social interaction, and cognitive function in NVH-lesioned rats. ST101 administration (0.5 mg/kg, p.o.) significantly restored the decreased CaMKII autophosphorylation (Thr-286) in the mPFC and hippocampal CA1 regions of NVH-lesioned rats when assessed by immunohistochemistry. Chronic ST101 administration (0.1 mg/kg, p.o.) improved the decline in phosphorylation levels of CaMKII (Thr-286), PKCα (Ser-657), α-amino-3-hydroxy-5-methyl-4-isoxazol- propionic acid (AMPA)-type glutamate receptor subunit 1 (GluA1: Ser-831), and N-methyl-d-aspartate (NMDA) receptor subunit 1 (GluN1: Ser-896) in the mPFC and hippocampal CA1 regions. Taken together, these results suggest that ST101 improves schizophrenia-like behaviors and cognitive impairment by enhancing CaMKII/PKCα signaling in the mPFC and hippocampus in NVH-lesioned rats.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Transtornos Cognitivos/tratamento farmacológico , Hipocampo/efeitos dos fármacos , Indanos/farmacologia , Proteína Quinase C-alfa/metabolismo , Esquizofrenia/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Compostos de Espiro/farmacologia , Animais , Animais Recém-Nascidos , Antipsicóticos/farmacologia , Cognição/efeitos dos fármacos , Transtornos Cognitivos/metabolismo , Feminino , Hipocampo/metabolismo , Masculino , Memória/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Risperidona/farmacologia , Esquizofrenia/metabolismo
6.
Biosci Biotechnol Biochem ; 83(9): 1676-1682, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31094294

RESUMO

Protein kinase C (PKC) is a class of phospholipid-dependent serine/threonine kinases that contribute to cell survival, migration, and invasion. Previous studies demonstrated that PKC participates in insulin secretion. However, the role of PKC in glucose-stimulated insulin secretion (GSIS) remains unclear. Herein, we demonstrated that PKC is an important mediator of insulin secretion and revealed a close relationship between PKC activation and insulin secretion in INS-1E cells. Meanwhile, the presence of PKCα was found to induce TRPC1 phosphorylation in INS-1E cells. TRPC1 phosphorylation levels increased by activating PKCα activity. Inhibition of PKCα activity reduced TRPC1 phosphorylation. Finally, we showed that TRPC1 could reverse the decrease in intracellular Ca2+ levels and reduced insulin secretion induced by treatment with PKCα inhibitor under high glucose conditions. In conclusion, our findings indicated that TRPC1 and PKCα are involved in promoting insulin secretion and that PKCα promotes insulin secretion via TRPC1 phosphorylation in INS-1E cells.


Assuntos
Secreção de Insulina , Proteína Quinase C-alfa/metabolismo , Canais de Cátion TRPC/metabolismo , Linhagem Celular , Glucose/metabolismo , Humanos , Fosforilação , Transdução de Sinais
7.
Biochim Biophys Acta Proteins Proteom ; 1867(7-8): 710-721, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31096047

RESUMO

Members of the casein kinase 1 (CK1) family are involved in regulation of crucial cellular pathways including chromosomal segregation, DNA repair, and apoptosis. Therefore, the activity of CK1 isoforms needs to be tightly regulated in order to avoid pathogenesis of proliferative diseases. Regulation of cellular CK1 activity is mainly mediated by (auto-) phosphorylation within its C-terminal regulatory domain. Cellular kinases, among them protein kinase A (PKA), checkpoint kinase 1 (Chk1), protein kinase C α (PKCα), and cyclin-dependent kinases (CDKs) have already been identified to C-terminally phosphorylate CK1δ, thereby modulating its kinase activity. In the present study we analyzed the CK1δ kinase domain for phosphorylation sites targeted by PKCα. Several phosphorylation sites were identified in vitro by initially using GST-CK1δ wild type and phosphorylation-site mutant protein fragments originating from the CK1δ kinase domain. Residues S53, T176, and S181 could finally be confirmed as targets for PKCα. Determination of kinetic parameters of full-length wild type and mutant GST-CK1δ-mediated substrate phosphorylation revealed that integrity of residue T176 is crucial for maintaining CK1δ kinase activity. Functional biochemical and cell culture-based analysis discovered that site-specific phosphorylation of CK1δ by PKCα contributes to fine-tuning of CK1δ kinase activity. In summary, our work for the first time demonstrates the effects of PKCα-mediated site-specific phosphorylation in the CK1δ kinase domain and enhances our knowledge about the regulation of the disease-associated CK1 kinase family.


Assuntos
Proteína Quinase C-alfa/metabolismo , Proteína Quinase C-delta/metabolismo , Substituição de Aminoácidos , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Fosforilação/genética , Domínios Proteicos , Proteína Quinase C-alfa/genética , Proteína Quinase C-delta/genética
8.
Cell Biol Int ; 43(6): 678-694, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30977575

RESUMO

We sought to determine the mechanism by which angiotensin II (ANGII) stimulates NADPH oxidase-mediated superoxide (O2 .- ) production in bovine pulmonary artery smooth muscle cells (BPASMCs). ANGII-induced increase in phospholipase D (PLD) and NADPH oxidase activities were inhibited upon pretreatment of the cells with chemical and genetic inhibitors of PLD2, but not PLD1. Immunoblot study revealed that ANGII treatment of the cells markedly increases protein kinase C-α (PKC-α), -δ, -ε, and -ζ levels in the cell membrane. Pretreatment of the cells with chemical and genetic inhibitors of PKC-ζ, but not PKC-α, -δ, and -ε, attenuated ANGII-induced increase in NADPH oxidase activity without a discernible change in PLD activity. Transfection of the cells with p47phox small interfering RNA inhibited ANGII-induced increase in NADPH oxidase activity without a significant change in PLD activity. Pretreatment of the cells with the chemical and genetic inhibitors of PLD2 and PKC-ζ inhibited ANGII-induced p47phox phosphorylation and subsequently translocation from cytosol to the cell membrane, and also inhibited its association with p22phox (a component of membrane-associated NADPH oxidase). Overall, PLD-PKCζ-p47phox signaling axis plays a crucial role in ANGII-induced increase in NADPH oxidase-mediated O2 .- production in the cells.


Assuntos
Angiotensina II/farmacologia , NADPH Oxidases/metabolismo , Fosfolipase D/metabolismo , Angiotensina II/metabolismo , Angiotensina II/fisiologia , Animais , Bovinos , Técnicas de Cultura de Células/métodos , Membrana Celular/metabolismo , Miócitos de Músculo Liso/metabolismo , NADPH Oxidases/fisiologia , Oxirredução , Fosfolipase D/antagonistas & inibidores , Fosfoproteínas/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Proteína Quinase C-alfa/metabolismo , Artéria Pulmonar/metabolismo , Transdução de Sinais/efeitos dos fármacos , Superóxidos/metabolismo
9.
Bioelectromagnetics ; 40(3): 180-187, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30920672

RESUMO

Previously, we found that exposure to a 50-Hz magnetic field (MF) could induce human amniotic epithelial (FL) cell proliferation and sphingosine kinase 1 (SK1) activation, but the mechanism was not clearly understood. In the present study, the possible signaling pathways which were involved in SK1 activation induced by 50-Hz MF exposure were investigated. Results showed that MF exposure increased intracellular Ca2+ which was dependent on the L-type calcium channel, and induced Ca2+ -dependent phosphorylation of extracellular regulated protein kinase (ERK), SK1, and protein kinase C α (PKCα). Also, treatment with U0126, an inhibitor of ERK, could block MF-induced SK1 phosphorylation, but had no effect on PKCα phosphorylation. Also, the inhibitor of PKCα, Gö6976, had no effect on MF-induced SK1 activation in FL cells. In addition, the activation of ERK and PKCα could be abolished by SKI II, the inhibitor of SK1. In conclusion, the intracellular Ca2+ mediated the 50-Hz MF-induced SK1 activation which enhanced PKCα phosphorylation, and there might be a feedback mechanism between SK1 and ERK activation in responding to MF exposure in FL cells. Bioelectromagnetics. 9999:XX-XX, 2019. © 2019 Bioelectromagnetics Society.


Assuntos
Cálcio/metabolismo , Campos Magnéticos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transdução de Sinais , Canais de Cálcio Tipo L/metabolismo , Linhagem Celular , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Espaço Intracelular/metabolismo , Fosforilação , Proteína Quinase C-alfa/metabolismo
10.
Int J Biochem Cell Biol ; 110: 91-102, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30849522

RESUMO

Lysophosphatidic acid (LPA) is a ubiquitous lysophospholipid that induces a wide range of cellular processes such as wound healing, differentiation, proliferation, migration, and survival. LPA signaling is increased in a number of cancers. In Glioblastoma (GBM), the most aggressive brain tumor, autotaxin the enzyme that produces LPA and its receptor LPA1 are overexpressed. LPA1 is preferentially couple to Gαq proteins in these tumors that in turn activates PKCs. PKCs are involved in many cellular processes including proliferation and metastasis. In this study, we aimed to determine if a classical PKC (α isozyme), could be activated through LPA1 in GBM cell lines and if this activation impacts on cell number. We found that LPA1 induces PKCα translocation to the nucleus, but not to the cell membrane after LPA treatment and the cell number diminished when LPA1/PKCα signaling was blocked, suggesting a relevant role of LPA1 and PKCα in GBM growth.


Assuntos
Núcleo Celular/metabolismo , Glioblastoma/patologia , Proteína Quinase C-alfa/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transporte Ativo do Núcleo Celular , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Diester Fosfórico Hidrolases/metabolismo
11.
Curr Top Med Chem ; 19(2): 116-122, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30834833

RESUMO

Several phenolic compounds bind to proteins (such as enzymes) and interfere in their catalytic mechanism. Interaction studies of natural polyphenol; Resveratrol with various targets like with tubulin, protein kinase C alpha (PKCα), phosphodiesterase-4D, human oral cancer cell line proteins, DNA sequences having AATT/TTAA segments, protein kinase C alpha, lysine-specific demethylase 1 have been reviewed in this article. Simulation studies indicate that resveratrol and its analogs/ derivatives show good interaction with the target receptor through its hydroxyl groups by forming hydrogen bonds and hydrophobic interactions with amino acid residues at the binding site. Binding geometry and stability of complex formed by resveratrol show that it is a good inhibitor for many pathogenic targets. Further studies in this direction is, however, the need of the hour to develop many more ligands based on resveratrol skeleton which can further serve in the treatment of ailments.


Assuntos
Resveratrol/análise , Sítios de Ligação , Catálise , Linhagem Celular Tumoral , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Histona Desmetilases/metabolismo , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Proteína Quinase C-alfa/metabolismo , Resveratrol/química , Resveratrol/metabolismo , Tubulina (Proteína)/metabolismo
12.
J Biol Chem ; 294(13): 5082-5093, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30733340

RESUMO

Twist1 is a basic helix-loop-helix transcription factor that plays a key role in embryonic development, and its expression is down-regulated in adult cells. However, Twist1 is highly expressed during cancer development, conferring a proliferative, migratory, and invasive phenotype to malignant cells. Twist1 expression can be regulated post-translationally by phosphorylation or ubiquitination events. We report in this study a previously unknown and relevant Twist1 phosphorylation site that controls its stability. To identify candidate phosphorylation sites in Twist1, we first conducted an in silico analysis of the Twist1 protein, which yielded several potential sites. Because most of these sites were predicted to be phosphorylated by protein kinase C (PKC), we overexpressed PKCα in several cell lines and found that it phosphorylates Twist1 on Ser-144. Using a combination of immunoblotting, immunoprecipitation, protein overexpression, and CRISPR/Cas9-mediated PKCα knockout experiments, we observed that PKCα-mediated Twist1 phosphorylation at Ser-144 inhibits Twist1 ubiquitination and consequently stabilizes it. These results provide evidence for a direct association between PKCα and Twist1 and yield critical insights into the PKCα/Twist1 signaling axis that governs cancer aggressiveness.


Assuntos
Proteínas Nucleares/metabolismo , Proteína Quinase C-alfa/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Ubiquitinação , Transição Epitelial-Mesenquimal , Células HEK293 , Humanos , Modelos Moleculares , Proteínas Nucleares/química , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteína 1 Relacionada a Twist/química
13.
EBioMedicine ; 40: 151-162, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30711516

RESUMO

BACKGROUND: Programmed death-ligand 1 (PD-L1) is a T-cell inhibitory checkpoint molecule that suppresses antitumor immunity. Anti-PD-L1 antibodies have shown remarkable promise in treating tumors, but the patient response rate is low. Therefore, small-molecule checkpoint inhibitors blocking PD-L1 function are urgently needed. METHODS: Changes of protein expression and phosphorylation levels were determined by immunoblotting. The level of Membrane PD-L1 was examined by flow cytometer. Cytotoxicity of T cells and NK cells toward tumor cells were detected using LDH and cell index assays. Lysosome function was investigated by NAG assay. Changes in lysosomal-related genes were measured by RT-PCR. In vivo anti-NSCLC cancer effects were assessed using C57BL/6 mice bearing Lewis tumor xenografts. FINDINGS: We identified SA-49 as a new regulator of PD-L1 expression from a series of novel aloperine derivatives. SA-49 decreased the expression of PD-L1 in NSCLC cells and enhanced the cytotoxicity of co-cultured T and NK cells toward tumor cells. Importantly, lysosomal pathway contributed to SA-49-mediated down-regulation of PD-L1. SA-49 increased the biogenesis of lysosome and promoted translocation of PD-L1 to lysosome for proteolysis, which was associated with nuclear translocation of MITF. SA-49-induced MITF translocation acted through activation of PKCα and subsequently suppression of GSK3ß activity. Furthermore, SA-49 suppressed Lewis tumor xenograft growth by activating immune microenvironment in C57BL/6 mice. INTERPRETATION: Our data demonstrate that SA-49 can be used to regulate PD-L1 in cancer cells and trigger its degradation by activating lysosome function.


Assuntos
Antígeno B7-H1/metabolismo , Lisossomos/metabolismo , Fator de Transcrição Associado à Microftalmia/metabolismo , Piperidinas/farmacologia , Animais , Linhagem Celular Tumoral , Feminino , Glicogênio Sintase Quinase 3 beta/metabolismo , Xenoenxertos , Humanos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Modelos Biológicos , Piperidinas/química , Proteína Quinase C-alfa/metabolismo , Proteólise , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Microambiente Tumoral
14.
Cell Physiol Biochem ; 52(1): 76-93, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30790506

RESUMO

BACKGROUND/AIMS: Protein kinase C (PKC)- and RhoA/Rho-associated kinase (ROCK) play important roles in arterial sustained contraction. Although depolarization-elicited RhoA/ROCK activation is accepted, the role of PKC in depolarized vascular smooth muscle cells (VSMCs) is a subject of controversy. Our aim was to study the role of PKC in arterial contraction and its interaction with RhoA/ROCK. METHODS: Mass spectrometry was used to identify the PKC isoenzymes. PKCα levels and RhoA activity were analyzed by western blot and G-LISA, respectively, and isometric force was measured in arterial rings. RESULTS: In depolarized VSMCs RhoA and PKCα were translocated to the plasma membrane, where they colocalize and coimmunoprecipitate. Interestingly, depolarization-induced RhoA activation was downregulated by PKCα, effect reverted by PKCα inhibition. Phorbol 12,13-dibutyrate (PDBu) induced the translocation of PKCα to the plasma membrane, increased the level of RhoA in the cytosol and reduced RhoA/ROCK activity. These effects were reverted when PKC was inhibited. Pharmacological or siRNA inhibition of PKCα synergistically potentiated the vasorelaxant effect of RhoA/ROCK inhibition. CONCLUSION: The present study provides the first evidence that RhoA activity is downregulated by PKCα in depolarized and PDBu treated freshly isolated VSMCs and arteries, with an important physiological role on arterial contractility.


Assuntos
Membrana Celular/enzimologia , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Proteína Quinase C-alfa/metabolismo , Vasodilatação , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Masculino , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Dibutirato de 12,13-Forbol/farmacologia , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Wistar , Quinases Associadas a rho/metabolismo
15.
J Immunol ; 202(5): 1540-1548, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30683702

RESUMO

IL-17A is a critical proinflammatory cytokine for the pathogenesis of asthma including neutrophilic pulmonary inflammation and airway hyperresponsiveness. In this study, by cell type-specific deletion of IL-17R and adaptor Act1, we demonstrated that IL-17R/Act1 exerts a direct impact on the contraction of airway smooth muscle cells (ASMCs). Mechanistically, IL-17A induced the recruitment of Rab35 (a small monomeric GTPase) and DennD1C (guanine nucleotide exchange factor [GEF]) to the IL-17R/Act1 complex in ASMCs, resulting in activation of Rab35. Rab35 knockdown showed that IL-17A-induced Rab35 activation was essential for protein kinase Cα (PKCα) activation and phosphorylation of fascin at Ser39 in ASMCs, allowing F-actin to interact with myosin to form stress fibers and enhance the contraction induced by methacholine. PKCα inhibitor or Rab35 knockdown indeed substantially reduced IL-17A-induced stress fiber formation in ASMCs and attenuated IL-17A-enhanced, methacholine-induced contraction of airway smooth muscle. Taken together, these data indicate that IL-17A promotes airway smooth muscle contraction via direct recruitment of Rab35 to IL-17R, followed by PKCα activation and stress fiber formation.


Assuntos
Interleucina-17/metabolismo , Músculo Liso/metabolismo , Proteína Quinase C-alfa/antagonistas & inibidores , Receptores de Interleucina-17/metabolismo , Fibras de Estresse/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Interleucina-17/antagonistas & inibidores , Interleucina-17/deficiência , Camundongos , Camundongos Knockout , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Proteína Quinase C-alfa/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptores de Interleucina-17/antagonistas & inibidores , Fibras de Estresse/efeitos dos fármacos , Proteínas rab de Ligação ao GTP/antagonistas & inibidores
16.
Histochem Cell Biol ; 151(6): 521-530, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30604284

RESUMO

The retina is a complex neural circuit, which processes and transmits visual information from light perceiving photoreceptors to projecting retinal ganglion cells. Much of the computational power of the retina rests on signal integrating interneurons, such as bipolar cells. Commercially available antibodies against bovine and human conventional protein kinase C (PKC) α and -ß are frequently used as markers for retinal ON-bipolar cells in different species, despite the fact that it is not known which bipolar cell subtype(s) they actually label. In zebrafish (Danio rerio) five prkc genes (coding for PKC proteins) have been identified. Their expression has not been systematically determined. While prkcg is not expressed in retinal tissue, the other four prkc (prkcaa, prkcab, prkcba, prkcbb) transcripts were found in different parts of the inner nuclear layer and some as well in the retinal ganglion cell layer. Immunohistochemical analysis in adult zebrafish retina using fluorescent in situ hybridization and PKC antibodies showed an overlapping immunolabeling of ON-bipolar cells that are most likely of the BON s6 and BON s6L or RRod type. However, comparison of transcript expression with immunolabeling, implies that these antibodies are not specific for one single zebrafish conventional PKC, but rather detect a combination of PKC -α and -ß variants.


Assuntos
Proteína Quinase C beta/metabolismo , Proteína Quinase C-alfa/metabolismo , Retina/enzimologia , Peixe-Zebra/metabolismo , Animais , Hibridização in Situ Fluorescente , Proteína Quinase C beta/análise , Proteína Quinase C-alfa/análise , Retina/metabolismo
17.
J Endocrinol ; 240(2): 345-360, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30508412

RESUMO

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 & inibidores
18.
Infect Immun ; 87(1)2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30348826

RESUMO

Listeria monocytogenes is a foodborne bacterium that causes gastroenteritis, meningitis, or abortion. Listeria induces its internalization (entry) into some human cells through interaction of the bacterial surface protein InlB with its host receptor, the Met tyrosine kinase. InlB and Met promote entry, in part, through stimulation of localized exocytosis. How exocytosis is upregulated during entry is not understood. Here, we show that the human signaling proteins mTOR, protein kinase C-α (PKC-α), and RalA promote exocytosis during entry by controlling the scaffolding protein Filamin A (FlnA). InlB-mediated uptake was accompanied by PKC-α-dependent phosphorylation of serine 2152 in FlnA. Depletion of FlnA by RNA interference (RNAi) or expression of a mutated FlnA protein defective in phosphorylation impaired InlB-dependent internalization. These findings indicate that phosphorylation of FlnA by PKC-α contributes to entry. mTOR and RalA were found to mediate the recruitment of FlnA to sites of InlB-mediated entry. Depletion of PKC-α, mTOR, or FlnA each reduced exocytosis during InlB-mediated uptake. Because the exocyst complex is known to mediate polarized exocytosis, we examined if PKC-α, mTOR, RalA, or FlnA affects this complex. Depletion of PKC-α, mTOR, RalA, or FlnA impaired recruitment of the exocyst component Exo70 to sites of InlB-mediated entry. Experiments involving knockdown of Exo70 or other exocyst proteins demonstrated an important role for the exocyst complex in uptake of Listeria Collectively, our results indicate that PKC-α, mTOR, RalA, and FlnA comprise a signaling pathway that mobilizes the exocyst complex to promote infection by Listeria.


Assuntos
Proteínas de Bactérias/metabolismo , Endocitose , Exocitose , Filaminas/metabolismo , Interações Hospedeiro-Patógeno , Listeria monocytogenes/fisiologia , Proteínas de Membrana/metabolismo , Proteína Quinase C-alfa/metabolismo , Células HeLa , Humanos , Listeria monocytogenes/metabolismo , Mapas de Interação de Proteínas
19.
Surgery ; 165(1): 202-210, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30413320

RESUMO

BACKGROUND: Adrenocortical carcinoma is a rare and aggressive malignancy with poor survival. With limited treatment options and high risk of relapse, identifying improved targets and therapies for adrenocortical carcinoma is important. We hypothesized that analysis of the database of The Cancer Genome Atlas could identify important novel biomarkers for improved therapeutic targeting of adrenocortical carcinoma. METHODS: We utilized the University of Alabama interactive web resource to identify novel biomarkers observed in 79 adrenocortical carcinoma patients. Identified biomarkers were then examined for prognostic correlations using the cBioPortal and analyzed for statistical significance using STATA 13.0. RESULTS: The Cancer Genome Atlas data mining in the University of Alabama interactive web resource for pathways associated with poor survival of patients with adrenocortical carcinoma revealed significant upregulation of genes involved in DNA damage and regulation of cell-cycle pathways, such as AURKA, AURKB, CDK1, CDK4, CDK6, PLK1, CHEK1, CHEK2, CDC7, BUB3, and MCM3 (P < .001-.05). On outcome correlation, greater expression levels of all the genes except CDK4 were associated with worse survival compared with medium or low levels of gene expression (P < .001 all) irrespective of age orsex. Consistent with our University of Alabama interactive web resource findings, data mining in the cBioPortal also revealed upregulation of genes regulating DNA-damage and cell cycle-related genes in 82% of patients (z score = 1.5). CONCLUSION: Large data mining from the The Cancer Genome Atlas and cBioPortal databases identified overexpression of genes involved in DNA damage and those regulating pathways of the cell cycle, which correlated with poorer overall survival in adrenocortical carcinoma patients.


Assuntos
Neoplasias do Córtex Suprarrenal/metabolismo , Neoplasias do Córtex Suprarrenal/mortalidade , Carcinoma Adrenocortical/metabolismo , Carcinoma Adrenocortical/mortalidade , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Neoplasias do Córtex Suprarrenal/genética , Carcinoma Adrenocortical/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Proteínas de Ciclo Celular/genética , Conjuntos de Dados como Assunto , Regulação para Baixo , Fosfatases de Especificidade Dupla/genética , Fosfatases de Especificidade Dupla/metabolismo , Feminino , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Masculino , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Prognóstico , Proteína Quinase C-alfa/genética , Proteína Quinase C-alfa/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Regulação para Cima
20.
In Vitro Cell Dev Biol Anim ; 55(1): 36-44, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30413935

RESUMO

Flavonoids have been chronicles of the history of a long way journey in the cure of physiological or pathophysiological conditions in various diseases including cancer. Our previous findings suggest the extensive mechanism of quercetin (QUE) mediated regression of cell survival, cell proliferation, oxidative stress, inflammation, and angiogenesis via modulating PI3K and PKC signaling in lymphoma as well as hepatocellular carcinoma. PI3K-PKC pathway is a key monitor of mammalian cells regulated by its different isoenzymes, which may exert similar or opposite cellular effects by differential coupling of signaling pathways. Put forward the invention of selective inhibitors against various isoenzymes is beneficial to reduce the burden of inclusive deleterious effects of drug for normal physiological process. Therefore, we hypothesized the improved anticancer efficacy of QUE in combination with isoenzyme inhibitors-rottlerin (ROT-PKCδ inhibitor), G0 6983 (PKCα inhibitor), and PI-103 (p110α-class I PI3K inhibitor) in MCF-7 and RAW 264.7 cells. QUE significantly improves the cytotoxicity of ROT + G0 6983 ranged 30-55% and PI-103 ranged 24-63% after 24-48 h against MCF-7 cells. Additionally in the presence of QUE, the improved cytotoxicity of ROT + G0 6983 is observed to range 69-75% and PI-103 ranged 45-88% after 24-48 h in RAW 264.7 cells. This increment in cell deaths are positively correlated with enhanced morphological alteration observed in MCF-7 cells. Further, QUE significantly increases the attenuation of PKCα level approximately by 50% in combination with PI-103. Overall results of the current study suggested that QUE improves the synergistic anticancer efficacy in combination with PI-103, ROT, and G0 6983 in MCF-7 and RAW 264.7 cells.


Assuntos
Acetofenonas/farmacologia , Antineoplásicos/farmacologia , Benzopiranos/farmacologia , Furanos/farmacologia , Indóis/farmacologia , Maleimidas/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Quercetina/farmacologia , Acetofenonas/química , Animais , Apoptose/efeitos dos fármacos , Benzopiranos/química , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Furanos/química , Humanos , Indóis/química , Células MCF-7 , Maleimidas/química , Camundongos , Proteína Quinase C-alfa/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Piridinas/química , Pirimidinas/química , Quercetina/química , Células RAW 264.7
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