RESUMEN
Tumor cells have high-energetic and anabolic needs and are known to adapt their metabolism to be able to survive and keep proliferating under conditions of nutrient stress. We show that PKCζ deficiency promotes the plasticity necessary for cancer cells to reprogram their metabolism to utilize glutamine through the serine biosynthetic pathway in the absence of glucose. PKCζ represses the expression of two key enzymes of the pathway, PHGDH and PSAT1, and phosphorylates PHGDH at key residues to inhibit its enzymatic activity. Interestingly, the loss of PKCζ in mice results in enhanced intestinal tumorigenesis and increased levels of these two metabolic enzymes, whereas patients with low levels of PKCζ have a poor prognosis. Furthermore, PKCζ and caspase-3 activities are correlated with PHGDH levels in human intestinal tumors. Taken together, this demonstrates that PKCζ is a critical metabolic tumor suppressor in mouse and human cancer.
Asunto(s)
Adenocarcinoma/metabolismo , Adenoma/metabolismo , Neoplasias del Colon/metabolismo , Proteína Quinasa C/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Animales , Vías Biosintéticas , Transformación Celular Neoplásica , Glucosa/metabolismo , Humanos , Ratones , Serina/biosíntesis , Organismos Libres de Patógenos Específicos , Estrés FisiológicoRESUMEN
PKCß-null (Prkcb-/-) mice are severely immunodeficient. Here we show that mice whose B cells lack PKCß failed to form germinal centers and plasma cells, which undermined affinity maturation and antibody production in response to immunization. Moreover, these mice failed to develop plasma cells in response to viral infection. At the cellular level, we have shown that Prkcb-/- B cells exhibited defective antigen polarization and mTORC1 signaling. While altered antigen polarization impaired antigen presentation and likely restricted the potential of GC development, defective mTORC1 signaling impaired metabolic reprogramming, mitochondrial remodeling, and heme biosynthesis in these cells, which altogether overwhelmingly opposed plasma cell differentiation. Taken together, our study reveals mechanistic insights into the function of PKCß as a key regulator of B cell polarity and metabolic reprogramming that instructs B cell fate.
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Linfocitos B/inmunología , Diferenciación Celular/inmunología , Activación de Linfocitos/inmunología , Células Plasmáticas/inmunología , Proteína Quinasa C beta/inmunología , Animales , Hemo/biosíntesis , Ratones , Ratones Noqueados , Mitocondrias/inmunología , Mitocondrias/metabolismo , Células Plasmáticas/citologíaRESUMEN
Members of the Protein kinases D (PKD) family are described as regulators of T cell responses. From the two T cell-expressed isoforms PKD2 and PKD3, so far mainly the former was thoroughly investigated and is well understood. Recently, we have investigated also PKD3 using conventional as well as conditional T cell-specific knockout models. These studies suggested PKD3 to be a T cell-extrinsic regulator of the cells' fate. However, these former model systems did not take into account possible redundancies with the highly homologous PKD2. To overcome this issue and thus properly unravel PKD3's T cell-intrinsic functions, here we additionally used a mouse model overexpressing a constitutively active isoform of PKD3 specifically in the T cell compartment. These transgenic mice showed a slightly higher proportion of central memory T cells in secondary lymphoid organs and blood. This effect could not be explained via differences upon polyclonal stimulation in vitro, however, may be connected to the observed developmental aberrances in the CD8 single positive compartment during thymic development. Lastly, the observed alterations in the CD8+ T cell compartment did not impact proper immune response upon immunization with ovalbumin or in a subcutaneous tumour model suggesting only a small to absent biological relevance. Taking together the knowledge of all our published studies on PKD3 in the T cell compartment, we now conclude that T cell-intrinsic PKD3 is a fine-tuner of central memory T cell as well as CD8 single positive thymocyte development.
Asunto(s)
Linfocitos T CD8-positivos , Diferenciación Celular , Células T de Memoria , Ratones Transgénicos , Proteína Quinasa C , Timocitos , Animales , Timocitos/inmunología , Timocitos/metabolismo , Ratones , Linfocitos T CD8-positivos/inmunología , Proteína Quinasa C/metabolismo , Proteína Quinasa C/genética , Células T de Memoria/inmunología , Células T de Memoria/metabolismo , Ratones Endogámicos C57BL , Memoria Inmunológica , Ratones NoqueadosRESUMEN
Cell immortalization, a hallmark of cancer development, is a process that cells can undergo on their path to carcinogenesis. Spontaneously immortalized mouse embryonic fibroblasts (MEFs) have been used for decades; however, changes in the global transcriptome during this process have been poorly described. In our research, we characterized the poly-A RNA transcriptome changes after spontaneous immortalization. To this end, differentially expressed genes (DEGs) were screened using DESeq2 and characterized by gene ontology enrichment analysis and protein-protein interaction (PPI) network analysis to identify the potential hub genes. In our study, we identified changes in the expression of genes involved in proliferation regulation, cell adhesion, immune response and transcriptional regulation in immortalized MEFs. In addition, we performed a comparative analysis with previously reported MEF immortalization data, where we propose a predicted gene regulatory network model in immortalized MEFs based on the altered expression of Mapk11, Cdh1, Chl1, Zic1, Hoxd10 and the novel hub genes Il6 and Itgb2.
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Fibroblastos , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Transcriptoma , Animales , Ratones , Fibroblastos/metabolismo , Mapas de Interacción de Proteínas/genética , Embrión de Mamíferos/metabolismo , Ontología de GenesRESUMEN
Clonal deletion of autoreactive B cells is crucial for the prevention of autoimmunity, but the signaling mechanisms that regulate this checkpoint remain undefined. Here we characterize a previously unrecognized Ca(2+)-driven pathway for activation of the kinase Erk, which was proapoptotic and biochemically distinct from Erk activation induced by diacylglycerol (DAG). This pathway required protein kinase C-δ (PKC-δ) and the guanine nucleotide-exchange factor RasGRP and depended on the concentration of the Ca(2+) sensor STIM1, which controls the magnitude of Ca(2+) entry. Developmental regulation of these proteins was associated with selective activation of the pathway in B cells prone to negative selection. This checkpoint was impaired in PKC-δ-deficient mice, which developed B cell autoimmunity. Conversely, overexpression of STIM1 conferred a competitive disadvantage to developing B cells. Our findings establish Ca(2+)-dependent Erk signaling as a critical proapoptotic pathway that mediates the negative selection of B cells.
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Linfocitos B/inmunología , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Factores de Intercambio de Guanina Nucleótido/inmunología , Glicoproteínas de Membrana/inmunología , Proteína Quinasa C-delta/inmunología , Animales , Apoptosis/inmunología , Linfocitos B/enzimología , Canales de Calcio , Línea Celular , Activación Enzimática/inmunología , Citometría de Flujo , Regulación del Desarrollo de la Expresión Génica , Immunoblotting , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación/inmunología , Transducción de Señal , Molécula de Interacción Estromal 1RESUMEN
Survival of chronic lymphocytic leukemia (CLL) cells critically depends on the support of an adapted and therefore appropriate tumor microenvironment. Increasing evidence suggests that B-cell receptor-associated kinases such as protein kinase C-ß (PKCß) or Lyn kinase are essential for the formation of a microenvironment supporting leukemic growth. Here, we describe the impact of PKCß on the glucose metabolism in bone marrow stromal cells (BMSC) upon CLL contact. BMSC get activated by CLL contact expressing stromal PKCß that diminishes mitochondrial stress and apoptosis in CLL cells by stimulating glucose uptake. In BMSC, the upregulation of PKCß results in increased mitochondrial depolarization and leads to a metabolic switch toward oxidative phosphorylation. In addition, PKCß-deficient BMSC regulates the expression of Hnf1 promoting stromal insulin signaling after CLL contact. Our data suggest that targeting PKCß and the glucose metabolism of the leukemic niche could be a potential therapeutic strategy to overcome stroma-mediated drug resistance.
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Células de la Médula Ósea/metabolismo , Glucosa/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Proteína Quinasa C beta/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Comunicación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/patología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Proteína Quinasa C beta/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Microambiente Tumoral/efectos de los fármacosRESUMEN
BACKGROUND: The Protein kinase D3 (PKD3) has been implicated in signal transduction downstream of the T cell receptor (TCR). However, its role for the activation of primary T lymphocytes has not been elucidated so far. METHODS: Expression of PKD isoforms in primary murine T cells was determined by RT-PCR and SDS-Page. A germline PKD3-knockout mouse line was analyzed for its immune response to OVA/alum intraperitoneal immunization. Phenotyping of the T cell compartment ex vivo as well as upon stimulation in vitro was performed by flow cytometry. Additionally, cytokine expression was assessed by flow cytometry, RT-PCR and Luminex technology. RESULTS: PKD expression in T cells is modulated by TCR stimulation, leading to a rapid down-regulation on mRNA and on protein level. PKD3-deficient mice respond to immunization with enhanced T follicular helper cell generation. Furthermore, peripheral PKD3-deficient CD4+ T cells express more interleukin-2 than wild type CD4+ T cells upon TCR stimulation ex vivo. However, purified naïve CD4+ T cells do not differ in their phenotype upon differentiation in vitro from wild type T cells. Moreover, we observed a shift towards an effector/memory phenotype of splenic T cells at steady state, which might explain the contradictory results obtained with pan-T cells ex vivo and naïve-sorted T cells. CONCLUSION: While PKD3-deficiency in vivo in mice leads to a skewing of the T cell compartment towards a more activated phenotype, this kinase seems to be dispensable for naïve CD4+ T cell differentiation in vitro. Video Abstract.
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Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Linfocitos T , Animales , Linfocitos T CD4-Positivos , Ratones , Ratones Noqueados , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismoRESUMEN
Transforming growth-factor ß (TGFß) has been implicated in T helper 17 (Th17) cell biology and in triggering expression of interleukin-17A (IL-17A), which is a key Th17 cell cytokine. Deregulated TGFß receptor (TGFßR) signaling has been implicated in Th17-cell-mediated autoimmune pathogenesis. Nevertheless, the full molecular mechanisms involved in the activation of the TGFßR pathway in driving IL-17A expression remain unknown. Here, we identified protein kinase C α (PKCα) as a signaling intermediate specific to the Th17 cell subset in the activation of TGFßRI. We have shown that PKCα physically interacts and functionally cooperates with TGFßRI to promote robust SMAD2-3 activation. Furthermore, PKCα-deficient (Prkca(-/-)) cells demonstrated a defect in SMAD-dependent IL-2 suppression, as well as decreased STAT3 DNA binding within the Il17a promoter. Consistently, Prkca(-/-) cells failed to mount appropriate IL-17A, but not IL-17F, responses in vitro and were resistant to induction of Th17-cell-dependent experimental autoimmune encephalomyelitis in vivo.
Asunto(s)
Interleucina-17/metabolismo , Proteína Quinasa C-alfa/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Animales , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Regulación de la Expresión Génica , Interleucina-17/inmunología , Ratones , Ratones Noqueados , Glicoproteína Mielina-Oligodendrócito/efectos adversos , Fragmentos de Péptidos/efectos adversos , Proteína Quinasa C-alfa/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Proteínas Smad/metabolismo , Especificidad por SustratoRESUMEN
C-type lectin receptors (CLRs) that couple with the kinase Syk are major pattern recognition receptors for the activation of innate immunity and host defense. CLRs recognize fungi and other forms of microbial or sterile danger, and they induce inflammatory responses through the adaptor protein Card9. The mechanisms relaying CLR proximal signals to the core Card9 module are unknown. Here we demonstrated that protein kinase C-δ (PKCδ) was activated upon Dectin-1-Syk signaling, mediated phosphorylation of Card9 at Thr231, and was responsible for Card9-Bcl10 complex assembly and canonical NF-κB control. Prkcd(-/-) dendritic cells, but not those lacking PKCα, PKCß, or PKCθ, were defective in innate responses to Dectin-1, Dectin-2, or Mincle stimulation. Moreover, Candida albicans-induced cytokine production was blocked in Prkcd(-/-) cells, and Prkcd(-/-) mice were highly susceptible to fungal infection. Thus, PKCδ is an essential link between Syk activation and Card9 signaling for CLR-mediated innate immunity and host protection.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Dendríticas/enzimología , Células Dendríticas/inmunología , Inmunidad Innata , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lectinas Tipo C/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Proteínas Adaptadoras de Señalización CARD , Candida albicans/inmunología , Ratones , Ratones Noqueados , Proteína Quinasa C-delta/metabolismo , Transducción de Señal , Quinasa SykRESUMEN
BACKGROUND: The protein kinase C theta (PKCθ) has an important and non-redundant function downstream of the antigen receptor and co-receptor complex in T lymphocytes. PKCθ is not only essential for activation of NF-κB, AP-1 and NFAT and subsequent interleukin-2 expression, but also critical for positive selection and development of regulatory T lymphocytes in the thymus. Several domains regulate its activity, such as a pseudosubstrate sequence mediating an auto-inhibitory intramolecular interaction, the tandem C1 domains binding diacylglycerol, and phosphorylation at conserved tyrosine, threonine as well as serine residues throughout the whole length of the protein. To address the importance of the variable domain V1 at the very N-terminus, which is encoded by exon 2, a mutated version of PKCθ was analyzed for its ability to stimulate T lymphocyte activation. METHODS: T cell responses were analyzed with promoter luciferase reporter assays in Jurkat T cells transfected with PKCθ expression constructs. A mouse line expressing mutated instead of wild type PKCθ was analyzed in comparison to PKCθ-deficient and wild type mice for thymic development and T cell subsets by flow cytometry and T cell activation by quantitative RT-PCR, luminex analysis and flow cytometry. RESULTS: In cell lines, the exon 2-replacing mutation impaired the transactivation of interleukin-2 expression by constitutively active mutant form of PKCθ. Moreover, analysis of a newly generated exon 2-mutant mouse line (PKCθ-E2mut) revealed that the N-terminal replacement mutation results in an hypomorph mutant of PKCθ combined with reduced PKCθ protein levels in CD4+ T lymphocytes. Thus, PKCθ-dependent functions in T lymphocytes were affected resulting in impaired thymic development of single positive T lymphocytes in vivo. In particular, there was diminished generation of regulatory T lymphocytes. Furthermore, early activation responses such as interleukin-2 expression of CD4+ T lymphocytes were significantly reduced even though cell viability was not affected. Thus, PKCθ-E2mut mice show a phenotype similar to conventional PKCθ-deficient mice. CONCLUSION: Taken together, PKCθ-E2mut mice show a phenotype similar to conventional PKCθ-deficient mice. Both our in vitro T cell culture experiments and ex vivo analyses of a PKCθ-E2-mutant mouse line independently validate the importance of PKCθ downstream of the antigen-receptor complex for activation of CD4+ T lymphocytes.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Activación de Linfocitos , Mutación , Proteína Quinasa C-theta/genética , Animales , Células HEK293 , Humanos , Células Jurkat , Ratones , Ratones Endogámicos C57BL , Fenotipo , Proteína Quinasa C-theta/metabolismoRESUMEN
BACKGROUND: Protein kinase C θ has been established as an important signaling intermediate in T-effector-cell activation and survival pathways by controlling activity of the key transcription factors NF-κB and NFAT. Previous studies identified an activation-induced auto-phosphorylation site at Thr-219, located between the tandem C1 domains of the regulatory fragment in PKCθ, as a structural requirement for its correct membrane translocation and the subsequent transactivation of downstream signals leading to IL-2 production in a human T cell line. METHODS: The present work aimed to define the role of this phosphorylation switch on PKCθ in a physiological context through a homozygous T219A knockin mouse strain. T cell activation was analyzed by H3-thymidine uptake (proliferative response), qRT-PCR and luminex measurements (cytokine production). NFAT and NF-κB transactivation responses were estimated by Gel mobility shift and Alpha Screen assays. Frequencies of T cell subsets were analyzed by flow cytometry. RESULTS: Despite a normal T cell development, in vitro activated effector T cells clearly revealed a requirement of Thr-219 phosphorylation site on PKCθ for a transactivation of NF-κB and NFAT transcription factors and, subsequently, robust IL-2 and IFN-γ expression. CONCLUSION: This phenotype is reminiscent of the PKCθ knockout T cells, physiologically validating that this (p) Thr-219 auto-phosphorylation site indeed critically regulates PKCθ function in primary mouse T cells.
Asunto(s)
Técnicas de Sustitución del Gen , Fenotipo , Proteína Quinasa C-theta/genética , Proteína Quinasa C-theta/metabolismo , Animales , Citocinas/metabolismo , Ratones , Linfocitos T/citología , Linfocitos T/metabolismoRESUMEN
Dysfunctional mitochondria contribute to the development of many diseases and pathological conditions through the excessive production of reactive oxygen species (ROS), and, where studied, ablation of p66Shc (p66) was beneficial. p66 translocates to the mitochondria and oxidizes cytochrome c to yield H2O2, which in turn initiates cell death. PKCß-mediated phosphorylation of serine 36 in p66 has been implicated as a key regulatory step preceding mitochondrial translocation, ROS production, and cell death, and PKCß thus may provide a target for therapeutic intervention. We performed a reassessment of PKCß regulation of the oxidoreductase activity of p66. Although our experiments did not substantiate Ser36 phosphorylation by PKCß, they instead provided evidence for Ser139 and Ser213 as PKCß phosphorylation sites regulating the pro-oxidant and pro-apoptotic function of p66. Mutation of another predicted PKCß phosphorylation site also located in the phosphotyrosine binding domain, threonine 206, had no phenotype. Intriguingly, p66 with Thr206 and Ser213 mutated to glutamic acid showed a gain-of-function phenotype with significantly increased ROS production and cell death induction. Taken together, these data argue for a complex mechanism of PKCß-dependent regulation of p66 activation involving Ser139 and a motif surrounding Ser213.
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Proteína Quinasa C beta/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/metabolismo , Células 3T3 , Animales , Muerte Celular , Eliminación de Gen , Células HEK293 , Humanos , Peróxido de Hidrógeno/metabolismo , Ratones , Mitocondrias/genética , Mitocondrias/metabolismo , Modelos Moleculares , Estrés Oxidativo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Fosforilación , Mutación Puntual , Proteína Quinasa C beta/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Serina/genética , Serina/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/genéticaRESUMEN
Blood vessel polarization in the apical-basal axis is important for directed secretion of proteins and lumen formation; yet, when and how polarization occurs in the context of angiogenic sprouting is not well understood. Here, we describe a novel topology for endothelial cells at the tip of angiogenic sprouts in several mammalian vascular beds. Two cells that extend filopodia and have significant overlap in space and time were present at vessel tips, both in vitro and in vivo. The cell overlap is more extensive than predicted for tip cell switching, and it sets up a longitudinal cell-cell border that is a site of apical polarization and lumen formation, presumably via a cord-hollowing mechanism. The extent of cell overlap at the tip is reduced in mice lacking aPKCζ, and this is accompanied by reduced distal extension of both the apical border and patent lumens. Thus, at least two polarized cells occupy the distal tip of blood vessel sprouts, and topology, polarization and lumenization along the longitudinal border of these cells are influenced by aPKCζ.
Asunto(s)
Vasos Sanguíneos/citología , Vasos Sanguíneos/embriología , Células Endoteliales/citología , Proteína Quinasa C/metabolismo , Animales , Femenino , Masculino , Ratones , Microscopía ConfocalRESUMEN
Integrins are transmembrane receptors composed of one α subunit and one ß subunit and are involved in cellular growth, differentiation, and apoptosis. The collagen-binding integrins α1ß1 and α2ß1 have been shown to regulate wound and tumor vascularization by different mechanisms. In this study, we assessed wound and tumor vascularization in mice with genetic ablation of both integrin subunits α1 and α2, which resulted in loss of integrins α1ß1 and α2ß1. Wound angiogenesis was investigated in excisional wounds that were inflicted on the back skin of control and mice lacking integrin α1ß1 and α2ß1. Mutant mice displayed reduced wound angiogenesis, which correlated with decreased macrophage numbers at 3 and 7 days after injury, and showed significantly attenuated vascularization of sponge implants. Angiogenesis induced by tumors arising from intradermal injection of B16 F1 melanoma cells was also reduced in comparison to controls 7 days after injection. This reduction in angiogenesis correlated with increased levels and activity of circulating matrix metalloproteinase 9 and elevated angiostatin levels in plasma of mutant mice, which reduced endothelial cell proliferation. Ex vivo mutant aortic ring explants developed significantly fewer and thinner aortic sprouts with fewer branch points than controls because of impaired endothelial cell proliferation. In conclusion, the loss of integrins α1ß1 and α2ß1 in mice results in reduced wound and tumor angiogenesis by cell-autonomous and extrinsic mechanisms.
Asunto(s)
Integrina alfa1beta1/metabolismo , Integrina alfa2beta1/metabolismo , Neoplasias/irrigación sanguínea , Cicatrización de Heridas/fisiología , Heridas y Lesiones/patología , Animales , Modelos Animales de Enfermedad , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Integrina alfa1beta1/genética , Integrina alfa2beta1/genética , Melanoma/irrigación sanguínea , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias/etiología , Neoplasias/patología , Neovascularización Patológica , Piel/irrigación sanguínea , Piel/lesiones , Piel/metabolismo , Piel/patología , Neoplasias Cutáneas/irrigación sanguínea , Heridas y Lesiones/etiologíaRESUMEN
We previously defined that the mitochondria-localized PKCδ signaling complex stimulates the conversion of pyruvate to acetyl-coenzyme A by the pyruvate dehydrogenase complex. We demonstrated in vitro and ex vivo that retinol supplementation enhances ATP synthesis in the presence of the PKCδ signalosome. Here, we tested in vivo if a persistent oversupply of retinol would further impair glucose metabolism in a mouse model of diet-induced insulin resistance. We crossed mice overexpressing human retinol-binding protein (hRBP) under the muscle creatine kinase (MCK) promoter (MCKhRBP) with the PKCδ(-/-) strain to generate mice with a different status of the PKCδ signalosome and retinoid levels. Mice with a functional PKCδ signalosome and elevated retinoid levels (PKCδ(+/+)hRBP) developed the most advanced stage of insulin resistance. In contrast, elevation of retinoid levels in mice with inactive PKCδ did not affect remarkably their metabolism, resulting in phenotypic similarity between PKCδ(-/-)hRBP and PKCδ(-/-) mice. Therefore, in addition to the well-defined role of PKCδ in the etiology of metabolic syndrome, we present a novel PKCδ signaling pathway that requires retinol as a metabolic cofactor and is involved in the regulation of fuel utilization in mitochondria. The distinct role in whole-body energy homeostasis establishes the PKCδ signalosome as a promising target for therapeutic intervention in metabolic disorders.
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Resistencia a la Insulina/fisiología , Obesidad/metabolismo , Proteína Quinasa C-delta/metabolismo , Vitamina A/metabolismo , Animales , Dieta/efectos adversos , Modelos Animales de Enfermedad , Glucosa/metabolismo , Homeostasis/fisiología , Humanos , Masculino , Síndrome Metabólico/metabolismo , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Regiones Promotoras Genéticas/fisiología , Complejo Piruvato Deshidrogenasa/metabolismo , Retinoides/metabolismo , Proteínas de Unión al Retinol/metabolismo , Transducción de Señal/fisiologíaRESUMEN
In this study, we used gene targeting in mice to identify the in vivo functions of PKD1. In addition to phenotypically characterizing the resulting knock-out animals, we also used mouse embryonic fibroblasts to investigate the associated signaling pathways in detail. This study is the first to use genetic deletion to reveal that PKD1 is a key regulator involved in determining the threshold of mitochondrial depolarization that leads to the production of reactive oxygen species. In addition, we also provide clear evidence that PKCδ is upstream of PKD1 in this process and acts as the activating kinase of PKD1. Therefore, our in vivo data indicate that PKD1 functions not only in the context of aging but also during nutrient deprivation, which occurs during specific phases of tumor growth.
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Fibroblastos/metabolismo , Potencial de la Membrana Mitocondrial/genética , Mitocondrias/metabolismo , Proteína Quinasa C-delta/genética , Transducción de Señal/genética , Canales Catiónicos TRPP/genética , Animales , Embrión de Mamíferos , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Regulación de la Expresión Génica , Peróxido de Hidrógeno/farmacología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Estrés Oxidativo , Cultivo Primario de Células , Proteína Quinasa C-delta/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Canales Catiónicos TRPP/deficienciaRESUMEN
All class I phosphoinositide 3-kinases (PI3Ks) associate tightly with regulatory subunits through interactions that have been thought to be constitutive. PI3Kγ is key to the regulation of immune cell responses activated by G protein-coupled receptors (GPCRs). Remarkably we find that PKCß phosphorylates Ser582 in the helical domain of the PI3Kγ catalytic subunit p110γ in response to clustering of the high-affinity IgE receptor (FcεRI) and/or store-operated Ca²âº- influx in mast cells. Phosphorylation of p110γ correlates with the release of the p84 PI3Kγ adapter subunit from the p84-p110γ complex. Ser582 phospho-mimicking mutants show increased p110γ activity and a reduced binding to the p84 adapter subunit. As functional p84-p110γ is key to GPCR-mediated p110γ signaling, this suggests that PKCß-mediated p110γ phosphorylation disconnects PI3Kγ from its canonical inputs from trimeric G proteins, and enables p110γ to operate downstream of Ca²âº and PKCß. Hydrogen deuterium exchange mass spectrometry shows that the p84 adaptor subunit interacts with the p110γ helical domain, and reveals an unexpected mechanism of PI3Kγ regulation. Our data show that the interaction of p110γ with its adapter subunit is vulnerable to phosphorylation, and outline a novel level of PI3K control.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Proteína Quinasa C beta/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Calcio/metabolismo , Dominio Catalítico , Degranulación de la Célula/efectos de los fármacos , Fosfatidilinositol 3-Quinasa Clase Ib/química , Activación Enzimática/efectos de los fármacos , Estabilidad de Enzimas/efectos de los fármacos , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Mastocitos/efectos de los fármacos , Mastocitos/enzimología , Mastocitos/fisiología , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Modelos Moleculares , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Transducción de Señal/efectos de los fármacos , Tapsigargina/farmacologíaRESUMEN
Studies showing reduced PKCζ expression or enzymatic activity in different types of human cancers support the clinical relevance of PKCζ as a tumor suppressor. However, the in vivo role of PKCζ and its mechanisms of action in prostate cancer remain unclear. Here we demonstrate that the genetic inactivation of PKCζ in mice results in invasive prostate carcinoma in vivo in the context of phosphatase and tensin homolog deficiency. Bioinformatic analysis of human prostate cancer gene-expression sets revealed increased c-Myc transcriptional activity in PKCζ-inactive cells, which correlated with increased cell growth, invasion, and metastasis. Interestingly, PKCζ knockdown or the overexpression of a kinase-inactive mutant resulted in enhanced cell proliferation and invasion in vitro through increased c-Myc mRNA and protein levels and decreased Ser-373 phosphorylation of c-Myc. Analysis of prostate cancer samples demonstrated increased expression and decreased phosphorylation of c-Myc at Ser-373 in PKCζ knockout tumors. In vivo xenograft studies revealed that c-Myc phosphorylation by PKCζ is a critical event in the control of metastasis. Collectively, these results establish PKCζ as an important tumor suppressor and regulator of c-Myc function in prostate cancer.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Metástasis de la Neoplasia/fisiopatología , Neoplasias de la Próstata/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Animales , Western Blotting , Biología Computacional , Luciferasas , Masculino , Ratones , Análisis por Micromatrices , Fosfohidrolasa PTEN/metabolismo , Fosforilación , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
BACKGROUND: Itch-producing compounds stimulate receptors expressed on small diameter fibers that innervate the skin. Many of the currently known pruritogen receptors are Gq Protein-Coupled Receptors (GqPCR), which activate Protein Kinase C (PKC). Specific isoforms of PKC have been previously shown to perform selective functions; however, the roles of PKC isoforms in regulating itch remain unclear. In this study, we investigated the novel PKC isoform PKCδ as an intracellular modulator of itch signaling in response to histamine and the non-histaminergic pruritogens chloroquine and ß-alanine. RESULTS: Behavioral experiments indicate that PKCδ knock-out (KO) mice have a 40% reduction in histamine-induced scratching when compared to their wild type littermates. On the other hand, there were no differences between the two groups in scratching induced by the MRGPR agonists chloroquine or ß-alanine. PKCδ was present in small diameter dorsal root ganglion (DRG) neurons. Of PKCδ-expressing neurons, 55% also stained for the non-peptidergic marker IB4, while a smaller percentage (15%) expressed the peptidergic marker CGRP. Twenty-nine percent of PKCδ-expressing neurons also expressed TRPV1. Calcium imaging studies of acutely dissociated DRG neurons from PKCδ-KO mice show a 40% reduction in the total number of neurons responsive to histamine. In contrast, there was no difference in the number of capsaicin-responsive neurons between KO and WT animals. Acute pharmacological inhibition of PKCδ with an isoform-specific peptide inhibitor (δV1-1) also significantly reduced the number of histamine-responsive sensory neurons. CONCLUSIONS: Our findings indicate that PKCδ plays a role in mediating histamine-induced itch, but may be dispensable for chloroquine- and ß-alanine-induced itch.
Asunto(s)
Regulación hacia Abajo/genética , Histamina/efectos adversos , Proteína Quinasa C-alfa/metabolismo , Prurito/inducido químicamente , Prurito/metabolismo , Animales , Péptido Relacionado con Gen de Calcitonina , Calcio/metabolismo , Capsaicina/farmacología , Células Cultivadas , Cloroquina/efectos adversos , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Ganglios Espinales/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa C-alfa/genética , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , beta-Alanina/efectos adversosRESUMEN
The Wnt signaling network, an ancient signaling system governing ontogeny and homeostatic processes, has recently been identified to exert immunoregulatory functions in a variety of inflammatory and infectious disease settings including tuberculosis. In this study, we show that Wnt6 is expressed in granulomatous lesions in the lung of Mycobacterium tuberculosis-infected mice. We identified foamy macrophage-like cells as the primary source of Wnt6 in the infected lung and uncovered a TLR-MyD88-NF-κB-dependent mode of induction in bone marrow-derived macrophages. Analysis of Wnt6-induced signal transduction revealed a pertussis toxin-sensitive, ERK-mediated, but ß-catenin-independent induction of c-Myc, a master regulator of cell proliferation. Increased Ki-67 mRNA expression levels and enhanced thymidine incorporation in Wnt6-treated macrophage cultures demonstrate a proliferation-promoting effect on murine macrophages. Further functional studies in M. tuberculosis-infected macrophages using Wnt6 conditioned medium and Wnt6-deficient macrophages uncovered a Wnt6-dependent induction of macrophage Arginase-1 and downregulation of TNF-α. This identifies Wnt6 as a novel factor driving macrophage polarization toward an M2-like phenotype. Taken together, these findings point to an unexpected role for Wnt6 in macrophage differentiation in the M. tuberculosis-infected lung.