RESUMEN

BACKGROUND: Variations in untranslated regions (UTR) alter regulatory pathways impacting phenotype, disease onset, and course of disease. Protein kinase C Zeta (PRKCZ), a serine-threonine kinase, is implicated in cardiovascular, neurological and oncological disorders. Due to limited research on PRKCZ, this study aimed to investigate the impact of UTR genetic variants' on binding sites for transcription factors and miRNA. RNA secondary structure, eQTLs, and variation tolerance analysis were also part of the study. METHODS: The data related to PRKCZ gene variants was downloaded from the Ensembl genome browser, COSMIC and gnomAD. The RegulomeDB database was used to assess the functional impact of 5' UTR and 3'UTR variants. The analysis of the transcription binding sites (TFBS) was done through the Alibaba tool, and the Kyoto Encyclopaedia of Genes and Genomes (KEGG) was employed to identify pathways associated with PRKCZ. To predict the effect of variants on microRNA binding sites, PolymiRTS was utilized for 3' UTR variants, and the SNPinfo tool was used for 5' UTR variants. RESULTS: The results obtained indicated that a total of 24 variants present in the 3' UTR and 25 variants present in the 5' UTR were most detrimental. TFBS analysis revealed that 5' UTR variants added YY1, repressor, and Oct1, whereas 3' UTR variants added AP-2alpha, AhR, Da, GR, and USF binding sites. The study predicted TFs that influenced PRKCZ expression. RNA secondary structure analysis showed that eight 5' UTR and six 3' UTR altered the RNA structure by either removal or addition of the stem-loop. The microRNA binding site analysis highlighted that seven 3' UTR and one 5' UTR variant altered the conserved site and also created new binding sites. eQTLs analysis showed that one variant was associated with PRKCZ expression in the lung and thyroid. The variation tolerance analysis revealed that PRKCZ was an intolerant gene. CONCLUSION: This study laid the groundwork for future studies aimed at targeting PRKCZ as a therapeutic target.

Asunto(s)

Regiones no Traducidas 3' , MicroARNs , Proteína Quinasa C , ARN Mensajero , Humanos , Regiones no Traducidas 3'/genética , Regiones no Traducidas 5'/genética , Sitios de Unión , MicroARNs/genética , Conformación de Ácido Nucleico , Polimorfismo de Nucleótido Simple , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Estabilidad del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regiones no Traducidas/genética

RESUMEN

NADPH oxidases (NOXs) are a family of membrane proteins responsible for intracellular reactive oxygen species (ROS) generation by facilitating electron transfer across biological membranes. Despite the established activation of NOXs by protein kinase C (PKC), the precise mechanism through which PKC triggers NOX activation during breast cancer invasion remains unclear. The present study aimed to investigate the role of NOX1 and NOX5 in the invasion of MCF­7 human breast cancer cells. The expression and activity of NOXs and matrix metalloprotease (MMP)­9 were assessed by reverse transcription­quantitative PCR and western blotting, and the activity of MMP­9 was monitored using zymography. Cellular invasion was assessed using the Matrigel invasion assay, whereas ROS levels were quantified using a FACSCalibur flow cytometer. The findings suggested that NOX1 and NOX5 serve crucial roles in 12­O­tetradecanoylphorbol­13­acetate (TPA)­induced MMP­9 expression and invasion of MCF­7 cells. Furthermore, a connection was established between PKC and the NOX1 and 5/ROS signaling pathways in mediating TPA­induced MMP­9 expression and cellular invasion. Notably, NOX inhibitors (diphenyleneiodonium chloride and apocynin) significantly attenuated TPA­induced MMP­9 expression and invasion in MCF­7 cells. NOX1­ and NOX5­specific small interfering RNAs attenuated TPA­induced MMP­9 expression and cellular invasion. In addition, knockdown of NOX1 and NOX5 suppressed TPA­induced ROS levels. Furthermore, a PKC inhibitor (GF109203X) suppressed TPA­induced intracellular ROS levels, MMP­9 expression and NOX activity in MCF­7 cells. Therefore, NOX1 and NOX5 may serve crucial roles in TPA­induced MMP­9 expression and invasion of MCF­7 breast cancer cells. Furthermore, the present study indicated that TPA­induced MMP­9 expression and cellular invasion were mediated through PKC, thus linking the NOX1 and 5/ROS signaling pathways. These findings offer novel insights into the potential mechanisms underlying their anti­invasive effects in breast cancer.

Asunto(s)

Neoplasias de la Mama , Metaloproteinasa 9 de la Matriz , NADPH Oxidasa 1 , NADPH Oxidasa 5 , Proteína Quinasa C , Especies Reactivas de Oxígeno , Acetato de Tetradecanoilforbol , Humanos , Metaloproteinasa 9 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Especies Reactivas de Oxígeno/metabolismo , NADPH Oxidasa 1/metabolismo , NADPH Oxidasa 1/genética , NADPH Oxidasa 5/metabolismo , NADPH Oxidasa 5/genética , Proteína Quinasa C/metabolismo , Células MCF-7 , Femenino , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/genética , Acetato de Tetradecanoilforbol/farmacología , NADPH Oxidasas/metabolismo , NADPH Oxidasas/genética , Invasividad Neoplásica , Movimiento Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Transducción de Señal

RESUMEN

BACKGROUND: Epithelial ovarian cancer, especially high grade serous ovarian cancer (HGSOC) is by far, the most lethal gynecological malignancy with poor prognosis and high relapse rate. Despite of availability of several therapeutic interventions including poly-ADP ribose polymerase (PARP) inhibitors, HGSOC remains unmanageable and identification of early detection biomarkers and therapeutic targets for this lethal malady is highly warranted. Aberrant expression of protein kinase C iota (PKCί) is implicated in many cellular and physiological functions involved in tumorigenesis including cell proliferation and cell cycle deregulation. METHODS AND RESULTS: Two high grade serous ovarian cancer cells SKOV3 and COV362 were employed in this study. PKCί was genetically knocked down or pharmacologically inhibited and several functional and biochemical assays were performed. We report that PKCί is overexpressed in HGSOC cells and patient tissue samples with a significant prognostic value. Pharmacological inhibition of PKCί by Na-aurothiomalate or its shRNA-mediated genetic knockdown suppressed HGSOC cell proliferation, EMT and induced apoptosis. Moreover, PKCί positively regulated GLUT1 and several other glycolytic genes including HK1, HK2, PGK1, ENO1 and LDHA to promote elevated glucose uptake and glycolysis in HGSOC cells. Mechanistically, PKCί drove glycolysis via PI3K/AKT/mTOR signalling. Na-aurothiomalate and highly selective, dual PI3K/mTOR inhibitor dactolisib could serve as novel anti-glycolytic drugs in HGSOC. CONCLUSION: Taken together, our results indicate PKCί/PI3K/AKT/mTOR signalling cascade could be a novel therapeutic target in a lethal pathology like HGSOC.

Asunto(s)

Proliferación Celular , Glucólisis , Isoenzimas , Neoplasias Ováricas , Fosfatidilinositol 3-Quinasas , Proteína Quinasa C , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Serina-Treonina Quinasas TOR , Humanos , Femenino , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Línea Celular Tumoral , Proteína Quinasa C/metabolismo , Proteína Quinasa C/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proliferación Celular/genética , Isoenzimas/metabolismo , Isoenzimas/genética , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patología , Apoptosis/genética , Regulación Neoplásica de la Expresión Génica , Carcinoma Epitelial de Ovario/metabolismo , Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/patología , Pronóstico

RESUMEN

Chronic fibrotic tissue disrupts various organ functions. Despite significant advances in therapies, mortality and morbidity due to heart failure remain high, resulting in poor quality of life. Beyond the cardiomyocyte-centric view of heart failure, it is now accepted that alterations in the interstitial extracellular matrix (ECM) also play a major role in the development of heart failure. Here, we show that protein kinase N (PKN) is expressed in cardiac fibroblasts. Furthermore, PKN mediates the conversion of fibroblasts into myofibroblasts, which plays a central role in secreting large amounts of ECM proteins via p38 phosphorylation signaling. Fibroblast-specific deletion of PKN led to a reduction of myocardial fibrotic changes and cardiac dysfunction in mice models of ischemia-reperfusion or heart failure with preserved ejection fraction. Our results indicate that PKN is a therapeutic target for cardiac fibrosis in heart failure.

Asunto(s)

Fibroblastos , Fibrosis , Insuficiencia Cardíaca , Miocardio , Miofibroblastos , Proteína Quinasa C , Animales , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/genética , Miofibroblastos/metabolismo , Miofibroblastos/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Ratones , Miocardio/patología , Miocardio/metabolismo , Proteína Quinasa C/metabolismo , Proteína Quinasa C/genética , Masculino , Humanos , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Ratones Noqueados , Matriz Extracelular/metabolismo , Fosforilación , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Transducción de Señal

RESUMEN

Obesity causes a range of tissue dysfunctions that increases the risk for morbidity and mortality. Protein kinase D (PKD) represents a family of stress-activated intracellular signalling proteins that regulate essential processes such as cell proliferation and differentiation, cell survival, and exocytosis. Evidence suggests that PKD regulates the cellular adaptations to the obese environment in metabolically important tissues and drives the development of a variety of diseases. This review explores the role that PKD plays in tissue dysfunction in obesity, with special consideration of the development of obesity-mediated cardiomyopathy, a distinct cardiovascular disease that occurs in the absence of common comorbidities and leads to eventual heart failure and death. The downstream mechanisms mediated by PKD that could contribute to dysfunctions observed in the heart and other metabolically important tissues in obesity, and the predicted cell types involved are discussed to suggest potential targets for the development of therapeutics against obesity-related disease.

Asunto(s)

Obesidad , Proteína Quinasa C , Humanos , Proteína Quinasa C/metabolismo , Proteína Quinasa C/genética , Obesidad/metabolismo , Obesidad/patología , Obesidad/genética , Animales , Miocardio/metabolismo , Miocardio/patología , Transducción de Señal , Cardiomiopatías/metabolismo , Cardiomiopatías/genética , Cardiomiopatías/patología

RESUMEN

Protein kinase D (PKD) is a family of serine/threonine kinases that play important roles in various signalling pathways in cells, including neuronal cells. In the nervous system, PKD has been shown to be involved in learning and memory formation by regulating neurotransmitter release, neurite outgrowth and dendrite development, synapse formation and synaptic plasticity. In addition, PKD has been implicated in pain perception or neuroprotection during oxidative stress. Dysregulation of PKD expression and activity has been linked to several neurological disorders, including autism and epilepsy. In this review, we summarize the current knowledge on the function of the PKD family members in neuronal cells, including the spatial regulation of their downstream signalling pathways. We will further discuss the potential role of PKD in the pathogenesis of neurological disorders.

Asunto(s)

Neuronas , Proteína Quinasa C , Transducción de Señal , Humanos , Proteína Quinasa C/metabolismo , Proteína Quinasa C/genética , Neuronas/metabolismo , Animales , Plasticidad Neuronal

RESUMEN

Glycogen storage, conversion and utilization in astrocytes play an important role in brain energy metabolism. The conversion of glycogen to lactate through glycolysis occurs through the coordinated activities of various enzymes and inhibition of this process can impair different brain processes including formation of long-lasting memories. To replenish depleted glycogen stores, astrocytes undergo glycogen synthesis, a cellular process that has been shown to require transcription and translation during specific stimulation paradigms. However, the detail nuclear signaling mechanisms and transcriptional regulation during glycogen synthesis in astrocytes remains to be explored. In this report, we study the molecular mechanisms of vasoactive intestinal peptide (VIP)-induced glycogen synthesis in astrocytes. VIP is a potent neuropeptide that triggers glycogenolysis followed by glycogen synthesis in astrocytes. We show evidence that VIP-induced glycogen synthesis requires CREB-mediated transcription that is calcium dependent and requires conventional Protein Kinase C but not Protein Kinase A. In parallel to CREB activation, we demonstrate that VIP also triggers nuclear accumulation of the CREB coactivator CRTC2 in astrocytic nuclei. Transcriptome profiles of VIP-induced astrocytes identified robust CREB transcription, including a subset of genes linked to glucose and glycogen metabolism. Finally, we demonstrate that VIP-induced glycogen synthesis shares similar as well as distinct molecular signatures with glucose-induced glycogen synthesis, including the requirement of CREB-mediated transcription. Overall, our data demonstrates the importance of CREB-mediated transcription in astrocytes during stimulus-driven glycogenesis.

Asunto(s)

Astrocitos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Glucógeno , Péptido Intestinal Vasoactivo , Astrocitos/metabolismo , Glucógeno/metabolismo , Glucógeno/biosíntesis , Animales , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Péptido Intestinal Vasoactivo/metabolismo , Transcripción Genética , Células Cultivadas , Proteína Quinasa C/metabolismo , Regulación de la Expresión Génica , Ratones , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Núcleo Celular/metabolismo

RESUMEN

Human dental follicle cells (DFCs) as multipotent stem cells are currently investigated within the field of regenerative medicine considering their potential for the regeneration of dental tissues, bone defects caused by periodontal or degenerative diseases and the treatment of craniofacial disorders. However, molecular mechanisms of the differentiation into mineralizing cells are still inadequately understood. Previous studies have shown that GÖ6976, an inhibitor of classical isoforms of protein kinase C (PKC), enhanced ostogenic differentiation of DFCs. A possible mechanism for increased osteogenic differentiation could be the regulation of ossification inhibitors. This study therefore investigated whether the osteogenic differentiation inhibitor sclerostin (SOST) is regulated by GÖ6976 and whether the addition of sclerostin attenuates the stimulating effect of the PKC inhibitor. We demonstrated that the expression of the sclerostin gene decreased after PKC inhibition by GÖ6976 and that its gene expression is likely maintained by PKC via the BMP signaling pathway. Furthermore, supplementation of osteogenic differentiation medium with sclerostin impairs GÖ6976-induced differentiation of DFCs. Our data suggest that regulation of sclerostin mediates PKC inhibition-induced mineralization of DFCs.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Carbazoles , Diferenciación Celular , Saco Dental , Osteogénesis , Proteína Quinasa C , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Diferenciación Celular/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Proteína Quinasa C/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Saco Dental/citología , Saco Dental/efectos de los fármacos , Saco Dental/metabolismo , Carbazoles/farmacología , Marcadores Genéticos , Transducción de Señal/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Morfogenéticas Óseas/metabolismo , Células Cultivadas

RESUMEN

Diacylglycerol O-acyltransferase 1 (DGAT1) is a key enzyme for fat absorption step in the enterocytes. We previously reported that DGAT1 inhibition increased plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in corn oil-loaded rats via protein kinase C (PKC) activation. In the present study, we investigated the mechanism with respect to the morphology and permeability of the small intestine, focusing on PKC function, and found that shortening of the intestinal villi and a decrease in the number of tdT-mediated dUTP-biotin nick-end labeling-positive cells in the tips of the villi were observed in the jejunum of DGAT1 inhibitor-treated rats loaded with corn oil. These results suggested that the tips of the villi were shed into the intestinal lumen. Next, fluorescein isothiocyanate-dextran, 110 kDa (FD-110) was administered intraduodenally to DGAT1 inhibitor-treated rats loaded with corn oil and we found that plasma FD-110 concentrations increased, indicating that the intestinal permeability to molecules with a molecular weight of approximately 110,000 (e.g., ALT and AST) increased. Taken together, the present results suggested that DGAT1 inhibitor-treatment in combination with corn oil causes ALT and AST to leak from the enterocytes into the blood by shedding the tips of the intestinal villi and increasing intestinal permeability.

Asunto(s)

Alanina Transaminasa , Aspartato Aminotransferasas , Aceite de Maíz , Diacilglicerol O-Acetiltransferasa , Mucosa Intestinal , Permeabilidad , Animales , Alanina Transaminasa/sangre , Masculino , Aspartato Aminotransferasas/sangre , Diacilglicerol O-Acetiltransferasa/antagonistas & inhibidores , Diacilglicerol O-Acetiltransferasa/metabolismo , Permeabilidad/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Ratas , Dextranos , Proteína Quinasa C/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Fluoresceína-5-Isotiocianato/análogos & derivados , Yeyuno/efectos de los fármacos , Yeyuno/metabolismo , Absorción Intestinal/efectos de los fármacos , Ratas Sprague-Dawley , Ratas Wistar , Funcion de la Barrera Intestinal

RESUMEN

Cardiomyopathy is particularly common in septic patients. Our previous studies have shown that activation of the alpha 1 adrenergic receptor (α1-AR) on cardiomyocytes inhibits sepsis-induced myocardial dysfunction. However, the role of cardiac endothelial α1-AR in septic cardiomyopathy has not been determined. Here, we identified α1-AR expression in mouse and human endothelial cells and showed that activation of α1-AR with phenylephrine (PE) improved cardiac function and survival by preventing cardiac endothelial injury in septic mice. Mechanistically, activating α1-AR with PE decreased the expression of ICAM-1, VCAM-1, iNOS, E-selectin, and p-p38MAPK, while promoting PKC and ERK1/2 phosphorylation in LPS-treated endothelial cells. These effects were abolished by a PKC inhibitor or α1-AR antagonist. PE also reduced p65 nuclear translocation, but this suppression is not blocked by PKC inhibition. Treatment with U0126 (a specific ERK1/2 inhibitor) reversed the effects of PE on p38MAPK phosphorylation. Our results demonstrate that cardiac endothelial α1-AR activation prevents sepsis-induced myocardial dysfunction in mice by inhibiting the endothelial injury via PKC-ERK/p38MAPK signaling pathway and a PKC-independent inhibition of p65 nuclear translocation. These findings offer a new perspective for septic patients with cardiac dysfunction by inhibiting cardiac endothelial cell injury through α1-AR activation.

Asunto(s)

Células Endoteliales , Ratones Endogámicos C57BL , Fenilefrina , Proteína Quinasa C , Receptores Adrenérgicos alfa 1 , Sepsis , Proteínas Quinasas p38 Activadas por Mitógenos , Animales , Sepsis/tratamiento farmacológico , Sepsis/metabolismo , Humanos , Receptores Adrenérgicos alfa 1/metabolismo , Proteína Quinasa C/metabolismo , Masculino , Ratones , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Fenilefrina/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Agonistas de Receptores Adrenérgicos alfa 1/farmacología , Agonistas de Receptores Adrenérgicos alfa 1/uso terapéutico , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Cardiomiopatías/tratamiento farmacológico , Cardiomiopatías/etiología , Cardiomiopatías/metabolismo , Transducción de Señal/efectos de los fármacos , Células Cultivadas

RESUMEN

The PKC-related kinases (PRKs, also termed PKNs) are important in cell migration, cancer, hepatitis C infection, and nutrient sensing. They belong to a group of protein kinases called AGC kinases that share common features like a C-terminal extension to the catalytic domain comprising a hydrophobic motif. PRKs are regulated by N-terminal domains, a pseudosubstrate sequence, Rho-binding domains, and a C2 domain involved in inhibition and dimerization, while Rho and lipids are activators. We investigated the allosteric regulation of PRK2 and its interaction with its upstream kinase PDK1 using a chemical biology approach. We confirmed the phosphoinositide-dependent protein kinase 1 (PDK1)-interacting fragment (PIF)-mediated docking interaction of PRK2 with PDK1 and showed that this interaction can be modulated allosterically. We showed that the polypeptide PIFtide and a small compound binding to the PIF-pocket of PRK2 were allosteric activators, by displacing the pseudosubstrate PKL region from the active site. In addition, a small compound binding to the PIF-pocket allosterically inhibited the catalytic activity of PRK2. Together, we confirmed the docking interaction and allostery between PRK2 and PDK1 and described an allosteric communication between the PIF-pocket and the active site of PRK2, both modulating the conformation of the ATP-binding site and the pseudosubstrate PKL-binding site. Our study highlights the allosteric modulation of the activity and the conformation of PRK2 in addition to the existence of at least two different complexes between PRK2 and its upstream kinase PDK1. Finally, the study highlights the potential for developing allosteric drugs to modulate PRK2 kinase conformations and catalytic activity.

Asunto(s)

Proteína Quinasa C , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Humanos , Regulación Alostérica , Proteína Quinasa C/metabolismo , Proteína Quinasa C/genética , Proteína Quinasa C/química , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/genética , Dominio Catalítico , Simulación del Acoplamiento Molecular , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/química , Proteínas Quinasas Dependientes de 3-Fosfoinosítido/metabolismo , Proteínas Quinasas Dependientes de 3-Fosfoinosítido/genética , Proteínas Quinasas Dependientes de 3-Fosfoinosítido/química , Unión Proteica

RESUMEN

The exchange protein directly activated by cAMP (EPAC) has been implicated in cardiac proarrhythmic signaling pathways including spontaneous diastolic Ca2+ leak from sarcoplasmic reticulum and increased action potential duration (APD) in isolated ventricular cardiomyocytes. The action potential (AP) lengthening following acute EPAC activation is mainly due to a decrease of repolarizing steady-state K+ current (IKSS) but the mechanisms involved remain unknown. This study aimed to assess the role of EPAC1 and EPAC2 in the decrease of IKSS and to investigate the underlying signaling pathways. AP and K+ currents were recorded with the whole cell configuration of the patch-clamp technique in freshly isolated rat ventricular myocytes. EPAC1 and EPAC2 were pharmacologically activated with 8-(4-chlorophenylthio)-2'-O-methyl-cAMP acetoxymethyl ester (8-CPTAM, 10 µmol/L) and inhibited with R-Ce3F4 and ESI-05, respectively. Inhibition of EPAC1 and EPAC2 significantly decreased the effect of 8-CPTAM on APD and IKSS showing that both EPAC isoforms are involved in these effects. Unexpectedly, calmodulin-dependent protein kinase II (CaMKII) inhibition by AIP or KN-93, and Ca2+ chelation by intracellular BAPTA, did not impact the response to 8-CPTAM. However, inhibition of PLC/PKC and nitric oxide synthase (NOS)/PKG pathways partially prevents the 8-CPTAM-dependent decrease of IKSS. Finally, the cumulative inhibition of PKC and PKG blocked the 8-CPTAM effect, suggesting that these two actors work along parallel pathways to regulate IKSS upon EPAC activation. On the basis of such findings, we propose that EPAC1 and EPAC2 are involved in APD lengthening by inhibiting a K+ current via both PLC/PKC and NOS/PKG pathways. This may have pathological implications since EPAC is upregulated in diseases such as cardiac hypertrophy.NEW & NOTEWORHTY Exchange protein directly activated by cAMP (EPAC) proteins modulate ventricular electrophysiology at the cellular level. Both EPAC1 and EPAC2 isoforms participate in this effect. Mechanistically, PLC/PKC and nitric oxide synthase (NO)/PKG pathways are involved in regulating K+ repolarizing current whereas the well-known downstream effector of EPAC, calmodulin-dependent protein kinase II (CaMKII), does not participate. This may have pathological implications since EPAC is upregulated in diseases such as cardiac hypertrophy. Thus, EPAC inhibition may be a new approach to prevent arrhythmias under pathological conditions.

Asunto(s)

Potenciales de Acción , Factores de Intercambio de Guanina Nucleótido , Ventrículos Cardíacos , Miocitos Cardíacos , Proteína Quinasa C , Transducción de Señal , Animales , Factores de Intercambio de Guanina Nucleótido/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/enzimología , Proteína Quinasa C/metabolismo , Ratas , Potenciales de Acción/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/citología , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismo , Fosfolipasas de Tipo C/antagonistas & inhibidores , Masculino , Ratas Wistar , Potasio/metabolismo , AMP Cíclico/metabolismo

RESUMEN

BACKGROUND: Diabetes mellitus (DM) affects up to one-third of breast cancer (BC) patients. Patients with co-existing BC and DM (BC-DM) have worsened BC prognosis. Nevertheless, the molecular mechanisms orchestrating BC-DM prognosis remain poorly understood. tRNA-derived fragments (tRFs) have been shown to regulate cancer progression. However, the biological role of tRFs in BC-DM has not been explored. METHODS: tRF levels in tumor tissues and cells were detected by tRF sequencing and qRT-PCR. The effects of tRF on BC cell malignancy were assessed under euglycemic and hyperglycemic conditions in vitro. Metabolic changes were assessed by lactate, pyruvate, and extracellular acidification rate (ECAR) assays. Diabetic animal model was used to evaluate the impacts of tRF on BC tumor growth. RNA-sequencing (RNA-seq), qRT-PCR, Western blot, polysome profiling, luciferase reporter assay, and rescue experiments were performed to explore the regulatory mechanisms of tRF in BC-DM. RESULTS: We identified that tRF-Cys-GCA-029 was downregulated in BC-DM tissues and under hyperglycemia conditions in BC cells. Functionally, downregulation of tRF-Cys-GCA-029 promoted BC cell proliferation and migration in a glucose level-dependent manner. tRF-Cys-GCA-029 knockdown also enhanced glycolysis metabolism in BC cells, indicated by increasing lactate/pyruvate production and ECAR levels. Notably, injection of tRF-Cys-GCA-029 mimic significantly suppressed BC tumor growth in diabetic-mice. Mechanistically, tRF-Cys-GCA-029 regulated BC cell malignancy and glycolysis via interacting with PRKCG in two ways: binding to the coding sequence (CDS) of PRKCG mRNA to regulate its transcription and altering polysomal PRKCG mRNA expression to modify its translation. CONCLUSIONS: Hyperglycemia-downregulated tRF-Cys-GCA-029 enhances the malignancy and glycolysis of BC cells. tRF-Cys-GCA-029-PRKCG-glycolysis axis may be a potential therapeutic target against BC-DM.

Asunto(s)

Neoplasias de la Mama , Regulación Neoplásica de la Expresión Génica , Glucólisis , Hiperglucemia , Humanos , Femenino , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Hiperglucemia/metabolismo , Hiperglucemia/genética , Ratones , Proliferación Celular , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Línea Celular Tumoral , Carcinogénesis/genética , Regulación hacia Abajo , Proteína Quinasa C/metabolismo , Proteína Quinasa C/genética , Regulación hacia Arriba , Pronóstico

RESUMEN

Protein kinase C is a family of kinases that play important roles in carcinogenesis. Medicinal plants from Plectranthus spp. (Lamiaceae) are a well-known source of interesting abietanes, such as 7α-acetoxy-6ß-hydroxyroyleanone (Roy). This study aimed to extract and isolate Roy from P. grandidentatus Gürke, comparing two extraction methods (CO2 supercritical and ultrasound-assisted acetonic extraction), and design new royleanone derivatives for PKC modulation focusing on breast cancer therapy. The concentration of Roy in the extracts was determined by HPLC-DAD. The supercritical extraction method yielded 3.6% w/w, with the presence of 42.7 µg mg-1 of Roy (yield of 0.13%), while ultrasound-assisted acetonic extraction yielded 2.3% w/w, with the presence of 55.2 µg mg-1 of Roy (yield of 0.15%). The reactivity of Roy was investigated aiming at synthetizing new ester derivatives through standard benzoylation and esterification reactions. The benzoylated (Roy-12-Bz) and acetylated (Roy-12-Ac) derivatives in the C12 position were consistently prepared with overall good yields (33-86%). These results indicate the 12-OH position as the most reactive for esterification, affording derivatives under mild conditions. The reported di-benzoylated (RoyBz) and di-acetylated (RoyAc) derivatives were also synthesized after increasing the temperature (50 °C), reaction time, and using an excess of reagents. The cytotoxic potential of Roy and its derivatives was assessed against breast cancer cell lines, with RoyBz emerging as the most promising compound. Derivatization at position C-12 did not offer advantages over di-esterification at positions C-12 and C-6 or over the parent compound Roy and the presence of aromatic groups favored cytotoxicity. Evaluation of royleanones as PKC-α, ßI, δ, ε, and ζ activators revealed DeRoy's efficacy across all isoforms, while RoyPr showed promising activation of PKC-δ but not PKC-ζ, highlighting the influence of slight structural changes on isoform selectivity. Molecular docking analysis emphasized the importance of microenvironmental factors in isoform specificity, underscoring the complexity of PKC modulation and the need for further exploration.

Asunto(s)

Proteína Quinasa C , Humanos , Proteína Quinasa C/metabolismo , Simulación del Acoplamiento Molecular , Extractos Vegetales/química , Extractos Vegetales/farmacología , Isoenzimas/metabolismo , Células MCF-7 , Línea Celular Tumoral , Diterpenos

RESUMEN

Emerging evidence indicates that gut dysbiosis is involved in the pathogenesis of visceral hypersensitivity (VH). However, how gut microbiota contributes to the development of VH is unknown. Here, we sought to examine the signal transduction pathways from gut to dorsal root ganglion (DRG) responsible for this. Therefore, abdominal withdrawal reflex (AWR) scores, fecal output, fecal water content, and total gastrointestinal transit time (TGITT) were assessed in Con rats, VH rats, rats treated with NaB, and VH rats treated with VSL#3. Fecal microbiota and its metabolite (short-chain fatty acids, SCFAs), mast cell degranulation in colon, lincRNA-01028, miR-143, and protease kinase C (PKC) and TRPV1 expression in DRGs were further detected. VH rats showed an increased fecal water content, a shortened TGITT, an increased abundance of Clostridium sensu stricto 1 and increased butyrate in fecal samples, an increased mast cell degranulation, an increased expression of lincRNA-01028, PKC, and TRPV1, and a decreased expression of miR-143 in DRGs compared with control rats, which could be restored by the application of probiotic VSL#3. The above-mentioned detection in rats treated with butyrate was similar to that of VH rats. We further confirm whether butyrate sensitized DRG neurons by a lincRNA-01028, miR-143, and PKC-dependent mechanism via mast cell in vitro. In co-cultures, MCs treated with butyrate elicited a higher TRPV1 current, a higher expression of lincRNA-01028, PKC, and a lower expression of miR-143 in DRG neurons, which could be inhibited by a lincRNA-01028 inhibitor. These findings indicate that butyrate promotes visceral hypersensitivity via mast cell-derived DRG neuron lincRNA-01028-PKC-TRPV1 pathway.IMPORTANCEIrritable bowel syndrome (IBS), characterized by visceral hypersensitivity, is a common gastrointestinal dysfunction syndrome. Although the gut microbiota plays a role in the pathogenesis and treatment of irritable bowel syndrome (IBS), the possible underlying mechanisms are unclear. Therefore, it is of critical importance to determine the signal transduction pathways from gut to DRG responsible for this in vitro and in vivo assay. This study demonstrated that butyrate sensitized TRPV1 in DRG neurons via mast cells in vivo and in vitro by a lincRNA-01028, miR-143, and PKC-dependent mechanism. VH rats similarly showed an increased abundance of Clostridium sensu stricto 1, an increased fecal butyrate, an increased mast cell degranulation, and increased expression of TRPV1 compared with control rats, which could be restored by the application of VSL#3. In conclusion, butyrate produced by the altered intestinal microbiota is associated with increased VH.

Asunto(s)

Butiratos , Modelos Animales de Enfermedad , Ganglios Espinales , Síndrome del Colon Irritable , Mastocitos , Proteína Quinasa C , Ratas Sprague-Dawley , Canales Catiónicos TRPV , Animales , Ganglios Espinales/metabolismo , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/genética , Ratas , Mastocitos/metabolismo , Mastocitos/efectos de los fármacos , Masculino , Butiratos/metabolismo , Butiratos/farmacología , Proteína Quinasa C/metabolismo , Síndrome del Colon Irritable/metabolismo , Síndrome del Colon Irritable/microbiología , Microbioma Gastrointestinal/efectos de los fármacos , Transducción de Señal , Neuronas/metabolismo , Neuronas/efectos de los fármacos

RESUMEN

The Pcdhg gene cluster encodes 22 γ-Protocadherin (γ-Pcdh) cell adhesion molecules that critically regulate multiple aspects of neural development, including neuronal survival, dendritic and axonal arborization, and synapse formation and maturation. Each γ-Pcdh isoform has unique protein domains-a homophilically interacting extracellular domain and a juxtamembrane cytoplasmic domain-as well as a C-terminal cytoplasmic domain shared by all isoforms. The extent to which isoform-specific versus shared domains regulate distinct γ-Pcdh functions remains incompletely understood. Our previous in vitro studies identified protein kinase C (PKC) phosphorylation of a serine residue within a shared C-terminal motif as a mechanism through which γ-Pcdh promotion of dendrite arborization via myristoylated alanine-rich C-kinase substrate (MARCKS) is abrogated. Here, we used CRISPR/Cas9 genome editing to generate two new mouse lines expressing only non-phosphorylatable γ-Pcdhs, due either to a serine-to-alanine mutation (PcdhgS/A) or to a 15-amino acid C-terminal deletion resulting from insertion of an early stop codon (PcdhgCTD). Both lines are viable and fertile, and the density and maturation of dendritic spines remain unchanged in both PcdhgS/A and PcdhgCTD cortex. Dendrite arborization of cortical pyramidal neurons, however, is significantly increased in both lines, as are levels of active MARCKS. Intriguingly, despite having significantly reduced levels of γ-Pcdh proteins, the PcdhgCTD mutation yields the strongest phenotype, with even heterozygous mutants exhibiting increased arborization. The present study confirms that phosphorylation of a shared C-terminal motif is a key γ-Pcdh negative regulation point and contributes to a converging understanding of γ-Pcdh family function in which distinct roles are played by both individual isoforms and discrete protein domains.

Asunto(s)

Proteínas Relacionadas con las Cadherinas , Cadherinas , Corteza Cerebral , Dendritas , Proteína Quinasa C , Animales , Corteza Cerebral/metabolismo , Corteza Cerebral/citología , Cadherinas/metabolismo , Cadherinas/genética , Fosforilación/fisiología , Dendritas/metabolismo , Ratones , Proteína Quinasa C/metabolismo , Proteína Quinasa C/genética , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/metabolismo , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/genética , Secuencias de Aminoácidos/fisiología , Ratones Transgénicos

RESUMEN

The HMGB1 protein typically serves as a DNA chaperone that assists DNA-repair enzymes and transcription factors but can translocate from the nucleus to the cytoplasm or even to extracellular space upon some cellular stimuli. One of the factors that triggers the translocation of HMGB1 is its phosphorylation near a nuclear localization sequence by protein kinase C (PKC), although the exact modification sites on HMGB1 remain ambiguous. In this study, using spectroscopic methods, we investigated the HMGB1 phosphorylation and its impact on the molecular properties of the HMGB1 protein. Our nuclear magnetic resonance (NMR) data on the full-length HMGB1 protein showed that PKC specifically phosphorylates the A-box domain, one of the DNA binding domains of HMGB1. Phosphorylation of S46 and S53 was particularly efficient. Over a longer reaction time, PKC phosphorylated some additional residues within the HMGB1 A-box domain. Our fluorescence-based binding assays showed that the phosphorylation significantly reduces the binding affinity of HMGB1 for DNA. Based on the crystal structures of HMGB1-DNA complexes, this effect can be ascribed to electrostatic repulsion between the negatively charged phosphate groups at the S46 side chain and DNA backbone. Our data also showed that the phosphorylation destabilizes the folding of the A-box domain. Thus, phosphorylation by PKC weakens the DNA-binding affinity and folding stability of HMGB1.

Asunto(s)

ADN , Proteína HMGB1 , Pliegue de Proteína , Proteína Quinasa C , Proteína HMGB1/metabolismo , Proteína HMGB1/química , Fosforilación , ADN/metabolismo , ADN/química , Proteína Quinasa C/metabolismo , Proteína Quinasa C/química , Estabilidad Proteica , Humanos , Unión Proteica , Animales , Resonancia Magnética Nuclear Biomolecular , Modelos Moleculares , Dominios Proteicos

RESUMEN

Tumor-associated macrophages (TAMs) secrete cytokines, chemokines, and growth factors in the tumor microenvironment (TME) to support cancer progression. Higher TAM infiltration in the breast TME is associated with a poor prognosis. Previous studies have demonstrated the role of macrophages in stimulating long-range intercellular bridges referred to as tunneling nanotubes (TNTs) in cancer cells. Intercellular communication between cancer cells via TNTs promotes cancer growth, invasion, metastasis, and therapy resistance. Given the important role of TNTs and macrophages in cancer, the role of macrophage-induced TNTs in chemotherapy drug doxorubicin resistance is not known. Furthermore, the mechanism of macrophage-mediated TNT formation is elusive. In this study, it is shown that the macrophage-conditioned medium (MΦCM) partially mimicked inflammatory TME, induced an EMT phenotype, and increased migration in MCF-7 breast cancer cells. Additionally, secreted proteins in MΦCM induced TNT formation in MCF-7 cells, which led to increased resistance to doxorubicin. Transcriptomic analysis of MΦCM-treated MCF-7 cells showed enrichment of the NF-κB and focal adhesion pathways, as well as upregulation of genes involved in EMT, extracellular remodeling, and actin cytoskeleton reorganization. Interestingly, inhibitors of PKC, Src, NF-κB, and p38 decreased macrophage-induced TNT formation in MCF-7 cells. These results reveal the novel role of PKC and Src in inducing TNT formation in cancer cells and suggest that inhibition of PKC and Src activity may likely contribute to reduced macrophage-breast cancer cell interaction and the potential therapeutic strategy of cancer.

Asunto(s)

Neoplasias de la Mama , FN-kappa B , Proteína Quinasa C , Proteínas Quinasas p38 Activadas por Mitógenos , Humanos , FN-kappa B/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Medios de Cultivo Condicionados/farmacología , Células MCF-7 , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Femenino , Proteína Quinasa C/metabolismo , Doxorrubicina/farmacología , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Familia-src Quinasas/metabolismo , Microambiente Tumoral , Nanotubos/química , Transducción de Señal/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos

RESUMEN

We synthesized a phenolic hydroxy group-bearing version (1) of a simplified analog of aplysiatoxin comprising a carvone-based conformation-controlling unit. Thereafter, we evaluated its antiproliferative activity against human cancer cell lines and its binding affinity to protein kinase C (PKC) isozymes. The antiproliferative activity and PKC-binding ability increased with the introduction of the phenolic hydroxy group. The results of molecular dynamics simulations and subsequent relative binding free-energy calculations conducted using an alchemical transformation procedure showed that the phenolic hydroxy group in 1 could form a hydrogen bond with a phospholipid and the PKC. The former hydrogen bonding formation facilitated the partitioning of the compound from water to the phospholipid membrane and the latter compensated for the loss of hydrogen bond with the phospholipid upon binding to the PKC. This information may facilitate the development of rational design methods for PKC ligands with additional hydrogen bonding groups.

Asunto(s)

Enlace de Hidrógeno , Toxinas de Lyngbya , Simulación de Dinámica Molecular , Proteína Quinasa C , Humanos , Proteína Quinasa C/metabolismo , Línea Celular Tumoral , Toxinas de Lyngbya/química , Toxinas de Lyngbya/farmacología , Fenoles/química , Fenoles/farmacología , Proliferación Celular/efectos de los fármacos , Unión Proteica , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Fosfolípidos/química

RESUMEN

AIMS: Niacin (NIA) supplementation showed effectiveness against Parkinson's disease (PD) in clinical trials. The depletion of NAD and endoplasmic reticulum stress response (ERSR) are implicated in the pathogenesis of PD, but the potential role for NAD precursors on ERSR is not yet established. This study was undertaken to decipher NIA molecular mechanisms against PD-accompanied ERSR, especially in relation to PKC. METHODS: Alternate-day-low-dose-21 day-subcutaneous exposure to rotenone (ROT) in rats induced PD. Following the 5th ROT injection, rats received daily doses of either NIA alone or preceded by the PKC inhibitor tamoxifen (TAM). Extent of disease progression was assessed by behavioral, striatal biochemical and striatal/nigral histopathological/immunohistochemical analysis. KEY FINDINGS: Via activating PKC/LKB1/AMPK stream, NIA post-treatment attenuated the ERSR reflected by the decline in ATF4, ATF6 and XBP1s to downregulate the apoptotic markers, CHOP/GADD153, p-JNK and active caspase-3. Such amendments congregated in motor activity/coordination improvements in open field and rotarod tasks, enhanced grid test latency and reduced overall PD scores, while boosting nigral/striatal tyrosine hydroxylase immunoreactivity and increasing intact neurons (Nissl stain) in both SNpc and striatum that showed less neurodegeneration (H&E stain). To different extents, TAM reverted all the NIA-related actions to prove PKC as a fulcrum in conveying the drug neurotherapeutic potential. SIGNIFICANCE: PKC activation is a pioneer mechanism in the drug ERSR inhibitory anti-apoptotic modality to clarify NIA promising clinical and potent preclinical anti-PD efficacy. This kinase can be tagged as a druggable target for future add-on treatments that can assist dopaminergic neuronal aptitude against this devastating neurodegenerative disease.

Asunto(s)

Estrés del Retículo Endoplásmico , Niacina , Animales , Estrés del Retículo Endoplásmico/efectos de los fármacos , Ratas , Niacina/farmacología , Masculino , Proteína Quinasa C/metabolismo , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Rotenona/farmacología , Ratones , Apoptosis/efectos de los fármacos , Ratas Wistar , Modelos Animales de Enfermedad