RESUMO
BACKGROUND: The epithelial isoform of the fibroblast growth factor receptor 2 (FGFR2b) controls the entire program of keratinocyte differentiation via the sequential involvement of protein kinase C (PKC) δ and PKCα. In contrast, the FGFR2 isoform switch and the aberrant expression of the mesenchymal FGFR2c isoform leads to impairment of differentiation, epithelial-mesenchymal transition (EMT) and tumorigenic features. Aim of our present study was to contribute in clarifying the complex network of signaling pathways involved in the FGFR2c-mediated oncogenic outcomes focusing on PKCε, which appears to be involved in the induction of EMT and tumorigenesis in several epithelial contexts. METHODS: Biochemical and molecular analysis, as well as in vitro invasion assays, combined with the use of specific small interfering RNA (siRNA), were performed in human keratinocytes stably expressing FGFR2c or FGFR2b isoforms. RESULTS: Our results showed that aberrant expression and signaling of FGFR2c, but not those of FGFR2b, in human keratinocytes induced a strong phosphorylation/activation of PKCε. The use of siRNA approach showed that PKCε is the hub signaling downstream FGFR2c responsible for the modulation of EMT markers and for the induction of the EMT-related transcription factors STAT3, Snail1 and FRA1, as well as for the acquisition of the invasive behavior. Moreover, experiments of depletion of ESRP1, responsible for FGFR2 splicing in epithelial cells, indicated that the activation of PKCε is the key molecular event triggered by FGFR2 isoform switch and underlying EMT induction. CONCLUSIONS: Overall, our results point to the identification of the downstream PKC isoform responsible for the FGFR signaling deregulation occurring in epithelial tissues from the physiological oncosoppressive to the pathological oncogenic profile. Video Abstract.
Assuntos
Transição Epitelial-Mesenquimal , Neoplasias/metabolismo , Proteína Quinase C-épsilon/fisiologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Diferenciação Celular , Células HaCaT , HumanosRESUMO
The failure of insulin to suppress glucose production by the liver is a key aspect of the insulin resistance seen in type 2 diabetes. Lipid-activated protein kinase C epsilon has long been identified as an important mediator of diet-induced glucose intolerance and hepatic insulin resistance and the current view emphasizes a mechanism involving phosphorylation of the insulin receptor by the kinase to inhibit downstream insulin action. However, the significance of this direct effect in the liver has now been challenged by tissue-specific deletion of PKCε, which demonstrated a more prominent role for the kinase in adipose tissue to promote glucose intolerance. New insights regarding the role of PKCε therefore contribute to the understanding of indirect effects on hepatic glucose metabolism.
Assuntos
Tecido Adiposo/metabolismo , Glucose/metabolismo , Homeostase/fisiologia , Resistência à Insulina/fisiologia , Fígado/metabolismo , Proteína Quinase C-épsilon/fisiologia , Animais , HumanosRESUMO
At concentrations found in humans after ingestion of one to two cups of green tea, epicatechin-3-gallate (ECG) modulates Na/K-ATPase conformation and activity. Akin to ouabain, an archetypal Na/K-ATPase ligand of the cardiotonic steroid (CTS) family, ECG also activates protein kinase C epsilon type (PKCε) translocation and increases the force of contraction of the rat heart. This study evaluated whether, like ouabain, ECG also modulates Na/K-ATPase/Src receptor function and triggers pre- and postconditioning against ischemia/reperfusion (I/R) injury. In vitro, ECG activated the purified Na/K-ATPase/Src complex. In Langendorff-perfused rat hearts, submicromolar concentrations of ECG administered either before or after ischemia reduced infarct size by more than 40%, decreased lactate dehydrogenase release, and improved the recovery of cardiac function. ECG protection was blocked by PKCε inhibition and attenuated by mitochondrial KATP channel inhibition. In a unique mammalian cell system with depleted Na/K-ATPase α1 expression, ECG-induced PKCε activation persisted but protection against I/R was blunted. Taken together, these results reveal a Na/K-ATPase- and PKCε-dependent mechanism of protection by ECG that is also distinct from the mechanism of action of ouabain. These ECG properties likely contribute to the positive impact of green tea consumption on cardiovaascular health and warrant further investigation into the role of cardiac Na/K-ATPase signaling in the cardioprotective effect of green tea consumption. SIGNIFICANCE STATEMENT: Consumption of green tea, the richest dietary source of ECG, is associated with a reduced risk of cardiac mortality. Antioxidant effects of ECG and other tea polyphenols are well known, but reported for concentrations well above dietary levels. Therefore, the mechanism underlying the cardioprotective effect of green tea remains incompletely understood. This study provides experimental evidence that ECG concentrations commonly detected in humans after consumption of a cup of tea trigger the Na/K-ATPase/Src receptor in a cell-free system, activate a CTS-like signaling pathway, and provide PKCε-dependent protection against ischemia/reperfusion injury in rat hearts. Mechanistic studies in mammalian cells with targeted Na/K-ATPase depletion revealed that although Na/K-ATPase does not mediate ECG-induced PKCε activation, it is required for ECG-induced protection against ischemia/reperfusion injury.
Assuntos
Catequina/análogos & derivados , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Animais , Catequina/farmacologia , Células Cultivadas , Masculino , Extratos Vegetais/farmacologia , Canais de Potássio/fisiologia , Proteína Quinase C-épsilon/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Suínos , CháRESUMO
The spatial organization of plasma membrane proteins is a key factor in the generation of distinct signal outputs, especially for PKC/Ras/ERK signalling. Regulation of activation of the membrane-bound Ras, critical for neuronal differentiation and highly specialized functions, is controlled by exchanges in nucleotides catalyzed by nucleotide exchange factors (GEFs) for GTP loading and Ras activation, and by Ras GTPase Activated Proteins (RasGAPs) that lead to activation of the intrinsic GTPase activity of Ras and thus its inactivation. PKCs are potent Ras activators yet the mechanistic details of these interactions, or the involvement of specific PKC isoforms are now beginning to be addressed. Even less known is the topology where RasGAPs terminate Ras activation. Towards this aim, we isolated lipid rafts from chick embryo neural tissue and primary neuronal cultures when PKCε is the prominent isoform and in combination with in vitro kinase assays, we now show that, in response the PKCε-specific activating peptide ψεRACK, an activated PKCε is recruited to lipid rafts; similar mobility was established when PKCε was physiologically activated with the Cannabinoid receptor 1 (CB1) agonist methanandamide. Activation of H-Ras for both agents was then established for the first time using in vivo RasGAP activity assays, which showed similar temporal profiles of activation and lateral mobility. Moreover, we found that the GEF SOS1, and the major neuronal RasGAP neurofibromin, a specific PKCε substrate, were both transiently significantly enriched in the rafts. Finally, our in silico analysis revealed a highly probable, conserved palmitoylation site adjacent to a CARC motif on neurofibromin, both of which are included only in the RasGAP related domain type I (GRDI) with the known high H-RasGAP activity. Taken together, these results suggest that PKCε activation regulates the spatial plasma membrane enrichments of both SOS1 and neurofibromin, thus controlling the output of activated H-Ras available for downstream signalling in neurons.
Assuntos
Microdomínios da Membrana/metabolismo , Neurofibromina 1/metabolismo , Neurônios/metabolismo , Proteína Quinase C-épsilon/fisiologia , Proteína SOS1/metabolismo , Proteínas Ativadoras de ras GTPase/genética , Fatores ras de Troca de Nucleotídeo Guanina/genética , Animais , Ácidos Araquidônicos/farmacologia , Membrana Celular/genética , Membrana Celular/metabolismo , Embrião de Galinha , Simulação por Computador , Proteínas Ativadoras de GTPase , Guanosina Trifosfato/metabolismo , Ácido Palmítico/metabolismo , Cultura Primária de Células , Receptor CB1 de Canabinoide/agonistas , Telencéfalo/efeitos dos fármacos , Telencéfalo/metabolismoRESUMO
Protein kinase C epsilon (PKCÉ) activation in the liver is proposed to inhibit insulin action through phosphorylation of the insulin receptor. Here, however, we demonstrated that global, but not liver-specific, deletion of PKCÉ in mice protected against diet-induced glucose intolerance and insulin resistance. Furthermore, PKCÉ-dependent alterations in insulin receptor phosphorylation were not detected. Adipose-tissue-specific knockout mice did exhibit improved glucose tolerance, but phosphoproteomics revealed no PKCÉ-dependent effect on the activation of insulin signaling pathways. Altered phosphorylation of adipocyte proteins associated with cell junctions and endosomes was associated with changes in hepatic expression of several genes linked to glucose homeostasis and lipid metabolism. The primary effect of PKCÉ on glucose homeostasis is, therefore, not exerted directly in the liver as currently posited, and PKCÉ activation in this tissue should be interpreted with caution. However, PKCÉ activity in adipose tissue modulates glucose tolerance and is involved in crosstalk with the liver.
Assuntos
Tecido Adiposo/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Fígado/metabolismo , Proteína Quinase C-épsilon/fisiologia , Animais , Dieta Hiperlipídica , Técnicas de Inativação de Genes , Intolerância à Glucose , Resistência à Insulina , Metabolismo dos Lipídeos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase C-épsilon/genéticaRESUMO
Global cerebral ischemia that accompanies cardiac arrest is a major cause of morbidity and mortality. Protein Kinase C epsilon (PKCε) is a member of the novel PKC subfamily and plays a vital role in ischemic preconditioning. Pharmacological activation of PKCε before cerebral ischemia confers neuroprotection. The role of endogenous PKCε after cerebral ischemia remains elusive. Here we used male PKCε-null mice to assess the effects of PKCε deficiency on neurodegeneration after transient global cerebral ischemia (tGCI). We found that the cerebral vasculature, blood flow, and the expression of other PKC isozymes were not altered in the PKCε-null mice. Spatial learning and memory was impaired after tGCI, but the impairment was attenuated in male PKCε-null mice as compared to male wild-type controls. A significant reduction in Fluoro-Jade C labeling and mitochondrial release of cytochrome C in the hippocampus was found in male PKCε-null mice after tGCI. Male PKCε-null mice expressed increased levels of PKCδ in the mitochondria, which may prevent the translocation of PKCδ from the cytosol to the mitochondria after tGCI. Our results demonstrate the neuroprotective effects of PKCε deficiency on neurodegeneration after tGCI, and suggest that reduced mitochondrial translocation of PKCδ may contribute to the neuroprotective action in male PKCε-null mice.
Assuntos
Hipocampo/metabolismo , Ataque Isquêmico Transitório/metabolismo , Proteína Quinase C-épsilon/deficiência , Proteína Quinase C-épsilon/fisiologia , Animais , Encéfalo/patologia , Citosol/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Proteína Quinase C-épsilon/metabolismo , Aprendizagem Espacial , Memória EspacialRESUMO
For the past century, vitamin A has been considered to serve as a precursor for retinoids that facilitate vision or as a precursor for retinoic acid (RA), a signaling molecule that modulates gene expression. However, vitamin A circulates in plasma at levels that far exceed the amount needed for vision or the synthesis of nanomolar levels of RA, and this suggests that vitamin A alcohol (i.e. retinol) may possess additional biological activity. We have pursued this question for the last 20 years, and in this chapter, we unfold the story of our quest and the data that support a novel and distinct role for vitamin A (alcohol) action. Our current model supports direct binding of vitamin A to the activation domains of serine/threonine kinases, such as protein kinase C (PKC) and Raf isoforms, where it is involved in redox activation of these proteins. Redox activation of PKCs was first described by the founders of the PKC field, but several hurdles needed to be overcome before a detailed understanding of the biochemistry could be provided. Two discoveries moved the field forward. First, was the discovery that the PKCδ isoform was activated by cytochrome c, a protein with oxidoreduction activity in mitochondria. Second, was the revelation that both PKCδ and cytochrome c are tethered to p66Shc, an adapter protein that brings the PKC zinc-finger substrate into close proximity with its oxidizing partner. Detailed characterization of the PKCδ signalosome complex was made possible by the work of many investigators. Our contribution was determining that vitamin A is a vital co-factor required to support an unprecedented redox-activation mechanism. This unique function of vitamin A is the first example of a general system that connects the one-electron redox chemistry of a heme protein (cytochrome c) with the two-electron chemistry of a classical phosphoprotein (PKCδ). Furthermore, contributions to the regulation of mitochondrial energetics attest to biological significance of vitamin A alcohol action.
Assuntos
Mitocôndrias/metabolismo , Proteína Quinase C-delta/metabolismo , Vitamina A/fisiologia , Animais , Citocromos c/metabolismo , Metabolismo Energético , Ativação Enzimática/efeitos dos fármacos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/fisiologia , Previsões , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/fisiologia , Humanos , Mitocôndrias/enzimologia , Oxirredução , Estresse Oxidativo , Ligação Proteica , Isoformas de Proteínas/metabolismo , Proteína Quinase C-épsilon/fisiologia , Proteínas Adaptadoras da Sinalização Shc/fisiologia , Vitamina A/análogos & derivados , Dedos de Zinco , Quinases raf/metabolismoRESUMO
AIMS: Previous studies have reported that decreased serine 208 phosphorylation of troponin T (TnTpSer208) is associated with ischaemic heart failure (HF), but the molecular mechanisms and functional consequences of these changes are unknown. The aim of this study was to characterize the balance between serine phosphorylation and O-N-acetylglucosaminylation (O-GlcNAcylation) of TnT in HF, its mechanisms, and the consequences of modulating these post-translational modifications. METHODS AND RESULTS: Decreased TnTpSer208 levels in the left ventricles of HF male Wistar rats were associated with reduced expression of PKCε but not of other cardiac PKC isoforms. In both isolated perfused rat hearts and cultured neonatal cardiomyocytes, the PKCε inhibitor εV1-2 decreased TnTpSer208 and simultaneously decreased cardiac contraction in isolated hearts and beating amplitude in neonatal cardiomyocytes (measured by atomic force microscopy). Down-regulating PKCε by silencing RNA (siRNA) also reduced TnTpSer208 in these cardiomyocytes, and PKCε-/- mice had lower TnTpSer208 levels than the wild-type. In parallel, HF increased TnT O-GlcNAcylation via both increased O-GlcNAc transferase and decreased O-GlcNAcase activity. Increasing O-GlcNAcylation (via O-GlcNAcase inhibition with Thiamet G) decreased TnTpSer208 in isolated hearts, while reducing O-GlcNAcylation (O-GlcNAc transferase siRNA) increased TnTpSer208 in neonatal cardiomyocytes. Mass spectrometry and NMR analysis identified O-GlcNAcylation of TnT on Ser190. CONCLUSION: These data demonstrate interplay between Ser208 phosphorylation and Ser190 O-GlcNAcylation of TnT in ischaemic HF, linked to decreased activity of both PKCε and O-GlcNAcase and increased O-GlcNAc transferase activity. Modulation of these post-translational modifications of TnT may be a new therapeutic strategy in HF.
Assuntos
Insuficiência Cardíaca/metabolismo , Isquemia Miocárdica/metabolismo , N-Acetilglucosaminiltransferases/fisiologia , Troponina T/metabolismo , Animais , Masculino , Contração Miocárdica , Fosforilação , Proteína Quinase C-épsilon/fisiologia , Ratos , Ratos WistarRESUMO
Nociceptors and neurons in the central nervous system (CNS) that receive nociceptive input show remarkable plasticity in response to injury. This plasticity is thought to underlie the development of chronic pain states. Hence, further understanding of the molecular mechanisms driving and maintaining this plasticity has the potential to lead to novel therapeutic approaches for the treatment of chronic pain states. An important concept in pain plasticity is the presence and persistence of "hyperalgesic priming." This priming arises from an initial injury and results in a remarkable susceptibility to normally subthreshold noxious inputs causing a prolonged pain state in primed animals. Here we describe our current understanding of how this priming is manifested through changes in signaling in the primary nociceptor as well as through memory like alterations at CNS synapses. Moreover, we discuss how commonly utilized analgesics, such as opioids, enhance priming therefore potentially contributing to the development of persistent pain states. Finally we highlight where these priming models draw parallels to common human chronic pain conditions. Collectively, these advances in our understanding of pain plasticity reveal a variety of targets for therapeutic intervention with the potential to reverse rather than palliate chronic pain states.
Assuntos
Nociceptores/efeitos dos fármacos , Animais , Fator Neurotrófico Derivado do Encéfalo/fisiologia , Humanos , Hiperalgesia/fisiopatologia , Plasticidade Neuronal , Nociceptores/fisiologia , Peptídeos Opioides/fisiologia , Proteína Quinase C-épsilon/fisiologia , Receptores Opioides mu/fisiologiaRESUMO
As one of the most effective and frequently used chemotherapeutic agents, paclitaxel produces peripheral neuropathy (paclitaxel-induced peripheral neuropathy or PIPN) that negatively affects chemotherapy and persists after cancer therapy. The mechanisms underlying this dose-limiting side effect remain to be fully elucidated. This study aimed to investigate the role of nociceptor protein kinase C (PKC) isoforms in PIPN. Employing multiple complementary approaches, we have identified a subset of PKC isoforms, namely ßII, δ, and ϵ, were activated by paclitaxel in the isolated primary afferent sensory neurons. Persistent activation of PKCßII, PKCδ, and PKCϵ was also observed in the dorsal root ganglion neurons after chronic treatment with paclitaxel in a mouse model of PIPN. Isoform-selective inhibitors of PKCßII, PKCδ, and PKCϵ given intrathecally dose-dependently attenuated paclitaxel-induced mechanical allodynia and heat hyperalgesia. Surprisingly, spinal inhibition of PKCßII and PKCδ, but not PKCϵ, blocked the spontaneous pain induced by paclitaxel. These data suggest that a subset of nociceptor PKC isoforms differentially contribute to spontaneous and evoked pain in PIPN, although it is not clear whether PKCϵ in other regions regulates spontaneous pain in PIPN. The findings can potentially offer new selective targets for pharmacological intervention of PIPN.
Assuntos
Nociceptores/fisiologia , Paclitaxel/toxicidade , Dor/enzimologia , Proteína Quinase C beta/fisiologia , Proteína Quinase C-delta/fisiologia , Proteína Quinase C-épsilon/fisiologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Nociceptores/efeitos dos fármacos , Dor/induzido quimicamente , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/fisiologia , Proteína Quinase C beta/antagonistas & inibidores , Proteína Quinase C-delta/antagonistas & inibidores , Proteína Quinase C-épsilon/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-DawleyAssuntos
Reprogramação Celular/fisiologia , Nociceptores/fisiologia , Bradicinina/fisiologia , Membrana Celular/fisiologia , Ativação Enzimática/fisiologia , Epinefrina/fisiologia , Estrogênios/fisiologia , Humanos , Dinâmica não Linear , Limiar da Dor/fisiologia , Proteína Quinase C-épsilon/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Protein Kinase C delta and epsilon are mediators of important cellular events, such as cell proliferation, migration or apoptosis. The formation of blood vessels, i.e., vasculo- and angiogenesis, is a process where these isoforms have also been shown to participate. However, mice deficient in either Protein Kinase C delta or epsilon are viable and therefore their individual contribution to the formation of the vasculature appeared so far dispensable. In this study, we show that double null mutation of Protein Kinase C delta and epsilon causes embryonic lethality at approximately E9.5. At this stage, whole mount staining of the endothelial marker CD31 in double null embryos revealed defective blood vessel formation. Moreover, culture of double deficient mouse allantois showed impaired endothelial cell organization, and analyses of double deficient embryo sections showed dilated vessels, decreased endothelial-specific adherent junctions, and decreased contact of endothelial cells with mural cells. Protein kinase C delta and epsilon also appeared essential for vascular smooth muscle cell differentiation, since α-smooth muscle actin, a classical marker for vascular smooth muscle cells, was almost undetectable in double deficient embryonic aorta at E9.5. Subsequent qPCR analyses showed decreased VE-cadherin, Vegfr2, Cd31, Cdh2, Ets1, and Fli-1, among other angiogenesis related transcripts in double deficient embryos. Taken together, these data suggest for the first time an in vivo redundant role between members of the novel Protein Kinase C subfamily that allows for mutual compensation during mouse embryonic development, with vasculogenesis/angiogenesis as an obvious common function of these two Protein Kinase Cs. Protein Kinase C delta and epsilon might therefore be useful targets for inhibiting vasculo- and/or angiogenesis.
Assuntos
Embrião de Mamíferos/metabolismo , Proteína Quinase C-delta/fisiologia , Proteína Quinase C-épsilon/fisiologia , Animais , Antígenos CD/genética , Caderinas/genética , Feminino , Camundongos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Gravidez , Proteína Quinase C-delta/genética , Proteína Quinase C-épsilon/genética , Proteína Proto-Oncogênica c-ets-1/genética , Proteína Proto-Oncogênica c-fli-1/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
To investigate the role of NADPH oxidase homolog Nox1 at an early step of cell transformation, we utilized human gingival mucosal keratinocytes immortalized by E6/E7 of human papillomavirus (HPV) type 16 (GM16) to generate progenitor cell lines either by chronic ethanol exposure or overexpression with Nox1. Among several cobblestone epithelial cell lines obtained, two distinctive spindle cell lines - FIB and NuB1 cells were more progressively transformed exhibiting tubulogenesis and anchorage-independent growth associated with increased invasiveness. These spindle cells acquired molecular markers of epithelial mesenchymal transition (EMT) including mesenchymal vimentin and simple cytokeratins (CK) 8 and 18 as well as myogenic alpha-smooth muscle actin and caldesmon. By overexpression and knockdown experiments, we showed that Nox1 on a post-translational level regulated the stability of CK18 in an ROS-, phosphorylation- and PKCepilon-dependent manner. PKCepilon may thus be used as a therapeutic target for EMT inhibition. Taken together, Nox1 accelerates neoplastic progression by regulating structural intermediate filaments leading to EMT of immortalized human gingival epithelial cells.
Assuntos
Transformação Celular Neoplásica/patologia , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/fisiologia , NADPH Oxidases/fisiologia , Proteínas de Neoplasias/fisiologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Transformada , Transformação Celular Neoplásica/efeitos dos fármacos , Progressão da Doença , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Etanol/farmacologia , Gengiva , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Filamentos Intermediários/fisiologia , Queratina-18/metabolismo , NADPH Oxidase 1 , Invasividade Neoplásica , Proteínas Oncogênicas Virais/fisiologia , Proteínas E7 de Papillomavirus/fisiologia , Fosforilação , Proteína Quinase C-épsilon/fisiologia , Processamento de Proteína Pós-Traducional , Espécies Reativas de Oxigênio , Proteínas Repressoras/fisiologia , Seleção GenéticaRESUMO
Nonalcoholic fatty liver disease (NAFLD), hepatic insulin resistance, and type 2 diabetes are all strongly associated and are all reaching epidemic proportions. Whether there is a causal link between NAFLD and hepatic insulin resistance is controversial. This review will discuss recent studies in both humans and animal models of NAFLD that have implicated increases in hepatic diacylglycerol (DAG) content leading to activation of novel protein kinase Cϵ (PKCϵ) resulting in decreased insulin signaling in the pathogenesis of NAFLD-associated hepatic insulin resistance and type 2 diabetes. The DAG-PKCϵ hypothesis can explain the occurrence of hepatic insulin resistance observed in most cases of NAFLD associated with obesity, lipodystrophy, and type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/fisiopatologia , Fígado Gorduroso/fisiopatologia , Resistência à Insulina/fisiologia , Fígado/fisiopatologia , Animais , Diglicerídeos/fisiologia , Modelos Animais de Doenças , Humanos , Metabolismo dos Lipídeos/fisiologia , Hepatopatia Gordurosa não Alcoólica , Proteína Quinase C-épsilon/fisiologiaRESUMO
BACKGROUND: Sepsis is usually accompanied by cardiomyocyte apoptosis and myocardial depression. Protein kinase C (PKC) has been reported to be important in regulating cardiac function and apoptosis; however, which PKC isoform is involved in sepsis-induced myocardial apoptosis remains unknown. MATERIALS AND METHODS: A rat model of sepsis by cecal ligation and puncture was used. Early and late sepsis refers to those rats sacrificed at 9 and 18 h after cecal ligation and puncture, respectively. Ventricular septum (Sep), left ventricle (LV), and right ventricle were fractionated into membrane, mitochondrial, and cytosolic fractions, individually. The protein levels of PKC isoforms (-α, -ß, -δ, -ε, -ζ, -ι, -λ, and -µ) and mitochondrial translocation of Bad were quantified by Western blot analysis. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP in situ nick-end labeling. The morphology of mitochondria was examined by electron microscopy. RESULTS: The membrane/cytosol ratio of PKCε was predominantly higher in the Sep, LV, and right ventricle under physiological conditions. At early sepsis, the membrane/cytosol ratio of PKCε was significantly decreased in Sep and LV. At late sepsis, cardiomyocyte apoptosis associated with severe mitochondrial swelling and crista derangement were observed in Sep and LV at late sepsis. Additionally, mitochondria/cytosol ratio of Bad was significantly increased in Sep and LV. CONCLUSIONS: The early inactivation of PKCε in the ventricle may affect the mitochondrial translocation of Bad and subsequent mitochondrial disruption and apoptosis at late sepsis. This finding opens up the prospect for a potential therapeutic strategy targeting PKCε activation to prevent myocardial depression in septic patients.
Assuntos
Apoptose , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Proteína Quinase C-épsilon/fisiologia , Sepse/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismo , Animais , Ventrículos do Coração , Masculino , Miócitos Cardíacos/patologia , Proteína Quinase C-épsilon/antagonistas & inibidores , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Sepse/patologiaRESUMO
The INrf2 (Keap1)-Nrf2 cell sensor complex has a crucial role in protection against chemical- and radiation-induced oxidative stress and cellular transformation. INrf2, in association with Cul3-Rbx1, ubiquitylates and degrades Nrf2. Exposure to stressors leads to stabilization of Nrf2 and the coordinated activation of cytoprotective proteins and cellular protection. However, the molecular signal(s) that regulate control of Nrf2 by INrf2 remain elusive. In this report, we demonstrate that phosphorylation of INrf2 at Ser599 and Ser602 by the oncoprotein PKCε is essential for INrf2-Nrf2 interaction, and the subsequent ubiquitylation and degradation of Nrf2. Inhibition of PKCε, knockdown of PKCε and the INrf2S602A mutant all failed to phosphorylate INrf2, leading to loss of the INrf2-Nrf2 interaction, Nrf2 degradation and enhanced cytoprotection and drug resistance. Molecular modeling analyses revealed that phosphorylation of S599 exposes the deeply buried S602 for phosphorylation and enhanced INrf2-Nrf2 interaction. Analysis of human lung and liver tumor protein arrays showed lower PKCε and higher Nrf2 levels, which presumably promoted cancer cell survival and drug resistance. In conclusion, phosphorylation of INrf2 by PKCε leads to regulation of Nrf2, with significant implications for the survival of cancer cells, which often express lower levels of PKCε.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Proteína Quinase C-épsilon/fisiologia , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Elementos de Resposta Antioxidante , Antioxidantes/farmacologia , Sobrevivência Celular , Resistencia a Medicamentos Antineoplásicos , Regulação da Expressão Gênica , Células Hep G2 , Humanos , Hidroquinonas/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteína 1 Associada a ECH Semelhante a Kelch , Camundongos , Modelos Moleculares , Oncogenes , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteólise , Transdução de SinaisRESUMO
AIM: Emerging evidence has pointed to the participation of protein kinase C (PKC) in insulin-regulated trafficking of the glucose transporter GLUT4. The present study investigated the effect of the PKC activator diacylglycerol (DAG) on GLUT4 trafficking and glucose uptake. MAIN METHODS: 3T3L1-GLUT4myc fibroblast cells expressing GLUT4myc were differentiated into adipocytes. Western blotting, glucose assay, and real-time RT-PCR were carried out in 3T3L1-GLUT4myc adipocytes. PKCλ/ι, -ζ, -ε, and -γ were knocked-down by transfecting each siRNA. Activity of PKC isozymes was assayed under the cell-free conditions. KEY FINDINGS: Insulin increased cell surface localization of GLUT4 in 3T3L1-GLUT4myc adipocytes, and a similar effect was obtained with 1,2-dioleoyl-sn-glycerol (DO-DAG), 1-oleoyl-2-acetyl-sn-glycerol (OA-DAG), or 1,2-dipalmitoyl-sn-glycerol (DP-DAG). Like insulin, DO-DAG stimulated glucose uptake into adipocytes, but no significant synergistic increase in the glucose uptake was found with co-treatment with insulin and DO-DAG. Insulin activated Akt in adipocytes, but no Akt activation was induced by any investigated DAG. In the cell-free PKC assay, DAGs examined here activated PKCα, -ßI, -ßII, -γ, -δ, and -ε, but the atypical PKC isozymes PKCλ/ι and -ζ were not activated. Insulin-induced GLUT4 translocation to the cell surface was inhibited by knocking-down PKCλ/ι and -ζ, but not PKCγ or -ε. In contrast, DO-DAG-induced GLUT4 translocation to the cell surface was clearly prevented by knocking-down PKCε. SIGNIFICANCE: The results of the present study indicate that DAG stimulates GLUT4 translocation to the cell surface by activating PKCε, regardless of PKCλ/-ι and -ζ.
Assuntos
Diglicerídeos/fisiologia , Transportador de Glucose Tipo 4/metabolismo , Isoenzimas/fisiologia , Proteína Quinase C-épsilon/fisiologia , Proteína Quinase C/fisiologia , Células 3T3 , Animais , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Humanos , Camundongos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologiaRESUMO
In RAW 264.7 cells, PKC-ε regulates FcγR-mediated phagocytosis. BMDM behave similarly; PKC-ε concentrates at phagosomes and internalization are reduced in PKC-εâ»/â» cells. Two questions were asked: what is the role of PKC-ε? and what domains are necessary for PKC-ε concentration? Function was studied using BMDM and frustrated phagocytosis. On IgG surfaces, PKC-εâ»/â» macrophages spread less than WT. Patch-clamping revealed that the spreading defect is a result of the failure of PKC-εâ»/â» macrophages to add membrane. The defect is specific for FcγR ligation and can be reversed by expression of full-length (but not the isolated RD) PKC-ε in PKC-εâ»/â» BMDM. Thus, PKC-ε function in phagocytosis requires translocation to phagosomes and the catalytic domain. The expression of chimeric PKC molecules in RAW cells identified the εPS as necessary for PKC-ε targeting. When placed into (nonlocalizing) PKC-δ, εPS was sufficient for concentration, albeit to a lesser degree than intact PKC-ε. In contrast, translocation of δ(εPSC1B) resembled that of WT PKC-ε. Thus, εPS and εC1B cooperate for optimal phagosome targeting. Finally, cells expressing εK437W were significantly less phagocytic than their PKC-ε-expressing counterparts, blocked at the pseudopod-extension phase. In summary, we have shown that εPS and εC1B are necessary and sufficient for targeting PKC-ε to phagosomes, where its catalytic activity is required for membrane delivery and pseudopod extension.
Assuntos
Membrana Celular/metabolismo , Imunoglobulina G/farmacologia , Macrófagos/metabolismo , Fagocitose/fisiologia , Fagossomos/metabolismo , Proteína Quinase C-épsilon/fisiologia , Pseudópodes/fisiologia , Animais , Células da Medula Óssea/metabolismo , Citometria de Fluxo , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Técnicas de Patch-Clamp , Plasmídeos , Transporte Proteico , Receptores de IgG/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
BACKGROUND: Protein kinase C (PKC) serves as the receptor for tumor-promoting phorbol esters, which are potent activators of conventional (c) and novel (n) PKCs. We recently showed that these activators induced selective upregulation of PKCη in breast cancer cells. The objective of this study is to understand unique regulation of PKCη and its importance in breast cancer. METHODS: The levels of PKC isozymes were monitored in breast cancer cells following treatment with inhibitors of kinases, proteasome and proteases by Western blotting. PKCε was introduced by adenoviral delivery. PKCη and PDK1 were depleted by siRNA silencing. Cell growth was determined by the MTT or clonal assay. RESULTS: The general PKC inhibitors Gö 6983 and bisindolylmaleimide but not cPKC inhibitor Gö 6976 led to substantial PKCη downregulation, which was partly rescued by the introduction of nPKCε. Inhibition of phosphoinositide-dependent kinase-1 (PDK1) by Ly294002 or knockdown of PDK1 also led to downregulation of basal PKCη but had no effect on PKC activator-induced upregulation of PKCη. Proteasome inhibitors blocked PKCη downregulation triggered by PDK1 inhibition/depletion but not by Gö 6983. PKCη level increased in malignant but not in non-tumorigenic or pre-malignant cells in the progressive MCF-10A series associated with activated PDK1, and knockdown of PKCη inhibited breast cancer cell growth and clonogenic survival. CONCLUSION: Upregulation of PKCη contributes to breast cancer cell growth and targeting either PKCε or PDK1 triggers PKCη downregulation but involves two distinct mechanisms. GENERAL SIGNIFICANCE: The status of PKCη may serve as a potential biomarker for breast cancer malignancy.