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Oncogenic transformation is associated with profound changes in cellular metabolism, but whether tracking these can improve disease stratification or influence therapy decision-making is largely unknown. Using the iKnife to sample the aerosol of cauterized specimens, we demonstrate a new mode of real-time diagnosis, coupling metabolic phenotype to mutant PIK3CA genotype. Oncogenic PIK3CA results in an increase in arachidonic acid and a concomitant overproduction of eicosanoids, acting to promote cell proliferation beyond a cell-autonomous manner. Mechanistically, mutant PIK3CA drives a multimodal signaling network involving mTORC2-PKCζ-mediated activation of the calcium-dependent phospholipase A2 (cPLA2). Notably, inhibiting cPLA2 synergizes with fatty acid-free diet to restore immunogenicity and selectively reduce mutant PIK3CA-induced tumorigenicity. Besides highlighting the potential for metabolic phenotyping in stratified medicine, this study reveals an important role for activated PI3K signaling in regulating arachidonic acid metabolism, uncovering a targetable metabolic vulnerability that largely depends on dietary fat restriction. VIDEO ABSTRACT.
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Ácido Araquidónico/análisis , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Eicosanoides/metabolismo , Animales , Ácido Araquidónico/metabolismo , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I/genética , Citosol/metabolismo , Eicosanoides/fisiología , Activación Enzimática , Femenino , Humanos , Metabolismo de los Lípidos/fisiología , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Redes y Vías Metabólicas/genética , Redes y Vías Metabólicas/fisiología , Ratones Endogámicos BALB C , Ratones Desnudos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfolipasas A2/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
B7 ligands (CD80 and CD86), expressed by professional antigen-presenting cells (APCs), activate the main co-stimulatory receptor CD28 on T cells in trans. However, in peripheral tissues, APCs expressing B7 ligands are relatively scarce. This raises the questions of whether and how CD28 co-stimulation occurs in peripheral tissues. Here, we report that CD8+ T cells displayed B7 ligands that interacted with CD28 in cis at membrane invaginations of the immunological synapse as a result of membrane remodeling driven by phosphoinositide-3-kinase (PI3K) and sorting-nexin-9 (SNX9). cis-B7:CD28 interactions triggered CD28 signaling through protein kinase C theta (PKCθ) and promoted CD8+ T cell survival, migration, and cytokine production. In mouse tumor models, loss of T cell-intrinsic cis-B7:CD28 interactions decreased intratumoral T cells and accelerated tumor growth. Thus, B7 ligands on CD8+ T cells can evoke cell-autonomous CD28 co-stimulation in cis in peripheral tissues, suggesting cis-signaling as a general mechanism for boosting T cell functionality.
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Antígenos CD28 , Linfocitos T CD8-positivos , Ratones , Animales , Antígenos CD28/metabolismo , Antígenos CD/metabolismo , Ligandos , Membranas Sinápticas/metabolismo , Antígeno B7-2 , Glicoproteínas de Membrana/metabolismo , Antígeno B7-1/metabolismo , Moléculas de Adhesión Celular , Activación de LinfocitosRESUMEN
Serrated adenocarcinoma, an alternative pathway for colorectal cancer (CRC) development, accounts for 15%-30% of all CRCs and is aggressive and treatment resistant. We show that the expression of atypical protein kinase C ζ (PKCζ) and PKCλ/ι was reduced in human serrated tumors. Simultaneous inactivation of the encoding genes in the mouse intestinal epithelium resulted in spontaneous serrated tumorigenesis that progressed to advanced cancer with a strongly reactive and immunosuppressive stroma. Whereas epithelial PKCλ/ι deficiency led to immunogenic cell death and the infiltration of CD8+ T cells, which repressed tumor initiation, PKCζ loss impaired interferon and CD8+ T cell responses, which resulted in tumorigenesis. Combined treatment with a TGF-ß receptor inhibitor plus anti-PD-L1 checkpoint blockade showed synergistic curative activity. Analysis of human samples supported the relevance of these kinases in the immunosurveillance defects of human serrated CRC. These findings provide insight into avenues for the detection and treatment of this poor-prognosis subtype of CRC.
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Mucosa Intestinal/inmunología , Neoplasias Intestinales/inmunología , Isoenzimas/inmunología , Proteína Quinasa C/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/inmunología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/metabolismo , Femenino , Humanos , Vigilancia Inmunológica/genética , Vigilancia Inmunológica/inmunología , Mucosa Intestinal/enzimología , Mucosa Intestinal/patología , Neoplasias Intestinales/enzimología , Neoplasias Intestinales/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones Noqueados , Ratones Transgénicos , Persona de Mediana Edad , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismoRESUMEN
Activity of central amygdala (CeA) PKCδ expressing neurons has been linked to appetite regulation, anxiety-like behaviors, pain sensitivity, and addiction-related behaviors. Studies of the role that CeA PKCδ+ neurons play in these behaviors have largely been carried out in mice, and genetic tools that would allow selective manipulation of PKCδ+ cells in rats have been lacking. Here, we used a CRISPR/Cas9 strategy to generate a transgenic Prkcd-cre knock-in rat and characterized this model using anatomical, electrophysiological, and behavioral approaches in both sexes. In the CeA, Cre was selectively expressed in PKCδ+ cells. Anterograde projections of PKCδ+ neurons to cortical regions, subcortical regions, several hypothalamic nuclei, the amygdala complex, and midbrain dopaminergic regions were largely consistent with published mouse data. In a behavioral screen, we found no differences between Cre+ rats and Cre- wild-type littermates. Optogenetic stimulation of CeA PKCδ+ neurons in a palatable food intake assay resulted in an increased latency to first feeding and decreased total food intake, once again replicating published mouse findings. Lastly, using a real-time place preference task, we found that stimulation of PKCδ+ neurons promoted aversion, without affecting locomotor activity. Collectively, these findings establish the novel Prkcd-Cre rat line as a valuable tool that complements available mouse lines for investigating the functional role of PKCδ+ neurons.
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Proteína Quinasa C-delta , Animales , Proteína Quinasa C-delta/genética , Proteína Quinasa C-delta/metabolismo , Ratas , Masculino , Femenino , Ratas Transgénicas , Neuronas/fisiología , Núcleo Amigdalino Central/fisiología , Integrasas/genética , Optogenética/métodos , Ratas Sprague-DawleyRESUMEN
Protein secretion in cancer cells defines tumor survival and progression by orchestrating the microenvironment. Studies suggest the occurrence of active secretion of cytosolic proteins in liver cancer and their involvement in tumorigenesis. Here, we investigated the identification of extended-synaptotagmin 1 (E-Syt1), an endoplasmic reticulum (ER)-bound protein, as a key mediator for cytosolic protein secretion at the ER-plasma membrane (PM) contact sites. Cytosolic proteins interacted with E-Syt1 on the ER, and then localized spatially inside SEC22B+ vesicles of liver cancer cells. Consequently, SEC22B on the vesicle tethered to the PM via Q-SNAREs (SNAP23, SNX3, and SNX4) for their secretion. Furthermore, inhibiting the interaction of protein kinase Cδ (PKCδ), a liver cancer-specific secretory cytosolic protein, with E-Syt1 by a PKCδ antibody, decreased in both PKCδ secretion and tumorigenicity. Results reveal the role of ER-PM contact sites in cytosolic protein secretion and provide a basis for ER-targeting therapy for liver cancer.
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Neoplasias Hepáticas , Proteínas R-SNARE , Sinaptotagmina I , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Transporte de Proteínas , Proteínas R-SNARE/metabolismo , Sinaptotagmina I/metabolismo , Microambiente TumoralRESUMEN
BACKGROUND: Single nucleotide polymorphisms (SNPs) have been linked with prostate cancer (PCa) and have shown potential as prognostic markers for advanced stages. Loss of function mutations in PKCι have been linked with increased risk of malignancy by enhancing tumor cell motility and invasion. We have evaluated the impact of two coding region SNPs on the PKCι gene (PRKCI) and their prognostic potential. METHODS: Genotypic association of non-synonymous PKCι SNPs rs1197750201 and rs1199520604 with PCa was determined through tetra-ARMS PCR. PKCι was docked with interacting partner Par-6 to determine the effect of these variants on PKCι binding capabilities. Molecular dynamic simulations of PKCι docked with Par-6 were performed to determine variant effects on PKCι protein interactions. The possible impact of changes in PKCι protein interactions on epithelial cell polarity was hypothesized. RESULTS: PKCι rs1199520604 mutant genotype TT showed association with PCa (p = 0.0055), while rs1197750201 mutant genotype AA also showed significant association with PCa (P = 0.0006). The binding interaction of PKCι with Par-6 was altered for both variants, with changes in Van der Waals energy and electrostatic energy of docked structures. CONCLUSION: Genotypic analysis of two non-synonymous PKCι variants in association with PCa prognosis was performed. Both variants in the PB1 domain showed potential as a prognostic marker for PCa. In silico analysis of the effect of the variants on PKCι protein interactions indicated they may be involved in PCa progression through aberration of epithelial cell polarity pathways.
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In the obesity context, inflammatory cytokines secreted by adipocytes lead to insulin resistance and are key to metabolic syndrome development. In our previous study, we found that the transcription factor KLF7 promoted the expression of p-p65 and IL-6 in adipocytes. However, the specific molecular mechanism remained unclear. In the present study, we found that the expression of KLF7, PKCζ, p-IκB, p-p65, and IL-6 in epididymal white adipose tissue (Epi WAT) in mice fed a high-fat diet (HFD) was significantly increased. In contrast, the expression of PKCζ, p-IκB, p-p65, and IL-6 was significantly decreased in Epi WAT of KLF7 fat conditional knockout mice. In 3T3-L1 adipocytes, KLF7 promoted the expression of IL-6 via the PKCζ/NF-κB pathway. In addition, we performed luciferase reporter and chromatin immunoprecipitation assays, which confirmed that KLF7 upregulated the expression of PKCζ transcripts in HEK-293T cells. Collectively, our results show that KLF7 promotes the expression of IL-6 by upregulating PKCζ expression and activating the NF-κB signaling pathway in adipocytes.
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Trastornos del Metabolismo de la Glucosa , FN-kappa B , Animales , Ratones , Células 3T3-L1 , Adipocitos/metabolismo , Dieta Alta en Grasa/efectos adversos , Trastornos del Metabolismo de la Glucosa/metabolismo , Proteínas I-kappa B/metabolismo , Inflamación/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , FN-kappa B/metabolismoRESUMEN
Postoperative cognitive dysfunction (POCD) is a kind of serious postoperative complication in surgery with general anesthesia and it may affect patients' normal lives. Activated microglia are thought to be one of the key factors in the regulation of POCD process. Once activated, resident microglia change their phenotype and secrete kinds of cytokines to regulate inflammatory response in tissues. Among these secretory factors, brain-derived neurotrophic factor (BDNF) is considered to be able to inhibit inflammation response and protect nervous system. Therefore, the enhancement of BDNF expression derived from resident microglia is suggested to be potential treatment for POCD. In our study, we focused on the role of C8-ceramide (a kind of interventional drug) and assessed its regulatory effect on improving the expression of BDNF secreted from microglia to treat POCD. According to the results of our study, we observed that C8-ceramide stimulated primary microglia to up-regulate the expression of BDNF mRNA after being treated with lipopolysaccharide (LPS) in vitro. We proved that C8-ceramide had ability to effectively improve POCD of mice after being accepted carotid artery exposure and their abnormal behavior recovered better than that of mice from the surgery group. Furthermore, we also demonstrated that C8-ceramide enhanced the cognitive function of mice via the PKCδ/NF-κB signaling pathway. In general, our study has confirmed a potential molecular mechanism that led to the occurrence of POCD caused by surgery and provided a new clinical strategy to treat POCD.
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Factor Neurotrófico Derivado del Encéfalo , Ceramidas , Microglía , FN-kappa B , Complicaciones Cognitivas Postoperatorias , Proteína Quinasa C-delta , Transducción de Señal , Animales , Microglía/efectos de los fármacos , Microglía/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Ratones , FN-kappa B/metabolismo , Complicaciones Cognitivas Postoperatorias/metabolismo , Complicaciones Cognitivas Postoperatorias/prevención & control , Ceramidas/metabolismo , Proteína Quinasa C-delta/metabolismo , Masculino , Ratones Endogámicos C57BLRESUMEN
Breast carcinoma (BC) is one of the most common malignant cancers in females, and metastasis remains the leading cause of death in these patients. Chemotaxis plays an important role in cancer cell metastasis and the mechanism of breast cancer chemotaxis has become a central issue in contemporary research. PKCζ, a member of the atypical PKC family, has been reported to be an essential component of the EGF-stimulated chemotactic signaling pathway. However, the molecular mechanism through which PKCζ regulates chemotaxis remains unclear. Here, we used a proteomic approach to identify PKCζ-interacting proteins in breast cancer cells and identified VASP as a potential binding partner. Intriguingly, stimulation with EGF enhanced this interaction and induced the translocalization of PKCζ and VASP to the cell membrane. Further experiments showed that PKCζ catalyzes the phosphorylation of VASP at Ser157, which is critical for the biological function of VASP in regulating chemotaxis and actin polymerization in breast cancer cells. Furthermore, in PKCζ knockdown BC cells, the enrichment of VASP at the leading edge was reduced, and its interaction with profilin1 was attenuated, thereby reducing the chemotaxis and overall motility of breast cancer cells after EGF treatment. In functional assays, PKCζ promoted chemotaxis and motility of BC cells through VASP. Our findings demonstrate that PKCζ, a new kinase of VASP, plays an important role in promoting breast cancer metastasis and provides a theoretical basis for expanding new approaches to tumor biotherapy.
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Neoplasias de la Mama , Quimiotaxis , Proteína Quinasa C , Femenino , Humanos , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Quimiotaxis/genética , Factor de Crecimiento Epidérmico/farmacología , Factor de Crecimiento Epidérmico/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , ProteómicaRESUMEN
Insulin-resistant diabetes is a common metabolic disease with serious complications. Treatments directly addressing the underlying molecular mechanisms involving insulin resistance would be desirable. Our laboratory recently identified a proteolytic-resistant cystine-dense microprotein from huáng qí (Astragalus membranaceus) called α-astratide aM1, which shares high sequence homology to leginsulins. Here we show that aM1 is a cell-penetrating insulin mimetic, enters cells by endocytosis, and activates the PI3K/Akt signaling pathway independent of the insulin receptor leading to translocation of glucose transporter GLUT4 to the cell surface to promote glucose uptake. We also showed that aM1 alters gene expression, suppresses lipid synthesis and uptake, and inhibits intracellular lipid accumulation in myotubes and adipocytes. By reducing intracellular lipid accumulation and preventing lipid-induced, PKCθ-mediated degradation of IRS1/2, aM1 restores glucose uptake to overcome insulin resistance. These findings highlight the potential of aM1 as a lead for developing orally bioavailable insulin mimetics to expand options for treating diabetes.
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Resistencia a la Insulina , Humanos , Proteínas Proto-Oncogénicas c-akt , Fosfatidilinositol 3-Quinasas , Insulina/farmacología , Transducción de Señal , Glucosa , Lípidos , MicropéptidosRESUMEN
INTRODUCTION: To investigate the role of a novel type of protein kinase C delta (PKCδ) in the neuroinflammation of Alzheimer's disease (AD). METHODS: We analyzed PKCδ and inflammatory cytokines levels in cerebrospinal fluid (CSF) of AD and normal controls, as well as their correlations. The cellular expression pattern of PKCδ and the effects of PKCδ modulation on microglia-mediated neuroinflammation were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), western blot, RNA sequencing (RNA-seq), and immunofluorescence staining. RESULTS: PKCδ levels were increased dramatically in the CSF of AD patients and positively correlated with cytokines. PKCδ is expressed mainly in microglia in the brain. Amyloid beta (Aß) stimulation increased PKCδ expression and secretion, which led to upregulation of the nuclear factor kappa B (NF-κB) pathway and overproduction of proinflammatory cytokines. Downregulation or inhibition of PKCδ attenuated Aß-induced microglial responses and improved cognitive function in an AD mouse model. DISCUSSION: Our study identifies PKCδ as a potential biomarker and therapeutic target for microglia-mediated neuroinflammation in AD. HIGHLIGHTS: Protein kinase C delta (PKCδ) levels increase in cerebrospinal fluid (CSF) of patients with Alzheimer's disease (AD), and positively correlate with elevated inflammatory cytokines in human subjects. PKCδ is expressed mainly in microglia in vivo, whereas amyloid beta (Aß) stimulation increases PKCδ expression and secretion, causing upregulation of the nuclear factor kappa B (NF-κB) pathway and production of inflammatory cytokines. Downregulation or inhibition of PKCδ attenuates Aß-enhanced NF-κB signaling and cytokine production in microglia and improves cognitive function in AD mice. PKCδ serves as a potential biomarker and therapeutic target for microglia-mediated neuroinflammation in AD.
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Porcine epidemic diarrhea virus (PEDV), a member of the Alpha-coronavirus genus in the Coronaviridae family, induces acute diarrhea, vomiting, and dehydration in neonatal piglets. This study aimed to investigate the genetic dependencies of PEDV and identify potential therapeutic targets by using a single-guide RNA (sgRNA) lentiviral library to screen host factors required for PEDV infection. Protein kinase C θ (PKCθ), a calcium-independent member of the PKC family localized in the cell membrane, was found to be a crucial host factor in PEDV infection. The investigation of PEDV infection was limited in Vero and porcine epithelial cell-jejunum 2 (IPEC-J2) due to defective interferon production in Vero and the poor replication of PEDV in IPEC-J2. Therefore, identifying suitable cells for PEDV investigation is crucial. The findings of this study reveal that human embryonic kidney (HEK) 293T and L929 cells, but not Vero and IPEC-J2 cells, were suitable for investigating PEDV infection. PKCθ played a significant role in endocytosis and the replication of PEDV, and PEDV regulated the expression and phosphorylation of PKCθ. Apoptosis was found to be involved in PEDV replication, as the virus activated the PKCθ-B-cell lymphoma 2 (BCL-2) ovarian killer (BOK) axis in HEK293T and L929 cells to increase viral endocytosis and replication via mitochondrial apoptosis. This study demonstrated the suitability of HEK293T and L929 cells for investigating PEDV infection and identified PKCθ as a host factor essential for PEDV infection. These findings provide valuable insights for the development of strategies and drug targets for PEDV infection.
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Virus de la Diarrea Epidémica Porcina , Enfermedades de los Porcinos , Animales , Humanos , Porcinos , Chlorocebus aethiops , Virus de la Diarrea Epidémica Porcina/genética , Proteína Quinasa C-theta/genética , Sistemas CRISPR-Cas , Células HEK293 , ARN Guía de Sistemas CRISPR-Cas , Células Vero , Enfermedades de los Porcinos/genética , Replicación Viral/genéticaRESUMEN
AIMS/HYPOTHESIS: Athletes exhibit increased muscle insulin sensitivity, despite increased intramuscular triacylglycerol content. This phenomenon has been coined the 'athlete's paradox' and is poorly understood. Recent findings suggest that the subcellular distribution of sn-1,2-diacylglycerols (DAGs) in the plasma membrane leading to activation of novel protein kinase Cs (PKCs) is a crucial pathway to inducing insulin resistance. Here, we hypothesised that regular aerobic exercise would preserve muscle insulin sensitivity by preventing increases in plasma membrane sn-1,2-DAGs and activation of PKCε and PKCθ despite promoting increases in muscle triacylglycerol content. METHODS: C57BL/6J mice were allocated to three groups (regular chow feeding [RC]; high-fat diet feeding [HFD]; RC feeding and running wheel exercise [RC-EXE]). We used a novel LC-MS/MS/cellular fractionation method to assess DAG stereoisomers in five subcellular compartments (plasma membrane [PM], endoplasmic reticulum, mitochondria, lipid droplets and cytosol) in the skeletal muscle. RESULTS: We found that the HFD group had a greater content of sn-DAGs and ceramides in multiple subcellular compartments compared with the RC mice, which was associated with an increase in PKCε and PKCθ translocation. However, the RC-EXE mice showed, of particular note, a reduction in PM sn-1,2-DAG and ceramide content when compared with HFD mice. Consistent with the PM sn-1,2-DAG-novel PKC hypothesis, we observed an increase in phosphorylation of threonine1150 on the insulin receptor kinase (IRKT1150), and reductions in insulin-stimulated IRKY1162 phosphorylation and IRS-1-associated phosphoinositide 3-kinase activity in HFD compared with RC and RC-EXE mice, which are sites of PKCε and PKCθ action, respectively. CONCLUSIONS/INTERPRETATION: These results demonstrate that lower PKCθ/PKCε activity and sn-1,2-DAG content, especially in the PM compartment, can explain the preserved muscle insulin sensitivity in RC-EXE mice.
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Resistencia a la Insulina , Ratones , Animales , Resistencia a la Insulina/fisiología , Proteína Quinasa C-theta/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Cromatografía Liquida , Fosfatidilinositol 3-Quinasas/metabolismo , Ratones Endogámicos C57BL , Espectrometría de Masas en Tándem , Insulina/metabolismo , Músculo Esquelético/metabolismo , Triglicéridos/metabolismo , Ceramidas/metabolismoRESUMEN
AIMS/HYPOTHESIS: The loss of pericytes surrounding the retinal vasculature in early diabetic retinopathy underlies changes to the neurovascular unit that lead to more destructive forms of the disease. However, it is unclear which changes lead to loss of retinal pericytes. This study investigated the hypothesis that chronic increases in one or more inflammatory factors mitigate the signalling pathways needed for pericyte survival. METHODS: Loss of pericytes and levels of inflammatory markers at the mRNA and protein levels were investigated in two genetic models of diabetes, Ins2Akita/+ (a model of type 1 diabetes) and Leprdb/db (a model of type 2 diabetes), at early stages of diabetic retinopathy. In addition, changes that accompany gliosis and the retinal vasculature were determined. Finally, changes in retinal pericytes chronically incubated with vehicle or increasing amounts of IFNγ were investigated to determine the effects on pericyte survival. The numbers of pericytes, microglia, astrocytes and endothelial cells in retinal flatmounts were determined by immunofluorescence. Protein and mRNA levels of inflammatory factors were determined using multiplex ELISAs and quantitative reverse transcription PCR (qRT-PCR). The effects of IFNγ on the murine retinal pericyte survival-related platelet-derived growth factor receptor ß (PDGFRß) signalling pathway were investigated by western blot analysis. Finally, the levels of cell death-associated protein kinase C isoform delta (PKCδ) and cleaved caspase 3 (CC3) in pericytes were determined by western blot analysis and immunocytochemistry. RESULTS: The essential findings of this study were that both type 1 and 2 diabetes were accompanied by a similar progression of retinal pericyte loss, as well as gliosis. However, inflammatory factor expression was dissimilar in the two models of diabetes, with peak expression occurring at different ages for each model. Retinal vascular changes were more severe in the type 2 diabetes model. Chronic incubation of murine retinal pericytes with IFNγ decreased PDGFRß signalling and increased the levels of active PKCδ and CC3. CONCLUSIONS/INTERPRETATION: We conclude that retinal inflammation is involved in and sustains pericyte loss as diabetic retinopathy progresses. Moreover, IFNγ plays a critical role in reducing pericyte survival in the retina by reducing activation of the PDGFRß signalling pathway and increasing PKCδ levels and pericyte apoptosis.
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Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Retinopatía Diabética , Ratones , Animales , Retinopatía Diabética/genética , Retinopatía Diabética/metabolismo , Modelos Animales de Enfermedad , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Células Endoteliales/metabolismo , Gliosis/complicaciones , Gliosis/metabolismo , Diabetes Mellitus Experimental/metabolismo , Retina/metabolismo , Inflamación/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Pericitos/metabolismoRESUMEN
A significant number of babies present transiently with low protein kinase C zeta (PKCζ) levels in cord blood T cells (CBTC), associated with reduced ability to transition from a neonatal Th2 to a mature Th1 cytokine bias, leading to a higher risk of developing allergic sensitisation, compared to neonates whose T cells have 'normal' PKCζ levels. However, the importance of PKCζ signalling in regulating their differentiation from a Th2 to a Th1 cytokine phenotype propensity remains undefined. To define the role of PKCζ signalling in the regulation of CBTC differentiation from a Th2 to a Th1cytokine phenotype we have developed a neonatal T cell maturation model which enables the cells to develop to CD45RA- /CD45RO+ T cells while maintaining the Th2 immature cytokine bias, despite having normal levels of PKCζ. The immature cells were treated with phytohaemagglutinin, but in addition with phorbol 12-myristate 13-acetate (PMA), an agonist which does not activate PKCζ. This was compared to development in CBTC in which the cells were transfected to express constitutively active PKCζ. The lack of PKCζ activation by PMA was monitored by western blot for phospho-PKCζ and translocation from cell cytosol to the membrane by confocal microscopy. The findings demonstrate that PMA fails to activate PKCζ in CBTC. The data show that CBTC matured under the influence of the PKC stimulator, PMA, maintain a Th2 cytokine bias, characterised by robust IL-4 and minimal interferon gamma production (IFN-γ), and lack of expression of transcriptional factor, T-bet. This was also reflected in the production of a range of other Th2/Th1 cytokines. Interestingly, introduction of a constitutively active PKCζ mutant into CBTC promoted development towards a Th1 profile with high IFN-γ production. The findings demonstrate that PKCζ signalling is essential for the immature neonatal T cells to transition from a Th2 to a Th1 cytokine production bias.
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Interferón gamma , Células TH1 , Recién Nacido , Humanos , Interferón gamma/metabolismo , Células TH1/metabolismo , Sangre Fetal , Citocinas/metabolismo , Diferenciación Celular , Antígenos Comunes de Leucocito , Células Th2/metabolismoRESUMEN
KRIT1 is a scaffolding protein that regulates multiple molecular mechanisms, including cell-cell and cell-matrix adhesion, and redox homeostasis and signaling. However, rather little is known about how KRIT1 is itself regulated. KRIT1 is found in both the cytoplasm and the nucleus, yet the upstream signaling proteins and mechanisms that regulate KRIT1 nucleocytoplasmic shuttling are not well understood. Here, we identify a key role for protein kinase C (PKC) in this process. In particular, we found that PKC activation promotes the redox-dependent cytoplasmic localization of KRIT1, whereas inhibition of PKC or treatment with the antioxidant N-acetylcysteine leads to KRIT1 nuclear accumulation. Moreover, we demonstrated that the N-terminal region of KRIT1 is crucial for the ability of PKC to regulate KRIT1 nucleocytoplasmic shuttling, and may be a target for PKC-dependent regulatory phosphorylation events. Finally, we found that silencing of PKCα, but not PKCδ, inhibits phorbol 12-myristate 13-acetate (PMA)-induced cytoplasmic enrichment of KRIT1, suggesting a major role for PKCα in regulating KRIT1 nucleocytoplasmic shuttling. Overall, our findings identify PKCα as a novel regulator of KRIT1 subcellular compartmentalization, thus shedding new light on the physiopathological functions of this protein.
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Transporte Activo de Núcleo Celular , Proteína KRIT1/metabolismo , Proteína Quinasa C-alfa , Células HeLa , Humanos , Fosforilación , Proteína Quinasa C-alfa/genética , Acetato de TetradecanoilforbolRESUMEN
The CCCH-type zinc finger antiviral protein (ZAP) can recognize and induce the degradation of mRNAs and proteins of certain viruses, as well as exerting its antiviral activity by activating T cells. However, the mechanism of ZAP that mediates T cell activation during virus infection remains unclear. Here, we found a potential function of ZAP that relieves immunosuppression of T cell induced by avian leukosis virus subgroup J (ALV-J) via a novel signaling pathway that involves norbin-like protein (NLP), protein kinase C delta (PKC-δ), and nuclear factor of activated T cell (NFAT). Specifically, ZAP expression activated T cells by promoting the dephosphorylation and nuclear translocation of NFAT. Furthermore, knockdown of ZAP weakened the reactivity and antiviral response of T cells. Mechanistically, ZAP reduced PKC-δ activity by upregulating and reactivating NLP by competitively binding with viral protein. Knockdown of NLP decreased the dephosphorylation of PKC-δ by ZAP expression. Moreover, we show that knockdown of PKC-δ reduced the phosphorylation levels of NFAT and enhanced its nuclear translocation. Taken together, these data revealed that ZAP relieves immunosuppression caused by ALV-J and mediates T cell activation through the NLP-PKC-δ-NFAT pathway. IMPORTANCE The evolution of the host defense system is driven synchronously in the process of resisting virus invasion. Accordingly, host innate defense factors effectively work to suppress virus replication. However, it remains unclear whether the host innate defense factors are involved in antiviral immune responses against the invasion of immunosuppressive viruses. Here, we found that CCCH-type zinc finger antiviral protein (ZAP) effectively worked in resistance to immunosuppression caused by avian leukosis virus subgroup J (ALV-J), a classic immunosuppressive virus. Evidence showed that ZAP released the phosphatase activity of NLP inhibited by ALV-J and further activated NFAT by inactivating PKC-δ. This novel molecular mechanism, i.e., ZAP regulation of the antiviral immune response by mediating the NLP-PKC-δ-NFAT pathway, has greatly enriched the understanding of the functions of host innate defense factors and provided important scientific ideas and a theoretical basis for research on immunosuppressive viruses and antiviral immunity.
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Virus de la Leucosis Aviar/inmunología , Factores de Transcripción NFATC/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteína Quinasa C-delta/metabolismo , Proteínas de Unión al ARN/metabolismo , Linfocitos T/inmunología , Animales , Pollos , Interacciones Huésped-Patógeno , Tolerancia Inmunológica , Activación de Linfocitos , Fosforilación , Unión Proteica , Proteínas de Unión al ARN/genética , Transducción de Señal , Linfocitos T/metabolismo , Linfocitos T/virología , Proteínas Virales/metabolismoRESUMEN
Metabolic-associated fatty liver disease (MAFLD) is a global concern, often undetected until reaching an advanced stage. Palmitic acid (PA) is a type of fatty acid that increases and leads to liver apoptosis in MAFLD. However, there is currently no approved therapy or compound for MAFLD. Recently, branched fatty acid esters of hydroxy fatty acids (FAHFAs), a group of bioactive lipids, have emerged as promising agents to treat associated metabolic diseases. This study utilizes one type of FAHFA, oleic acid ester of 9-hydroxystearic acid (9-OAHSA), to treat PA-induced lipoapoptosis in an in vitro MAFLD model using rat hepatocytes and a high-fat high-cholesterol high-fructose (HFHCHFruc) diet in Syrian hamsters. The results indicate that 9-OAHSA rescues hepatocytes from PA-induced apoptosis and attenuates lipoapoptosis and dyslipidemia in Syrian hamsters. Additionally, 9-OAHSA decreases the generation of mitochondrial reactive oxygen species (mito-ROS) and stabilizes the mitochondrial membrane potential in hepatocytes. The study also demonstrates that the effect of 9-OAHSA on mito-ROS generation is at least partially mediated by PKC-δ signaling. These findings suggest that 9-OAHSA shows promise as a therapy for MAFLD.
Asunto(s)
Enfermedad del Hígado Graso no Alcohólico , Ácido Palmítico , Cricetinae , Ratas , Animales , Ácido Palmítico/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Mesocricetus , Fructosa/toxicidad , Hepatocitos , Ácidos Grasos/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Colesterol/metabolismo , Dieta Alta en Grasa/efectos adversosRESUMEN
Diabetes mellitus, known for its complications, especially vascular complications, is becoming a globally serious social problem. Atherosclerosis has been recognized as a common vascular complication mechanism in diabetes. The diacylglycerol (DAG)-protein kinase C (PKC) pathway plays an important role in atherosclerosis. PKCs can be divided into three subgroups: conventional PKCs (cPKCs), novel PKCs (nPKCs), and atypical PKCs (aPKCs). The aim of this review is to provide a comprehensive overview of the role of the PKCδ pathway, an isoform of nPKC, in regulating the function of endothelial cells, vascular smooth muscle cells, and macrophages in diabetic atherosclerosis. In addition, potential therapeutic targets regarding the PKCδ pathway are summarized. Video Abstract.
Asunto(s)
Diabetes Mellitus , Células Endoteliales , Humanos , Células Endoteliales/metabolismo , Proteína Quinasa C/metabolismo , Isoformas de Proteínas , BiologíaRESUMEN
Oxidative stress, reactive oxygen species generation, and overexpression of VEGF are signatory events in diabetic retinopathy. The downregulation of VEGF and anti-inflammatory action pave the way for diabetic retinopathy (DR) therapy. In that, lower absorption kinetics of melatonin limits its immense therapeutic potential. Hence, we have demonstrated a reverse microemulsion method to synthesize melatonin-loaded polydopamine nanoparticles to replenish both at a single platform with an improved melatonin delivery profile. The study has evaluated in vitro and in vivo protection efficiency of biocompatible melatonin-loaded polydopamine nanoparticles (MPDANPs). The protection mechanism was explained by downregulation of VEGF, CASPASE3, and PKCδ against high-glucose/streptozotocin (STZ)-induced insults, in vitro and in vivo. The anti-inflammatory and antiangiogenic effect and potential of MPDANPs to enhance melatonin in vivo stability with prolonged circulation time have proved MPDANPs as a potential therapeutic candidate in DR management. The DR therapeutic potential of MPDANPs has been arbitrated by improving the bioavailability of melatonin and inhibition of VEGF-PKCδ crosstalk in vivo.